052808 asr ch8 asr ecotox phase1

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September 2005

PHASE 1 REPORT

Screening-Level Method DevelopmentPreliminary Investigation of the Ecotoxicological Effects of

Recovered ASR Water on Receiving Aquatic Ecosystems

Using Pilot Project Groundwater and/or Recovered Water

Prepared by: Isabel C. Johnson

Golder Associates Inc.

6241 NW 23rd

Street, Suite 500

Gainesville, FL 32653

U.S. Army Corps of Engineers South Florida WaterJacksonville District Management District

SFWMD Contract C-C13401P-WO07

In cooperation with PBS & J

COMPREHENSIVE EVERGLADESRESTORATION PLAN



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TABLE OF CONTENTS

Golder Associates

Executive Summary................................................................................................................................ 1

SECTION PAGE

1.0 INTRODUCTION..................................................................................................................... 4

2.0 Background ...............................................................................................................................5

2.1 Ecotoxicology Project – Phase 1 .................................................................................. 5

2.2 Ecotoxicology Project – Phase 2 .................................................................................. 6

3.0 Phase 1 Study ............................................................................................................................ 7

3.1 Project Initiation........................................................................................................... 7

3.2 Project Background...................................................................................................... 7

3.3 Scope of Work.............................................................................................................. 7

3.3.1 Task 1 – Program Management....................................................................... 8 3.3.2 Task 2 – Acquisition and Storage of Surface Water ....................................... 9 3.3.3 Task 3 – Acquisition and Storage of Groundwater ....................................... 10 3.3.4 Task 4 – Range-finding Toxicity Tests of Mixtures ..................................... 10 3.3.5 Task 5 – Evaluation of the Appropriateness of Using Standard Test

Organisms...................................................................................................... 10 4.0 Range-finding Tests (Task 4) .................................................................................................. 12

4.1 Aquatic Toxicity Tests ............................................................................................... 12

4.2 Samples Tested........................................................................................................... 13

4.3 Tests Conducted ......................................................................................................... 14

4.4 Test Results ................................................................................................................ 14

4.5 Range-finding Test Conclusions ................................................................................ 17

5.0 Screening Study – Evaluation of Standard Test Organisms.................................................... 18

5.1 Aquatic Toxicity Scoping Studies.............................................................................. 18

5.2 Samples Tested........................................................................................................... 19

5.3 Aquatic Toxicity Tests ............................................................................................... 19

5.4 Test Results ................................................................................................................ 19

5.4.1 Ceriodaphnia dubia ...................................................................................... 19

5.4.2 Daphnia magna ............................................................................................. 21 5.4.3 Pimephales promelas .................................................................................... 22 5.4.4 FETAX.......................................................................................................... 23

5.5 Alternate Dilution Series ............................................................................................ 24

5.6 Water Analysis ........................................................................................................... 24

5.7 Aquatic Scoping Toxicity Study Conclusions............................................................ 25

6.0 Bioconcentration Scoping Study............................................................................................. 26



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6.1 Bioconcentration Methods ......................................................................................... 26

6.2 Samples Tested........................................................................................................... 26

6.3 Tests Conducted ......................................................................................................... 26

6.4 Test Species................................................................................................................ 27

6.5 Treatments.................................................................................................................. 28

6.6 Bioconcentration Tests............................................................................................... 28

6.6.1 Feeding.......................................................................................................... 29 6.6.2 Test Completion ............................................................................................ 29 6.6.3 Statistical Analysis ........................................................................................ 29

6.7 Test Results ................................................................................................................ 30

6.7.1 Dry Season Results........................................................................................ 30 6.7.2 “Wet Season” Results.................................................................................... 32

6.8 Trace Metals and Radionuclides in Waters – Discussion........................................... 33 6.9 Bioconcentration Study Conclusions ......................................................................... 35

REFERENCES..................................................................................................................................6-33

Appendices

APPENDIX A BENCH-SCALE ASR TREATMENT SYSTEM

APPENDIX B CALOOSAHATCHEE RIVER ASR PILOT PROJECT – WATER QUALITY

SUMMARY

APPENDIX C RESPONSE TO COMMENTS



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LIST OF TABLES

Table 1 Range-finding – 7-day Chronic Daphnid, Ceriodaphnia dubia, Survival and

Reproduction Test, EPA Test Method 1002.0

Table 2 Range-finding 7-day Chronic Fathead Minnow, Pimephales promelas, Embryo-larval

Survival and Teratogenicity Test, EPA Method 1001.0

Table 3 Range-finding 21-day Daphnia magna Life-cycle Toxicity Test

Table 4 Range-finding Frog Embryo Teratogenesis Assay-Xenopus (FETAX)

Table 5 “Dry Season” – 7 day Chronic Daphnid, Ceriodaphnia dubia, Survival and

Reproduction, Test Method 1002.0

Table 6 “Wet Season” – 7-day Chronic Daphnid, Ceriodaphnia dubia, Survival and


Table 7 “Dry Season” – Daphnia magna Life-cycle Toxicity Test

Table 8 “Wet Season” – Daphnia magna Life-cycle Toxicity Test

Table 9 “Dry Season” – 7-day Chronic Fathead Minnow, Pimephales promelas,

Embryo-larval Survival and Teratogenicity, Test Method 1001.0

Table 10 “Wet Season” – 7-day Chronic Fathead Minnow, Pimephales promelas, Embryo-

larval Survival and Teratogenicity, Test Method 1001.0

Table 11 “Dry Season” – Frog Embryo Teratogenesis Assay- Xenopus (FETAX)

Table 12 “Wet Season” – Frog Embryo Teratogenesis Assay- Xenopus (FETAX)

Table 13 “Dry Season” – Trace Metals Results in Water Samples

Table 14 “Wet Season” - Trace Metals Results in Water

Table 15 “Dry Season” – Conductivity (April 12 through May 3, 2005)

Table 16 “Wet Season” – Conductivity (June 16 through July 7, 2005)

Table 17 “Dry Season” – pH (April 12 through May 3, 2005

Table 18 “Wet Season” – pH (June 16 through July 7, 2005)

Table 19 “Dry Season” – Radium Results in Water

Table 20 “Wet Season” – Radium Results in Water

Table 21 Trace Metals Results in Food Supply (Algae and Fish Feed)

Table 22 “Dry Season” – Trace Metals Background Results in Tissue Samples

Table 23 “Dry Season” – Bioconcentration – Fish Tissue Post-Exposure Results

Table 24 “Dry Season” – Bioconcentration – Mussel Tissue Post-Exposure Results

Table 25 “Dry Season” – Radium Results in Mussel Tissues

Table 26 Background Radium in Mussel Tissues



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Table 27 “Wet Season” – Trace Metals Background Results in Tissue Samples

Table 28 “Wet Season” – Bioconcentration – Fish Tissue Results

Table 29 “Wet Season” – Bioconcentration – Mussel Tissue Results

Table 30 “Wet Season” – Radium Results in Tissues

LIST OF FIGURES

Figure 1 Flow Chart for Preliminary Investigation of Ecotoxicological Effects of Recovered

ASR Water

Figure 2 Photograph of ASR Bench-scale Treatment System

Figure 3 UV Sterilization and pH Adjustment Systems

Figure 4 Caloosahatchee Pilot Project Site Facility Layout Showing Sampling Locations

Figure 5 Background Surface Water Sampling Location

Figure 6 Floridian Aquifer Well (ASR-1) at the Caloosahatchee Pilot Project Site

Figure 7 Fathead Minnow and Ceriodaphnia dubia Chronic Test

Figure 8 Frog Embryo Teratogenesis Assay – Xenopus (FETAX)

Figure 9 Modified Bench-scale Sand Filter

Figure 10 Test Species Used in the Bioconcentration Scoping Study

Figure 11 Bioconcentration Study Static-renewal (Continuous Flow) Exposure System



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EXECUTIVE SUMMARY

This report summarizes the work conducted under the “Preliminary Investigation of the

Ecotoxicological Effects of Recovered ASR Water on Receiving Water Ecosystems Using Pilot

Project Groundwater and/or Recovered Water: Phase 1 – Screening-Level Method Development”

project (Ecotoxicology Project). The need for this study is documented in the South Florida Water

Management District’s (SFWMD) and U.S. Army Corps of Engineers (USACE) Project Management

Plan (PMP) for the ASR Regional Study (ASRRS). The aquatic toxicity tests and bioconcentration

studies described in this report are based on U.S. Environmental Protection Agency (EPA) methods

and Florida Department of Environmental Protection (FDEP) guidance. The results of this study will

be used to evaluate the need for additional method development as well as to define the final

screening-level testing of ASR pilot projects once the ASR wells are functional.

Ultimately, these ecotoxicological methods and relevant physical, chemical, and biological data and

modeling results generated from other water quality and ecological studies recommended in the

ASRRS PMP, may be used in the preparation of a regional-scale probabilistic ecological risk

assessment for the ASR projects.

The overall project objective was to develop a set of screening-level aquatic toxicity tests methods to

evaluate the toxicity and/or bioconcentration potential of ASR-stored waters that will be discharged

into surface waters as part of the ASRRS. A set of assays were selected and evaluated during Phase 1

of the Ecotoxicology Project. The methods were applied during both the “dry” and “wet” seasons,

representing conditions during periods of low precipitation and high precipitation, respectively.

Chronic toxicity studies using sensitive standard fish, frog, and invertebrate species were conducted;

bioconcentration studies using fish and freshwater mussels were also evaluated.

Phase 1, Tasks 1 through 5, was conducted to address concerns that the use of standard aquatic plant

and animal test organisms and methods in the evaluation of ASR waters could result in the standard

test organisms exhibiting toxic responses to the ASR-treated water, as well as the original background

surface water, and/or the groundwater as compared to laboratory control waters. The source of this

potential toxicity could be from contaminants present in the waters tested, high ionic content, and/or

ionic imbalance of these waters (in particular groundwater). Therefore, the first step in Phase 1 was

to test selected standard aquatic species to evaluate their response and applicability to the ASR

conditions. To address these toxicity concerns, a scoping toxicity study of the response of standard



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test organisms, conditions, and endpoints to background surface water, “treated” surface waters, and

untreated groundwater collected from the Caloosahatchee ASR Pilot Project site was conducted.

Similar toxicity concerns existed regarding the potential bioconcentration potential of ASR waters

and the use of standard bioconcentration protocols and test species. A scoping bioconcentration study

of the response of standard test organisms, conditions, and exposure regimens to background surface

water, “treated” surface water, and untreated groundwater was also conducted. In particular, a

concern existed that metals and radionuclide parameters present in groundwater could be released to

surface waters in sufficiently high concentrations resulting in significant bioconcentration in aquatic

organisms.

Aquatic Toxicity Scoping Studies

Aquatic toxicity tests and method development were completed successfully. The bench-scale ASR

“treated” water did not have any quantifiable toxic effect on survival, reproduction or embryological

development on any test species or endpoint measured.

The primary treatment that affected the species tested under these short chronic assays was the full-

strength groundwater. However, since there is no scenario in the CERP ASR program where

100-percent groundwater is discharged from an ASR well, this observation of toxicity is benign and

not unexpected (groundwaters typically have higher conductivity than surface waters and this can

affect the survival and/or reproduction of aquatic organisms through ionic imbalance) . In all cases,

once the groundwater was diluted with 50-percent bench-scale “treated” water, no effect was

quantifiable for any species on any sample tested. The bench-scale “treated” water did not have any

effect on any species tested. All tests met the guidelines required by the cited methods and were

acceptable. No changes are recommended to this battery of tests.

Bioconcentration Scoping Studies

Trace metals and radium (226/228) did not bioconcentrate in fish or mussel tissues during the two 28-

day bioconcentration studies conducted during the “dry” and “wet” seasons. It should be noted that

the relatively low environmental background concentrations for these parameters in the water samples

to some extent inhibited our ability to quantify the bioconcentration phenomenon in the test species.

The methods used met all criteria applicable to these tests.



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The mussel species used in this study, E. buckleyi, was a good choice for the bioconcentration studies,

as it adapted well to laboratory conditions, accepted an algal diet, and was easily handled for the tests.

