United States
Environmental Protection
Agency
Office Of Water
(4203)
EPA 833-B-99-002
August 1999
vyEPA
Toxicity Reduction
Evaluation Guidance For
Municipal Wastewater
Treatment Plants
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EPA/833B-99/002
August 1999
Toxicity Reduction Evaluation Guidance
for Municipal Wastewater Treatment Plants
Office of Wastewater Management
U.S. Environmental Protection Agency
Washington, D.C. 20460
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Notice and Disclaimer
The U.S. Environmental Protection Agency, through its Office of Water, has funded, managed, and
collaborated in the development of this guidance, which was prepared under order 7W-1235-NASX to
Aquatic Sciences Consulting; order 5W-2260-NASA to EA Engineering, Science and Technology, Inc.; and
contracts 68-03-3431, 68-C8-002, and 68-C2-0102 to Parsons Engineering Science, Inc. It has been
subjected to the Agency's peer and administrative review and has been approved for publication.
The statements in this document are intended solely as guidance. This document is not intended, nor can it
be relied on, to create any rights enforceable by any party in litigation with the United States. EPA and State
officials may decide to follow the guidance provided in this document, or to act at variance with the
guidance, based on an analysis of site-specific circumstances. This guidance may be revised without public
notice to reflect changes in EPA policy.
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Foreword
This document is intended to provide guidance to permittees, permit writers, and consultants on the general
approach and procedures for conducting toxicity reduction evaluations (TREs) at municipal wastewater
treatment plants. TREs are important tools for Publicly Owned Treatment Works (POTWs) to use to identify
and reduce or eliminate toxicity in a wastewater discharge. TREs may be required by the discharger's
National Pollutant Discharge Elimination System (NPDES) permit or through state or federal enforcement
actions. Dischargers can use the guidance to evaluate the nature and sources of effluent toxicity before a
TRE becomes a regulatory requirement. Whether the TRE is voluntary or mandated, this guidance can be
helpful in preparing and executing a plan to address effluent toxicity.
This guidance describes the general approaches that have been successfully used in municipal TREs. Each
TRE will be different; therefore, the strategy for conducting TREs should be tailored to address site-specific
conditions. The components of a TRE may include the collection and review of pertinent data; an evaluation
of the treatment facility to identify conditions that may contribute to effluent toxicity; identification of
effluent toxicants using toxicity identification evaluation (TIE) procedures (USEPA 1991a, 1992a, 1993a,
1993b, 1996); location of the sources of toxicants and/or toxicity using chemical analysis or refractory
toxicity assessment (RTA) procedures; and the evaluation, selection, and implementation of toxicity control
measures. Dischargers are encouraged to develop a TRE plan that describes the initial components to
perform in the TRE. Following initial testing, the results can be used to provide direction for further testing
to identify the cause(s) and source(s) of toxicity and evaluate and select methods for toxicity control.
This document is an update of the municipal TRE protocol that was published in 1989 (USEPA, 1989a).
Much experience has been gained since 1989, including the use of a number of freshwater and estuarine/
marine species in acute and chronic TRE studies and the development of additional procedures for TIE and
RTA studies. In most cases, the approaches and methods described in the municipal TRE protocol have been
validated through TRE studies and other municipal TREs (Amato et al., 1992; Bailey et al, 1995; Botts et
al., 1990, 1992, 1993, 1994; Collins et al., 1991; Fillmore et al., 1990; Lankford and Eckenfelder, 1990;
Morris et al., 1990, 1992). Important lessons have been learned and this information has been incorporated
in this guidance where possible. Additions to this guidance include considerations in evaluating the
operation and performance of current publicly owned treatment works (POTW) technology, descriptions of
current TIE procedures for acute and short-term chronic toxicity (USEPA 199la, 1992a, 1993a, 1993b,
1996), updated methods for tracking sources of acute and chronic toxicity in POTW sewer collection
systems, and additional recent case studies on acute and chronic TREs using freshwater and estuarine/marine
species. Information is also provided on sampling requirements, equipment and facilities, quality
assurance/quality control, and health and safety.
The updated TIE guidance procedures are important tools for conducting TREs including Toxicity
Identification Evaluation: Characterization of Chronically Toxic Effluents, Phase I (USEPA, 1992a),
Methods for Aquatic Toxicity Identification Evaluations: Phase I Toxicity Characterization Procedures,
Second Edition (USEPA, 199la), Marine Toxicity Identification Evaluation (TIE) Guidance Document,
Phase I (USEPA, 1996), Methods for Aquatic Toxicity Identification Evaluations: Phase II Toxicity
Identification Procedures for Samples Exhibiting Acute and Chronic Toxicity (USEPA, 1993 a), and Methods
for Aquatic Toxicity Identification Evaluations: Phase III Toxicity Confirmation Procedures for Samples
Exhibiting Acute and Chronic Toxicity (USEPA, 1993b). The acute and chronic whole effluent toxicity
testing manuals should also be reviewed during the TRE process (USEPA 1993c, 1994a, 1994b, 1995). These
manuals describe procedures for the toxicity tests that are the core of the TREs.
in
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Contents
Foreword iii
Acronyms, Abbreviations, and Symbols viii
Acknowledgments xii
Section 1 Introduction 1
Background 1
TRE Goals and Objectives 3
Components of the Municipal TRE 3
Limitations of the TRE Guidance 5
Organization of the TRE Guidance 6
Section 2 Information and Data Acquisition 7
Introduction 7
Review of Effluent Toxicity Data 7
Toxicants Identified in POTW Effluents 8
POTW Design and Operations Data 10
Pretreatment Program Data 12
Section 3 Facility Performance Evaluation 15
Introduction 15
POTW Performance Evaluation 15
Section 4 Toxicity Identification Evaluation 29
Introduction 29
Toxicity Tests 29
TIE Procedures 32
Additional Characterization Procedures for Evaluating the Effect of Ion Composition ... 34
TDS Toxicity 34
Ion Imbalance 35
Section 5 Toxicity Source Evaluation 38
Introduction 38
Sampling Approach 40
Sampling Conditions 40
Chemical-Specific Investigation 40
Refractory Toxicity Assessment 41
POTW Wastewater Profile 43
Findings of the Toxicity Source Evaluation 51
Section 6 Toxicity Control Evaluation 53
Introduction 53
Identifying Toxicity Control Options 53
Pretreatment Control Evaluation 56
In-Plant Control Evaluation 57
Toxicity Control Selection 61
IV
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Contents (continued)
Section 7 Toxicity Control Implementation 64
Introduction 64
Implementation 64
Follow-Up Monitoring 64
Section 8 Quality Assurance/Quality Control 65
Introduction 65
Sample Collection and Preservation 65
Chain-of-Custody 66
TRE Procedures 66
Equipment Maintenance 68
Documentation and Reporting of Data 68
Corrective Action 68
Section 9 Health and Safety 69
Introduction 69
Sample Collection and Handling 69
TRE Methods 69
General Precautions 70
Section 10 Facilities and Equipment 71
Introduction 71
Toxicity Identification Evaluations 71
Refractory Toxicity Assessment and Treatability Tests 71
General Analytical Laboratory Equipment 72
Section 11 Sample Collection and Handling 73
Introduction 73
Sampling Location 73
POTW Sampling 73
Sewer Discharge Sampling '. 74
Section 12 References 75
Section 13 Bibliography 82
Appendices
A Original Case: Histories: Lesson Learned 84
B TRE Case Study: Central Contra Costa Sanitary District, Martinez, California 93
C TRE Case Study: City of Reidsville, North Carolina 98
D TRE Case Study: City of Durham, North Carolina 105
E TRE Case Study: Michigan City Sanitary District, Indiana 113
F TRE Case Study: Central Contra Costa Sanitary District, Martinez, California, and Other
San Francisco Bay Area POTWs 119
G TRE Case Study: Linden Roselle Sewerage Authority, New Jersey 133
H Toxicity Control Options for Organophosphate Insecticides 142
I Pretreatment Program Chemical Review 147
J Refractory Toxicity Assessment Protocol: Step-by-Step Procedures 153
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Figures
Figure 1-1. TRE flow diagram for municipal wastewater treatment plants 4
Figure 3-1. Flow diagram for a facility performance evaluation 16
Figure 4-1. Flow diagram of a toxicity identification evaluation 30
Figure 5-1. Flow diagram for a toxicity source evaluation 39
Figure 5-2. Schematic of a refractory toxicity assessment test 42
Figure 5-3. Theoretical results of inhibition testing 51
Figure 6-1. Flow diagram for a toxicity control evaluation 54
Figure A-l. Acute LC50 of Hollywood effluent versus diazinon concentration 86
Figure A-2. Correlation of diazinon TUs versus whole effluent TUs 87
Figure A-3. Correlation of diazinon and CVP TUs versus whole effluent TUs 87
Figure C-l. Results of RTA (rounds 1 and 2) 101
Figure C-2. Results of RTA (round 3) 102
Figure D-l. Flow diagram for wastewater treatment situations 107
Figure E-l. Acute and chronic effluent toxicity: 1991 through 1992 117
Figure F-l. Effluent TUs versus diazinon TUs in the CCCSD effluent samples 122
Figure F-2. Percent mass contribution to sources to the CCCSD influent 125
Figure F-3. Mean diazinon and chlorpyrifos concentrations (ฑstd) in influent and effluent from
three Bay Area POTWs 127
Figure F-4. Mean chlorpyrifos and diazinon concentrations (ฑstd) in influent and effluent from
nine Bay Area POTWs during August 1997 128
Figure H-l. Diazonin removal as a function of SRT, HRT, and MLSS concentration 145
VI
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Tables
Table 2-1. Toxicants Identified in POTW Effluents r 9
Table 2-2. Example POTW Design and Operation Data 11
Table 2-3. Example Pretreatment Program Data 14
Table 5-1. POTW Wastewater Profile Analyses for a Refractory Toxicity Assessment 44
Table 5-2. Example of Bracketing the LC50 Concentration in the RTA Sewer Wastewater Test ... 46
Table 5-3. Comparison of Control Test and Industrial Wastewater Spiked Test Results 46
Table 6-1. An Example of the Comparison of In-Plant Ammonia Treatment Alternatives 55
Table 6-2. POTW In-Plant Control Technologies for Categories of Toxic Compounds 58
Table A-l. Comparison of Whole Effluent TUs and Methanol Fraction TUs 88
Table A-2. Summary of TIE Phase H Results 89
Table B-l. Summary of Results of Phase I TIE Conducted on Two Effluent Samples with
D. excentricus 94
Table B-2. NOECs Obtained for D. excentricus and 5. purpuratus Exposed to Different Metals ... 95
Table B-3. Comparison of Effluent Concentration of Selected Metals with NOECs Derived from
Laboratory Studies with D. excentricus : 95
Table B-4. Comparison of NOECs, LOECs, and Percent Fertilization Obtained with D. excentricus
Exposed to Effluent and Seawater Spiked with Cu 96
Table B-5. Percent Fertilization Obtained with D. excentricus Exposed to Effluent and Effluent
Spiked with Cu 96
Table C-l. Chronic Toxicity of Raw Industrial Wastewaters 99
Table C-2. Description of Industries Evaluated in the RTA 99
Table C-3. Comparison of Operating Conditions for the City of Reidsville POTW Processes
and RTA Simulation Tests 100
Table D-l. Farrington Road and Northside Simulation Operating Conditions 108
Table D-2. Comparison of Calibration Test Results to Permit Limitations and Design Criteria 109
Table D-3. Total Phosphorus Results for the Calibration Tests Conducted on April 10-11, 1990 .. 109
Table D-4. Comparison of Simulation Test Results to Performance Criteria Ill
Table D-5. Toxicity of Simulation Effluents to C. dubia Ill
Table E-l. Acute Toxicity Characterization Test Results from April 1991 Through June 1991 114
Table E-2. Toxicity Characterization Test Results form July 1991 Through October 9, 1991 115
Table E-3. Acute and Chronic Toxicity of MCWTP' s Effluent (with and without added EDTA)
from October 1991 Through January 1992 , 116
Table F-l. Matrix of Results of Phase I TIE Conducted on Five Effluent Samples with C. dubia .. 121
Table F-2. Summary of TIE Phase H Results 121
Table F-3. Summary of Follow-Up TIE Studies 123
Table F-4. Diazinon and Chlorpyrifos concentrations in Wastewater Samples from Selected
Residential and Commercial Sources in the CCCSD 124
Table G-l. TIE Phase m Results: Non-Polar Organic Compound Confirmation (LRSA POTW) .. 135
Table G-2. Results of Refractory Toxicity Assessment, July and October 1993 137
Table 1-1. PPCR Data Sheet 148
Table 1-2. Data Sheet for Regression Analysis 149
Table 1-3. Summary of the PPCR Chemical Optimization Procedures 150
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Acronyms, Abbreviations, and Symbols
AA Atomic absorption
AGO Administrative consent order
Ag Silver
Alum Aluminum sulfate
A/Oฎ Patented biological nutrient removal process
AQUIRE Aquatic Information Retrieval Toxicity Data Base
APE Alkylphenol ethoxylates
ASCE American Society of Civil Engineers
ASM American Society for Microbiology
ASTM American Society for Testing and Materials
ATP Adenosine triphosphate
BNR Biological nutrient removal
BOD Biochemical oxygen demand
BOD5 Five-day biochemical oxygen demand
BOD^) Twenty-day biochemical oxygen demand
ฐC Degrees centigrade
CaCO3 Calcium carbonate
CADPR California Department of Pesticide Registration
Cb Concentration of specified analyte in filtrate of return activated sludge biomass
CCCSD Central Contra Costa Sanitary District
C. dubia Ceriodaphnia dubia, cladoceran
Cd Cadmium
Ce Effluent concentration of the specified analyte
CERCLA Comprehensive Environmental Response, Compensation, and Liability Act
CFR Code of Federal Regulations
ChV Chronic value, geometric mean of the no observed effect concentration (NOEC) and
lowest observed effect concentration (LOEC)
Ci Influent concentration of the specified analyte
CO2 Carbon dioxide
COG Chain-of-custody
COD Chemical oxygen demand
Cpe Concentration of specified analyte in primary effluent
Cr Chromium
CSO Combined sewer overflow
CTAS Cobalt thiocyanate active substances
Cu Copper
CuSO4 Copper sulfate
cu ft/lb Cubic feet per pound
CV Coefficient of variation
CVP Chlorfenvinphos
Cw Concentration of specified analyte in sewer wastewater
CWA Clean Water Act
CIS Carbon-based resin used in solid phase extraction (SPE) columns to evaluate for the
presence of nonpolar organic toxicants
D. excentricus Dendraster excentricus, sand dollar
D. magnet Daphnia magna, cladoceran
D. pulex Daphnia pulex, cladoceran
DMR Discharge monitoring report
vni
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Acronyms, Abbreviations, and Symbols (continued)
DO
EC50
ECOTOX
EDTA
ELISA
EXTOXNET
F/M
F/Mb
Fw
g/L
gpd/sq ft
gpm/sf
GAC
GC
GC/MS
H&S
Hg
HNO3
H202
HPLC
HRT
ICP
ICp
IWC
IWS
L
LC50
LRSA
LOEC
m3/m2/day
MB AS
MCRT
MCWTP
mg/L
mg O2/hr/g
mg O2/L/
Dissolved oxygen
Effective concentration causing a 50% effect in the test species
Ecotoxicology Database Retrieval System
Ethylenediaminetetraacetate
Enzyme-linked immunosorbent assay
Extension Toxicology Network
Food to microorganism ratio
Food to microorganism ratio
Flow concentration factor for RTA testing
Grams per liter
Gallons per day per square foot
Gallons per minute per square foot
Granular activated carbon
Gas chromatography
Gas chromatograph/mass spectrophotometer
Health and safety
Mercury
Nitric acid
Hydrogen peroxide
High-performance liquid chromatography
Hydraulic retention time
Inductively coupled plasma spectrometry
Inhibition concentration causing a percent effect (p) in the test species (e.g., IC25, IC50)
Instream waste concentration
Industrial waste survey
Liter
Lethal concentration causing a 50% mortality in exposed test organisms
Linden Roselle Sewerage Authority
Lowest observed effect concentration
Cubic meters per square meter per day
Methylene blue active substances
Mean cell residence time
Michigan City Wastewater Treatment Plant
Milligram per liter
Milligram dissolved oxygen per hour per gram
Milligram dissolved oxygen per liter per gram mixed liquor volatile suspended solids
gMLVSS/min per minute
M. bahia Mysidopsis bahia, mysid shrimp
Milliliter
Mixed liquor suspended solids
Mixed liquor volatile suspended solids
Mass spectrometry
Material safety data sheet
million gallons per day
Sodium chloride
Disodium phosphate
Monosodium phosphate
North Carolina Division of Environmental Management
mL
MLSS
MLVSS
MS
MSDS
mgd
NaCl
Na2HPO4
NaH2PO4
NCDEM
IX
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Acronyms, Abbreviations, and Symbols (continued)
NETAC USEPA' s National Effluent Toxicity Assessment Center
NH3 Un-ionized ammonia
NH4* Ammonium ion
NH3-N Total ammonia as nitrogen
Ni Nickel
NIOSH National Institute of Occupational Safety and Health
NJDEP New Jersey Department of Environmental Protection
NOEC No observed effect concentration
NO3-N Nitrate as nitrogen
NJPDES New Jersey Pollutant Discharge Elimination System
NPDES National Pollutant Discharge Elimination System
NPO Non-polar organic
NTIS National Technical Information Service
O2 Oxygen
O. mykiss Oncorhynchus mykiss, trout
OSHA Occupational Safety and Health Administration
OUR Oxygen uptake rate, a measure of the activity of activated sludge biomass
P Phosphorus
PAC Powdered activated carbon
Pb Lead
PBO Piperonyl butoxide, a metabolic blocking agent used to detect for the presence of
organophosphate insecticides
pH Logarithm of the reciprocal of the hydrogen ion concentration
PO4-P Orthophosphate as phosphorus
POTW Publicly owned treatment works
P. promelas Pimephales promelas, fathead minnow
PPCR Pretreatment program chemical review
Qi POTW influent flow rate
Qw Sewer wastewater flow rate !
QA/QC Quality assurance/quality control
r value Correlation coefficient
RAS Return activated sludge
RBC Rotating biological contactor
RCRA Resource Conservation and Recovery Act
RREL Risk Reduction Engineering Laboratory
RTA Refractory toxicity assessment
RWQCP Regional Water Quality Control Plant
SARA Superfund Amendments and Reauthorization Act
SBOD5 Five-day soluble biochemical oxygen demand
SCOD Soluble chemical oxygen demand
sf square foot
SIC Standard industrial classification
SNAP Sewer network analysis program
SNH3-N Soluble ammonia - nitrogen
SO2 Sulfur dioxide
SOP Standard operating procedures
SOR Surface overflow rate
SOUR Specific oxygen uptake rate
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Acronyms, Abbreviations, and Symbols (continued)
S. purpurtus Strongylocentrotus purpurtus
SP Soluble phosphorus
SPI Simulated plant influent
SPE Solid phase extraction
SRT Sludge retention time
SS Suspended solids
SSO Sanitary sewer overflow
SSUR Specific substrate utilization rate
SVI Sludge volume index
TBOD5 Total five-day biochemical oxygen demand
TCIP Toxics control implementation plan
TCLP Toxicity characteristic leaching procedure
TDS Total dissolved solids
TIE Toxicity identification evaluation
TKN Total Kjeldahl nitrogen
TMP Toxicity management program
TOC Total organic carbon
TP Total phosphorus
TRC Total residual chlorine
TRE Toxicity reduction evaluation
TSD Technical Support Document
TSDF Treatment, storage, and disposal facility
TSS Total suspended solids
TU Toxic unit
TUa Acute toxic unit
TUc Chronic toxic unit
ug/L Microgram per liter
jam Micron
UMVUE Uniformly Minimum Variance Unbiased Estimator
USD Union Sanitary District
USEPA United States Environmental Protection Agency
UV Ultraviolet
UV-VIS Ultraviolet - visible spectrophotometer
Vb Volume of biomass for RTA testing
Vr Reactor volume for RTA testing
Vnb Volume of non-toxic biomass for RTA testing
Vpe Volume of primary effluent for RTA testing
Vsw Volume of synthetic wastewater for RTA testing
Vw Volume of sewer wastewater for RTA testing
VSS Volatile suspended solids
WEF Water Environment Federation
WPCF Water Pollution Control Federation (currently Water Environment Federation)
WWTP Wastewater treatment plant
xg Times gravity
Zn Zinc
ZSV Zone setting velocity
XI
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Acknowledgments
This guidance was prepared through the cooperative efforts of USEPA; Aquatic Sciences Consulting; EA
Engineering, Science and Technology, Inc.; Parsons Engineering-Science, Inc.; and Burlington Research,
Inc. The principal authors of the municipal TRE protocol (USEPA, 1989a) were John A. Botts, Jonathan
W. Braswell, Jaya Zyman, William L. Goodfellow, Samuel B. Moore, and Dolloff F. Bishop. Although
much of the information presented in the TRE protocol (USEPA, 1989a) is included herein, the primary
authors of this guidanceJohn A. Botts, William L. Goodfellow, Mark A. Collins, Timothy L. Morris, and
Richard A. Diehlhave updated the TRE protocol (USEPA, 1989a) using information from recent studies
and guidance. Contributors to specific sections of this guidance are listed below.
Sections 1 through 7 - Main Guidance:
John A. Botts, Aquatic Sciences Consulting, Woodbine, Maryland
Jonathan W. Braswell, Parsons Engineering-Science, Inc., Fairfax, Virginia
Timothy L. Morris, ENTRIX, Inc., Wilmington, Delaware
William L. Goodfellow, EA Engineering, Science and Technology, Inc., Sparks, Maryland
Dolloff F. B ishop, USEPA, Cincinnati, Ohio
Elizabeth Sullivan, CH2M Hill, Reston, Virginia
H. Jeffrey Elseroad, EA Engineering, Science and Technology, Inc., Sparks, Maryland
Samuel B. Moore, Burlington Research, Inc., Burlington, North Carolina
Richard A. Diehl, Burlington Research, Inc., Burlington, North Carolina
John Cannell, former USEPA employee, Portland, Oregon
Sections 8 through 11 - Laboratory Guidelines:
John A. Botts, Aquatic Sciences Consulting, Woodbine, Maryland
Elaine Wilson, Parsons Engineering-Science, Inc., Fairfax, Virginia
Jaya Zyman, CH2M Hill, Reston, Virginia
Appendices A through G - TRE Case Summaries:
John A. Botts, Aquatic Sciences Consulting, Woodbine, Maryland
Mark A. Collins, Parsons Engineering-Science, Inc., Fairfax, Virginia
Jeffrey Miller, AQUA-Science, Inc., Davis, California
Timothy L. Morris, ENTRIX, Inc., Wilmington, Delaware
Lauren Fillmore, Parsons Engineering-Science, Inc., Fairfax, Virginia
Mary Welch, Science Applications International Corporation, Denver, Colorado
James Salisbury, Parsons Engineering-Science, Inc., Fairfax, Virginia
Christina L. Cooper, Aquatic Sciences Consulting, Woodbine, Maryland
William Clement, Great Lakes Environmental Center, Columbus, Ohio
Bart Brandenburg, Central Contra Costa Sanitary District, Martinez, California
Bhupinder Dahliwal, Central Contra Costa Sanitary District, Martinez, California
Jim Kelly, Central Contra Costa Sanitary District, Martinez, California
Jerry Rothrock, City of Reidsville, Reidsville, North Carolina
A.T. Rolan, City of Durham, Durham, North Carolina
W.W. Sun, City of Durham, Durham, North Carolina
Vicki Westbrook, City of Durham, Durham, North Carolina
Susan Turbak, City of Durham, Durham, North Carolina
Gary Fare, Linden Roselle Sewage Authority, Linden, New Jersey
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Judy Spadone, Linden Roselle Sewage Authority, Linden, New Jersey
Appendix H - Toxicity Control Options for Organophosphate Insecticides
John A. Botts, Aquatic Sciences Consulting, Woodbine, Maryland
Carlos Victoria-Rueda, Parsons Engineering-Science, Inc., Austin, Texas
Lauren Fillmore, Parsons Engineering-Science, Inc., Fairfax, Virginia
Dennis Tierney, Novartis Crop Protection, Greensboro, North Carolina
Appendix I - Pretreatment Program Chemical Review:
Richard A. Diehl, Burlington Research, Inc., Burlington, North Carolina
Samuel B. Moore, Burlington Research, Inc., Burlington, North Carolina
Appendix J - Refractory Toxicitv Assessment Protocol: Step-bv-Step Procedures
John A. Botts, Aquatic Sciences Consulting, Woodbine, Maryland
Timothy L. Morris, ENTRDC, Inc., Wilmington, Delaware
Mark A. Collins, Parsons Engineering-Science, Inc., Fairfax, Virginia
The following individuals are gratefully acknowledged for their technical review and advice on this
guidance:
Stephen Bainter, USEPA, Region VI, Dallas, Texas
Stephen L. Bugbee, retired USEPA employee, Petersburg, Pennsylvania
Debra Denton, USEPA, Region K, San Francisco, California
Teresa J. Norberg-King, USEPA, Duluth, Minnesota
Laura Phillips, USEPA Office of Wastewater Management, Washington, D.C.
Donna Reed-Judkins, USEPA Office of Science and Technology, Washington, D.C.
Carl Potter and Richard Dobbs - USEPA Office of Research and Development, Cincinnati, Ohio
William J. Rue, EA Engineering, Science and Technology, Inc., Sparks, Maryland
G. Michael DeGraeve - Great Lakes Environmental Center, Traverse City, Michigan
Sheila Frace, USEPA Office of Science and Technology, Washington, D.C.
Jeffrey Lape, USEPA Office of Wastewater Management, Washington, D.C.
Linda Anderson-Carnahan, USEPA, Region IV, Atlanta, Georgia
Donald I. Mount, AScI Corporation, Duluth, Minnesota
Carlos Victoria-Rueda, Parsons Engineering-Science, Inc., Austin, Texas
Stephen Bainter, Debra Denton, and Teresa J. Norberg-King were technical advisors for the preparation of
this guidance (order 7W-1235-NASX to Aquatic Sciences Consulting). Ms. Laura Phillips and Mr. Stephen
Bugbee served as USEPA Technical Project Managers under order 7W-1235-NASX to Aquatic Sciences
Consulting and order 5W-2260-NASA to EA Engineering, Science and Technology, Inc., respectively.
Peer review was conducted following the USEPA's Science Policy Council Handbook for Peer Review
(January 1998). The review comments are gratefully acknowledged and the changes were incorporated, as
appropriate. USEPA's Office of Water, Office of Wastewater Management provided support for the
development of this guidance.
xm
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Section 1
Introduction
Background
The Clean Water Act (CWA) (United States Federal
Water Pollution Control Act Amendments, Public Law
92-500 of 1972) prohibits the discharge of "toxic
pollutants in toxic amounts." In the CWA, the
mechanism for regulating discharges to the Nation's
waterways is the National Pollutant Discharge
Elimination System (NPDES). Permits issued under
NPDES may contain effluent limits and other
requirements based on ambient water quality standards
for the protection of aquatic life and human health.
The water quality-based approach applies criteria for
both chemical specific parameters and whole effluent
toxicity to ensure that toxic pollutants are controlled
and water quality standards are maintained (Federal
Register 23868, 1989). This integrated approach to
water quality protection is described in detail in
USEPA's Technical Support Document for Water
Quality-Based Toxics Control (hereafter referred to as
the TSD, 199 Ib).
"Whole effluent toxicity" refers to the results of acute
and chronic aquatic toxicity tests used to monitor
discharges to surface waters. Acute toxicity is a
measure of primarily lethal effects that occur over a
short period of time (i.e., 96 hours or less). Chronic
toxicity refers to sublethal effects, such as inhibition of
fertilization, growth, and reproduction that occur over
a longer exposure period (e.g., 7 days). Acute and
chronic effects to ;aquatic species are measured using
standard procedures (40 CFR 136.3) as specified in
NPDES permits. USEPA has published manuals that
describe the toxicity test methods for freshwater and
estuarine/marine organisms (USEPA 1993c, 1994a,
1994b, 1995). On October 26, 1995, USEPA
promulgated a final rule under the CWA that adds
whole effluent toxicity testing methods to the list of
nationally applicable methods in 40 Code of Federal
Regulations (CFR) Part 136. These methods can be
accessed electronically along with all other approved
analytical methods on CD-ROM (USEPA, 1997).
Effluents from permitted facilities are monitored, and
where a reasonable potential exists to exceed numeric
toxicity criteria, NPDES permit limits for whole
effluent toxicity are established (40 CFR
122.44(d)(l)(iv)). Whole effluent toxicity limits may
also be established where there is reasonable potential
to exceed a narrative toxicity criterion in the receiving
water (40 CFR 122.44(a)(l)(v)). A toxicity reduction
evaluation (TRE) may be used to identify and reduce
or eliminate sources of effluent toxicity whether or not
there are whole effluent toxicity limits in the NPDES
permits. For example, where a permittee has no whole
effluent toxicity limits in its current permit but
discovers a toxicity problem, it may use a TRE to
reduce or eliminate effluent toxicity, ensure that there
is no reasonable potential that its discharge will exceed
toxicity criteria and possibly obviate the need for
whole effluent toxicity limits in a subsequent permit.
On the other hand, if a permit contains whole effluent
toxicity monitoring requirements or limits and
unacceptable toxicity is observed, the permitting
authority may require the permittee to perform a TRE
through special conditions in the permit or an
enforcement action.
The TSD defines a TRE as "a site specific study
conducted in a stepwise process designed to identify
the causative agents of effluent toxicity, isolate the
sources of toxicity, evaluate the effectiveness of
toxicity control options, and then confirm the reduction
in effluent toxicity" (USEPA, 1991b). USEPA has
developed procedures that can be used to conduct
TREs (USEPA 1989a, 1989b, 199 la, 1992a, 1993a,
1993b, 1996).
This document represents the first update of USEPA's
Toxicity Reduction Evaluation Protocol for Municipal
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Wastewater Treatment Plants (1989a). This guidance
provides a general framework for conducting TREs at
publicly owned treatment works (POTWs) and
describes the available methods and procedures that
experience to date has shown to be most useful. It is
designed for POTW staff, consultants, and regulatory
agency staff who are implementing TREs to identify
and reduce or eliminate sources of effluent toxicity.
Where possible, POTW staff are encouraged to use the
guidance before the discharge of whole effluent
toxicity is subject to regulatory review and action.
This guidance presents methods and procedures that
are useful to:
Develop and implement a TRE plan.
Evaluate the results and data generated during the
TRE.
Develop a sound scientific and engineering basis
for the selection and implementation of toxicity
control methods.
This guidance supports the strategy desribed in the
TSD (USEPA, 1991b) for integrated toxics control
using whole effluent toxicity and pollutant specific
limits. It is well recognized that while POTWs may
achieve effluent limits for conventional pollutants, the
discharge of effluent toxicity, volatilization of toxic
materials, and contamination of sewage sludges can
still occur. The focus of this guidance is the reduction
of whole effluent toxicity at municipal wastewater
treatment plants.
The methods and decision points that comprise a TRE
are described in the context of an overall generalized
approach.'Each municipality must address regulatory
issues and treatment operations that are unique to each
POTW; therefore, not all components of this guidance
will apply in every case. POTW staff may also select
components to address specific questions about the
causes and sources of effluent toxicity; however, the
decision to choose a particular step should be based on
technically sound information. Given the site-specific
nature of TREs, POTW staff will need to develop a
TRE plan that describes the overall approach and
components of the guidance to be implemented.
In most cases, 'the approaches and methods described
in the TRE protocol (USEPA, 1989a) have been
validated by USEPA TRE research studies and other
municipal TREs (Amato et al., 1992; Bailey et al.,
1995; Botts et al., 1990, 1992, 1993, 1994; Collins et
al., 1991; Fillmore et al., 1990; Lankford and
Eckenfelder, 1990; Morris et al., 1990, 1992).
Appendix A provides the original case studies from the
municipal TRE protocol (USEPA, 1989a). Additional
examples of successful TREs are presented in
Appendices B through H of this guidance. The TRE
guidance includes information learned from these
studies. Major changes include:
Information on toxicants commonly found in
POTW effluents and the conditions that influence
their toxicity (Section 2).
Considerations in evaluating the operation and
performance of POTWs with respect to conditions
that may contribute to effluent toxicity. Additional
information is provided on operations review of
biological nutrient removal (BNR) processes
(Section 3).
A brief description of the use of updated toxicity
identification evaluation (TIE) procedures for
acute and short-term chronic toxicity (Section 4).
The reader is referred to USEPA's guidance on
TIE procedures for further details (USEPA 1991a,
1992a, 1993a, 1993b, 1996).
Refined step-by-step guidance for tracking sources
of acute and chronic toxicity in POTW collection
systems (Section 5).
Additional recent TRE case studies that describe
approaches for identifying the causes and sources
of acute and chronic effluent toxicity and practical
methods for toxicity reduction (Appendices B
through H).
The methods and procedures described herein will
continue to be updated and refined based on the results
of further studies.
Professional judgment is required in selecting the
appropriate steps for identifying toxicants and for
evaluating options for controlling effluent toxicity.
USEPA has developed TIE procedures to use as tools
for TRE studies. These TIE manuals (USEPA 1991a,
1992a, 1993a, 1993b, 1996) describe procedures for
characterization, identification, and confirmation of
effluent toxicants. TIE procedures are a basic
component of the municipal TRE and the USEPA
guidance manuals should be obtained and reviewed
prior to implementing a TRE. USEPA also has
developed a generalized protocol for conducting
industrial TREs (USEPA, 1989b).
-------
TRE Goals and Objectives
It is the responsibility of POTW staff to conduct a TRE
to identify and reduce or eliminate sources of effluent
toxicity and to fully comply with applicable toxicity-
based NPDES permit limits. The goal of the TRE may
be to achieve compliance with a whole effluent toxicity
limit; however, POTW staff are encouraged to use the
guidance to evaluate effluent toxicity before it becomes
a regulatory issue. The TRE goal and implementation
schedule should be clearly defined with the regulatory
authority as part of the preparation of the TRE plan.
The regulatory authority will review the TRE plan and
carefully monitor the progress of the TRE, providing
direction as needed.
The following objectives may be defined to accomplish
the TRE goal:
Evaluate the operation and performance of the
POTW to identify and correct treatment
deficiencies contributing to effluent toxicity (e.g.,
operations problems, chemical additives, or
incomplete treatment).
Identify the compounds causing effluent toxicity.
Trace the effluent toxicants and/or toxicity to their
sources (e.g., industrial, commercial, or domestic).