Given the desire to test not only standard laboratory species but also species found in Florida habitats,

the choice of E. buckleyi for the subject bioconcentration testing and the results proved to be a robust

approach.

If bioconcentration studies are conducted as part of future ASR project evaluations, it is

recommended that these tests be conducted onsite using flow-through test conditions or in situ

exposures. Both of the species used in the development of the bioconcentration protocols are

acceptable and can be used in future onsite bioconcentration studies. Of the two, the mussel species

would lend itself to in situ cage studies exposing them to the background surface water and the ASR

discharge, thus allowing a direct comparison of background and “treated” water metal and

radionuclide bioavailability. Should mesocosm studies be considered as part of future ASR projects,

either fish or mussel species used in the current study would be suitable for inclusion for the

evaluation of potential trace metal and/or radionuclide bioconcentration.



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1.0 INTRODUCTION

The ecotoxicological effects of various mixtures of surface water and native groundwater collected

from the Caloosahatchee Aquifer Storage and Recovery (ASR) Pilot Project area were evaluated

through this preliminary investigation. This report summarizes the work conducted under the

“Preliminary Investigation of the Ecotoxicological Effects of Recovered ASR Water on Receiving

Water Ecosystems Using Pilot Project Groundwater and/or Recovered Water: Phase 1 – Screening-

Level Method Development” project (Ecotoxicology Project). Figure 1 summarizes the overall

ecotoxicological program as envisioned at the onset of Phase 1.

The need for this study is documented in the South Florida Water Management District’s (SFWMD)

and U.S. Army Corps of Engineers (USACE) Project Management Plan (PMP) for the ASR Regional

Study (ASRRS). The aquatic toxicity tests and bioconcentration studies described in this report are

based on U.S. Environmental Protection Agency (EPA) methods and Florida Department of

Environmental Protection (FDEP) guidance. The results of this study will be used to evaluate the

need for additional method development (Phase 2) as well as to define the final screening-level

testing of ASR pilot projects (Phase 3) once the ASR wells are functional.

Ultimately, these studies and relevant physical, chemical, and biological data and modeling results

generated from other water quality and ecological studies recommended in the ASRRS PMP, may be

used in the preparation of a regional-scale probabilistic ecological risk assessment for the ASR

projects.

The overall project objective was to develop a set of screening-level aquatic toxicity tests to evaluate

the toxicity and/or bioconcentration potential of ASR-stored waters that will be discharged into

surface waters as part of the ASRRS. A set of assays were selected and evaluated during Phase 1 of

the Ecotoxicology Project. Studies were conducted for both the “dry” and “wet” seasons,

representing conditions during periods of low precipitation and high precipitation, respectively.

Chronic toxicity studies using sensitive standard fish, frog, and invertebrate species were conducted;

the Phase 1 studies also included bioconcentration studies using fish and freshwater mussels.



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2.0 BACKGROUND

This project used the Caloosahatchee ASR Pilot Project pre-treatment system as the model for this

evaluation. This system design includes surface water treatment [i.e., filtration, ultraviolet (UV)

disinfection, and pH adjustment] prior to recharge into the Floridan aquifer system via an ASR well

for storage and later use.

2.1 Ecotoxicology Project – Phase 1

Phase 1, Tasks 1 through 5, was conducted to address concerns that the use of standard aquatic plant

and animal test organisms and methods in the evaluation of ASR waters could result in the standard

test organisms exhibiting toxic responses to the ASR treated water, as well as the original background

surface water, and/or the groundwater as compared to laboratory control waters. The source of this

potential toxicity could be from contaminants present in the waters tested, high ionic content, and/or

ionic imbalance of these waters (in particular groundwater). Therefore, the first step in Phase 1 was

to test selected standard test species to evaluate their response and applicability to the ASR

conditions. To address these toxicity concerns, a scoping toxicity study of the response of standard

test organisms, conditions, and endpoints to background surface water, “treated” surface waters, and

untreated groundwater collected from the Caloosahatchee ASR Pilot Project site was conducted

(Phase 1). This scoping toxicity study included both range-finding and definitive toxicity tests.

Similar toxicity concerns existed regarding the potential bioconcentration potential of ASR waters

and the use of standard bioconcentration protocols and test species. A scoping bioconcentration study

of the response of standard test organisms, conditions, and exposure regimens to background surface

water, “treated” surface water, and untreated groundwater was also conducted. The bioconcentration

concerns included that the uptake and/or depuration kinetics for these test conditions could result in

statistically significant differences between these treatments and the laboratory controls. In

particular, a concern existed that metals and radionuclide parameters present in groundwater could be

released to surface waters in sufficiently high concentrations resulting in significant bioconcentration

in aquatic organisms. These studies were conducted as part of Phase 1



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2.2 Ecotoxicology Project – Phase 2

Phase 2 of this project, and as originally identified in the PMP for the ASRRS, is designed to evaluate

the use of resident organisms for toxicity testing as well as acclimation procedures for standard

organisms should the standard organisms used in Phase 1 prove inadequate (Figure 1). The results of

the Phase 1 study will be used in conjunction with future ecotoxicological testing at the ASR Pilot

Project sites to determine, the need and scope of Phase 2 activities.



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3.0 PHASE 1 STUDY

3.1 Project Initiation

Phase 1 of the Ecotoxicology Project was initiated on August 25, 2004, with a meeting at the

SFWMD Headquarters in West Palm Beach, Florida. Meeting participants included representatives

of the SFWMD, FDEP, USACE, PBS&J, Hydrosphere Research, and Golder Associates Inc.

(Golder).

3.2 Project Background

The ASR pilot projects provide site-specific field data to assist in addressing issues raised regarding

the regional ASR program associated with the Comprehensive Everglades Restoration Plan (CERP).

This Phase 1 project used Floridan aquifer system groundwater from an existing exploratory well for

toxicological testing, along with source water supplies. At the time that this study was conducted,

ASR “treated” water was not available; therefore, bench-scale ASR water treatment for samples to be

tested mimicked the actual processes that will be taking place at the ASR Pilot Projects.

3.3 Scope of Work

The objective of Phase 1 of the Ecotoxicology Project was to develop screening-level methods for the

evaluation of the potential ecotoxicological effects of various mixtures of treated surface water and

native groundwater collected from the Caloosahatchee ASR Pilot Project area. Through this phase,

aquatic toxicology and bioconcentration protocols were selected and developed for the evaluation of

future ASR Pilot projects.

Figure 1 illustrates the phases of the overall project, as well as the five project tasks comprising Phase

1 of this project.

• Task 1 – Project Management

• Task 2 – Acquisition and Storage of Surface Water

• Task 3 – Acquisition and Storage of Groundwater

•

Task 4 – Range-finding Toxicity Tests of Mixtures

• Task 5 – Evaluation of the Appropriateness of Using Standard Test Organisms



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3.3.1

Task 1 – Program Management

Golder was selected by the SFWMD to lead this project, with peer review provided by PBS&J. The

following is a list of individuals involved in this project and their roles:

•

SFWMD:

Peter Kwiatkowski – Project Manager

Richard Pfeuffer – Project technical review

Larry Fink – Scope of Work Development

• Consultants:

Jon Shaw -- Project Manager, Golder

Isabel Johnson – Project Director and Principal Investigator, Golder

Michael Michaeu -- Contract Manager, PBS&J

Dennis Logan – QA/QC, PBS&J

• Laboratories:

Hydrosphere Research – aquatic toxicology

Fort Environmental Laboratories – frog assay (FETAX)

Frontier Geosciences – metal analysis

Elab, Inc. – radium analysis

• FDEP:

David Whiting – Project technical review

Jose Calas – Project technical review

• USACE:

Mark Shafer – Project technical review

3.3.2

Task 2 – Acquisition and Storage of Surface Water

“Treated” Surface Water

“Treated” water from the Caloosahatchee site was not available at the time this project was

conducted; therefore, with SFWMD approval, a bench-scale ASR treatment system was outlined by

Golder, approved by the SFWMD, and built at Hydrosphere Research. The specific details of the



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treatment system are presented in a letter from Golder to the SFWMD, dated December 10, 2004

(Appendix A).

The bench-scale treatment system (Figure 2) was modeled after the Caloosahatchee ASR Pilot Project

facility. The three main elements of the bench-scale system are:

• Sand filtration,

•

Ultraviolet (UV) desinfection, and

• pH adjustment.

The treatment of surface water was conducted in batch mode. The surface water sample was first

filtered through a column of sand, then passed through a UV chamber, and finally the pH was

adjusted. The sand filter was constructed using 6-inch PVC pipe and was filled with 39 inches of

sediment collected from the Caloosahatchee ASR Pilot Project site. A peristaltic pump supplied

surface water to the sand column at a flow rate of 500-milliters per minute (mL/min).

The sand column-filtered water sample was then pumped through an 8-watt UV disinfection chamber

(Figure 3a) using a second peristaltic pump assembly at a rate of 1 liter per minute (L/min). This

batch of treated sample was then pH-adjusted using carbon dioxide gas to a pH of 6.6 to 7.1 Standard

Units (SU) (Figure 3b).

Background Surface Water

The source of the background surface water was the Caloosahatchee Header Canal (Figures 4 and 5)

located at Berry Groves in western Hendry County, Florida.

Sand Sample

The sand sample used for the bench-scale ASR was collected from the Caloosahatchee ASR Pilot

Project site (Figure 4) in consultation with Lloyd Horvath of Water Resources Solutions. This sand

sample was representative of subsurface materials proposed for use in the filtration design of the

Caloosahatchee ASR Pilot Project.

3.3.3 Task 3 – Acquisition and Storage of Groundwater

An existing exploratory well – completed into the Floridan aquifer system at the Berry Groves site

[24-inch-diameter, cased depth to 640 feet (ft) below land surface (bls), open-hole to 900 feet bls]



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was used as the source of groundwater (Figure 6) to facilitate Phase 1 laboratory testing. Prior to use

for this investigation, approximately 47 million gallons of groundwater were pumped from the well as

part of the well development process. Initially, this ASR well was planned to be in production during

the ecotoxicological sample collection phase of this project. Since the well was not in production at

the time of groundwater sample collection and no piping was in place to withdraw the large volume

of water present in the casing (approximately 15,000 gallons), the well may not have been adequately

purged prior to each collection event. Even though the groundwater samples were collected after

field parameters for temperature, pH, specific conductance, dissolved oxygen and turbidity stabilized,

conditions may not have been representative of upper Floridan aquifer water. However, any

differences in groundwater quality would not compromise one of the main objectives of the study,

which was to determine if standard test organisms and methods were appropriate for testing surface

and groundwaters associated with the ASR Pilot Facilities. Should future method development work

include the evaluation of the toxicity of the groundwaters, subsequent testing will consider the

mechanical ability to purge the well prior to sampling such that groundwater is truly representative of

upper Floridan aquifer conditions.

3.3.4 Task 4 – Range-finding Toxicity Tests of Mixtures

Range-finding toxicity tests were conducted to evaluate the methods to be used in the definitive

toxicity and bioconcentration tests using standard species. Steve Gilbert, United States Fish and

Wildlife Service (USFWS), was contacted for the identification and source of mussel species that

could be used in the bioconcentration studies.

3.3.5 Task 5 – Evaluation of the Appropriateness of Using Standard Test Organisms

The definitive tests using standard species were conducted during the dry season (January through

February 2005) and the wet season (June through July 2005). Definitive tests are defined as valid

tests conducted using the full range of concentrations needed to evaluate the potential toxicity of the

waters being tested.

The next three report sections (Sections 4.0, 5.0, and 6.0) present detailed discussions of Tasks 4 and

5 methods, results and conclusions.



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4.0 RANGE-FINDING TESTS (TASK 4)

4.1 Aquatic Toxicity Tests

The aquatic species used in the range-finding tests were the fathead minnow, Pimephales promelas,

two species of daphnid, Ceriodaphnia dubia and Daphnia magna, and the South African clawed frog,

Xenopus laevis (Figures 7 and 8). The following adverse effects were evaluated in these toxicity

tests:

• Survival (P. promelas, C. dubia, D. magna,and X. laevis),

•

Terata or malformations (P. promelas and X. laevis),

• Reproduction (C. dubia and D. magna), and

•

Growth ( X. laevis).