Evaluate, select, and implement toxicity reduction
methods or technologies to control effluent
toxicity (i.e., in-plant or pretreatment control
options).
These objectives are applied to meet the TRE goal of
compliance with regulatory requirements.
Components of the Municipal TRE
A generalized flow diagram for a TRE program is
illustrated in Figure 1-1. A brief description of each
major TRE component is presented below along with
the section number in the guidance in which additional
information is provided.
Information and Data Acquisition (Section 2)
The first step in a TRE is the collection of information
and analytical data pertaining to effluent toxicity. This
information includes data on the operation and
performance of the POTW, such as plant design
criteria and discharge monitoring reports (DMRs), and
data from the POTW's pretreatment program, such as
industrial waste survey (IWS) information, permit
applications, and industrial user compliance reports.
The POTW performance data and pretreatment
program information are used in the second stage of
the TRE, as described below.
Facility Performance Evaluation (Section 3)
Operations and performance data can be reviewed in a
POTW performance evaluation to indicate possible in-
plantsources of toxicity or operational deficiencies that
may be contributing to the effluent toxicity. If a
treatment deficiency is causing noncompliance with
conventional pollutant permit limits, studies should be
conducted to evaluate treatment modifications before
proceeding further in the TRE. These studies should
evaluate the toxicity reduction that can be achieved by
correcting treatment deficiencies. If plant performance
is not a principal cause of toxicity or treatment
modifications do not reduce effluent toxicity, a logical
next step is to identify the cause(s) of toxicity using
TIE procedures.
Pretreatment program data also can be gathered to
prepare a data base on the wastewaters discharged to
the POTW collection system. These data can be used
in the latter stages of the TRE to assist in tracking the
sources of toxicity and/or toxicants that are
contributing to POTW effluent toxicity.
Toxicity Identification Evaluation (Section 4)
This section provides a brief overview of the TIE
procedures. TIE procedures are available to evaluate
the causes of acute and short-term chronic toxicity.
When implementing a TIE, the reader is advised to
consult USEPA's TIE procedures for freshwater
species (1991a, 1992a, 1993a, 1993b) or estuarine/
marine species (1996). The generic TIE protocol is
performed in three phases: toxicity characterization
(Phase I), toxicant identification (Phase II), and
toxicant confirmation (Phase HI). Phase I characterizes
the types of effluent toxicants by testing the toxicity of
aliquots of effluent samples that have undergone
bench-top manipulation (e.g., pH adjustment,
filtration). An evaluation of common POTW effluent
toxicants such as ammonia, chlorine, and
organophosphate insecticides may be included in
Phase I. Phases II and HI involve further treatments in
conjunction with chemical analyses to identify and
confirm the compounds causing effluent toxicity.
USEPA's Phase H and m procedures (1993a, 1993b)
for freshwater species are generally applicable for
estuarine/marine species.
Toxicity Source Evaluation (Section 5)
A toxicity source evaluation involves the sampling and
analysis of wastewaters discharged from sewer lines
-------
Voluntary or Mandatory TRE
TRE Test Plan
I
Information and Data Acquisition
I
Facility Performance Evaluation
Pretreatment Program
Review
POTW Performance
Evaluation
Additional
Information
Required?
No
TIE
Additional
Information
Required?
No
Toxicity Source Evaluation
Additional
Information
Required?
No
Yes
Toxicity Control Evaluation
Pretreatment Control
Evaluation
In-Plant Control
Evaluation
Select Control Options
Toxicity Control Implementation
and Follow-Up Monitoring
Figure 1-1. TRE flow diagram for municipal wastewater treatment plants.
4
-------
and indirect dischargers such as industrial users and
commercial facilities. Two types of source evaluation
studies may be performed: chemical tracking or
toxicity-based tracking.
Chemical-specific tracking is recommended when the
POTW effluent toxicants have been identified and
confirmed in the TEE, and can be readily traced to the
responsible sewer dischargers. Toxicity tracking is
used when TIE data indicate the type of effluent
toxicant, but the specific toxicant(s) is not identified.
Toxicity tracking involves treating the sewer samples
in a bench-scale treatment simulation prior to toxicity
measurements to account for the toxicity removal that
is provided by the POTW.
The sampling strategy for toxicity source evaluations
involves two tiers. Tier I focuses on sampling and
analysis of the main sewer lines in the collection
system. Tier n involves testing sewer lines and
indirect dischargers upstream of the main lines
identified as being toxic in Tier I. This tiered approach
can be used to identify the contributors of toxicity
and/or toxicants by eliminating segments of the
collection system that do not contribute toxicity/
toxicants.
Toxicity Control Evaluation (Section 6)
Using the results of each of the above TRE elements,
alternatives for effluent toxicity reduction are evaluated
and the most feasible option(s) is selected for
implementation. Effluent toxicity may be controlled
either through pretreatment regulations or in-plant
treatment modifications or additions. In some cases,
several control methods may be required to achieve the
desired toxicity reduction. Selection of control options
is usually based on technical and cost criteria.
If the toxicity source evaluation is successful in
locating the sources that are contributing the POTW
effluent toxicants, local limits can be developed and
implemented. If in-plant control appears to be a
feasible approach, treatability testing may be used to
evaluate methods for optimizing existing treatment
processes and to assess options for additional
treatment.
Toxicity Control Implementation (Section 7)
The toxicity control method or technology is
implemented and follow-up monitoring is conducted to
ensure that the control method achieves the TRE
objectives and meets permit limits.
Limitations of the TRE Guidance
This guidance describes procedures for evaluating and
implementing controls for reduction of whole effluent
toxicity. Procedures for the reduction of toxic
pollutants in residuals, biosolids, and air emissions at
POTWs are not discussed. The reader may consult the
Standards for the Use or Disposal of Sewage Sludge
(40 CFR Part 503) regarding the control of toxic
materials in biosolids.
The municipal TRE guidance was developed based on
the results and findings of TRE and TIE studies. The
following limitations have been identified in these
studies:
Intermittent or ephemeral toxicity may be
challenging to characterize using TEE/TRE
procedures. In these cases, modifications to TRE
procedures may be needed to achieve the best
possible results (see Sections 4 and 5).
Discussions with the regulatory authority also may
help to identify the most appropriate approach for
complying with effluent toxicity requirements.
As described in this guidance, alternative
procedures are available if traditional methods
such as TEE testing are not successful. Additional
TRE procedures, especially tools for toxicity
source evaluations, have not been widely used, but
may be helpful if careful consideration is given to
their design and application.
As more TRE studies are completed, more
information is available on the feasibility and
effectiveness of in-plant and pretreatment toxicity
control options. Examples of TREs in which
toxicity controls have been successfully
implemented are provided in Appendices B, C, D,
E, G, and H.
The TRE guidance is designed to help public
works managers select appropriate toxicity control
approaches. As such, it does not discuss
regulatory procedures that may be useful for
assessing the need for, or compliance with,
toxicity requirements, such as the determination of
reasonable potential, dilution factors, and permit
limits. The importance of these procedures in the
evaluation of whole effluent toxicity is mentioned
in Section 2 and is discussed more fully in
USEPA's TSD (1991b).
-------
Organization of the TRE Guidance
This guidance is organized according to the
components of the TRE flow diagram shown in Figure
1-1.
Sections 1 through 7 describe the primary TRE
elements noted above. Changes to the municipal TRE
protocol (USEPA, 1989a) include more information on
toxicants commonly identified in POTW effluents,
suggestions for evaluating the effect of POTW
operations on effluent toxicity, an overview of updated
TIE procedures for acute and short-term chronic
toxicity, and refined step-by-step procedures for
tracking sources of acute and chronic toxicity in
POTW collection systems.
Sections 8 through 11 provide information on the
TRE requirements for quality assurance/quality
control, health and safety, facilities and equipment, and
sample collection and handling.
Sections 12 and 13 list the references and bibliography
cited in this guidance.
Appendix A presents the original case histories (given
in the municipal TRE protocol, USEPA, 1989a) along
with commentary on how the TEE/TRE procedures
have been updated to better address toxicity observed
in future studies.
Appendices B through G provide new in-depth case
examples of municipal TREs. These new examples
include summaries of four chronic TRE studies and
two acute TRE studies.
Appendix H is a new appendix that describes
approaches for addressing effluent toxicity caused by
organophosphate insecticides.
Appendix I describes a chemical-specific approach for
TREs that may be applied in limited circumstances.
Appendix J is referenced in Section 5 (toxicity source
evaluation) and presents an updated step-by-step
procedure for tracking sources of toxicity in POTW
collection systems.
-------
Section 2
Information and Data Acquisition
Introduction
The first step in a TRE is to gather all information and
data that may relate to effluent toxicity and that might
prove useful in planning and conducting the TRE.
This information can be categorized as POTW
treatment plant data and pretreatment program data.
The pertinent POTW information includes historical
effluent toxicity data as well as information on the
treatment plant's1 design capabilities, treatment
performance, and operation and maintenance practices.
Appropriate pretreatment program information consists
of IWS data, industrial user permits, pretreatment
inspection reports, and monitoring and compliance
reports. If a pretreatment program is not in place,
POTW staff may need to collect monitoring data on the
POTW industrial users and, where necessary,
appropriate controls should be considered to ensure
good effluent quality.
Background information may provide insight into the
nature of effluent toxicity and can be used to select the
initial steps to take in the TRE. However, it is
important ttot to draw conclusions about the causes and
sources of toxicity in the beginning of the TRE unless
corroborative testing is performed. A summary of
information recommended for a TRE is provided in the
following subsections.
Review of Effluent Toxicity Data
Information and data acquisition should include a
careful review of recent effluent toxicity data. This
review should be used to confirm the effluent toxicity
results and the potential to cause adverse instream
effects. The data also can be used to evaluate general
toxicity characteristics, such as temporal variability,
species sensitivity, and whether the toxicant(s) is fast
or slow acting.
In some states, laboratories are required to be certified
to perform toxicity tests. Toxicity test data reports also
may be reviewed by regulatory staff to confirm that the
tests meet basic quality assurance/quality control
(QA/QC) requirements. However, this is usually the
exception; most state and regional regulatory agencies
do not have certification programs for toxicity testing
and reports may not be formally reviewed. As an
initial step in the TRE, POTW staff should conduct an
independent review of the toxicity test reports to verify
the quality of the reported data, especially results that
have triggered TRE requirements.
It often is beneficial to develop a profile on the
characteristics of effluent toxicity using the available
historical data. Information on toxicity variability, the
relative sensitivity of various test species to the
effluent, and effluent characteristics [e.g., pH,
alkalinity, hardness, conductivity, total residual
chlorine (TRC), and dissolved oxygen (DO)] can
provide important clues about the nature of the
toxicity. These characteristics can be compared to
POTW and pretreatment information to help determine
if effluent toxicity may be related to operational
practices or sewer discharges. This information also
can be used as part of the TIE (Section 4) to help
identify the causes of effluent toxicity.
The data review may show that some test conditions
such as pH may artificially change during testing.
Typically, the pH of toxicity test solutions tends to drift
upward over time, which can cause pH sensitive
compounds such as ammonia and metals to exhibit
toxicity. With the consent of the regulatory authority,
it may be possible to modify the test procedures to
control pH drift (USEPA 1993c and T. Davies,
USEPA, Office of Water, Memorandum on
Clarifications Regarding Flexibility in 40 CFR Part
136 Whole Effluent Test Methods, April 10, 1996).
Modifications may also be allowed to better reflect the
range of temperatures and hardness observed in the
receiving water. Depending on the temperatures to be
-------
considered, it is recommended to use a different test
species rather than modify the recommended
temperature range for a given test species.
Prior to a TRE, POTW staff or the regulatory agency
may evaluate the "reasonable potential" for exceeding
a toxicity-based water quality standard to determine if
a permit limit is required. If there is reasonable
potential to cause instream toxicity or contribute to an
excursion above a narrative criterion, a statistical
approach may be used to calculate a toxicity-based
permit limit. This approach may also be applied during
the course of a TRE to assess compliance with a permit
limit or a water quality standard. For example,
improvements in effluent quality resulting from the
TRE could lower effluent toxicity to a point where
there is no longer a reasonable potential to exceed the
permit limit. Or, these improvements may reduce
effluent variability. The reduced variability could
result in a smaller coefficient of variation (CV), which
would lessen the potential for excursions above the
TRE goal. The reader is referred to USEPA' s TSD for
details on these procedures (USEPA, 1991b).
The TSD also discusses the use of dilution, and
particularly the use of high-rate diff users, in achieving
compliance with toxicity-based water quality
standards. The dilution determination, if allowed by
applicable regulations, is one of the first steps in
characterizing the effluent for toxicity-based permitting
(USEPA, 1991b). Public works managers, who are
initiating a TRE, may choose to evaluate the
application of appropriate mixing zone allowances to
eliminate the potential for instream effects. A
shoreline outfall, for instance, may not qualify for any
dilution when determining an acute toxicity
requirement. Use of a diffuser constructed in deeper
water may allow the effluent to achieve sufficient
dilution in the rapid-mixing, near-field area to meet
permit requirements. Similar results may be obtained
by moving an outfall from a small or intermittent
stream, where no dilution is available under low flow
conditions, to a larger permanent stream, with greater
dilution. It should be noted, however, that less costly
toxicity control approaches than outfall relocation may
be identified during the course of the TRE. The
process of selecting the most feasible and practical
control option(s) is described in Section 6 of this
guidance.
Toxicants Identified in POTW Effluents
As noted, the occurrence of toxicity and the treatment
process operations are unique to each POTW;
therefore, the causes of effluent toxicity are likely to be
different for each case. Nonetheless, some toxicants
have been identified at many POTWs. A list of
toxicants commonly found in POTW effluents, the
levels of concern, and potential sources is presented in
Table 2-1. The levels of concern are to be used as a
general guide, not as absolute values. Due to the site-
specific nature of effluent toxicity, these data are
intended only as background information to consider in
the process of conducting a TRE. It is important to
stress that a direct comparison of chemical
concentrations to toxicity data reported in the literature
often provides misleading information. The toxicity of
effluent constituents is affected by many factors
including the effluent matrix and toxicity test
conditions. The most effective way to identify causes
of effluent toxicity is by applying the TIE procedures,
which are described in Section 4 of this guidance.
Some of the information that can be collected to help
evaluate the contribution of the toxicants to effluent
toxicity is provided in Table 2-1. Toxicity information
on specific parameters can be obtained fromUSEPA's
Aquatic Information Retrieval Toxicity Data Base
(AQUIRE, 1992), TIE manuals (USEPA 1991a,
1992a, 1993a, 1993b, 1996), Extension TOXicology
NETwork (EXTOXNET, 1998), peer-reviewedjournal
articles, and other sources. AQUIRE information can
be obtained through the National Technical
Information Service (NTIS) in Springfield, Virginia, or
through several commercial vendors. USEPA's Mid-
Continent Ecology Division (Duluth, Minnesota) will
be offering Internet access to AQUIRE data in early
1999 through its Ecotoxicology Database Retrieval
System (ECOTOX). The Extension TOXicology
NETwork is currently available on the Internet at
http://ace.orst.edu/info/extoxnet/. When reviewing
toxicological data, it is important to ensure that the
references for the data have been peer-reviewed and
the values given are considered to be accurate.
In some cases, toxicological data may be presented in
toxic units (TUs) instead of in lethal concentrations
causing a 50% mortality in exposed test organisms
(LC50) or no observed effect concentrations (NOEC).
TUs are the inverse of the percent concentration
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Table 2-1. Toxicants Identified in POTW Effluents
Toxicant Type
Chlorine
Ammonia
Non-polar organics,'
such as
organophosphate
insecticides (e.g.,
diazinon, malathion,
chlorpyrifos, and
chlorfenvinphos)
Metals [e.g., cadmium
(Cd), copper (Cu),
chromium (Cr), lead
(Pb), nickel (Ni), zinc
(Zn)]
Other treatment
chemical additives such
as dechlorination
chemicals and polymers
Surfactants
Total dissolved solids
(TDS)
Level of Concern*
0.05 to 1 milligram per
liter (mg/L)
5 mg/L as NH3-N
Diazinon: 0.12-0.58
microgram per liter
(ug/L)
Chlorpyrifos: 0.03 ug/L
Varies
Varies
Varies
1,000-6,000 uhmos/cm
depending on endpoint,
species tested, and TDS
constituents
Potential Source
POTW disinfection
process
Domestic and industrial
sources
POTW sludge
processing sidestreams
Homeowners,
apartments,
veterinarians, pest
control, lawn care, and
commercial businesses
Treatment additives in
POTW
Industrial users
Disinfection,
dechlorination, sludge
processing, and solids
clarification in the
POTW
Industrial users
Industrial users
Sludge processing
sidestreams
Information Needed
to Assess Toxicity
TRC, temperature, and pH upon
receipt of effluent sample and
during toxicity test
Toxicity degradation tests
TIE Phase I testsf
Ammonia-nitrogen upon receipt
of effluent sample
pH, temperature, and salinity
during toxicity test
TIE Phase I testsf
High resolution analysis of
organophosphate insecticides
TIE Phase I testsf
Dissolved metals, effluent
hardness (mg/L as CaCO3), and
alkalinity upon receipt of
sample
TIE Phase I testsf
Vendor information on toxicity
of products
Dosage rates
Effluent characteristics that
affect toxicity (e.g., pH)
TIE Phase I testsf
Methylene blue active
substances (MBAS) and cobalt
thiocyanate active substances
(CTAS)
TIE Phase I testsf
TDS, ion analysis, and anion/
cation balance
TIE Phase I testsf
* As referenced by USEPA (1992a) and D. Mount (personal communication, AScI Corp, Duluth, Minnesota, 1991) for
chlorine; USEPA (1992a) for ammonia; TRAC Laboratories (1992), Bailey et al. (1997) for diazinon and chlorpyrifos; and
USEPA (1992a) for TDS.
t The contribution of effluent constituents such as chlorine, ammonia, organic compounds, metals, and TDS to effluent
toxicity can be most effectively evaluated using the TIE Phase I procedures described in Sections 3 and 4 of this guidance
and the USEPA manuals (1991a, 1992a, 1996).