Surface water (background and treated), groundwater, and mixtures of these waters were tested; a

laboratory control water was also included.

The following are the EPA and American Society for Testing and Materials (ASTM) methods used to

conduct these range-finding tests:

•

7-day Chronic Fathead Minnow, P. promelas, Embryo-larval Survival and

Teratogenicity (gross morphological deformities), Test Method 1001.0 (EPA, 2002);

• 7-day Chronic Daphnid, C. dubia, Survival and Reproduction, Test Method 1002.0

(EPA, 2002);

• 21-day Daphnid, D. magna Life-cycle Toxicity Test, ASTM: E 1193-97; and

•

96-hour Frog Embryo Teratogenesis Assay- Xenopus (FETAX), ASTM: E1439-98.



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The battery of tests selected covers a broad range of toxicological effects using fish, amphibians and

invertebrates. The 7-day chronic fathead minnow test evaluates effects on fish embryos including

morphological deformities during embryological and larval development as well as survival. The 7-

day and 21-day daphnid chronic tests evaluate the survival of these sensitive freshwater invertebrates

as well as effects on reproductive ability. During the 7-day Ceriodaphnia test, young (>24 hours old

at test initiation) will mature and typically produce 3 broods of young; this life cycle allows the

evaluation of reproductive capacity and survival over a short test period. The advantage of the

Daphnia life cycle test (although longer) is that this species is more tolerant of high ion

concentrations in the water (as compared to Ceriodaphnia) and can be used under a broader range of

environmental conditions. FETAX tests are initiated using frog embryos, and during this short

exposure period (96 hours) effects on embryological development as well as embryo survival are

evaluated.

The exposure solutions (treatments) for the tests were:

•

Laboratory control water; moderately hard reconstituted freshwater (MHR*);

• Background surface water, 100-percent unaltered (BSW*);

• Treated surface water, 100-percent (TSW*) – treatment previously discussed;

•

20-percent groundwater diluted with treated surface water;

•

50-percent groundwater diluted with treated surface water;

• 80-percent groundwater diluted with treated surface water; and

• Groundwater, 100-percent unaltered (GW*).

* Abbreviations used in the data tables and laboratory bench sheets.

4.2 Samples Tested

Golder collected samples of surface water, groundwater, and sediments from the Caloosahatchee ASR

Pilot Project site from January 17 to February 4, 2005. Samples were collected on Mondays,

Wednesdays, and Fridays. Samples were shipped on ice via an overnight courier service to

Hydrosphere Research. Bioassays were conducted on the water samples; the sediment sample was

used for the bench-scale treatment system.



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4.3 Tests Conducted

Range-finding tests were initiated at Hydrosphere Research on January 18 and 20, 2005. A subset of

the water samples used was shipped on ice to Fort Environmental Laboratories (FEL) on January 25,

2005. FEL conducted the FETAX tests using these samples, and they were initiated on January 26,

2005. All studies were conducted as static-renewal tests; the test treatment solutions were replaced

with fresh solutions throughout the duration of the test. The tests with C. dubia, D. magna, and

P. promelas were renewed daily. The FETAX tests were renewed at 48-hours (Hydrosphere

Research, 2005a).

4.4 Test Results

Only one of the four bioassay tests conducted demonstrated an effect in the test endpoints evaluated.

The 7-day chronic bioassay using C. dubia (Table 1) demonstrated a statistically significant reduction

in reproduction in the background surface water (BSW) sample when compared to the laboratory

control (MHR), but these statistical benchmarks were very close (t Stat = 1.74 and t critical one-tailed

= 1.73), indicating that the background surface water had a marginal reduction in reproduction ability

in these daphnids.

Table 1. Range-finding -- 7-day Chronic Daphnid, Ceriodaphnia dubia, Survival and

Reproduction Test, EPA Test Method 1002.0

Sample ID

(%)

Final Survival

(%)

Three Brood Totals

(Average # of neonates

/ female)

MHR Control 100 25.8

BSW – 100% 90 20.5*

TSW – 100% 100 27.1

GW – 20% (80% TSW) 90 25.0

GW – 50% (50% TSW) 90 21.6

GW – 80% (20% TSW) 90 19.4*

GW – 100% 100 6.7*

* Indicates a significant difference between the treatments and the MHR control. (p=0.05)



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This test showed that the “treated” surface water (TSW) had no effect on this species (survival of 100

percent and the highest reproduction of any treatment or control). In other words, the “treatment” of

the surface water through the bench-scale ASR pilot removed the toxicity observed in the background

surface water.

The groundwater also had an effect on C. dubia. This 7-day chronic test demonstrated a reduction in

reproduction in the 80- and 100-percent groundwater samples compared to the control; this indicates

that the groundwater (full strength and diluted to 80 percent) affected the reproductive ability of C.

dubia. The groundwater, once diluted by 50 percent using “treated” surface water, did not show any

effects.

The remaining range-finding tests (P. promelas, D. magna, and FETAX) did not show any response

to the treatments.

Table 2 summarizes the data for the fathead minnow test (P. promelas); test results were based on

both mortality and gross morphological deformities (terata).

Table 2. Range-finding 7-day Chronic Fathead Minnow, Pimephales promelas,

Embryo-larval Survival and Teratogenicity Test, EPA Method 1001.0

Sample ID

(%)

Final Survival

(%)

Terata

(%)

Total Mortality

(%)MHR Control 95 7.5 12.5

BSW – 100% 92.5 7.5 15

TSW – 100% 97.5 5 7.5

GW – 20% (80% TSW) 85 12.5 27.5

GW – 50% (50% TSW) 90 2.5 12.5

GW – 80% (20% TSW) 92.5 7.5 15

GW – 100% 100 5 5

There is no significant difference between the treatments and the MHR control. (p=0.05)

Table 3 summarizes the D. magna test, which showed no effects as compared to the controls; in fact,

higher reproduction was observed in all treatment as compared to the controls.



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Table 3. Range-finding 21-day Daphnia magna Life-cycle Toxicity Test

ASTM: E 1193-97

Sample ID

(%)

Final Survival

(%)

Three Brood Totals


/ female)

MHR Control 90 180

BSW – 100% 100 246

TSW – 100% 100 215

GW – 20% (80% TSW) 90 206

GW – 50% (50% TSW) 90 215

GW – 80% (20% TSW) 100 238

GW – 100% 90 259


The FETAX test showed no significant mortality, malformations, or reduction in growth endpoint in

the treatments as compared to the controls.

Table 4. Range-finding Frog Embryo Teratogenesis Assay-Xenopus (FETAX)

ASTM: E 1439-98

Sample ID(%)

Mean

Mortality

(%)

Mean

Malformations

(%)

Mean

Growth

(%)

FETAX Control 0 0 100

BSW – 100% 5.0 2.6 98.7

TSW – 100% 0.0 0.0 101.5

GW – 20% (80% TSW) 2.5 2.6 101.1

GW – 50% (50% TSW) 0.0 5.0 101.5

GW – 80% (20% TSW) 2.5 7.7 101.3

GW – 100% 0.0 2.5 98.4




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4.5 Range-finding Test Conclusions

All range-finding tests were conducted successfully and demonstrated that the standard species

selected for Phase 1 of the Ecotoxicology Project are viable and that the test conditions and

treatments appear to be acceptable for testing of ASR waters.

The only effect observed was with the daphnid C. dubia. In other words, no acute survival effects

were observed for any of the species tested; and the only chronic endpoint affected was reproduction

in C. dubia when exposed to the background surface water (Header Canal) and groundwater (100-

and 80-percent groundwater). The background surface water toxicity observed was not evident

following “ASR bench-scale treatment” of the background surface water.

The C. dubia response to groundwater (100- and 80-groundwater) was not surprising as this species

has a narrow tolerance for ionic ratios in freshwater. The use of the D. magna test in addition to

C. dubia was included in this scope because high-anion concentrations (similar to those found in

Floridan aquifer groundwater) are known or reasonably anticipated to be toxic to C. dubia, but

D. magna is more tolerant of wider ionic concentrations and could tolerate the high ion

concentrations in groundwater and be able to detect toxic compounds present in these waters. A

response to anions can mask the response to other toxic compounds present in potentially toxic

concentrations [Mount et al., 1997]. Since the anions may be present in toxic amounts, and other

resident cladocera (daphnids), or related organisms, may also exhibit such sensitivities, it was

appropriate to include the two daphnid species in the range-finding tests.

Based on the results of the range-finding tests, it was decided to initiate Task 5 using the same test

species, conditions, and water treatments.



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5.0 SCREENING STUDY – EVALUATION OF STANDARD TEST ORGANISMS

Task 5 of the Ecotoxicology Project was comprised of three subtasks, subtask 5.1 Aquatic Toxicity

Scoping Study, 5.2 Alternative Dilution Series, and 5.3 Bioconcentration Scoping Study. The

Alternative Dilution Series subtask was used to address technical issues and additional analysis or

tests, as needed. The Aquatic Scoping Studies were conducted during the “dry season” and the “wet

season.” The “dry season” in South Florida is defined as the time period between November and

May, a period of time in Florida with lower precipitation rates; the “wet season” is defined as June

through October, a period with higher precipitation rates.

5.1 Aquatic Toxicity Scoping Studies

The range-finding study results (Task 4) defined the scope of these aquatic toxicity scoping studies

(Subtask 5.1); the test species, protocols, and treatments for Task 4 and Subtask 5.1 are similar. The

Aquatic Toxicity Scoping Studies included the:

•

7-day Chronic Fathead Minnow, P. promelas, Embryo-larval Survival and

Teratogenicity Test, Test Method 1001.0 (EPA, 2002);

•

7-day Chronic Daphnid, C. dubia, Survival and Reproduction Test, Test

Method 1002.0 (EPA, 2002);

•

21-day D. magna Life-cycle Toxicity Test, ASTM: E 1193-97; and

• FETAX, ASTM: E 1439-98.

The exposure solutions (treatments) for the tests were:

• Laboratory control water; moderately hard reconstituted freshwater (MHR);

• Background surface water, 100% unaltered (BSW);

• Treated surface water, 100% (TSW);

•

20% groundwater diluted with treated surface water;

• 50% groundwater diluted with treated surface water;

• 80% groundwater diluted with treated surface water; and

•

Groundwater, 100% unaltered (GW).



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5.2 Samples Tested

Golder collected samples of surface water, groundwater, and sediment from the Caloosahatchee ASR

Pilot Project site from March 28 to April 29, 2005 (dry season), and June 15 to July 11, 2005 (wet

season). Samples were collected on Mondays, Wednesdays, and Fridays. Samples were transported

on ice by Golder personnel to Hydrosphere Research. Samples were received in good condition and

were used within acceptable holding times. Tests were conducted on the water samples; the sediment

samples were used for the ASR bench-scale treatment system. Due to the large volume of “treated”

water required for these tests and the continuous-flow bioconcentration studies (Subtask 5.3), the

ASR bench-scale was modified to include two sediment columns in order to double the volume of

“treated” water produced per day (Figure 9).

5.3

Aquatic Toxicity Tests

The “dry season” toxicity tests were initiated during April 2005. A subset of the water samples was

shipped on ice from Hydrosphere to FEL for the FETAX tests. All studies were conducted under

static-renewal conditions. The “wet season” toxicity tests were initiated during June 2005. The tests

with C. dubia, D. magna, and P. promelas were daily renewals. The FETAX test was renewed at

48 hours (Hydrosphere Research, 2005b and 2005c).

5.4

Test Results

5.4.1 Ceriodaphnia dubia

The 7-day chronic tests with C. dubia (Tables 5 and 6) demonstrated a reduction in reproduction in

100-percent groundwater compared to the controls during the “dry” and “wet” seasons. The results

for both seasons were very similar. Both tests indicate that the full-strength groundwater reduces the

reproductive ability of C. dubia. The groundwater did not show any effects once it was diluted with

“treated” surface water. The “treated” surface waters had a survival of 100 percent and the highest

reproduction of any treatment or control during both seasons.



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Table 5. “Dry Season” 7-day Chronic Daphnid,Ceriodaphnia dubia, Survival and


Sample ID

(%)

Final Survival

(%)

Three Brood Totals


/ female)


BSW – 100% 100 33.8

TSW – 100% 100 35.6

GW – 20% (80% TSW) 100 33.3

GW – 50% (50% TSW) 90 29.2

GW – 80% (20% TSW) 100 25.8

GW – 100% 100 16.1*


Table 6. “Wet Season” 7-day Chronic Daphnid,Ceriodaphnia dubia, Survival and


Sample ID

(%)

Final Survival

(%)

Three Brood Totals


/ female)

MHR Control 90 22.4

BSW – 100% 100 26.9

TSW – 100% 100 30.7

GW – 20% (80% TSW) 100 28.1

GW – 50% (50% TSW) 100 24.1

GW – 80% (20% TSW) 100 19.8

GW – 100% 90 12.3*




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5.4.2 Daphnia magna

The D. magna exposed to the two highest concentrations of groundwater (80 and 100 percent)

demonstrated statistically significant reductions in survival during the “dry season” (Table 7). The

final survival was 10 and 0 percent in the 80- and 100-percent groundwater samples, respectively. No

effects were quantified during the “wet season” tests (Table 8).

Table 7. “Dry Season” Daphnia magna Life-cycle Toxicity Test

ASTM: E 1193-97

Sample ID

(%)

Final Survival

(%)

Brood Totals

(Average # of

neonates / female)


BSW – 100% 80 141.3

TSW – 100% 90 143.0

GW – 20% (80% TSW) 80 132.7

GW – 50% (50% TSW) 100 155.8

GW – 80% (20% TSW) 10* 104.5

GW – 100% 0* 26.8


Table 8. “Wet Season” Daphnia magna Life-cycle Toxicity Test

ASTM: E 1193-97

Sample ID

(%)

Final Survival

(%)

Brood Totals

(Average # of

neonates / female)


BSW – 100% 100 180.7

TSW – 100% 90 179.9

GW – 20% (80% TSW) 80 155.9

GW – 50% (50% TSW) 100 185.4

GW – 80% (20% TSW) 80 164.5

GW – 100% 70 138.3

There is no significant difference between the treatments and the lab control. (p=0.05)



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5.4.3 Pimephales promelas

The “dry season” fathead minnow test (P. promelas) did not show any differences between treatments

and controls (Table 9), but the “wet season” test showed an effect on survival when the embryos were

exposed to 100-percent groundwater (Table 10). No other effects were quantified.

Table 9 “Dry Season” 7-day Chronic Fathead Minnow, Pimephales promelas,


Sample ID

(%)

Final Survival

(%)

Terata

(%)

Total Mortality

(%)

MHR Control 100 2.5 2.5

BSW – 100% 95 2.5 7.5

TSW – 100% 87.5 5 17.5

GW – 20% (80% TSW) 87.5 2.5 15

GW – 50% (50% TSW) 90 0 10

GW – 80% (20% TSW) 97.5 0 2.5

GW – 100% 92.5 2.5 10




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Table 10. “Wet Season” 7-day Chronic Fathead Minnow, Pimephales promelas,


Sample ID

(%)

Final Survival

(%)

Terata

(%)

Total Mortality

(%)

MHR Control 92.5 5 12.5BSW – 100% 80 NA NA

TSW – 100% 87.5 7.5 20

GW – 20% (80% TSW) 82.5 0 17.5

GW – 50% (50% TSW) 90 10 20

GW – 80% (20% TSW) 87.5 10 22.5

GW – 100% 75* NA NA


NA – Not applicable

5.4.4 FETAX

The FETAX data for the “dry” and “wet” seasons are summarized in Tables 11 and 12. No

significant mortality, malformations or reduction in growth were observed.

Table 11. “Dry Season” Frog Embryo Teratogenesis Assay-Xenopus (FETAX)

ASTM: E 1439-98

Sample ID

(%)

MeanMortality

(%)

MeanMalformations

(%)

MeanGrowth

(%)

FETAX Control 0 2.5 100

BSW – 100% 12.5 0 98.6

TSW – 100% 12.5 2.9 100.3

GW – 20% (80% TSW) 7.5 2.7 101.0

GW – 50% (50% TSW) 10 2.8 100.6

GW – 80% (20% TSW) 17.5 0 99.0

GW – 100% 10 2.8 99.8




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Table 12. “Wet Season” Frog Embryo Teratogenesis Assay- Xenopus (FETAX)

ASTM: E 1439-98

Sample ID

(%)

Mean

Mortality

(%)

Mean

Malformations

(%)

Mean

Growth

(%)

FETAX Control 10.0 8.3 100

BSW – 100% 7.5 8.1 101.9

TSW – 100% 20.0* 12.5 100.8

GW – 20% (80% TSW) 20.0 15.6 97.8

GW – 50% (50% TSW) 5.0 10.5 99.0

GW – 80% (20% TSW) 20.0 3.1 100.0

GW – 100% 32.5 0.0 99.7


*Replicate B was considered an anomaly; these data represent Replicate A only

5.5 Alternate Dilution Series

Due to the large number of treatments, FEL conducted each set of samples (range-finding, “dry” and

“wet” season) as if they were two tests. This did not affect the scope of the work.

5.6 Water Analysis

Tables 13 and 14 summarize the trace metal analysis of water samples collected for the “dry”

(6 samples) and “wet season” (8 samples), respectively. The metals analyzed in these samples were

arsenic (As), selenium (Se), cadmium (Cd), total mercury (TMg), and methyl mercury (MeHg).

Averages were calculated using the detection limit when the metal was reported below the detection

limit.

Tables 15 and 16 summarize the conductivity measurements for the test treatments collected during

the 21-day D. magna “dry” and “wet season tests,” respectively. As anticipated, the conductivity of

the groundwater was very high [~2,700 microhmo (µmho/cm)], as compared to the controls and

surface water (~300 and ~420-464 µmho/cm), respectively. Tables 17 and 18 summarize the pH

measurements for the test treatments collected during the same tests, “dry” and “wet season”

respectively.



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5.7 Aquatic Scoping Toxicity Study Conclusions

All tests were conducted successfully. The bench-scale ASR “treated” water did not have any

quantifiable toxic effect on any test species or endpoint measured.

C. dubia’s results were consistent, showing no effect on survival, but showing a statistically

significant effect on reproduction when exposed to full strength groundwater (all three tests, range-

finding, “dry,” and “wet season”). This species was also shown to have a statistically lower

reproduction rate in background surface water, during the range-finding test only, when compared to

the controls; once the background surface water was “treated” through the bench-scale ASR system,

there was no effect on reproduction. One possible explanation for this observation is that the

disinfection process neutralized microorganisms that negatively affect reproduction.

The other daphnid tested, D. magna, was affected by the groundwater only and only during the “dry”

season”; the range-finding and “wet season” samples had no effect on this species. The effects

measured during the “dry season” were on survival in groundwater (80- and 100-percent

groundwater).

The fathead minnow, P. promelas, was affected by the 100-percent groundwater only and only during

the “wet season”; this effect was on reduced survival (75-percent) as compared to survival in the

controls (92.5-percent).

All FETAX tests were negative and did not quantify any effects from any of the treatments (range-

finding, “dry,” and “wet season”).

In conclusion, the primary treatment that affected the species tested under these short chronic assays

was the full-strength groundwater. However, since there is no scenario in the CERP ASR program

where 100-percent groundwater is discharged from an ASR well, this observation of toxicity is

benign and not unexpected (groundwaters typically have higher conductivity than surface waters andthis can affect the survival and/or reproduction of aquatic organisms through ionic imbalance) . In all

cases, once the groundwater was diluted with 50-percent bench-scale “treated” water, no effect was

quantifiable for any species on any sample tested. The bench-scale “treated” water did not have any

effect on any species tested. All tests met the guidelines required by the cited methods and were

acceptable. No changes are recommended to this battery of tests.



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6.0 BIOCONCENTRATION SCOPING STUDY

Subtask 5.3 of the Ecotoxicology Project is the Bioconcentration Scoping Study. Bioconcentration

studies were designed to evaluate the potential for bioconcentration of selected trace metals in fish

and mussel tissues. These studies also evaluated the potential for radium 226/228 bioconcentration in

freshwater mussels. These studies were conducted during the “dry” and “wet” season concurrently

and using the same water samples as Subtask 5.1, Aquatic Toxicity Scoping Study.

6.1 Bioconcentration Methods

The ASTM method “Standard Guide for Conducting Bioconcentration Tests with Fishes and

Saltwater Bivalve Mollusks, ASTM: E 1002-94” was used as a guide to conduct these tests. The tests

were conducted under static-renewal conditions (continuous flow) for 28 days.

6.2 Samples Tested

These tests used the same samples collected for Subtask 5.1 and summarized in Section 5.2, and

included in Tables 13 and 14. The samples collected from March 28 to April 26, 2005 were used for

the “dry season” test, and the samples collected from June 15 to July 11, 2005, for the “wet season”

tests.

6.3 Tests Conducted

The study design was based on the SFWMD Statement of Work, Subtask 5.3 “Bioconcentration

Scoping Study.” The tests were 28-day bioconcentration studies using a freshwater fish and a mussel.

In order to determine the potential bioconcentration of selected trace metals (As, Se, Cd, THg, and

MeHg) and radium (226/228) in these test species, tissue samples were collected and analyzed at the

beginning (background) and end of the study. Bioconcentration tests were initiated on March 29,

2005, for the “dry season” and June 16, 2005, for the “wet season” (Hydrosphere Research, 2005d

and 2005e, respectively).

Metals analyses were conducted by Frontier Geosciences Inc., Seattle, Washington, on fish and

mussel tissues and water samples. Radium analyses were conducted on water and mussel tissues at

Elab, Inc., Ormond Beach, Florida.



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6.4 Test Species

The aquatic vertebrate used in these tests was the fish Lepomis macrochirus, commonly known as a

bluegill. The aquatic invertebrate was a mussel, Elliptio buckleyi, also known as the Florida Shiney

Spike (Figure 10). Bluegill were purchased from a commercial supplier and acclimated to laboratory

conditions.

The selection of the freshwater mussel to be used for these bioconcentration studies was conducted in

cooperation with Dr. Steve Gilbert, USFWS, and Gary Warren, Florida Fish and Wildlife

Conservation Commission (FFWCC). Mr. Warren offered to collect two species of mussels for

Golder, and identified two candidate species: E. buckleyi and Utterbackia imbecillis. Golder and the

FFWCC staff made numerous attempts in north-central Florida and the Lake Okeechobee area to

collect these two species. Very few U. imbecillis were found at all locations sampled; therefore

following numerous attempts, it was agreed with Mr. Warren that E. buckleyi would be used for the

bioconcentration tests.

A group of E. buckleyi were collected and maintained in the laboratory for several weeks prior to the

“dry season” bioconcentration tests in order to monitor their survival and ability to be used for testing

under laboratory conditions. This freshwater mussel species was amenable to laboratory conditions,

and was selected for bioconcentration testing. Golder obtained the appropriate sampling permits from

the FFWCC and conducted all collections; acclimation and testing was conducted at Hydrosphere

Research. Reference toxicant tests were conducted using three different reference toxicants, as this

species had not been previously used for bioassays. The E. buckleyi used for Subtask 5.3 were

collected from ponds located on FFWCC property in Gainesville, Florida.

6.5 Treatments

The tests were conducted using the same treatments used in the Subtask 5.1 Aquatic Toxicity Scoping

Studies, except that only full-strength treatments were used. The four exposure treatments were:

• A test control solution of Lab Control Water (LCW*),

•

Background Surface Water, 100-percent unaltered (BSW*),

• Treated Surface Water, 100 percent (TSW*), and

• Groundwater, 100-percent unaltered (GW*).



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* Hydrosphere-designated abbreviations used in tables and laboratory bench sheets.