-------
values and are calculated by dividing 100% by acute or
chronic percent effluent values or chemical
concentration data. For example, a chronic TU (TUc)
of 2 is equivalent to an NOEC value of 50% effluent
(i.e., 100%/50%). Likewise, if the LC50 of a
compound is 20 pg/L, an effluent sample with 100
\igfL contains 5 acute TU (TUa) of the compound (i.e.,
100 ng/L/20 |ig/L). TU values are helpful in
understanding the relative contribution of toxicants to
effluent toxicity and are often used in interpreting TEE
data (Section 4). For example, if one of two
compounds is contributing to effluent toxicity (e.g., 4
TUc of compound A and 1.5 TUc of compound B), it
may be possible to focus on controlling the major
toxicant if compliance with the permit limit (e.g., 3
TUc) can be achieved. However, consideration should
be given to possible antagonistic effects between the
toxicants such that removal of one toxicant may cause
the other toxicant to exhibit greater toxicity. An
overview of this is provided in Section 4 and the TIE
manuals (USEPA 1991a, 1992a, 1993a, 1993b, 1996)
provide thorough guidance.
POTW Design and Operations Data
POTW design and operations information can indicate
possible in-plant sources of toxicity or operational
problems that might be contributing to treatment
interferences and the pass through of toxicity. In the
beginning of the THE, it is often helpful to briefly
review the operations and performance of the major
unit treatment processes. Notes can be added to
POTW data base about initial impressions and
potential problem areas that should be investigated
further in the POTW Performance Evaluation (see
Section 3).
The types of POTW data to be gathered include:
Background information on treatment plant design
and operation.
Data routinely collected for NPDES DMRs and
treatment process control.
Existing data on potential effluent toxicants,
including chlorine, ammonia, organophosphate
insecticides, surfactants, metals, and treatment
additives (e.g., polymers, chlorine, dechlorination
chemicals).
A list of useful POTW data is provided in Table 2-2.
The POTW data can be compared to the profile on
effluent toxicity characteristics to determine if toxicity
may be related to operation and performance. Several
questions can be posed, including:
Is toxicity apparent during certain operational
events, such as when treatment upsets are
observed, when treatment units are taken offline
for maintenance, or as a result of other operating
practices (e.g., excess chlorine addition)?
Does toxicity exhibit a weekly, monthly, or
seasonal pattern? For example, if the POTW is
operated to achieve seasonal ammonia removal, is
toxicity present in the period when ammonia
removal is not practiced? What process control
parameters may be related to toxicity? Is toxicity
apparent with changes in hydraulic and pollutant
loadings to the primary sedimentation process,
changes in biological treatment parameters
[e.g., mixed liquor suspended solids (MLSS)
concentration, DO concentration, sludge volume
index (SVI), mean cell residence time (MCRT)],
changes in filtration rates in filters, or changes in
application rates of chlorine and dechlorinating
agents?
Is toxicity apparent when the type and dose of
treatment additives change? For example, did
toxicity occur when a different polymer or other
coagulant/flocculent aid was used?
In the beginning of the TRE, emphasis should be
placed on effluent concentrations of ammonia and
chlorine, which are common toxicants in POTW
effluents (USEPA 1991a, 1992a, 1993a, and 1993b).
The toxicity of ammonia is dependent on effluent
characteristics such as the pH, temperature, and salinity
of the sample, as well as the sensitivity of the species
being tested. Therefore, it will be difficult to
determine the toxicity of ammonia based solely on a
comparison of literature values to effluent
concentrations. Likewise, the toxicity of chlorine will
depend on the form of chlorine, which may be in the
free form as chlorine, hypochlorous acid, or
hypochlorite ion, or in the combined form as
chloroamines or nitrogen trichloride. The sum of the
free and combined chlorine, termed total residual
chlorine or TRC, is matrix dependent. Chloramines,
which are formed by chlorine combining with
ammonia, can be more toxic than free chlorine
(AQUIRE, 1992).
Assessments of the contribution of ammonia, chlorine,
and other compounds to effluent toxicity can be made
using Table 2-1 as a guide. As stated in Table 2-1, if
10
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Table 2-2. Example POTW Design and Operation Data
1. NPDES permit requirements
a. Effluent limitations
b. Special conditions
c. Monitoring data and compliance history
d. Dilution studies or modeling results
2. POTW design criteria
a. Hydraulic loading capacities
b. Pollutant loading capacities
c. Biodegradation kinetics calculations and assumptions
3. Influent and effluent pollutant data
a. Ammonia
b. Residual chlorine
b. Other pollutants of concern such as non-polar organic compounds (e.g., organophosphate insecticides),
metals, and TDS (see Table 2-1)
c. Conventional pollutant data, including five-day biochemical oxygen demand (BOD5), chemical oxygen
demand (COD), total organic carbon (TOC), total suspended solids (TSS), volatile suspended solids (VSS),
total Kjeldahl nitrogen (TKN), ammonia-nitrogen (NH3-N), total phosphorus (TP), orthophosphate (PO4-P),
and nitrate-nitrogen (NO3-N),to evaluate treatment performance
d. Parameters, including pH, hardness, and alkalinity, to evaluate the toxicity of suspect compounds
(see Table 2-1)
4. Process control data
a. Chemical usage for each treatment process (e.g., coagulants for primary sedimentation, lime for biological
treatment, polymers for tertiary clarification; see Table 2-1)
b. Process control data for primary sedimentation (i.e., hydraulic loading capacity and BOD5 and TSS removal)
c. Process control data for activated sludge [e.g., food to microorganism (F/M) ratio, MCRT, MLSS, sludge
yield, removal efficiency of BOD5, COD, TKN, NH3-N, TP, PO4-P, NO3-N, and other pollutants specified
in the permit].
d. Process control data for secondary and tertiary clarification [e.g., hydraulic and solids loading capacity, SVI,
sludge blanket depth]
e. Number of process units online and number offline for maintenance
5. Operations Information
a. Reports on previous operation and maintenance evaluations, including engineering studies and USEPA and
state compliance inspections
b. Operating logs
c. Standard operating procedures
d. Operation and maintenance practices (e.g., filter backwash procedures)
6. Process sidestream characterization data
a. Chemical usage for sludge processing, including thickener, digester, and dewatering processes
b. Pollutant data for sludge processing sidestreams, including ammonia, metals, organophosphate insecticides,
and TDS (see Table 2-1)
c. Incinerator scrubber waste stream, including data on possible formation of cyanide (see discussion in
Section 3)
d. Tertiary filter backwash
e. Cooling water
7. Wastewater bypass, combined sewer overflow (CSO), and sanitary sewer overflow (SSO) for bypasses or
overflows that are discharged to the POTW effluent
a. Frequency
b. Volume
11
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chlorine is suspected of contributing to toxicity, TRC
should be measured when the sample arrives and when
toxicity tests are initiated because chlorine usually
dissipates rapidly. If ammonia is a concern, conditions
that affect its toxicity, including sample pH, should be
carefully monitored during toxicity tests. This
information is necessary to determine the concentration
of the toxic un-ionized form of ammonia (NH3) in the
toxicity test. In addition to toxicity data, USEPA's
AQUIRE data base (1992) includes information on the
conditions of the toxicity test that may have influenced
the reported toxicity of chemicals of concern.
However, not all conditions may be recognized or
reported, which may limit the utility for TIE tests.
Surfactants also have been identified as toxicants in
POTW effluents (Diehl and Moore, 1987; Ankley and
Burkhard, 1992; Botts et al., 1994). These studies
focused on characterizing the type or source of
surfactants rather than trying to identify the toxic
surfactant compound because analytical methods are
not readily available to detect and quantify surfactant
compounds in complex effluents. Municipal effluents
contain numerous substances that interfere with
surfactant analysis. Also, surfactants are actually
mixtures of many homologues and oligomers;
therefore, the composition and toxicity of surfactants is
complex and often variable (USEPA, 1993a). One
exception is a class of surfactants, referred to as alkyl
phenol ethoxylates (APEs), that can be analyzed by a
gaschomatograph/mass spectrometer (GC/MS) (Giger
et al., 1981). Also, it may be helpful to characterize
surfactants by the nature of their polar segment;
surfaqtants may be classified as nonionic, anionic,
cationic, and amphoteric. Many surfactants tend to
sorb to CIS resin and analyses of the methanol extracts
from solid phase extraction (SPE) columns can help to
indicate the type of surfactant causing toxicity
(USEPA, 1993a). The American Public Health
Association (1995) describes methods for determining
anionic surfactants as MB AS and nonionic surfactants
as CTAS.
The contribution of effluent constituents such as
ammonia and chlorine to effluent toxicity can be most
effectively evaluated using the TIE procedures
described in Sections 3 and 4. Non-polar organic
compounds (such as organophosphate insecticides),
metals, surfactants, and TDS also can be effectively
evaluated using these procedures. In Phase I of the
TIE, various sample manipulations and toxicity tests
are performed to determine how toxicity is affected by
removing or isolating a particular group of toxicants.
These procedures establish a cause and effect
relationship between toxicants and whole effluent
toxicity. If a toxicant is indicated through Phase I
testing, additional TIE procedures can be applied to
identify (Phase II) and confirm (Phase ID) the toxicant
(see Section 4).
Treatment additives can be screened by obtaining
toxicity data from product vendors or by performing
toxicity tests on samples that have been treated using
typical chemical dosages. If toxicity tests are
performed, it is important to simulate the conditions
occurring in the treatment process where additives are
being used because some portion of the additives is
usually removed in the treatment process. For
example, polymers are largely bound with suspended
solids in the wastewater being treated and only minor
amounts may pass through in the final effluent (Hall
andMirenda, 1991).
Once the toxicants are identified, the POTW data will
be useful in evaluating and selecting in-plant toxicity
control options (see Section 6).
Pretreatment Program Data
Pretreatment program information may provide
evidence that can be used to identify sources of
toxicants or toxicity in the wastewater collection
system. For this reason, pretreatment data should be
briefly reviewed in the beginning of the TRE. As an
initial step, pretreatment data can be compared to the
profile on POTW effluent toxicity characteristics to
determine if toxicity may be related to a particular type
of discharge. This review may attempt to answer
several questions, including:
What changes in POTW influent characteristics
may be observed during toxic periods (e.g., pH,
alkalinity, suspended solids, hardness,
conductivity, DO, color)? Also, does toxicity
occur during changes in hydraulic and pollutant
loadings to the POTW? Can these characteristics
be related to certain types of discharges?
Does toxicity occur when treatment upsets are
observed at the POTW? Can the upsets be related
to a particular discharge(s)?
Does toxicity exhibit a weekly, monthly, or
seasonal pattern that may be related to production
schedules of certain industries? For example, is
12
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toxicity observed when an industry is
manufacturing a particular type of product? Also,
does toxicity abate when the industry is shutdown
for maintenance or holidays?
If the POTW accepts hauled wastes, is toxicity
apparent when a particular hauler delivers wastes?
Appropriate pretreatment program information to
review includes the data on the industrial users of the
POTW [e.g., industrial manufacturers, Resource
Conservation and Recovery Act (RCRA) waste
disposers, and Comprehensive Environmental
Response, Compensation, and Liability Act
(CERCLA) dischargers] and the toxic pollutant data on
the POTW waste streams. A list of suggested
pretreatment data is shown in Table 2-3.
The POTW pretreatment program data can be
reviewed as part of a Pretreatment Program Review
(described in Section 3). The summarized data may be
useful in locating the sources of toxicants identified in
the TIE (see Section 4). In cases in which effluent
toxicants are not identified, the pretreatment program
data can be used to develop a sampling and analysis
program to track sources of toxicity in the collection
system (see Section 5).
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Table 2-3. Example Pretreatment Program Data
1. POTW influent and effluent characterization data
a. Toxicity
b. Priority pollutants ,
c. Hazardous pollutants
d. Pollutants listed in Superfund Amendments and Reauthorization Act (SARA) Title 313
c. Other chemical-specific monitoring results (e.g., industry raw materials and products)
2, Sewage residuals characterization data (e.g., raw, digested, thickened, and dewatered sludge, composted biosolids, and incinerator
ash) !
a. Toxicity characteristic leaching procedure (TCLP)
b. Chemical data
3. IWS
a. Information on industrial users with categorical standards or local limits and other significant non-categorical industrial users
- number of industrial users
- discharge flow
- chemical usage ',
b. Standard Industrial Classification (SIC) code
c. Wastewater flow
d. Types and concentrations of pollutants in the discharge j
c. Products manufactured
f. Description of pretreatment facilities and operating practices
4. Industrial User Permits
a. Pretreatment standards
categorical standards '
- local limits
prohibited discharge standards
b. Monitoring requirements |
5. Annual pretreatment program report ;
a. Schematic of sewer collection system
b. Industrial user monitoring and inspection data collected by POTW staff
- discharge characterization data ;
- spill prevention and control procedures
- hazardous waste generation '
c. Industrial user self-monitoring data
discharge characterization data ',
- flow measurements
- description of operations
compliance schedule (if out of compliance; e.g., notice of slug loading)
6. Headworks analysis for local limits
7. Industrial user compliance reports
8. Waste hauler monitoring data and manifests \ '
9. RCRA reports [if the POTW is considered a hazardous waste treatment, storage, and disposal facility (TSDF)]
a. Hazardous waste manifests
b. Operating record
c. Biennial report
d. Unmanifested waste report '
10. CERCLA reports (if the POTW accepts wastes from a superfund site)
a. Preliminary site assessment
b. Site investigations
c. Remedial investigations
d. Feasibility studies \
e. CERCLA decision documents
11. Information on POTW treatment interferences (e.g., biological process inhibition); example data include:
a. Evidence of slug loadings :
b. Decreased pollutant removal
c. Decreased oxygen uptake rates (OURs), SVI, and sludge yield in biological treatment process
d. Increased requirement for chemical usage (e.g., chlorine, coagulants, flocculents)
c. Decreased filtration rate and increased backwash frequency for filtration treatment
14
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Section 3
Facility Performance Evaluation
Introduction
POTW treatment deficiencies that cause poor
conventional pollutant removal can have an adverse
effect on toxicity reduction as well. As an initial step
in the TRE, effluent toxicity data (Table 2-1) and
POTW operations and performance data (Table 2-2)
should be evaluated to indicate potential toxicants of
concern and to identify treatment deficiencies or in-
plant sources of toxicity that may be responsible for all
or part of the effluent toxicity. POTW pretreatment
program data (Table 2-3) should also be reviewed to
indicate possible sources of toxicity and summarized
for use in later steps of the TRE such as the toxicity
source evaluation (Section 5).
POTW Performance Evaluation
A POTW performance evaluation can be conducted to
indicate conventional pollutant treatment deficiencies
or in-plant sources of toxicity that may be contributing
to effluent toxicity. Conventional pollutant treatment
deficiencies include the inability to meet permit limits
for BOD, TSS, and nutrients. These deficiencies
should be corrected before initiating a full TRE
because improved treatment also may reduce effluent
toxicity. In-plant sources of toxicity may be present
even if the POTW is meeting permit limits for
pollutants other than toxicity. An example of an in-
plant source of toxicity includes inadequate solids
separation in the final clarifier, which may result in the
discharge of toxic material bound to suspended solids.
Also, incomplete biological treatment may cause the
pass-through of biodegradable toxicants. Other in-
plant sources of toxicity may include treatment
additives used in toxic amounts or additives that
contain toxic impurities. If deficiencies are found in
the POTW performance evaluation, improvements can
be implemented to eliminate the causes of toxicity.
Several examples of operating conditions that have
contributed to effluent toxicity at POTWs and the steps
taken to correct the problem are included in the
following discussion of the POTW performance
evaluation process.
A flowchart for conducting a POTW performance
evaluation is presented in Figure 3-1. The POTW
performance evaluation involves a review of the major
treatment unit processes (e.g., primary sedimentation,
activated sludge, and secondary clarification) using
wastewater characterization data and process
operations information. A TIE Phase I analysis (as
described below and in Section 4) also can be
performed to indicate the presence of effluent toxicants
caused by incomplete treatment (e.g., ammonia),
routine operating practices (e.g., chlorine), or the
discharge of organophosphate pesticides in the POTW
collection system. Ammonia and chlorine are
commonly found to cause toxicity in POTW effluents
and should be evaluated at this stage of the POTW
performance evaluation. As noted in Section 2,
ammonia and chlorine may be of concern at
concentrations greater than 5 mg/L and 0.01 mg/L,
respectively, depending on the effluent matrix and the
species being tested. Levels of concern for other
relatively common effluent toxicants are listed in
Table 2-1. Special consideration also should be given
to chemicals used in the treatment process such as used
or reused waste materials and coagulants, which may
contribute to toxicity due to pass-through of residual
concentrations or impurities in the product.