The LCW source was dechlorinated municipal water from the City of Alachua, whose source is

groundwater from the Floridan aquifer system. Background surface water was collected from the

Header Canal and groundwater was collected from the exploratory well at the Caloosahatchee ASR

Pilot Project site. The samples used for the bioconcentration studies were the same samples used for

Subtask 5.1. Portions of the samples received were used on the day of receipt and the unused

portions were stored in a cold room at 4 degrees Celsius (°C). Sample processing was conducted on

the day that the samples were received. Sample usage for test renewals was done within 72 hours of

sample collection.

Tables 13 and 14 summarize the trace metal analysis conducted on the water samples used in these

studies. Tables 19 and 20 summarize the radium 226/228 measurements from water samples

collected at test initiation, mid-test, and test conclusion.

6.6 Bioconcentration Tests

The bioconcentration studies were conducted as static-renewal (continuous flow) exposures using

peristaltic pumps distributing fresh test solutions from head tanks to exposure vessels containing fish

and mussels (Figure 11). The test vessels were 10-gallon glass aquaria calibrated to contain 15 liters

(L) of test solution. Each test exposure was conducted in duplicate. Peristaltic pumps were used to

deliver test solutions to exposure vessels containing fish and mussels.

The objective of the bioconcentration tests was to evaluate the potential uptake of metals and radium

(mussels only) from the treatment solutions by the fish and mussels exposed during the 28-day

bioconcentration study. Bioconcentration studies are typically conducted as flow-through tests, but

due to the fact that ASR-treated water had to be prepared in batches in the laboratory, static-renewal

test conditions were evaluated. Loading rate (mass of tissue per volume of water) was also a critical

component in exposure design in order to balance the physiological needs of the test organisms and

the need for sufficient tissue samples at the end of the test for metal and radium analysis. All surface

water and groundwater had to be transported from the Caloosahatchee site to the toxicology

laboratory three times per week, which also added the volume turnover constraint in the design.

Based on all of these considerations and loading calculations, the bioconcentration studies were



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conducted under static-renewal (continuous) flow conditions, with one test vessel volume renewal per

day.

6.6.1 Feeding

Fish were fed Finfish Starter #2 twice daily, and mussels were fed algae (Selenastrum capricornutum)

every 7th day. Both of these food supplies were analyzed for metals (Table 21). The algae used for

the mussel test were also analyzed for radium (226/228).

6.6.2 Test Completion

After 28-days of exposure, the test organisms were removed from the exposure vessels. Muscle

tissue from the fish and the soft tissue from the mussels were collected for chemical analysis.

The analyses conducted were for arsenic, cadmium, selenium, total mercury, methyl-mercury and

radium 226/228 (mussels only). Background samples of fish tissue, mussel tissue, the feed used to

feed the fish and the green alga, Selenastrum capricornutum, used to feed the mussels were also

analyzed at the beginning of the study. Instruments used to process the tissue samples were dipped in

ultra pure water and submitted as blanks for the metals analyses.

6.6.3

Statistical Analysis

The objectives of these tests were to evaluate the potential accumulation of selected metals and

radium in the tissues of the test organisms exposed to the laboratory control water, surface water,

groundwater, and “treated” surface water. The tests were also used to determine if there was a

difference in trace metal concentrations between treatments and tissue concentrations, as well as

comparisons to tissue background concentrations (pre-exposure). Statistical comparisons were made

using Analysis of Variance (ANOVA) using the NCSS-2000 software. The level of statistical

significance was the standard alpha = 0.05 level.

6.7 Test Results

All tests were completed successfully and were considered valid and acceptable tests based on

method criteria. Fish and mussel tissue samples were analyzed by Frontier Geosciences for trace

metal analysis. Mussel tissues and water treatments were analyzed for radium 226/228 by Elab.



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6.7.1

Dry Season Results

Water Concentrations- Dry Season

The results for the water sample analysis for arsenic, cadmium, selenium, total mercury, and methyl

mercury are summarized in Table 13.

In general, all treatment concentrations were low and below 3 micrograms per liter (µg/L) except for

selenium in groundwater which averaged 12.24 µg/L. Statistical analysis showed that arsenic and

selenium concentrations in groundwater were significantly greater than all other waters analyzed. For

cadmium, “treated” surface water was significantly greater than all other waters tested. Total and

methyl mercury water concentrations for background surface water and “treated” surface water were

equal, but statistically greater than the laboratory control water and groundwater.

Fish and Mussel Tissue Trace Metal Concentrations – “Dry Season”

Table 22 summarizes the “dry” season background trace metal concentrations in fish and mussels.

Tables 23 and 24 summarize the tissue concentrations at the end of the 28-day exposure in the fish

and mussel tissues, respectively. Two sets of triplicate tissue analysis were conducted (three samples

from each of 2 replicate aquaria for each species). In general, mean metal tissue concentrations

between the pre (background) and post (end of 28-day test) exposures were very similar suggesting at

least qualitatively that little metal accumulation had occurred in both fish and mussels tissues.

Concentrations are reported as milligram per kilogram (mg/kg) wet weight, except for total and

methyl mercury where concentrations are reported as nanogram per gram (ng/g) wet weight.

Hydrosphere by error submitted only one tissue sample per species for the metal background analysis

(Table 22), three samples were specified in the Scope of Work; therefore, direct statistical

comparisons of background and post-exposure tissue concentrations was not possible for the “dry”

season. To determine if there was metal accumulation from pre-exposure (background) to post-

exposure, a comparison of background data with post-exposure data range and mean was made for

each metal. A statistical comparison using Analysis of Variance (ANOVA) was used to evaluate the

various treatments.

For fish tissues, the results showed for all cases the background data fell within the range of the post-

exposure data indicating that there was no measurable difference in tissue concentration between the

two conditions (pre- and post-exposure). For the post-exposure data, there were no statistical

significant differences at the alpha = 0.05 level between treatments (water types) and fish tissue



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concentrations. For cadmium, all fish tissue values were below detection limits for all treatments and

no statistical analysis was conducted.

For mussels tissues there was no statistically significant difference at the alpha = 0.05 level between

treatments (water types) and metal tissue concentrations. Comparison between background samples

and post-exposure showed for all cases that the background data fell within the range of the post

exposure data indicating that there was no measurable difference or increase in bioconcentration

between the two conditions. However, for arsenic, cadmium, and methyl mercury, there was a slight

decrease in tissue concentration between background and post-exposure for the laboratory control

water and background surface water, likely due to depuration.

Radium in Water and Mussel Tissue – Dry Season

In addition to the above metals, radium (226/228) analysis was conducted on both water and mussel

tissue samples. Table 19 summarizes the water concentrations for both radium 226 and 228

conducted on March 29, April 12, and April 25, 2005, for the various treatments. Radium 228 was

below detection limit in all but one water sample. Mussel tissue analysis was conducted for

laboratory control water (LCW) and groundwater (GW) exposures and they were collected in

duplicate from additional treatment replicates (C and D) in order to provide sufficient tissue for

radium analysis (Table 25, C1 and D1). Table 26 presents the background radium 226/228 in mussel

tissues. This study shows that there was no increase in radium 226/228 concentration in mussel tissue

in the LCW and GW tissue samples as compared to the background concentrations.

6.7.2 “Wet Season” Results

Water Concentrations – “Wet Season”

The results for the water sample analysis for arsenic, cadmium, selenium, total mercury, and methyl

mercury are summarized in Table 14.

In general, for all treatments metal concentrations were below 4 µg/L, except for selenium in

groundwater, which ranged from 10.7 to 15.6 µg/L. Several metals showed distinct temporal trends.

For example, arsenic in groundwater showed a decrease in concentration throughout the study

ranging from 3.83 to 0.26 µg/L. The greatest decrease occurred between the June 21 and 28, 2005,

samples. Inversely, selenium showed a general increase in groundwater concentrations over time.

Total mercury showed a general increase in lab control water over time.



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Fish and Mussel Tissue Trace Metal Concentrations — “Wet Season”

Metal tissue concentrations between the pre- (background tissue concentration) and post-experimental

conditions were very similar, suggesting at least qualitatively that little accumulation had occurred in

both the fish and mussels. Background triplicate metal concentrations in bluegill and mussel tissue

are summarized in Table 27.

Tissue concentrations for post-testing for the bioconcentration study are summarized in Tables 28 and

29 for fish and mussels, respectively. Two sets of triplicate tissue analysis were completed (three

samples from each of two replicate aquaria for each species). A statistical comparison using Analysis

of Variance (ANOVA) was used to evaluate the various treatments (water type, for the post

experiments).

The results showed that for all metals there was no difference in tissue concentration at the alpha =

0.05 level between water types (treatments) in the post-exposure fish and mussel data. In other

words, the environmental concentrations of the metals in the treatments were similar in the

background surface water, “treated” water, groundwater and laboratory controls.

To determine if there was metal accumulation from pre-exposure (background) to post-exposure, a

comparison of background tisue data with post-exposure tissue data range and mean was made for

each metal. A statistical comparison using Analysis of Variance (ANOVA) was used to evaluate

these data.

Comparison between background tissue samples and the post-experiment tissue samples did not show

any difference in mussel or fish tissue concentrations for any treatment.

Radium in Water and Mussel Tissue – “Wet Season”

Table 20 summarizes the water concentrations (treatments) and Table 30 summarizes the mussel

tissue concentrations at the end of the exposure for both radium 226 and 228. Table 26 includes the

background mussel tissue concentrations for radium, this is the background sample analyzed for the“dry season.” Hydrosphere Research inadvertedly did not analyze for radium background in the “wet

season.”

Statistical analysis of the water treatments showed that groundwater radium 226 concentration was

significantly higher than the LCW and BSW at the alpha = .05 level. No statistical difference was



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found between radium 226 concentrations in LCW and BSW. This difference was not observed when

comparing the radium 228 data for these treatments; in other words, the radium 228 concentrations of

the LCW, BSW, and GW were similar.

Tissue radium 226 and 228 concentrations for the mussels exposed in LCW and GW were not

statistically different from each other. When the radium 226 background mussel tissue concentrations

were compared to the exposed mussel tissue concentrations (LCW and GW) it was found that there is

no statistical difference between these tissue concentrations, no increase in radium 226/228 in the

tissues during the test.

The mussels for all tests were collected from the same ponds, with 3 weeks of testing.

6.8

Trace Metals and Radionuclides in Waters – Discussion

In accordance with the scope of work, water samples were analyzed for the following trace metals:

arsenic, selenium, cadmium, total mercury and methyl mercury. The results for samples collected

from lab control water, background surface water, treated surface water, and groundwater are shown

on Tables 13 and 14. Arsenic and selenium are trace elements that are present in water, generally in

the µg/L range. They are typically associated with agricultural use; however, solubilities are very

low at the pH found in most natural waters and they are readily removed from water by either

sedimentation or sorption removal processes. Arsenic concentrations in native groundwaters in the

Floridan aquifer are reported to be approximately 10 µg/L (Arthur et al., 2002).

Cadmium concentrations in water are generally well below saturation and typically occur at

concentrations below 2 µg/L (Hem, 1985).

Mercury has an equilibrium solubility of approximately 25 µg/L, but is generally found at much

lower concentrations due to its volatility. Concentrations of mercury in natural surface waters are

generally less than one-tenth part per billion. Organic complexes such as methyl mercury are

produced by methane-generating bacteria in contact with metallic mercury (McPherson et al., 2000).

Surface water samples were collected by the SFWMD (Appendix B) to examine the water quality of

the Caloosahatchee River and surface water quality at Berry Groves, the site of the Caloosahatchee

ASR Pilot Project. Arsenic concentrations in the Caloosahatchee River and Berry Groves surface



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water ranged from 3.2 to 5.0 µg/L and 3.2 to 8 µg/L, respectively, with a groundwater cleanup target

level (GCTL) of 10 µg/L. Selenium concentrations in the Caloosahatchee River and Berry Groves

surface water ranged from 2.1 to 7.4 µg/L and 2.1 to 5.0 µg/L, respectively with a GCTL of 50 µg/L.

Cadmium was not detected above the minimum detection level of 1 µg/L with a GCTL of 5 µg/L.