Based on the process review results and TIE
characterization data, options for improving operations
and performance may be selected and evaluated in
treatability studies. If treatability tests are successful in
identifying options for improving conventional
pollutant treatment and toxicity reduction, the TRE
proceeds to the selection and implementation of those
options (Section 6). If no treatment deficiencies or in-
plant sources of toxicity are observed, or the treatment
alternatives do not reduce effluent toxicity to
15
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Facility Performance Evaluation
Information and Data Acquisition
Phase I Toxicity
Characterization
Evaluation of POTW Operation and Performance
Evaluate Common Toxicants
Ammonia, Chlorine, Surfactants, Organophosphate
Pesticides, Metals, Treatment Additives, TDS
Evaluate Conventional Pollutant Treatment
Preliminary Treatment
Primary Sedimentation
- Biological Treatment
- Secondary/Tertiary Clarification
Filtration
Disinfection/Dechlorination ;
Process Sidestreams/Bypasses
Evaluate In-Plant Sources of Toxicity j
Disinfection Chemicals :
Coagulants/Flocculents
Toxic Impurities in Additives
No
Plant
Failure
Observed?
Bench-Scale
Conventional
Treatability Tests
Pilot-Scale
Conventional
Treatability Tests
TIE
Toxicity Adequately
Reduced by Modification of
Treatment/Operation?
Toxicity
Control
Selection
Figure 3-1. Flow diagram for a facility performance evaluation.
16
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acceptable levels, a complete effluent toxicity
characterization should be performed using the TIE
procedures described in Section 4.
POTWs are subject to both variable influent
characteristics and changing operating conditions that
may have a significant effect of effluent toxicity. The
POTW performance evaluation should be conducted
during a period when the influent loadings and facility
.operations are representative of average conditions. If
effluent toxicity varies seasonally or as a result of a
specific operational condition, the POTW performance
evaluation should be scheduled to coincide with the
expected toxic event. Due to the variability inherent in
POTW operations, it may be necessary to conduct
additional POTW performance evaluation
investigations during the course of the TRE. For
example, POTW performance evaluations may be
useful when performed before and after
implementation of facility modifications, changes in
industrial user activities, or variations in effluent
toxicity.
Operations and Performance Review
The operations and performance review involves the
evaluation of the maj or POTW unit processes using the
information described in Table 2-2. This review
focuses on the secondary treatment system because
secondary treatment is responsible for removing the
majority of the conventional and toxic pollutants from
municipal wastewater. Deficiencies in this system are
more likely to result in incomplete treatment of
wastewater toxicity. For example, problems with
nitrification treatment may cause toxic concentrations
of ammonia to pass through in the effluent. Other unit
processes to be evaluated include primary
sedimentation, disinfection, and advanced treatment
processes such as filtration.
Procedures for evaluating and improving POTW
operations and performance are described in USEPA' s
Handbook on Retrofitting POTWs (USEPA, 1989c).
This handbook describes a two-step process to improve
POTW performance: comprehensive performance
evaluation and a composite correction program
approach. The comprehensive performance evaluation
involves a thorough review of the POTW design and
operating conditions to identify problem areas. The
composite correction program involves the systematic
identification and implementation of improvements
with an emphasis on low-cost solutions. Other useful
sources of information include a joint publication by
Water Environment Federation and American Society
of Civil Engineers entitled Design of Municipal
Wastewater Treatment Plants (WEF/ASCE, 1992a,
1992b) and Metcalf and Eddy's Wastewater
Engineering Treatment, Disposal, and Reuse (1991).
Computer software programs, including USEPA's
POTW Expert (1990), have also been developed to
"troubleshoot" operations and performance problems.
In addition, USEPA (1993d) has a data base on
pollutant removal efficiencies (RREL Treatability Data
Base, Version 5) for various treatment processes.
Although this guidance does not specifically address
toxicity, correcting conventional pollutant treatment
problems and controlling in-plant toxicants may
improve toxicity reduction. In addition to the noted
guidance, public works managers are advised to use
the services of a professional engineer who has
experience with the POTW treatment system.
Preliminary Treatment
Preliminary treatment processes that may be used to
enhance toxicity control include equalization/storage
and oil and grease removal. Equalization basins can be
effective in dampening the effect of slug loads of
toxicity or to equalize flow and organic loadings to
achieve consistent subsequent treatment of the influent
wastewater. Oil and grease removal can assist in
removing toxicants associated with oil and grease and
to minimize the impact of oil and grease on the POTW.
TRE Example
A municipality in Texas experienced effluent toxicity
that was related to a volatile organic compound
entering the POTW. A pre-aeration system was to be
added to the influent headworks or the grit removal
system; however, before construction was started, a
city employee noticed a strong odor in a sewer line
that was related to the volatile toxicant. The source of
the volatile compound was identified and controlled.
As a result, effluent toxicity was eliminated
(S. Bainter, personal communication, USEPA, Dallas,
TX, 1998).
Primary Sedimentation
Primary treatment processes are designed to reduce the
loading of TSS, BOD5, and COD on the secondary
treatment system. Toxic pollutant removal also can
occur by sedimentation of insoluble or paniculate
wastewater constituents. Optimal removal of both
toxic and conventional pollutants in primary
17
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sedimentation ultimately reduces the amount of
material to be treated in the biological treatment
process.
Primary clarifier performance can be evaluated by
comparing BOD5 removal to the surface overflow rate
(SOR), which is the average daily flow divided by the
clarifier surface area. A clarifier operating at an SOR
of less than 24 cubic meters per square meter per day
(nrYnrVday) [600 gallons pre day per square foot
(gpd/sq ft)] should remove 35 to 45% of the influent
BODS. A clarifier operating at an SOR of 24 to
40 rrrVrrrVday (600-1,000 gpd/sq ft) should remove
25 to 35% of the influent BOD5 (USEPA, 1989c). In
most cases, COD removal performance is comparable
to the BODS removal performance. If the primary
clarifiers do not achieve the expected BOD5 or COD
removal, engineering studies should be initiated to
determine the need for additional clarifier capacity.
Removal of toxicity associated with TSS may be
enhanced by addition of coagulants to the primary
clarifiers. The optimum conditions for coagulation and
flocculation of toxicants can be determined by j ar tests.
These tests are used to establish the optimum type and
dosageof coagulant, the propermixing conditions, and
the flocculent settling rates for enhanced toxicant
removal (Adams et al., 1981).
A key operating parameter for controlling clarifier
performance is sludge removal. Primary clarifiers
generally function best with a minimum sludge
blanket. Sludge withdrawal should be adjusted to
maintain the primary sludge concentration in the range
of 3 to 6% total solids (USEPA, 1989c).
Biological Treatment
Biological treatment is a critical process at most
POTWs because it is the process that converts organic
matter and nutrients to settleable microorganisms.
Toxic pollutant removal during biological treatment
can occur by biodegradation, oxidation, volatilization,
and adsorption onto the biological floe. Key factors
affecting the removal of toxic pollutants are the rates of
biodegradation, tendency to volatilize, oxidize, or sorb
onto solids, and the degree to which the pollutants may
inhibit the treatment process.
Ammonia is a common cause of effluent toxicity at
POTWs that do not include nitrification treatment. As
noted by USEPA (1991a), ammonia is often present in
effluents in concentrations varying from 5 to 40 mg/L.
These concentrations can cause toxicity depending on
several factors that affect the toxicity of ammonia,
including pH, temperature, DO, and TDS. A simple
TIE procedure for checking whether effluent toxicity
may be related to ammonia is described below (see
"TIE Phase I Tests"). Literature data on ammonia
toxicity (USEPA, 1985a) should only be used as a
general guide because ammonia toxicity is significantly
affected by slight pH changes.
The most commonly used biological treatment systems
can be defined as either suspended growth processes,
such as conventional activated sludge, contact
stabilization, and extended aeration; or fixed film
processes, such as trickling filters, denitrification
filters, and rotating biological contactors (RBC). To
simplify the discussion of biological treatment, the
following subsections focus on evaluating the
performance of conventional activated sludge
processes and related BNR processes, which are the
systems most widely used in POTWs.
Conventional activated sludge treatment is an aerobic
process that can be accomplished in one stage or zone.
BNR processes integrate carbon oxidation, as achieved
in conventional activated sludge treatment, with
treatment stages designed for nitrification,
denitrification, and enhanced biological phosphorus
removal. These stages require specific treatment
conditions, including anaerobic, anoxic, and aerobic
zones in the mixed liquors. The stages may be
separated by physical divisions, non-discrete zones, or
by operating cycle (WEF/ASCE, 1992b). The
sequence and sizing of the BNR stages depend on the
effluent nitrogen and phosphorus concentrations that
must be achieved.
Conventional activated sludge processes remove
phosphorus and nitrogen in the course of converting
organic matter to new biomass. The typical
phosphorus content of microbial cells is 1.5 to 2% on
a dry-weight basis (WEF/ASCE, 1992b). BNR
processes enhance phosphorus removal by utilizing the
sequence of an anaerobic stage followed by an aerobic
stage, which results in the selection of a biomass
population capable of concentrating phosphorus from
4 to 12% of the microbial cell mass. Enhanced
nitrogen removal in BNR processes is a two stage
process: nitrification oxidizes ammonia to nitrite and
then to nitrate, and denitrification reduces the nitrate to
nitrogen gas. The nitrogen and phosphorus removal
processes can be used independently (e.g., oxidation
18
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ditches and A/Oฎ process, respectively) or can be
integrated into a combined nutrient removal process
(e.g., A2Oฎ and Bardenphoฎ processes). A wide
variety of BNR systems are in operation, some of
which are proprietary; therefore, specific information
on the process being studied may be obtained by
consultation with the system vendors.
TRE Example
A United States east coast municipality implemented
nitrification to achieve ,a seasonal NH3-N limit of
1 mg/L (Engineering Science, Inc., 1994). ThePOTW
typically achieved less than 1 mg/L NH3-N. As a
result, the POTW effluent eliminated chronic toxicity
to fathead minnows (Pimephales promelas) from May
1 through September 31 each year; however, the
effluent continued to be toxic during the remainder of
the year. In an effort to comply with the permit limit
for chronic toxicity, nitrification was extended for the
full year. This modification eliminated chronic
toxicity to fathead minnows throughout the year.
The parameters that are typically used to evaluate the
operational capability of an activated sludge system
include organic loading, oxygen requirement, and
MCRT. Additional important operating conditions
include the alkalinity requirement for nitrification, and
the BOD5 requirement for phosphorus removal and
denitrification. Operating values for these parameters
can be compared to design specifications or
recommended criteria to determine how well the
processes are being operated.
Organic Loading
Organic loading affects the organic removal efficiency,
oxygen requirement, and sludge production of
activated sludge processes. The most common
measure of organic loading in suspended growth
processes is the F/M ratio, which is the organic load
removed per unit of mixed liquor volatile suspended
solids (MLVSS) in the aeration basin per unit time.
High F/M ratios (i.e., high organic loading to MLVSS)
will result in a low organic removal efficiency, low
oxygen requirement, and high sludge production. Low
F/M ratios (i.e., low organic loading to MLVSS) will
lead to high organic removal efficiencies and low
sludge production, but high oxygen requirements.
If the suspected toxicants are biodegradable or partition
to activated sludge, the MLVSS of the treatment
process should be increased to the maximum levels that
can be maintained at the POTW. The maximum
MLVSS often will be limited by the available
secondary clarifier capacity. It is important to consider
the effect of increased MLVSS on secondary solids
separation and the TSS concentrations of the clarifier
effluent. The Patapsco Wastewater Treatment Plant
(WWTP) in Baltimore, Maryland, was operated at an
F/M ratio of 0.40 Ib BOD5/lb MLVSS-day instead of
the design F/M ratio of 0.55 Ib BOD5/lb MLVSS-day,
because the POTW could not achieve consistent
wastewater treatment at the higher organic loading.
The increased MLVSS levels were thought to be
necessary because of the toxic effect that industrial
wastewaters were having on the activated sludge
biomass (Slattery, 1987). For optimal treatment, it may
be necessary to maintain F/M ratios that are on the low
end of the range typically observed for biological
treatment processes. The F/M ratio in an activated
sludge system is generally maintained in the range of
0.2 to 0.4 Ib BOD5/lb MLVSS-day for conventional
activated sludge, 0.05 to 0.15 Ib BOD5/lb MLVSS-day
for extended aeration, and 0.2 to 0.6 Ib BOD5/lb
MLVSS-day for contact stabilization (Metcalf and
Eddy, 1991). The recommended F/M ratio for
integrated BNR processes is generally in the range of
0.1 to 0.25 Ib BOD5/lb MLVSS-day (Metcalf and
Eddy, 1991).
Influent BOD5 concentrations should be high relative
to phosphorus levels to ensure optimal phosphorus
uptake and removal in BNR processes. Although
optimum conditions vary according to the system
design, the ratio of total influent BOD5 (TBOD5) to
influent TP should be 20:1 to 25:1 to meet an effluent
TP level of 1.0 mg/L or less. More importantly, the
ratio of influent soluble BOD5 (SBOD5) to influent
soluble phosphorus (SP) should be 15:1 (WEF/ASCE,
1992b).
The presence of biodegradable material also is
necessary for denitrification in the first anoxic stage of
BNR processes. For example, the Water Research
Commission (1984) found that the TKN to COD ratio
should be less than 0.08 to accomplish complete
denitrification with the Bardenphoฎ process. In most
cases, carbon must be added to the anoxic stage either
by internal recycling of BOD5 in process streams (e.g.,
nitrified effluent) or by chemical addition (e.g.,
methanol or acetate).
19
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Oxygen Requirement
Microorganisms in the activated sludge system require
oxygen to metabolize organic material and nutrients
and breakdown biodegradable toxicants. Oxygen in
diffused air or pure oxygen systems also may oxidize
toxicants. Oxygen deficient conditions can result in
lower treatment efficiency and, as a result, a greater
potential for pass-through of toxic material. To ensure
an adequate supply of oxygen, the DO level for
conventional activated sludge (carbon oxidation)
should be at least 2 mg/L during average loading
conditions and 0.5 mg/L under peak loadings
(WEF/ASCE, 1992a). Typical air requirements are
1,500 cu ft/lb BOD5 load for conventional activated
sludge and contact stabilization, and 2,000 cu ft/lb
BODS load for extended aeration (USEPA, 1989c).
Air requirements for nitrification are higher because
4.2 to 4.6 mg of oxygen are required per mg of NH3-N
oxidized as compared to 0.6 to 1.1 mg of oxygen
needed per mg of BOD5 oxidized (WEF/ASCE,
1992b). DO concentrations of 2 to 2.5 mg/L are
needed for nitrification in activated sludge processes
with short retention times. In integrated activated
sludge/nutrient removal processes, sufficient oxygen
should be provided to achieve carbon oxidation and
complete nitrification at the maximum daily loading
rate.
Some of the oxygen consumed in the aerobic stage is
in the form of NO3-N. This oxygen source can be
recovered in the denitrification process, which is
generally located in an anoxic stage at the head of the
BNR system. Approximately 2.86 mg of oxygen is
recovered for each mg of NO3-N reduced by biological
denitrification. Internal recycling from the aerobic
stage to the anoxic stage can decrease the oxygen
required for nitrification by 50 to 60%. Carryover of
molecular oxygen from the aerobic stage to the anoxic
stage should be minimized by regulating the internal
recycle rate. Generally, the recycle rate should be no
more than three to four times the influent flow rate for
these systems (WEF/ASCE, 1992b).
The transfer of oxygen from the gas phase to the liquid
phase is a function of the aeration equipment and the
basin mixing conditions. USEPA (Handbook for
Retrofitting POTWs, 1989c) describes a procedure for
estimating the oxygen transfer capacity in aeration
basins based on equipment specifications. Another
estimate of oxygen transfer capacity involves
comparing OUR of the biomass to the calculated
theoretical oxygen demand for the aeration system
(USEPA, 1989c). If the OUR results indicate an
oxygen demand that is greater than the calculated
oxygen demand, the oxygen supply may be inadequate.
The opposite case (i.e., higher theoretical oxygen
demand than actual oxygen demand) is preferred;
however, a substantial difference may indicate
inhibition of biomass activity.
TRE Example
OUR measurements were used to document the start-
up performance of the activated sludge treatment
process at the Patapsco Wastewater Treatment Plant
(Botts et-al., 1987). During the start-up, the OUR of
the biomass averaged 20 milligrams O2 per liter per
hour per gram MLSS (mg O2/L/hr/g MLSS), and the
POTW frequently exceeded its conventional pollutant
permit limits. As the biological system became
acclimated to the wastewater, the effluent quality
improved and the biomass OUR increased to an
average of 50 mg O2/L/hr/g MLSS.
Oxygen is not desirable in denitrification processes and
the initial (anaerobic) stage of biological phosphorus
removal processes. Molecular oxygen must be absent
for denitrifying organisms to reduce NO3-N to nitrogen
gas. Phosphorus removing organisms require
anaerobic conditions to accomplish the initial
phosphorus release step (resynthesis occurs in the
subsequent aerobic zone). NO3-N and DO
concentrations in the anaerobic zone should be kept
below 1 mg/L (WEF/ASCE, 1992b). Although mixing
is usually required in anaerobic and anoxic basins, it is
minimized to prevent oxygen transfer to the mixed
liquors. Also, DO carry over from the aerobic zone to
the anoxic (denitrification) zone should be regulated
through independent control of aeration equipment at
the end of the aerobic zone.
Mean Cell Residence Time
In the course of biological treatment, activated sludge
microorganisms convert some of the organic matter
and nutrients in the wastewater to new cell mass.
Toxic constituents may also be degraded or adsorbed
onto the biomass. To achieve optimal treatment, the
biomass concentration in the aeration tank is held at a
constant level by routinely wasting the excess sludge.
Sludge mass control can be practiced by maintaining a
consistent average age of activated sludge (i.e.,
MCRT) in the system. The MCRT is calculated by
dividing the total sludge mass in the system by the
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amount of sludge that is wasted each day (i.e., Ib/lb per
day).
The optimal MCRT for toxicity reduction will depend
on the type of toxicant(s) in the wastewater. Some
compounds may be more efficiently removed by a
younger biomass (low MCRT) and other toxicants are
treated better with an older biomass (high MCRT)
(Metcalf and Eddy, 1991). In general, biodegradable
toxicants are more efficiently removed using a
relatively long MCRT (WEF/ASCE, 1992a); therefore,
the MCRT may be set at the high end of the range of
values typically used for biological treatment.
Hagelstein and Dauge (1984) also found improved
toxicity reduction of a petroleum waste at MCRTs
greater than 10 days. Typical MCRTs for aeration
processes are 6 to 12 days for conventional activated
sludge, 10 to 30 days for contact stabilization, and 20
to 40 days for extended aeration (USEPA, 1989c).
System MCRTs for nitrification and BNR processes
are generally long (20 to 40 days) because the growth
rates of nitrifying organisms are slower compared to
those for heterotrophic organisms found in
conventional activated sludge systems (WEF/ASCE,
1992b). Overly long MCRTs should be avoided
because subsequent denitrification treatment may be
adversely affected if essential carbon has been depleted
in the nitrification/carbon oxidation stage.
TRE Example
In 1992,Novartis Crop Protection, Inc., in cooperation
with Makhteshim-Agan of North America, Inc., the
two principal manufacturers of organophosphate
insecticides in North America, evaluated the removal
of diazinon and chlorpyrifos by various treatment
methods (Novartis, 1997). Anecdotal evidence from
other studies (Fillmore et al., 1990) and treatability
studies by Novartis suggested that adsorption onto
solids was the dominant mechanism for removal of
organophosphate insecticides. The treatability tests
performed in the 1997 study showed that about 30%
of the diazinon and 85 to 90% of the chlorpyrifos
present in POTW primary influent samples is adsorbed
onto primary influent solids and approximately 65 to
75% of the diazinon added to the mixed liquor is
adsorbed onto the biomass. Diazinon adsorption was
greater for a 30-day MCRT biomass than for a 15-day
biomass. Chlorpyrifos strongly adsorbed to the
biomass; none remained after biological treatment.
These results suggest that longer MCRTs may improve
removal of organophosphate insecticides.
Additional Considerations for BNR Process
Control
Additional considerations for BNR process control
include maintaining sufficient alkalinity, proper
management of sludge processing sidestreams, and
achieving efficient solids separation in the secondary
clarifier. Nitrification reduces the wastewater
alkalinity by 7.2 mg/L as CaCO3 for each mg of NH3-N
oxidized. However, about 40 to 50% of the alkalinity
destroyed by nitrification can be restored in
the denitrification process. The carbonate/CO2
equilibrium in the mixed liquor determines the pH. As
a general rule, alkalinity should be maintained above
50 mg/L in the nitrification process in order to keep the
pH high enough for optimal treatment.
Secondary Clarification
In order for activated sludge and BNR processes to
operate efficiently, the secondary clarifier must
effectively separate solids from the liquid phase and
concentrate the solids for subsequent return to the
aeration basin. In addition to clarifier design, solids-
liquid separation is influenced to a large degree by the
aeration basin operating conditions such as DO levels,
F/M ratio, and MCRT. If the MLVSS and MCRT of
the aeration basin is to be maximized for toxicity
control, it is important to consider the impact of this
change on secondary solids separation and effluent
TSS concentrations. Sludge settling characteristics are
affected by how the aeration basin is operated. Low or
high DO levels in the aeration basin can result in the
growth of filamentous bacteria (e.g., Norcardia spp.
and Sphaerotilus natans, respectively) that can hinder
solids settling, whereas DO levels of 2 to 4 mg/L
promote the growth of "zoogleal-type" bacteria, which
aggregate into fast settling floes. At very high organic
loadings (high F/M), the activated sludge can be
dispersed and will not settle well. This condition was
observed at the East Side Sewage Treatment Plant in
Os wego, New York, which experienced sludge bulking
due to high effluent organic loadings (USEPA, 1984a).
Sludge settleability was improved by increasing the
MCRT and the sludge return rate.
The performance of secondary clarifiers in solids-
liquid separation is dependent on a variety of factors
including clarifier configuration, SOR, clarifier depth
at the weirs, the type of sludge removal mechanism,
and the return sludge flow rate. USEPA's Handbook
on Retrofitting POTWs (1989c) describes a system for
scoring secondary clarifier performance based on these
factors. Design clarifier SORs for conventional
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activated sludge processes are typically in the range of
400 to 800 gpd/sq ft (Metcalf and Eddy, 1991). In
general, integrated BNR processes require larger
clarifier capacity than conventional activated sludge
processes, particularly where effluent quality must be
high.
Clarifier capacity is important for phosphorus removal,
because phosphorus is associated with the biomass,
which can be carried over into the final effluent as
residual solids. At peak sustained flow, a clarifier
SOR of 800 gpd/sq ft is recommended to achieve a
final effluent TP concentration of 2 mg/L. Lower
effluent TP limits may require chemical treatment
using a metal salt of iron, aluminum, or calcium, and,
perhaps, a follow-on filtration process. For example,
effluent phosphorus levels at the Jerry Sellers POTW
in Cocoa, Florida, were reduced from an average of 2.9
mg/L to less than 0.2 mg/L with the addition of
aluminum sulfate, commonly referred to as alum
(WEF/ASCE, 1992b). In general, effluent TP levels of
0.2 to 0.5 mg/L can be met through chemical addition
with a clarifier capacity of 500 gpd/sq ft (WEF/ASCE,
1992b).
Process Sidestreams and Wastewater Bypasses
Some wastewater and sludge treatment processes can
produce sidestream wastes that may have a deleterious
effect on the wastewater treatment system or might
contribute to effluent toxicity. In addition, raw or
partially treated wastewater that bypasses part or all of
the treatment system can add substantial toxicity to
POTW discharges (Mosure et al., 1987).
Examples of POTW sidestreams include sludge
processing wastewaters (from thickening, digestion,
and dewatering of sludges), cooling water blowdown,
incinerator scrubber blowdown, and backwash from
tertiary filters. Sidestreams from anaerobic digestion
and sludge dewatering can contain high concentrations
of BODj, COD, nitrogen, and phosphorus that can
represent a significant loading to the aeration basin.
Nitrogen and phosphorus in sidestreams are a concern
for BNR processes, which can be compromised unless
the loadings are removed, equalized, or separately
treated. Also, some sidestreams may contain toxic
materials such as metals and cyanide that may pass
through the POTW. For example, cyanide maybe
formed during incineration of biosolids. Once formed,
the cyanide may be captured in the incinerator
scrubbers and introduced into the treatment system via
the scrubber waste stream. If the treatment process
does not degrade the cyanide, toxic concentrations may
be discharged in the POTW effluent. Also, sufficient
amounts of cyanide may be present to cause inhibition
of the biological treatment process, which leads to the
release of more cyanide in the POTW effluent.
In some municipalities, storm water and sewage are
still collected in the same sewer system. When a large
storm event occurs, the CSO is often diverted away
from all or part of the treatment system to prevent
hydraulic over loading. In some cases, untreated
overflows or bypasses may be directed into the POTW
effluent, which can cause the discharge of relatively
high concentrations of toxic and conventional
pollutants.
The POTW performance evaluation should include a
review of data on process sidestreams, wastewater
bypasses, and overflows that are discharged into all or
part of the POTW or into the final effluent. Additional
analytical and toxicity data may be needed to
characterize the levels of toxic pollutants and toxicity
in these waste streams. Information on the frequency,
volume, and toxicity of sidestream discharges,
bypasses, and overflows also can be compared to
historical effluent toxicity data to evaluate possible
trends or relationships. This information can be used
to determine if the discharges are a significant source
of pollutants or toxicity, and whether current treatment
practices are sufficient to remove toxicity. If
necessary, consideration may be given to enhanced
treatment of process sidestreams to remove toxicants
such as metals. Enhanced treatment may involve
changing the dosage of currently used coagulants
applied for solids separation or adding new coagulant
and flocculent aids. Bench-scale jar tests can be
performed to determine the optimum type and dosage
of coagulant and the appropriate treatment conditions.
Advanced Treatment Processes
Advanced treatment processes may be included in
some POTWs to achieve pollutant removal beyond
what is provided by biological treatment. These
processes may include filtration, adsorption, chemical
treatment, air stripping, and breakpoint chlorination.
Of these processes, chemical treatment and filtration
are most commonly used in POTWs, particularly for
enhanced phosphorus removal.
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Chemical Treatment
Chemical treatment can contribute to toxicity where
toxic residual concentrations or contaminants in the
product are present in the final effluent Chemicals
used in the latter stages of wastewater treatment are of
particular concern because final treatment processes
(e.g., tertiary clarification, chlorination) are less likely
to remove residual concentrations (Note: potentially
toxic disinfection byproducts, including residual
chlorine, are discussed in a following subsection).
When used wisely, treatment additives such as
coagulants, flocculent aids, and hydrogen peroxide
(H2O2) can also improve toxicity treatment.
Chemicals of concern in POTWs include chemicals
formerly classified as hazardous waste. Under RCRA,
a hazardous waste sold to a POTW is no longer
considered a hazardous waste. According to 40 CFR
Part 261.21(c)(5)(ii):
"A material is 'used' or 'reused' if it is...
employed in a particular function or
application as an effective substitute for a
commercial product (for example, spent
pickle liquor used as phosphorus precipitant
and sludge conditioner in wastewater
treatment)."
TRE Example
An example of the potential problems that may occur
with process chemicals was the use of a dechlorination
agent at several City of Houston wastewater treatment
plants (S. Bainter, personal communication, USEPA,
Dallas, TX, 1998). The City had routinely passed
effluent toxicity tests until a dechlorination chemical
was obtained from a new vendor. When the chemical
was applied, effluent toxicity was observed at each of
the POTWs. At the time, the City did not know the
chemical may be a problem and proceeded to retain
consultants to conduct TREs at the facilities. In the
meantime, the supply of the dechlorination chemical
was depleted and the city turned to a new source of the
chemical. When the new chemical Was applied, the
POTWs started to pass the effluent toxicity tests.
POTW staff can avoid similar problems if vendors are
queried about potential contaminants in the waste
chemicals or toxicity tests are performed on product
samples to verify their suitability.
lime, alum, sodium aluminate, ferric chloride, and
ferrous sulfate. These coagulants have generally not
been found to be toxic at the concentrations typically
used for phosphorus removal. For example, alum
dosages as high as 20 mg/L did not cause chronic
effluent toxicity in treatability tests conducted at the
City of Durham, North Carolina (Appendix D).
Nonetheless, steps should be taken to prevent
excessive and inadvertent chemical use. Also, each
chemical additive used for treatment should be
evaluated as a potential source of toxicity, not just
suspect chemicals.
If toxicity is associated with suspended solids,
chemical treatment conditions may be modified to
enhance toxicity removal. The optimum conditions for
coagulation can be determined by conducting jar tests.
These tests can be used to establish the optimum type
and dosage of coagulant, the proper mixing conditions,
and the flocculent settling rates for improved
phosphorus and/or toxicity removal (Adams et al.,
1981).
Chemical treatment is often practiced for phosphorus
removal at POTWs. Typical coagulant aids include
TRE Examples
Studies have been performed to evaluate the reduction
of organophosphate insecticides by chemical treatment
(Novartis, 1997). Chemical precipitation using ferric
chloride and polymer was found to only slightly
reduce diazinon levels. No major change in diazinon
concentrations was observed whether the coagulants
were added to primary wastewater or secondary
treated wastewater prior to clarification. Chlorination
treatment was effective in reducing diazinon from
secondary clarifier effluent; however, chronic toxicity
was unchanged. Qualitative results suggest that the
chlorine oxidized diazinon to diazoxon, a byproduct
that exhibits similar toxic effects as diazinon. The
results of additional treatments for diazinon are given
in Appendix H.
A study conducted for San Francisco Bay area
POTWs also evaluated the effect of chlorine on
organophosphate insecticide concentrations
(AQUA-Science, 1995). This study evaluated the use
of household bleach as a measure that residential
customers could use to degrade diazinon in spray
container rinsate and chlorpyrifos from pet flea washes
prior to disposal into the sewer. Samples of tap water
were spiked with diazinon (60.0 ug/L) and
chlorpyrifos (10.0 ug/L) and treated with either 0.005
or 5% solutions of household bleach for 24 hours.
Results showed that both bleach concentrations
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reduced concentrations of the insecticides by 86 to
92%. The study suggested that household bleach may
be an effective pretreatment for waste solutions of the
two insecticides prior to disposal. Additional studies
are planned to further define bleach exposure times
and concentrations under actual use conditions and to
characterize the chemical oxidation products produced
by the chlorine treatment. Additional information on
this study is presented in Appendix F.
H2O2 has been used by a North Carolina municipality
to control toxicity associated with non-polar organic
toxicants (Aquatic Sciences Consulting, 1997).
Although the specific toxic compounds were not
identified; jar tests with H2O2 showed a substantial
reduction in chronic toxicity to Ceriodaphnia dubia
(C. dubia) at dosages ranging from 1 to 10 mg/L H2O2.
Since the City began adding H2O2 to the POTW
effluent (final concentration of 5 to 7 mg/L), results of
a single C. dubia test show that the effluent NOEC
was reduced from <15 to 90%.
Granular Media Filtration
Granular media filtration is usually applied after
biological treatment to remove residual suspended
solids, paniculate BOD5, or insoluble phosphorus.
Rlter influent is often chemically pretreated to enhance
removal of suspended solids and phosphorus. In
addition to the metal salts noted above, polyelectrolytes
may be added to improve coagulation and flocculation
of chemically treated influents. Typical polymer
dosages are 0.5 to 1.5 mg/L for settling of flocculent
suspensions before filtration and 0.05 to 0.15 mg/L
when added directly to the filter influent. Some
polymers can be toxic to aquatic life (Hall and
Mirenda, 1991); therefore, polymers used in the
filtration process should be evaluated for the potential
to contribute to effluent toxicity.
Poor filter performance should be investigated,
especially the pass-through of potentially toxic
material. Loss of suspended solids and other pollutants
may result from high hydraulic and solids loadings,
excessively long filtration cycles, and incomplete
backwashing and cleaning of the filter.
Disinfection
Disinfection is generally achieved by treating the
secondary effluent with chlorine and allowing a
sufficient contact period prior to discharge.
Alternative disinfection practices such as ultraviolet
(UV) radiation are becoming more popular because of
concerns about the effects of chlorine on aquatic life
and human health. :
The chlorine disinfection process should be carefully
evaluated because residual chlorine and other by-
products of chlorination (i.e., mono- and dichloro-
amines, nitrogen trichloride) are toxic to aquatic life
(Brungs, 1973). Chlorine dosages are usually based on
the level of residual chlorine to be maintained in the
final effluent as specified in the NPDES permit. The
POTW performance evaluation should focus on the
minimum amount of chlorine that can be applied to
achieve the required residual chlorine concentration. In
some cases, the TRC level specified in the NPDES
permit may be sufficient to cause effluent toxicity. In
general, TRC concentrations above 0.05 mg/L are a
concern (D. Mount personal communication, AScI
Corp, Duluth, Minnesota, 1991), although its toxicity
will depend on the effluent matrix and the species used
for effluent monitoring. Residual chlorine levels can
be compared to toxicity data reported in the literature
(USEPA, 1984b) to determine if chlorine may be a
potential cause of effluent toxicity. If dechlorination
is practiced following chlorination, information on the
type and amount of oxidant-reducing material also
should be obtained.
A chlorination process that is not continuously adjusted
to varying flow and chlorine demand may cause
effluent toxicity. Fortunately, this problem can be
corrected easily by more frequent monitoring of the
chlorine residual in effluent samples and more frequent
adjustments in the addition of chlorine and
dechlorination chemical. Flow-proportional feed
equipment for chlorine and dechlorinating agents
should be used to minimize the potential for excess
chemical addition.
TIE Phase I Tests
TIE Phase I tests (USEPA 1991a, 1992a) can be
conducted in parallel with the above operations and
performance review to obtain information on the types
of compounds causing effluent toxicity. An overview
of the Phase I procedure is described in Section 4 of
this guidance.
TIE Phase I testing in the POTW performance
evaluation focuses on characterizing toxicants that may
be present in the effluent because of inadequate
treatment performance or routine operating practices.
Phase I results, when taken together with the POTW
performance evaluation data, may provide important
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clues about possible in-plant toxicants. Based on this
information, treatability tests may be designed to
evaluate methods for removing the suspected effluent
toxicants.