Total Mercury concentrations in the Caloosahatchee River and Berry Groves surface water ranged

from .5 to 2.87 nanograms per liter (ng/L) and 0.096 to 3.52 ng/L, respectively with a GCTL of 2,000

ng/L. Methyl Mercury concentrations in the Caloosahatchee River and Berry Grove surface water

ranged from 0.064 to 0.5 ng/L and 0.032 to 0.86 ng/L, respectively with a GCTL of 70 ng/L.

Background surface water quality data for the parameters collected from the Header Canal in Berry

Groves for this investigation are shown in Table 14 for “wet season” samples collected from June 16,

2005 to July 12, 2005. Arsenic concentrations ranged from 1.89 to 2.92 µg/L. Selenium

concentrations ranged from 0.40 to 1.30 µg/L. Cadmium concentrations ranged from below detection

limit to 0.023 µg/L. Total mercury and methyl mercury ranged from 1.56 to 3.45 and 0.058 to 0.276

ng/L, respectively.

Groundwater samples were collected from the exploratory well at the Caloosahatchee ASR Pilot

Project Site during this Phase 1 investigation and analyzed for these trace metals as part of this

investigation. Background Floridan aquifer system data was collected by the SFWMD using the Pilot

ASR well, on June 21, 2004 and is compared to the data gathered from this study in June/July 2005

(Appendix B).

The June/July 2005 data for the wet season indicated that arsenic was well below the GCTL of

10 µg/L and ranged from 0.26 to 3.83 µg/L. Selenium, which was also below the GCTL of 50 µg/L,

was found at concentrations ranging from 10.7 to 15.6 µg/L. Cadmium was not detected above the

method detection limit in any of the samples. Total mercury ranged from below detection limit to

0.35 ng/L. Methyl mercury was not detected above the minimum detection level of 0.025 ng/L.

Data collected by the SFWMD in June 2004, indicated that arsenic, selenium and cadmium werefound at concentrations below their respective minimum detection levels of 2.6 ug/L, 2.1 ug/L and

0.7 ug/L. Total mercury ranged from below detection limit to 0.32 ng/L. Methyl mercury was not

detected above the minimum detection level of 0.021 ng/L.



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Radium results for the wet season are shown in Table 20, for laboratory control water, background

surface water and groundwater from the Pilot ASR well. Average Radium 228 concentrations were

similar for both the surface water and the groundwater, as compared to the laboratory control water.

The concentration of Radium-226 in surface water is also similar to the laboratory control water.

However, Radium-226 concentration in groundwater was slightly elevated with a combined Radium

226/228 concentration of 5.61 Pico Curies per Liter (pCi/L), which slightly exceeds the GCTL of 5.0

pCi/L.

Data collected by the SFWMD for the Pilot ASR well in June 2004 also had an elevated

concentration of Radium 226/226. The combined concentration in groundwater was reported to be

8.99 pCi/L.

6.9

Bioconcentration Study Conclusions

Trace metals and radium (226/228) did not bioconcentrate in fish or mussel tissues during the two 28-

day bioconcentration studies conducted during the “dry” and “wet” seasons. It should be noted that

the relatively low environmental background concentrations for these parameters in the water samples

to some extent inhibited our ability to quantify the bioconcentration phenomenon in the test species.

The methods used met all criteria applicable to these tests.

The mussel species used in this study, E. buckleyi, was a good choice for the bioconcentration studies,

as it adapted well to laboratory conditions, accepted an algal diet, and was easily handled for the tests.

Given the desire to test not only standard laboratory species but also species found in Florida habitats,

the choice of E. buckleyi for the subject bioconcentration testing and the results proved to be a robust

approach.

The laboratory control water used for the tests was also groundwater, chosen because of the large

volume of water needed to conduct these bioconcentration studies under offsite laboratory conditions.

Future bioconcentration studies will use a different source of water, such as surface water, for

controls. That said, its choice did not impede our ability to draw the conclusions we did regarding

method development for future bioconcentration studies of metals and radionuclides using the test

species.



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If bioconcentration studies are conducted as part of future ASR project evaluations, it is

recommended that these tests be conducted onsite using flow-through test conditions or in situ

exposures. This approach would not require the transport of large volumes of water to a laboratory

and the control water for these studies should be the background surface water used for the ASR.

Both of the species used in the development of the bioconcentration protocols are acceptable and can

be used in future onsite bioconcentration studies. Of the two, the mussel species would lend itself to

in situ cage studies exposing them to the background surface water and the ASR discharge, thus

allowing a direct comparison of background and “treated” water metal and radionuclide

bioavailability. Should mesocosm studies be considered as part of future ASR projects, either fish or

mussel species would be suitable for inclusion for the evaluation of potential trace metal and/or

radionuclide bioconcentration.



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REFERENCES

Arthur, Jonathan D. , Adel A. Dabous, and James B. Cowart, Mobilization of arsenic and other trace

elements during aquifer storage and recovery, southwest Florida, In George R. Aiken and Eve

L. Kuniansky, editors, 2002, U.S. Geological Survey Artificial Recharge Workshop

Proceedings, Sacramento, California, April 2-4, 2002: USGS Open-File Report 02-89.

ASTM International. 2004. Standard Guide for Conducting Daphnia magna Life-Cycle Toxicity

Tests. Designation: E 1193-97 (Reapproved 2004).

ASTM International. Standard Guide for Conducting the Frog Embryo Teratogenesis Assay-

Xenopus. Designation: E 1439-98

Fink, L. and I. Johnson. 2004. Draft Preliminary Investigation of the Ecotoxicological Effects of

Recovered ASR Water on Receiving Water Ecosystems using Pilot Project Groundwater

and/or Recovered Water. Phase 1 – Screening-level Method Development, June 4, 2004.

Hem, John D. 1985, Study and Interpretation of the Chemical Characteristics of Natural Water, U.S.

Geological Survey, Water Supply Paper 2254.

Ho, K. and D. Caudle. 1997. Letter to the Editor. Ion Toxicity and Produced Water. Environmental

Toxicology and Chemistry, Vol. 16, No. 10. pp. 1993-1195.

Hydrosphere Research. 2005a. Task 4 Range-Finding Toxicity Tests. Prepared for Golder

Associates Inc. Test Number GLD-AS 4338.

Hydrosphere Research. 2005b. Task 5.1 Aquatic Scoping Study (Dry Season). Prepared for Golder


Hydrosphere Research. 2005c. Task 5.1 Aquatic Scoping Study (Wet Season). Prepared for Golder


McPherson, Benjamin F., Ronald L. Miller, Kim H. Haag, and Anne Bradner, 2000, Water Quality in

Southern Florida, 1996–98, U.S. Geological Survey Circular 1207.

Mount, D.R., Gulley, D.D., Hackett, J.R., Garrison, T.D., and Evans, J.M. 1997. Statistical Models

to Predict the Toxicity of Major Ions to Ceriodaphnia dubia, Daphnia magna, and Pimphales promelas (Fathead Minnows). Environmental Toxicology and Chemistry, Vol. 16, No. 10,

pp. 2009-2019.

Project Management Plan (PMP) for the ASR Regional Study (ASRRS).



Phase 1 -- 2005 - 37 - 023-3937C

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Sauer, T.C., Costa, H.J., Brown, J.S., and Ward, T.J. 1997. Toxicity Identification Evaluations of

Produced-Water Effluents. Environmental Toxicology and Chemistry, Vol. 16, No. 10, pp.

2020-2028.

U.S. Environmental Protection Agency (EPA). 2002. Short-Term Methods for Estimating the

Chronic Toxicity of Effluents and Receiving Water to Freshwater Organisms. Fourth Edition.EPA-821-R-02-013. October 2002.

X://0233937C-0005/Final/Report.doc



October 2005

Date Collected: March 29, 2005 April 5, 2005 April 12, 2005 April 14, 20005 April 19,

Analyte

Lab Control Water Arsenic 1.52 1.44 1.46 1.48 1.46

(LCW) Selenium 0.60 0.57 1.03 1.12 1.07Cadmium 0.013 0.014 0.013 0.014 0.013

Total Mercury (ng/L) 0.27 1.42 0.17 0.26 0.37

Methyl Mercury (ng/L) <0.025 <0.025 <0.0252 <0.025 <0.02

Background Surface Water Arsenic 2.38 2.28 2.20 1.52 1.64

(BSW) Selenium 0.96 0.96 1.21 0.99 1.02

Cadmium 0.011 0.009 0.010 <0.008 0.008


Methyl Mercury (ng/L) 0.196 0.084 0.095 0.126 0.088

Treated Surface Water Arsenic 1.98 1.96 1.92 1.55 1.53

(TSW) Selenium 0.97 0.85 1.13 1.00 1.07

Cadmium 0.089 0.082 0.064 0.059 0.059


Methyl Mercury (ng/L) 0.145 0.079 0.079 0.091 0.060

Groundwater Arsenic 3.02 2.75 2.71 2.73 2.76

(GW) Selenium 12.9 11.8 11.8 11.8 12.2

Cadmium <0.008 <0.008 <0.008 <0.008 <0.00

Total Mercury (ng/L) 0.19 <0.15 <0.015 <0.15 <0.15

Methyl Mercury (ng/L) <0.025 <0.025 <0.025 <0.025 <0.02

Note: ng/L = nanograms per liter.

µg/L = micrograms/liter.

Source: Frontier Geosciences, 2005.

Sample Type

Table 13

"Dry Season" - Trace Metals Results in Water Samples

Concentration (ug/L unless noted)

X:\0233937C-0005-7\Tables\Table 13.xls.xls Golder Associates



October 2005

Date Collected: June 16, 2005 June 21, 2005 June 23, 2005 June 28, 2005 June 30, 2005 July 5, 2005

Analyte

Lab Control Water Arsenic 1.39 1.34 1.26 1.47 1.53 1.57

(LCW) Selenium 0.64 0.46 0.46 0.50 0.71 0.56

Cadmium 0.015 0.014 0.009 0.015 0.019 0.019

Total Mercury (ng/L) 0.39 0.46 0.21 0.71 0.42 1.37

Methyl Mercury (ng/L) < 0.025 < 0.025 < 0.025 < 0.025 0.029 <0.025

Background Surface Water Arsenic 2.92 1.89 2.00 2.69 2.66 2.50

(BSW) Selenium 0.80 0.87 0.70 1.30 1.41 1.27

Cadmium 0.023 < 0.016 < 0.016 0.010 < 0.008 0.009


Methyl Mercury (ng/L) 0.166 0.276 0.252 0.256 0.274 0.189

Treated Surface Water Arsenic 2.94 2.02 2.19 2.53 2.50 2.38

(TSW) Selenium 1.02 1.03 0.95 1.37 1.33 1.20

Cadmium 0.120 0.088 0.072 0.096 0.086 0.081


Methyl Mercury (ng/L) 0.180 0.261 0.219 0.200 0.139 0.173

Groundwater Arsenic 3.83 3.54 3.28 0.41 0.26 0.29

(GW) Selenium 12.5 11.3 10.7 15.4 14.7 14.8

Cadmium < 0.008 < 0.008 < 0.008 < 0.008 < 0.008 <0.008

Total Mercury (ng/L) 0.26 0.19 < 0.15 0.19 0.17 0.34

Methyl Mercury (ng/L) < 0.025 < 0.025 < 0.025 < 0.025 < 0.025 <0.025

Note: ng/L = nanograms per liter.

µg/L = micrograms per liter.

Source: Frontier Geosciences Inc., 2005.

Sample Type

Table 14

"Wet Season" - Trace Metals Results in Water

Concentration (µg/L unless noted)

X:\0233937C-0005-7\Tables\Table 14.xls Golder Associates



October 2005

Sample ID Percent 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

MHR Control 0 300 300 295 295 295 293 294 300 295 300 290 300 293 292 296 299 293

Background

Surfacewater 100 485 450 375 380 400 427 395 400 400 400 395 355 422 416 394 399 432

Treated

Surfacewater 100 470 455 375 380 405 429 392 400 395 400 400 385 392 385 386 398 434

Treated

Surfacewater:

Groundwater

80:20 925 915 875 935 935 978 921 900 865 875 880 880 882 882 867 888 884

Treated

Surfacewater:

Groundwater

50:50 1580 1530 1540 1590 1630 1722 1607 1550 1575 1560 1580 1610 1608 1576 1615 1614 1542

Treated

Surfacewater:

Groundwater

20:80 2215 2140 2150 2250 2360 2500 2320 2170 2220 2190 2250 2320 2320 2250 2350 2280 2210

Groundwater 100 2625 2465 2590 2630 2750 3040 2770 2620 2660 2640 2660 2750 2780 2680 2880 2760 2400

Conductivity (µmho/cm)

Table 15

Source: Hydrosphere Research, 2005b.