TIE Phase I testing includes several characterization
steps that can be used to indicate the presence of "in-
plant toxicants" such as ammonia and chlorine. One
step involves pH adjustment of the effluent sample to
three distinct pHs, such as pH 6, 7, and 8 prior to
toxicity testing to indicate the effect of pH changes on
effluent toxicity. The pH adjustment will shift the
equilibrium concentration of ammonia between its
toxic form (NH3) and its essentially nontoxic form
(NH4). As pH increases, the percentage of total
ammonia (NH3 and NH4) present as NH3 increases. If
adjusting the effluent sample pH to 8 increases the
toxicity and if lowering the effluent sample pH to 6
decreases the toxicity, the identity of the effluent
toxicant would be consistent with ammonia (Section
4). Another Phase I step is designed to indicate
whether wastewater oxidants, such as TRC (i.e., free
chlorine and mono- and dichloroamines), are causing
toxicity. Sodium thiosulfate, a reducing agent, is
added to eliminate TRC and other oxidants. The
thiosulfate is added to serial dilutions of the effluent
sample with 1 or 2 levels added across the dilutions.
Toxicity tests on samples with and without thiosulfate
treatment are used to indicate if oxidants such as TRC
may be causing effluent toxicity.
It is important to note that each of the TIE Phase I
characterization steps described above addresses a
broad class of toxicants rather than specific effluent
constituents, such as ammonia and TRC (USEPA,
1991a). For example, the toxicant affected by pH
adjustment may be a pH sensitive compound that
behaves in the same manner as ammonia. Also, the
oxidants that are neutralized in the thiosulfate
treatment step include bromine, iodine, and manganous
ions in addition to TRC. Also, some cations, including
selected heavy metals, are complexed by thiosulfate
and may be rendered nontoxic (Hockett and Mount,'
1996). Therefore, the Phase I results should be
compared with information from the POTW operations
and performance review to substantiate the evidence
for a particular toxicant. Using the previous example,
the assumption that TRC is causing oxidant toxicity
would be corroborated if operations data show that
toxic concentrations of chlorine are maintained in the
final effluent (see Table 2-1 for levels of concern).
Organophosphate insecticides also have been identified
as causes of effluent toxicity at POTWs (Ankley et al.,
1992; Amato et al., 1992; Bailey et al., 1997; Botts et
al., 1990; Burkhard and Jenson, 1993). TIE Phase I
procedures that affect Organophosphate insecticides
include CIS SPE and treatment with a metabolic
blocker, piperonyl butoxide (PBO). PBO can be added
to effluent samples or methanol eluates from C18 SPE
columns to block the toxicity of metabolically activated
toxicants like Organophosphate insecticides. PBO has
been shown to block the acute toxicity of diazinon,
parathion, methyl parathion, and malathion to
cladocerans, but does not decrease the acute effects of
dichlorvos, chlorfenvinphos, and mevinphos (Ankley
etal., 1996). A reduction in toxicity by PBO treatment
together with toxicity removal by the C18 SPE column,
recovery from the CIS SPE column, and effluent
concentration data can provide strong evidence for the
presence of Organophosphate insecticides. An
exception is chlorpyrifos, which is not recovered well
from C18 SPE columns (see Appendix F).
Conventional Wastewater Treatability Testing
The operations and performance information may
identify areas in the POTW where improvements in
conventional pollutant treatment may reduce the
pass-through of toxicity. This information and
the optional TIE Phase I data also may indicate in-
plant sources of toxicants such as process
sidestreams or over chlorination. Using these data, a
wastewater treatability program may be devised and
implemented to assess in-plant options for improving
conventional treatment and eliminating in-plant
sources of toxicity.
Treatability studies are recommended prior to
comprehensive TEE testing (Section 4) in situations
where improvements in treatment operations and
performance are needed to attain acceptable
conventional pollutant treatment. Otherwise, TIE
testing of poor quality effluents could lead to erroneous
conclusions about the nature of effluent toxicity. For
example, inadequate conventional pollutant treatment
could cause toxic materials to pass through the POTW
that would otherwise be removed. In the POTW
performance evaluation, treatability studies should
focus on conventional pollutant treatment deficiencies
that are suspected of contributing to effluent toxicity.
The scope of the treatability studies program should be
based on clear evidence of a consistent treatment
deficiency causing toxicity over time. If sufficient
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information is not available to develop a
straightforward treatability program, additional data
must be gathered in the subsequent stages of the TRE
before in-plant toxicity control (Section 6) can be
evaluated.
Treatability studies can vary from a simple evaluation
such as testing the effect of TRC reduction on effluent
toxicity to an extensive effort involving long-term
bench- and pilot-scale work. Prior to beginning these
studies, the POTW operations and performance data
and the optional TEE Phase I results should be carefully
reviewed and an appropriate treatability test program
should be developed using best professional judgment.
The nature and variability of effluent toxicity must be
completely assessed (Section 4) prior to implementing
an extensive treatability effort.
A treatability program can be devised to evaluate
modifications in existing treatment processes.
Evaluating new or additional treatment units should be
attempted only after further effluent characterization
studies (i.e., TIE) have been performed. POTW
performance evaluation treatability testing may involve
physical/chemical treatment approaches, such as
coagulation and precipitation, solids sedimentation,
granular media filtration, powdered activated carbon
adsorption, or biological treatment approaches, such as
activated sludge or sludge digestion.
Toxicity control is the ultimate goal of the TRE;
therefore, toxicity tests should be performed in addition
to the conventional pollutant analyses normally
conducted in treatability studies. Toxicity tests are
used to assess the capability of the treatment
modifications for toxicity reduction. In some cases, the
waste streams to be tested may exert a high oxygen
demand and aeration may be needed to maintain a
minimum DO level of 4 mg/L in the toxicity test.
Aeration may affect toxicant characteristics; therefore,
it may be necessary to use an alternative test method,
such as Microtoxฎ, that is not affected by low DO.
Side-by-side testing with alternative methods or
species and the definitive test can be used to select a
procedure that correlates well with the definitive test.
This initial testing will help to ensure that the
alternative test method or species is sensitive to the
effluent toxicants of concern.
The following subsections briefly describe some of the
treatability tests that can be used to determine if
improvements in existing conventional pollutant
treatment will reduce effluent toxicity. If bench-scale
tests suggest that toxicity can be reduced, follow-up
pilot-scale or full-scale testing is recommended to
confirm the initial results. As shown in Figure 3-1, if
this testing is successful in identifying improvements
in conventional pollutant treatment that will achieve
acceptable levels of effluent toxicity, the TRE proceeds
to the selection and implementation of those options
(Sections 6 and 7). If, however, the treatability data
indicate that improved in-plant treatment will not
reduce effluent toxicity to acceptable levels, other
approaches must be investigated, including TIE testing
(Section 4).
Chemical Treatment
Chemical treatment may be applied in primary
sedimentation, secondary clarification, filtration, and
sidestream treatment processes. As noted above, jar
tests can be used to determine the optimum type and
dosage of chemical, the proper mixing conditions, and
the flocculent settling rates for improved conventional
pollutant and toxicity removal (Adams et al., 1981).
As noted, some chemical additives, including polymers
(Hall and Mirenda, 1991), can be toxic; therefore, the
toxicity of the chemicals should be evaluated as part of
treatability testing.
Sedimentation
Sedimentation processes remove suspended solids or
flocculent suspensions from the wastewater. In
general, sedimentation in POTWs is characterized by
flocculent settling for wastewater (i.e., primary
clarification) and zone settling for mixed liquors (i.e.,
secondary clarification) and sewage sludges (sludge
thickening).
Flocculent settling rates can be converted to a clarifier
SOR by measuring the flocculent percent removal with
time in a settling column test (Adams et al., 1981). If
coagulants are needed, the optimum conditions for
flocculation can be determined from jar tests, as noted
above. A series of settling column tests can then be
performed to compare particle settling profiles for
various coagulant doses and mixing conditions.
Zone settling also can be evaluated in settling column
tests. The settling velocity of mixed liquor or sludge is
determined by measuring the subsidence of the liquid-
solids interface over time (Adams et al., 1981). A
series of tests are performed using the anticipated
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range of suspended solids loadings to the clarifier.
Test results are used to calculate a solids flux curve
that can be used for clarifier design.
Activated Sludge
Continuous flow and batch biological reactor tests can
be used to assess pollutant and toxicity treatability, and
to predict the process kinetics of an activated sludge
system. A series of bioreactors are generally operated
under a range of MCRT values to determine optimum
operating conditions (Adams et al., 1981).
The operational performance of bioreactors can be
evaluated by measuring pollutant removals, OUR,
MLVSS, and the zone settling velocity (ZSV) of the
sludge. These measurements are used to determine the
biodegradation kinetics of the wastewater, the potential
for treatment inhibition, and the preferred sludge
settling conditions. Samples of the influent,
intermediate treatment stages, and effluent of the
bioreactors can also be tested for toxicity to evaluate
the system's toxicity reduction capability. Appendix D
provides an example of the use of batch treatability
tests to evaluate toxicity reduction in a BNR process.
If results of bench-scale treatability tests suggest that
full-scale treatment will reduce effluent toxicity,
follow-up pilot-scale or full-scale tests are
recommended to confirm the results.
Granular Media Filtration
Toxicity removal by filtration can be evaluated in
bench-scale tests or in full-scale tests of existing
processes. The main parameters to be evaluated in
filtration testing include hydraulic loading rate, media
type and configuration, and, if necessary, type and dose
of chemical coagulant (Adams et al., 1981). Filtration
testing results can be used to correlate removal of
suspended solids and toxic compounds with loss of
toxicity. These results are ultimately used to establish,
the optimum design and operational conditions for
conventional pollutant and toxicity removal, including
filter type and loading rates, media characteristics,
backwashing, and headless development. Examples of
the use of filtration in toxicity treatability studies are
presented in Appendices C and D.
Activated Carbon Adsorption
Activated carbon may be applied in powdered form to
the activated sludge process or may be used in granular
form in a post treatment process (e.g., columns). The
capability of carbon adsorption for treatment of organic
wastewater constituents or toxicity is determined by
conducting batch isotherm tests and continuous-flow
tests (Adams et al., 1981).
The effectiveness of carbon in removing BOD5,
selected organic contaminants (e.g., phenols), or
toxicity is predicted by adding varying amounts of
powdered activated carbon (PAC) to wastewater
samples and measuring removal of the organic
constituents or toxicity. The equilibrium relationship
between a wastewater and carbon usually can be
described either by a Langmier or Freundlich isotherm.
A plot of equilibrium concentration versus carbon
capacity is used to select the required PAC
concentration to add to activated sludge processes.
Continuous-flow tests are required to confirm the batch
isotherm results. PAC tests involve adding PAC to
bench- or pilot-scale biological reactors and monitoring
the removal of the organic wastewater constituents or
toxicity.
TRE Example
Activated carbon was investigated as a toxicity control
option in the TRE at the Linden Roselle Sewerage
Authority's (LRSA) POTW in New Jersey (Appendix
G). Both PAC and granular activated carbon (GAC)
were expected to remove non-polar organic toxicity in
the effluent; however, the costs were determined to be
prohibitive. It was also anticipated that carbon would
concentrate the toxicants in the mixed liquor and cause
unacceptable sludge quality.
Pretreatment Program Review
POTW pretreatment program data (Table 2-3) may
provide information that can be used in subsequent
steps of the TRE such as the toxicity source evaluation
(Section 5). Information on the main trunk lines and
the types of indirect dischargers in the sewer collection
system can be used to devise a sampling strategy for
tracking the sources of toxicants or toxicity. In some
cases, the pretreatment program data may be sufficient
to identify the sources of effluent toxicants identified
in the TIE. In most cases, however, additional data,
such as wastewater flow and toxicant concentrations in
indirect discharges, will be needed to track the sources
of toxicants or toxicity.
The information needed to conduct a toxicity source
evaluation is presented in Section 5. In a USEPA TRE
27
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research study conducted in Linden, New Jersey,
pretreatment program data on wastewater
characteristics of the main sewer lines and industrial
dischargers were used to develop a comprehensive
toxicity tracking program (see Appendix G). Sources
of toxicity were successfully identified by devising a
sampling schedule that accounted for periods of normal
industry activity and periods of temporary shut-down
for industry maintenance. The level of toxicity from
the industries was found to vary with the industry
production schedules.
It may be possible, in a few cases, to identify the toxic
sources by comparing chemical-specific data on the
POTVV effluent to information on suspected sources of
the toxic pollutants. This pretreatment program
chemical review (PPCR) approach is recommended
only in situations where the POTW has only a few
indirect dischargers that have relatively non-complex
wastewaters.
PPCR methods are described in Appendix I. These
methods involve a direct comparison of industry
chemical data to POTW effluent toxicity. It is
important to emphasize that drawing preliminary
conclusions based on PPCR results can be misleading
because pretreatment monitoring information could be
incomplete, analytical techniques may not be sensitive
to low levels of effluent toxicants, and the estimated
toxicity of individual compounds may not reflect the
whole effluent toxicity. Domestic sources of toxicants
TRE Example
The PPCR approach was applied at the Mt. Airy
POTW in North Carolina that receives industrial
wastewater from only a few sources, all of which are
textile industries (Diehl and Moore, 1987). Detailed
information on the manufacturing processes and
wastewater discharges of the industries was gathered,
including data on the toxicity and biodegradability of
raw and manufactured chemicals as provided in
material safety data sheets (MSDS) and the scientific
literature. This information was used to identify
industrial chemicals with a relatively high potential to
cause toxicity. Subsequent chemical analysis of the
POTW effluent was performed to evaluate the
presence of the suspected industrial toxicants.
Effluent results were then compared to literature
toxicity values for individual compounds. Using this
approach, APE surfactants, largely attributed to textile
industries, were identified as the primary causes of
POTW effluent toxicity.
such as organophosphate insecticides also may be
responsible for effluent toxicity (see Appendices A
and F). In summary, comparisons of toxic pollutant
concentrations to effluent toxicity may yield false
correlations. Whenever possible, results of TIE testing
should be used in lieu of PPCR results because the TIE
establishes a cause and effect relationship between
toxicants and effluent toxicity.
28
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Section 4
Toxicity Identification Evaluation
Introduction
The TEE is an integral tool in the TRE process and is
applied to evaluate the acute and short-term chronic
toxicity of effluents and other samples. Toxicity is the
trigger for TREs; therefore, the toxicity test is used in
the TIE as the detector for chemicals causing effluent
toxicity. Many types of test species and test
conditions, including lethal (acute) and sublethal
(chronic) measures for both freshwater and saltwater,
can be adapted for use in the TIE. The use of modified
effluent monitoring procedures, which incorporate the
permit test species or a suitable surrogate, will help to
ensure that the toxicants identified are the ones that
specifically affect the species of concern. In the TIE,
the toxicity test is used to track changes in the presence
and magnitude of toxicity as the effluent is
manipulated to isolate, remove, or render biologically
unavailable specific types of constituents (e.g., volatile,
filterable, oxidizable). These procedures relate toxicity
to the wastewater' s physical/chemical characteristics to
determine the compound(s) causing effluent toxicity.
This section of the guidance is intended to be a general
guide for TIEs. For specific guidance on how to
conduct TIEs, the reader should consult USEPA' s TIE
manuals (USEPA 1991a, 1992a, 1993a, 1993b, 1996).
The TIE procedures consist of three phases: Phase I
involves characterization of the toxic wastewater
components, Phase n is designed to specifically
identify the toxicants of concern, and Phase IH is
conducted to confirm the causes of toxicity. Figure 4-1
presents the logical progression of these three phases
within the framework of a municipal TRE. USEPA
has published guidance documents for performing each
phase of the TIE procedures. Phase I procedures are
available to characterize acute (USEPA 1991a, 1996)
and short-term chronic toxicity (USEPA 1992a, 1996).
Phase H procedures (USEPA, 1993a) and Phase m
procedures (USEPA, 1993b) are used to identify and
confirm the causes of acute or chronic toxicity,
respectively. Each TIE is unique and a strategy should
be developed for each study that accounts for site-
specific conditions and allows flexibility in the study
design, including the use of alternative tools and
techniques noted in the TIE documents.
Several effluent samples should be tested to
characterize the magnitude and variability of effluent
toxicity over time. Failure to understand the variability
in whole effluent toxicity and individual toxicants
could lead to the selection of controls that do not
consistently reduce toxicity to compliance levels.
Sampling requirements for TIEs are described in
Section 11. In addition to effluent testing, the TIE
procedures can be applied in toxicity source
evaluations (Section 5) to obtain information about the
causes of toxicity in sewer wastewater or industrial
discharges.
Toxicity Tests
The choice of acute or short-term chronic tests in the
TIE should be determined based on discharge permit
requirements and the toxicity exhibited by the effluent.
Modifications to the whole effluent toxicity test
procedures specified in the permit (USEPA 1993c,
1994a, 1994b, 1995) have been made to streamline the
TIE process. These modifications are described in the
respective TIE characterization, identification, and
confirmation manuals and include smaller test
volumes, shorter test duration, smaller number of
replicates, reduced number of test concentrations, and
reduced frequency of sample renewal (USEPA 1991a,
1992a, 1996). In addition, it is often more useful to
evaluate only one effluent sample in chronic TIEs
instead of multiple samples (e.g., two, three, or seven)
as is typically used for chronic toxicity monitoring.
Reducing the scale of the toxicity tests improves the
efficiency of processing the large number of
subsamples usually generated in the TIE. During the
29
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Pretreatment
Program
Data
Toxicity
Control
Evaluation
TIE
Evidence of Effluent Toxicity
Phase I - Toxicity Characterization Procedures
Initial Toxicity (Acute Phase I)
Baseline Toxicity
pH Adjustment (Tier 2 in Chronic Phase I)
Aeration
Filtration
Cl8 SPE Treatment ;
Sodium Thiosulfate
EDTA Additions
Graduated pH Adjustments
Additional
TIE Information
Required?