"Dry Season" - Conductivity (April 12 through May 3, 2005)

X://0233937C-0005/Final Report/Life-Cycle Toxicity Test.xls/Cond 2712.xls Golder Associates



October 2005

Sample ID Percent 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1

MHR Control 0 303 300 300 297 308 301 304 304 300 315 303 305 302 302 300 295 30

Background

Surfacewater 100 490 459 455 464 461 480 496 493 545 425 417 423 449 440 450 470 45

Treated

Surfacewater 100 469 481 460 473 477 466 500 507 525 425 420 522 421 446 450 475 46

Treated

Surfacewater:

Groundwater

80:20 943 917 930 907 933 925 953 963 995 870 909 972 884 899 895 950 94

Treated

Surfacewater:

Groundwater

50:50 1682 1617 1560 1572 1609 1569 1652 1636 1670 1555 1574 1672 1565 1589 1570 1610 15

TreatedSurfacewater:

Groundwater

20:80 2360 2260 2220 2230 2280 2220 2350 2310 2300 2210 2280 2360 2230 2300 2200 2240 21

Groundwater 100 2790 2680 2670 2660 2740 2700 2780 2720 2740 2760 2760 2780 2700 2700 2640 2700 25

Conductivity (µmho/cm)

Table 16

Source: Hydrosphere Research, 2005c.

"Wet Season" - Conductivity (June 16 through July 7, 2005)

X://0233937C-0005/Final Report/Life-Cycle Toxicity Test.xls/Cond 2784.xls Golder Associates



October 2005

Sample ID % 0

N O N O N O N O N O N O N O N O N O N O N O N O N O N O N O N O N

MHR Control 0 7.8 8 8 7.8 7.5 7.9 8 8.2 8.1 7.9 7.9 7.8 7.9 7.5 8.1 7.8 8 7.6 8 7.9 8.1 7.8 8 7.8 8 7.7 7.9 7.6 7.8 7.7 7.8 7.5 7.8

Background

Surfacewater 100 7.6 8 7.9 8 7.7 7.8 8 8.2 7.5 7.8 7.6 7.8 7.6 7.6 7.6 7.8 7.8 7.7 8 7.9 7.8 7.8 7.7 7.8 7.6 7.7 7.6 7.7 7.8 7.6 7.6 7.5 7.5

Treated

Surfacewater 100 7 8.1 7.1 7.5 6.8 7.9 7 8.2 6.8 7.9 6.9 7.8 7 7.3 6.9 7.9 7 7.7 7 8 7 7.9 6.7 7.8 6.7 7.8 6.7 7.7 6.7 7.7 6.9 7.4 6.5

Treated

Surfacewater:

Groundwater

80:20 7 8.1 7.1 7.8 6.7 7.8 7 8 6.8 7.9 6.8 7.8 6.9 7.5 6.8 7.8 7 7.7 7 7.9 6.9 7.5 6.8 7.7 6.7 7.7 6.6 7.6 6.7 7.6 7 7.4 6.5

Treated

Surfacewater:

Groundwater

50:50 6.9 8.1 7.1 7.6 6.5 7.8 7 7.9 6.8 7.8 6.8 7.7 6.9 7.3 6.7 7.7 7 7.6 7 7.8 6.9 7.8 6.7 7.6 6.6 7.6 6.7 7.5 6.7 7.5 7 7.3 6.6

Treated

Surfacewater:

Groundwater

20:80 6.9 8 7.1 7.5 6.5 7.7 7 7.8 7 7.7 7 7.6 7.1 7.2 6.8 7.6 6.9 7.3 7 7.7 6.9 7.7 6.9 7.4 6.7 7.4 6.8 7.4 6.8 7.5 7.2 7.3 6.8

Groundwater 100 8.1 7.5 8.4 7.4 8.4 7.7 8 7 .7 8.5 7.6 8.4 7.5 8.4 7.2 8.3 7.5 8.4 7.3 8 7 .6 8.3 7.6 8.3 7.4 8.2 7.4 8.2 7.3 8.1 7.4 8.4 7.2 8.3

Note: N = New Solution.O = Old Solution.

1612

Source: Hydrosphere Research, 2005b.

13 14 158 9 10 11

"Dry Season" - pH (April 12 through May 3, 2005)

Table 17

pH

1 2 3 4 5 6 7

X://0233937C-0005/Final Report/Life-Cycle Toxicity Test.xls/pH 2712.xls Golder Associates



October 2005

0

Sample ID Percent N O N O N O N O N O N O N O N O N O N O N O N O N O N O N O N O N

MHR Control 0 7.9 7.9 7.8 7.9 8.1 7.9 8.1 7.8 8.5 7.8 8.4 7.8 8.7 7.7 8.3 7.7 8.6 7.9 8.3 7.7 8.3 7.7 8.4 7.6 8.1 7.6 8.3 7.7 8.4 7.6 7.4 7.6 7.

Background

Surfacewater 100 7.3 8.1 7.3 7.9 7.5 8.2 7.5 8 7.5 8 7.5 8 7.6 8 7.5 8.1 7.6 8.2 7.6 7.9 7.4 7.9 7.6 7.8 7.4 7.8 7.4 7.8 7.5 8 7.4 7.9 7.5

Treated

Surfacewater 100 6 .6 8.1 6.7 7.9 6 .7 8.2 6.9 8.1 6.9 8 .1 6.9 8 7 .2 8 6 .8 8 7 8 .2 6.6 7 .9 6.5 7.9 7 .1 8 6 .7 7.7 6.7 7 .8 6.7 7 .8 6.7 7.5 6 .7

Treated

Surfacewater:

Groundwater

80:20 6.6 8.1 6.7 8 6.8 8.1 7 8 6.9 8 6.9 8 7.2 7.8 6.8 7.9 7.1 7.9 6.6 7.7 6.5 7.8 7.1 7.7 6.7 7.7 6.8 7.8 6.9 7.6 6.7 7.4 6.7

Treated

Surfacewater:

Groundwater

50:50 6.7 8 6.7 8 6.8 8 6.9 7.9 6.9 7.9 6.9 7.9 7.2 7.7 6.8 7.7 7.1 7.8 7.8 7.7 6.5 7.7 7.2 7.7 6.8 7.6 6.8 7.7 6.5 7.6 6.7 7.6 6.7

Treated

Surfacewater:

Groundwater

20:80 6.8 7.9 6.8 8 6.9 7.9 7.1 7.6 6.9 7.6 6.9 7.7 7.4 7.5 7.1 7.5 7.3 7.6 7.8 7.9 6.7 7.6 7.4 7.5 6.9 7.5 7 7.6 7 7.5 6.8 7.4 6.

Groundwater 100 8.2 7.7 8 7.8 7.9 7.7 7.9 7.5 7.6 7.4 7.8 7.3 7.7 7.3 8.3 7.3 8.5 7.4 8.5 7.3 8.3 7.5 8.5 7.3 8.3 7.3 8.3 7.4 8.3 7.3 8.4 7.2 8.4

Note: N = New solution.

O = Old solution.

1511

Source: Hydrosphere Research, 2005C.

16 12 13 147 8 9 10

"Wet Season" - pH June 16 through July 7, 2005)

Table 18

pH

1 2 3 4 5 6

X://0233937C-0005/Final Report/Life-Cycle Toxicity Test.xls/pH 2784.xls Golder Associates



October 2005

Treatment Analyte March 29, 2005 April 12, 2005 April 25Latoratory Control Water Radium 226 0.753 +/- 0.499 0.558 +/- 0.202 0.7 +/-

(LCW) Radium 228 <1.63 +/- 0.816 U <0.974 +/- 0.544 U <1.0 +/-

Background Surface Water Radium 226 2.15 +/- 0.637 0.999 +/- 0.254 0.7 +/-

(BSW) Radium 228 <1.45 +/- 0.693 U <1.37 +/- 0.758 U <1.0 +/-

Groundwater Radium 226 3.08 +/- 0.734 3.09 +/- 0.434 1.9 +/-

(GW) Radium 228 <1.60 +/- 0.817 U <1.07 +/- 0.588 U 1.6 +/-

Treated Surface Water Radium 226 0.290 +/- 0.148

(TSW) Radium 228 <1.47 +/- 0.796 U

U = below detection limit.

Source: ELAB, 2005.

Table 19

"Dry Season" - Radium Results in Water

(picocuries per liter)

Date Collected

X:\0233937C-0005-7\Table 19.xls Golder Associates



October 2005

Treatment Analyte June 16, 2005 June 30, 2005 July 14, 20

Laboratory Control Water Radium 226 0.883U +/- 0.567 0.613U +/- 0.423 U 1.08U +/- 0.7

(LCW) Radium 228 1.41U +/- 0.731 2.27U +/- 1.16 U 1.07U +/- 0.5

Background Surface Water Radium 226 0.571 +/- 0.370 1.20 +/- 0.559 0.717U +/- 0.

(BSW) Radium 228 1.34U +/- 0.686 1.21U +/- 0.56 U 1.25U +/- 0.6

Groundwater Radium 226 4.54 +/- 0.829 4.58 +/- 0.969 2.86 +/- 0.9

(GW) Radium 228 2.33U +/- 1.21 1.26U +/- 0.647 U 1.26 +/- 0.5

Note: U = below detection limit.

Source: ELAB, 2005.

Table 20

"Wet Season" - Radium Results in Water

(picocuries per liter)

Date Collected

X:\0233937C-0005-7\Table 20.xls.xls Golder Associates



October 2005 023-3937C

Analyte Concentration

Algae Arsenic (µg/L) <0.15Selenium (µg/L) <0.30

Cadmium (µg/L) <0.008

Total Mercury (ng/L) 1.1

Methyl Mercury (ng/L) 0.2

Radium 226 (picocuries) <0.665 ± 0.402*

Radium 228 (picocuries) <2.32 ± 1.33*

Fish Feed Arsenic (mg/kg) 2.3

Selenium (mg/kg) 1.63

Cadmium (mg/kg) 0.054

Total Mercury (ng/g) (B3) 40.1


Methyl Mercury (ng/g) 33.8

Note: mg/kg = micrograms per kilogram (wet weight).

ng/g = nanograms per gram (wet weight).

µg/L = microgram per liter.

B3 and B5. Total mercury analysis were conducted twice per sample by the laboratory

due to QA/QC requirements.

* = dry weight

Source: Frontier Geosciences, 2005 (metals).

ELAB, 2005 (radium).

Sample Type

Table 21

Trace Metals Results in Food Supply (Algae and Fish Feed)

Used for Bioconcentrations Studies

X:\0233937C-0005-7\4.2\Table 21.xls.xls Golder Associates



October 2005 023-3937C

Date Collected: March 31, 2005

Concentration

Sample Type Analyte (mg/kg unless noted)Bluegill Arsenic 0.40

Selenium 0.77

Cadmium < 0.010



Methyl Mercury (ng/g) 61.4

Mussel Arsenic 1.38

Selenium 0.75

Cadmium 0.566


Total Mercury (ng/g) (B5) 20.7Methyl Mercury (ng/g) 5.19

Note: mg/kg = micrograms per kilogram (wet weight).


B3 and B5. Total mercury analysis were conducted twice per sample by the

analytical laboratory due to QA/AC requirements.


Table 22

"Dry Season" - Trace Metals Background Results in Tissue Samples




October 2005

Sample Type Sample ID/Analyte A1 A2 A3 B1 BLab Control Water Arsenic 0.26 < 0.25 0.36 < 0.25 0.2

(LCW) Selenium 1.37 2.44 1.37 1.40 1.8

Cadmium < 0.010 < 0.010 < 0.010 < 0.010 < 0.

Total Mercury (ng/g) 60.5 43.1 41.8 51.0 42

Methyl Mercury (ng/g) 59.6 46.7 41.2 51.6 45

Background Surface Water Arsenic < 0.25 0.26 0.26 < 0.25 0.3

(BSW) Selenium 1.52 1.56 1.63 1.78 2.0

Cadmium < 0.010 < 0.010 < 0.010 < 0.010 < 0.


Methyl Mercury (ng/g) 43.9 98.8 51.2 117 55

Treated Surface Water Arsenic < 0.25 0.26 0.29 0.32 0.4(TSW) Selenium 1.77 1.71 1.58 1.18 0.7

Cadmium < 0.010 < 0.010 < 0.010 < 0.010 < 0.

Total Mercury (ng/g) 54.3 143 79.5 47.6 46

Methyl Mercury (ng/g) 66.4 174 68.5 56.0 57

Groundwater Arsenic 0.28 0.43 0.26 < 0.25 < 0

(GW) Selenium 1.14 0.72 1.51 1.83 1.4

Cadmium < 0.010 < 0.010 < 0.010 < 0.010 < 0.



Note: mg/kg = milligrams per kilogram (wet weight).



Replicates Sampled

Table 23

"Dry Season" - Bioconcentration -- Fish Tissue Post-Exposure Results

(Concentrations in mg/kg unless noted.)

X:\0233937C-0005-7\Final ReportTable 23.xls Golder Associates



October 2005

Treatment Analyte A1 A2 A3 B1Lab Control Water Arsenic 1.01 1.63 0.89 1.08 0

(LCW) Selenium 0.62 0.95 0.62 0.69 0

Cadmium 0.130 0.365 0.238 0.336 0

Total Mercury (ng/g) 15.1 11.3 28.5 31.2


Background Surface Water Arsenic 0.81 1.08 0.87 1.46

(BSW) Selenium 0.60 0.74 0.81 0.96 0

Cadmium 0.429 0.388 0.266 0.773 0



ASR Treated Arsenic 1.37 0.89 1.13 0.99 0

Selenium 0.54 0.77 0.69 0.66 0

Cadmium 0.683 0.545 0.762 0.324 0



Groundwater Arsenic 1.41 0.96 1.93 0.79 0

(GW) Selenium 0.58 0.63 0.88 0.81 0

Cadmium 0.957 0.644 0.322 0.054 0






Replicate Sampled

Table 24

"Dry Season" - Bioconcentration -- Mussel Tissue Post-Exposure Results

(Concentrations in mg/kg unless noted.)

X:\0233937C-0005-7\Tables\Table 24.xls Golder Associates



October 2005 023-3937

Treatment Analyte Post-Exposure

Mussel LCW C1 Radium 226 9.16 +/- 0.965

Radium 228 1.69 +/- 0.666

Mussel LCW D1 Radium 226 12.3 +/- 1.6

Radium 228 1.83 +/- 0.478

Mussel GW C1 Radium 226 12.8 +/- 1.52

Radium 228 1.78 +/- 0.862

Mussel GW D1 Radium 226 13.6 +/- 0.543

Radium 228 1.72 +/- 0.607

C1 and D1 are the replicate treatments used for radium bioconcentration

Source: ELAB, 2005.

Table 25

"Dry Season" - Radium Results in Mussel Tissues

(picocuries per gram dry weight)




October 2005 023-3937

Measured Analyte Post-Exposure

March 31, 2005 Radium 226 6.95 +/- 0.766

Radium 228 1.44 +/- 0.659

Source: ELAB, 2005.

Table 26

Background Radium in Mussel Tissues





October 2005

Sample Type Analyte

Fish Arsenic <0.25 <0.25

Selenium 1.32 1.54Cadmium <0.010 <0.010

Total Mercury (ng/g) 74 53.5

Methyl Mercury (ng/g) 70.3 41.2

Mussel Arsenic 1.4 1.34

Selenium 0.58 0.59

Cadmium 0.691 1.113

Total Mercury (ng/g) 31.99 32.49

Methyl Mercury (ng/g) 8.072 0.607

*Result reported is the average.

Note: ng/g = nanograms per gram (wet weight). mg/kg = milligram per kilogram wet weight.


Table 27

"Wet Season" - Trace Metals Background Results in Tissue Samples

Concentration (mg/kg unless n

X:\0233937C-0005-7\Table 27 revised.xls Golder Associates



October 2005 023-3937C

`

Treatment Analyte A1 A2 A3 B1 B2 B3 Average

Lab Control Water Arsenic < 0.25 < 0.25 < 0.25 < 0.25 < 0.25 < 0.25 < 0.25

Selenium 1.41 1.75 1.07 1.61 1.42 1.23 1.42Cadmium < 0.010 < 0.010 < 0.010 < 0.010 < 0.010 < 0.010 < 0.010

Total Mercury (ng/g) 58.3 60.6 36.2* 51.3 49.3 91.5 57.87

Methyl Mercury (ng/g) 46.1 57.1 51.8* 35.4 37.2 50.8 46.4

Background Surface Arsenic < 0.25 < 0.25 < 0.25 < 0.25 < 0.25 < 0.25 < 0.25

Water Selenium 1.67 1.35 1.43 1.63 1.18 1.16 1.40

Cadmium < 0.010 < 0.010 < 0.010 < 0.010 < 0.010 < 0.010 < 0.010

Total Mercury (ng/g) 51.7 87.9 140 135 47.3 53.5 85.9

Methyl Mercury (ng/g) 33.0 43.9 109 65.2 39.5 36.4 54.50

ASR Treated Arsenic < 0.25 < 0.25 < 0.25 < 0.25 < 0.25 < 0.25 < 0.25

Surface Water Selenium 1.25 1.14 1.27 1.38 1.35 1.81 1.37

Cadmium < 0.010 < 0.010 < 0.010 < 0.010 < 0.010 < 0.010 < 0.010

Total Mercury (ng/g) 122 48.3 63.0 85.2 76.7 56.6 75.3

Methyl Mercury (ng/g) 89.4 34.9 39.4 49.4 48.5 38.8 50.07

Groundwater Arsenic < 0.25 < 0.25 < 0.25 < 0.25 < 0.25 < 0.25 < 0.25Selenium 1.60 1.56 1.20 1.42 1.00 1.06 1.31

Cadmium < 0.010 < 0.010 < 0.010 < 0.010 < 0.010 < 0.010 < 0.010

Total Mercury (ng/g) 42.2 74.5 50.2 56.5 123 156 83.73

Methyl Mercury (ng/g) 28.6 23.7 21.6 15.2 34.2 55.1 29.73

Note: mg/L = milligrams per kilogram (wet weight).



Replicates Sampled

Table 28

"Wet Season" - Bioconcentration -- Fish Tissue Results

Concentrations in mg/kg unless noted.

X:\0233937C-0005-7\Table 28.xls.xls Golder Associ ates



October 2005

Treatment Analyte A1 A2 A3 B1Lab Control Water Arsenic 1.49 1.11 0.99 1.01

(LCW) Selenium 0.80 0.74 0.68 0.51

Cadmium 1.04 0.678 0.858 0.570 0


Methyl Mercury (ng/g) 9.08 8.06 6.19 11.0

Background Surface Water Arsenic 1.80 0.95 1.52 0.91

(BSW) Selenium 0.90 0.53 0.76 0.52

Cadmium 1.37 0.837 0.968 0.664 0



ASR Treated Surface Water Arsenic 1.18 0.95 1.05 0.83Selenium 0.52 0.62 0.49 0.61

Cadmium 1.02 0.469 0.627 0.783 0



Groundwater Arsenic 0.94 0.96 1.14 1.00

(GW) Selenium 0.58 0.61 0.51 0.56

Cadmium 0.961 0.600 1.06 0.728 0






Replicates Sampled

Table 29

"Wet Season" - Bioconcentration -- Mussel Tissue Results

Concentrations in mg/kg unless noted.




October 2005 023-3937

Treatment Analyte Test Completion Average

Mussel LCW C1 Radium 226 8.48 +/- 1.04 8.32Radium 228 2.5 +/- 0.932 Radium 226

Mussel LCW D1 Radium 226 8.16 +/- 1.1 2.7

Radium 228 2.9 +/- 1.06 Radium 228

Mussel GW C1 Radium 226 8.66 +/- 1.17 8.1

Radium 228 2.65 +/- 1.13 Radium 226

Mussel GW D1 Radium 226 7.55 +/- 0.946 2.7

Radium 228 2.76 +/- 0.939 Radium 228

C1 and D1 are the replicate treatments used for radium bioconcentration.

Source: ELAB, 2005.


"Wet Season" - Radium Results in Tissues

Table 30




FIGURES



//2002/0233937-0005/Final Report/Figure 1.vsd

igure 1.

low Chart for Preliminary Investigation

f Ecotoxico logical Effects of Recovered ASR Water

2. Acquire

Surface Water

3. Acquire

Ground Water

4. Prepare Mix

of Surface and

Ground Water

5. Evaluation of

Standard Test

Organisms

6. Evaluation of

Resident Test

Organisms

7. Develop

AcclimatedStandard Test

Organisms

1. Program

Management

Screening-Level Method Development

Reports

Meetings

Kickoff

Meeting

Day 14

Midcourse

Meeting

Day 194

Semi-annual Team Meetings

To Be Scheduled

Interim Report Interim Report

Phase 1

ReportDay 345

Phase 1

Meeting

Day 360

Kickoff

Report

Day 21

Interim Report

Day 180

Midcourse Meeting

Report

Day 200

Phase 1 Phase 2

eptember 2005



December 2005 023-3937C

Figure 2

Photograph of ASR Bench-scale Treatment System

X://2002/0233937C-0005/4.2/Final Report/Figure 2.doc.doc

UV TREATMENT

pH ADJUSTMENT

SAND

FILTER



12/7/2005 0233937C-0005/4.2/Figure 3.doc.do

Figure 3

UV Sterilization and pH Adjustment Systems

Source: Golder, 2005.

Photo 3a. UV Sterilization System

Photo 3b. pH Adjustment System

CO2 Gas

pH

controller

pH

Probe

Stir Plate

UV Sterilizer

Sand Filtered

Sufacewater

Peristaltic Pump



December 2005

X://2002/0233937C-0005-7/4.2/Final Report/Figure 4.doc.doc

Figure 4

Caloosahatchee Pilot Project Site Facility Layout Showing Sampling Locations (ASR-1 Well, Header Canal, and Sedime

HEADER CANAL

ASR-1

SEDIMENT

SAMPLING

SITE

PUMP STATION NO. 1

SW-1



December 2005 023-3937C


Figure 5

Background Surface Water Sampling Location – Header Canal,

Caloosahatchee Pilot Project Site

Photo 5a. Pump Station Number 1 (SW-1)

Photo 5b. Header Canal



December 2005 023-3937C

X://2002/0233937C-0005/4.2Final Report/Figure 6.doc.doc

Figure 6

Floridan Aquifer Well (ASR-1) at the Caloosahatchee Pilot Project Site



December 2005 023-3937C


Figure 7

Fathead Minnow and Ceriodaphnia dubia Chronic Test

Photo 7a. Fathead Minnow (Pimephales promelas)

Photo 7b. Daphnid Assay



December 2005


Figure 8

Frog Embryo Teratogenesis Assay – Xenopus (FETAX)

Source: Fort Environmental Laboratories website.



December 2005 023-3937C

X://2002/0233937C/4.2/Final Report/Figure 9.doc.doc

Figure 9

Modified Bench-scale Sand Filter



December 2005 023-3937C


Figure 10

Test Species Used in the Bioconcentration Scoping Study

Photo 10a. Freshwater mussel, Elliptio buckleyi

Photo 10b. Bluegill, Lepomis macrochirus



December 2005


Figure 11

Bioconcentration Study Static-renewal (Continuous Flow) Exposure System

Flow-through System with Head Tanks (top row), Pumps (middle-left row), and Exposure Tanks (middle and bottom row

PUMPS

052808 asr ch8 asr ecotox phase1

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