No
Phase II - Toxicant Identification
Phase III Toxicant Confirmation
No
Additional
Information
Required?
::
:]
Toxicity Source
Evaluation
Tier 1
POTW In-Plant
Control
Evaluation
Figure 4-1. Flow diagram of a toxicity identification evaluation.
30
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confirmation stage .of the TIE (Phase ffl), whole
effluent toxicity test methods are applied to confirm
that the toxicant(s) identified in Phases I and n is the
cause of the observed effluent toxicity.
TEE procedures have been designed to utilize both
freshwater and estuarine/marine species in acute and
short-term chronic tests (USEPA 1991a, 1993a, 1993b,
1996). Most POTW discharges to freshwater are
monitored with the cladoceran, C. dubia, and/or
P. promelas or, less commonly, the cladocerans,
Daphnia magna or Daphnia pulex, and the trout,
Oncorhynchus mykiss. C. dubia and P. promelas were
used in the development of the TEE procedures and
many subsequent TIEs have been performed
successfully with these species (USEPA 1991a, 1992a,
1993a, 1993b), including the case studies presented in
Appendices A, E, and F. TIEs also have been
performed with trout (Goodfellow et al., 1994) and the
green alga, Selenastrum capricornutum (Walsh and
Garnas, 1983).
In addition, USEPA has provided guidance for the use
of Atlantic, Pacific, and Gulf coast estuarine/marine
species in TIEs (USEPA, 1996). A compilation of
marine TIE studies has been prepared (Burgess,
personal communication, USEPA, Narragansett,
Rhode Island, August, 1998). TEEs have been
performed using mysid shrimp, Mysidopsis bahia
(Morris et al., 1990; Collins, et al., 1994; Burgess et
al., 1995; Douglas et al., 1996), the grass shrimp,
Paleomonetes pugio (Goodfellow and McCulloch,
1993), the mussels, Mytilus edulis (Edile et al., 1995)
and Mytilus califomianus (Higashi et al., 1992), the
sheephead minnow, Cyprinodon variegatus
(Goodfellow and McCulloch, 1993; Burgess et al.,
1995; Douglas et al., 1996), the inland silverside,
Menida beryllina (Burgess et al., 1995), the purple
urchin, Strongylocentrotus purpurtus (Bailey et al.
1995; Jiriketal., 1998), the urchin, Arbaciapunctulata
(Burgess et al., 1995), the sand dollar, Dendraster
excentricus (Bailey et al. 1995), the red abalone,
Haliotis rufenscens (Griffin et al., 1993), the alga,
Champiaparvula (Burgess et al., 1995), the giant kelp,
Macrocystis pyrifera (Higashi et al., 1992; Griffin et
al., 1993), and other estuarine/marine species (Higashi
et al., 1992; Weis et al., 1992). Case studies that
utilized the echinoderms, S. purpuratus and D.
excentricus, and the mysid shrimp, M. bahia, are
presented in Appendices B and G, respectively.
Although many species can be used in TIEs, the use of
the species that is specified in ,the NPDES permit or
that triggered the TRE is encouraged to ensure that the
toxicants identified are the ones that affect the species
of concern. Also, NPDES permit species are more
widely used in TEEs; therefore, extensive published
data are generally available to help characterize and
identify the toxicants affecting these species.
The TEE should incorporate modifications in toxicity
test procedures that are specified in the permit, to the
extent practicable. If pH control in the toxicity tests is
allowed (T. Davies, USEPA, Office of Water,
Memorandum on Clarifications Regarding Flexibility
in 40 CFR Part 136 Whole Effluent Test Methods,
April 10,1996), the effects of pH should be addressed
when evaluating effluent toxicants. However,
procedural modifications should be limited to steps that
are easy and practical to implement.
Effluents with intermittent and ephemeral toxicity may
be challenging to characterize using TEE procedures.'
Intermittent toxicity may require adjustments in the
TIE such as performing frequent toxicity screening
tests over time to ensure that toxic samples are
collected. Some effluents also may exhibit toxicity that
dissipates after the samples are received and the initial
and baseline toxicity tests are performed. If possible,
this ephemeral toxicity may be characterized by
conducting both the baseline test and TIE treatments
immediately upon sample receipt. Also, it may be
possible to shorten the time between sample collection
and testing (i.e., <36 hours) or use grab samples in
addition to composite samples. Depending on the level
of effluent toxicity, it also may be challenging to
discern differences in toxicity following the various
TIE treatments. Steps that may improve
characterization of these samples include adding more
replicates and/or effluent concentrations in toxicity
tests used in the TIE and testing more samples to
evaluate trends in the toxicity characteristics.
Additional information on this topic is given in
USEPA's TIE manuals (1991a, 1992a, 1996).
Effluent monitoring data often includes information on
the relative sensitivity of test organisms. It is generally
recommended that initial TEE testing be performed
using the test species that has been shown to be most
sensitive to the effluent. In cases where equal
sensitivity is observed, the organism that is easiest to
use in the TIE should be selected. Phase Ed
confirmation tests should utilize each of the species
required by the discharge permit to ensure that all
toxicants of concern have been determined.
31
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A brief description of the TIE procedures is provided
below. Examples of TIE applications in municipal
TREs are presented in Appendices A through G.
TIE Procedures
Acute Toxicity Characterization (Phase I)
The first step in the TEE is to characterize effluent
toxicity using the Phase I approach (USEPA, 199 la).
This procedure involves several bench-top treatment
steps to indicate the general types of compounds that
are causing effluent toxicity. An initial toxicity test is
performed to determine if the sample is acutely toxic.
Simple manipulations for removal or alteration of
effluent toxicity are then performed and the resulting
treated samples and the original sample are tested for
toxicity. The physical/chemical characteristics of the
toxicants are indicated by the treatment steps that
reduced toxicity relative to the baseline test.
The Phase I characterization includes the following
tests:
Initial toxicity (unaltered effluent)
Baseline toxicity(unaltered effluent)
pH adjustment (pH 3 and 11)
Filtration/pH adjustment (pH 3 and 11)
Aeration/pH adjustment (pH 3 and 11)
CIS SPE/pH adjustment (pH 3 and 11)
Sodium thiosulfate additions
Ethylenediaminetetraacetate (EDTA) additions
Graduated pH adjustments.
USEPA recommends performing the full suite of Phase
I procedures on initial effluent samples (USEPA
199 la, 1996). As information is obtained on the nature
and variability of toxicity, additional Phase I tests may
focus on the steps that are successful in affecting
toxicity. The aeration procedure is used to determine
if toxicity is associated with volatile or oxidizable
compounds. The filtration procedure is designed to
evaluate whether toxicity is in the suspended
particulate phase or in the soluble fraction. Aeration
and filtration, in conjunction with pH adjustments, are
used to evaluate the volatility and solubility of
toxicants such as ammonia, hydrogen sulfide, and
metals. The toxicity of oxidants and certain metals is
evaluated by adding sodium thiosulfate.
Cationic metal toxicity is determined by
ethylenediaminetetraacetate EDTA additions and,
possibly, by the graduated pH procedure. The
graduated pH step is used to evaluate for the presence
of pH sensitive compounds such as ammonia. An
aliquot of the effluent sample also is used to evaluate
the presence of pH sensitive compounds such as
ammonia. In addition, an aliquot of the effluent
sample is passed through a CIS SPE column that
selectively removes non-polar organic compounds
(USEPA, 1991a).
In general, the TIE procedures used for marine species
are similar to those used for freshwater species, except
that samples used in marine TIEs must be adjusted to
the salinity appropriate to the species being tested
(USEPA, 1996). As part of the development of the
marine TIE procedures, USEPA found that marine
species can tolerate EDTA and sodium thiosulfate
additions at concentrations that can affect toxicants of
concern. Marine species can also tolerate methanol at
concentrations that are necessary to evaluate non-polar
organic compounds with the CIS SPE column.
However, there are exceptions to the methods used for
freshwater species for TIE steps. Due to the strong
carbonate buffering capacity of seawater, it is difficult
to characterize pH dependent toxicants using acids,
bases, and organic buffers. The only efficient method
for maintaining pH in the pH manipulation procedures
is to use controlled atmospheric chambers. Also, a
higher range of pH values is used in the graduated pH
procedure because of the sensitivity of some marine
species to lower pH.
When characterizing toxicity to marine species,
USEPA recommends adjusting the salinity of samples
before performing Phase I manipulations (USEPA,
1996; Ho et al., 1995). However, if a Phase I TIE is
being conducted to help identify potential treatment
options for the POTW, the salinity of the samples may
be adjusted after the TIE manipulations are performed.
This approach is necessary to ensure that toxicity
removal in the TIE reflects the conditions that would
occur in the POTW (i.e., mimics treatment before
discharge to saline waters).
Subsequent tests are recommended to further
characterize effluent toxicity. These tests are described
in the acute Phase I document (USEPA, 199la) in the
"Interpretation of Results/Subsequent Tests" sections
for each procedure. Some of these procedures include
elution of the CIS SPE column with methanol to retain
possible toxicants for further testing. If Phase I does
not adequately characterize the toxicants, other
techniques can be used, such as ion exchange resins for
anions and cations; XAD (a commercially available ion
exchange resin) and activated carbon for various
32
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inorganic and organic compounds; and molecular
sieves, such as Sephadex resins that separate
compounds by molecular weight (Walsh and Garnas,
1983; Lankford and Eckenfelder, 1990; Burgess et al.,
1997). TDS is an example of a toxicant that may not
be well characterized in the TIE. Methods for
characterizing and identifying TDS toxicity are
presented below.
The characterization procedures are relatively broad
and can indicate more than one class of toxicant.
Additional tests are needed to delineate the nature of
the toxicity if significant toxicity changes occur
following the Phase I tests. For example, the CIS SPE
column procedure, which is designed to determine if
non-polar organic compounds contribute to toxicity,
also can remove other compounds such as metals.
USEPA (1991a, 1992a) reports that aluminum, nickel,
and zinc concentrations maybe adsorbed onto the CIS
SPE resin. Confirmation that the CIS SPE column
removed non-polar organic compounds is obtained by
eluting the column with methanol to try to recover the
toxicity. If toxicity can be recovered in the methanol
eluate, then a non-polar organic toxicant is likely
causing toxicity because metals do not elute with
methanol. If toxicity adsorbed by the CIS SPE column
is not recovered by the methanol elution, the column
may have removed toxicants other than non-polar
organic compounds, such as metals, or the non-polar
organic compounds may have a higher affinity for the
SPE column resin than methanol. Appendix E
provides a case example in which toxicity due to
metals was removed by the CIS SPE procedure.
When the primary toxicant is present in high
concentrations, it may mask other potential toxicants,
making it difficult to detect changes in toxicity
following the TIE treatments. Modified procedures
can be designed to control or account for the toxicity of
the primary toxicant. Ammonia is a common example
of a toxicant that may need to be controlled in the TIE
(e.g., pH control) in order to evaluate secondary
toxicants (see Appendix G).
Pretreatment program data and chemical-specific
effluent data may provide useful information to assist
in the Phase I characterization. By reviewing available
information, compounds that are known to be
problematic can be compared to the Phase I results to
assist in indicating the effluent toxicants. This data
comparison should not, however, replace the Phase II
and HI analyses.
After successful completion of Phase I, it may not be
necessary to proceed to Phases n and IE. If the
effluent toxicity can be isolated to a class of
compounds, POTW staff may opt to evaluate the
treatment of effluent toxicity. These studies may
involve bench-scale or pilot-scale testing procedures
described in Section 6. However, if toxicity remains
following implementation of toxicity control methods,
the TIE should begin again with Phase I. In most
cases, a complete TIE using all three phases will
provide results that will lead to a more cost-effective
evaluation of toxicity control approaches.
Chronic Toxicity Characterization (Phase I)
The chronic TIE Phase I procedures (USEPA 1992a,
1996) are similar to the acute Phase I procedures and
include aeration, filtration, CIS SPE treatment,
chelation with EDTA, oxidant reduction and/or
precipitation with sodium thiosulfate, and graduated
pH testing. The chronic test measures sublethal
effects, such as reproduction, fertilization, cyst
development, and/or growth. These measurements
may be affected by the TIE manipulations.
Adjustments have been made in the TIE procedures to
limit toxicity artifacts. As in acute TIEs, additional
steps are recommended to evaluate potential toxicity
artifacts, including use of system blanks and replicate
tests.
The same freshwater species typically used in acute
TIEs (i.e., C. dubia, P. promelas, and, less commonly,
D. magna or D. pulex) can be applied in chronic TIEs.
Species that have been used in chronic marine TIEs
include those noted above in the section titled
"Toxicity Tests."
Two tiers of the Phase I characterization are
recommended for the chronic TIE. Tier 1 is performed
without major pH adjustments. Consistent,
representative blank tests with reconstituted water are
not readily obtained at higher pHs; therefore, the pH
adjustment procedures used in the acute TIE are
separated into Tier 2. Tier 2 is performed only when
Tier 1 does not provide sufficient information about
the types of compounds causing toxicity, and includes
adjusting the effluent sample to pH 3 and 10 as part of
the filtration and aeration steps and pH 9 for CIS SPE
treatment.
Tier I of the chronic Phase I characterization consists
of the following:
33
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Baseline toxicity
Aeration
Filtration
CIS SPE treatment (including tests on post CIS
SPE treatment and methanol eluate)
Sodium thiosulfate additions
EDTA additions
Graduated pH adjustments.
The Tier 2 tests are to be conducted when Tier 1 does
not provide sufficient information and consists of
filtration, aeration, and the CIS treatment technique of
Tier 1 with an effluent sample adjusted to both pH 3
and 10. Tier 2 of the chronic Phase I characterization
consists of the following:
pH adjustment
Aeration and pH adjustment
Filtration and pH adjustment
CIS SPE treatment and pH adjustment (including
tests on post CIS SPE treatment and methanol
eluate).
Additional Characterization Procedures for
Evaluating the Effect of Ion Composition
Although toxicity caused by ion composition is more
commonly found in industrial effluents, ion-based
toxicity has been reported at POTWs (Rodgers 1989a,
1989b; Douglas and Home, 1997; Dawson et al.,
1997). Ion composition can cause toxicity in two
ways: relatively high levels of TDS can inhibit
osmotic regulation in freshwater species, and an
imbalance in ion composition, particularly calcium
carbonate levels, can adversely affect marine
crustaceans (Ward, 1989; MacGregor et al., 1996;
Mickley et al., 1996). The later mechanism primarily
affects crustaceans such as the mysid shrimp, M. bahia,
which require minimum concentrations of calcium
carbonate for survival, growth, and reproduction.
Procedures for evaluating toxicity caused by ion
composition are available (USEPA 1991a, 1992a;
Goodfellow et al., 1998). The following summary is
intended to provide an overview of procedures that can
be used to evaluate ion-based toxicity.
TDS Toxicity
As a general guide, TDS may contribute to acute
toxicity when conductivity exceeds 3,000 and 6,000
jihmos/cm at the LC50 for C. dubia and fathead
minnows, respectively (USEPA, 1991a). For chronic
toxicity, TDS may be a concern when conductivity
exceeds 1,000 and 3,000 |jhmos/cm at the lowest
observed effect concentration (LOEC) for C. dubia and
P. promelas, respectively (USEPA, 1992a). The
conductivity of 100% effluent is not the relevant
reading, but rather the conductivity at the
concentrations bracketing the effluent LC50 and
NOEC.
C. dubia's higher sensitivity to TDS as compared to
P. promelas can provide additional evidence for TDS
toxicity. Also, the cladoceran, D. magna, exhibits less
sensitivity to TDS than the cladocerans, C. dubia and
D. pulex (API, 1998). These species generally show
similar sensitivities to most toxicants (Mount and
Gulley, 1992); therefore, the difference in sensitivity to
TDS can be useful in characterizing TDS toxicity. It
is the toxicity of the individual ions that actually
constitutes TDS toxicity; therefore, it is important to
review the literature for toxicity data on specific ions.
A thorough review of the toxicity of common ions to
freshwater and marine organisms was recently
published by the American Petroleum Institute (API,
1998).
An approach for evaluating TDS toxicity may consist
of the following steps:
1. Monitor the effluent for TDS and if the
conductivity exceeds the levels given above,
measure the major cations (calcium, magnesium,
sodium, potassium) and anions (carbonate,
bicarbonate, sulfate, chloride). A cation/anion
balance should be performed to ensure that all
major ions have been accounted for.
2. For freshwater effluents, conduct toxicity tests
using D. magna, C. dubia, and D. pulex. A
greater sensitivity by C. dubia and D. pulex
compared to D. magna, together with high
conductivity readings, provides a weight of
evidence for TDS toxicity.
3. If TDS toxicity is suspected, review the ion
analysis data gathered above and prepare a stock
solution of the ions in proportion to the amounts
typically observed in toxic effluent samples.
Collect an effluent sample and immediately
measure the constituent cations and anions.
Prepare a mock effluent by adding the solution to
deionized water to yield the same cation/anion
concentrations observed in the effluent sample.
Measure the toxicity of the effluent sample and
mock effluent. If the toxicity is similar, additional
evidence is provided for TDS toxicity.
34
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4. If TDS toxicity is indicated, additional procedures
can be used to determine the extent to which TDS
contributes to effluent toxicity. A sample of the
effluent can be prepared for toxicity testing by
setting up an appropriate dilution series and then
adjusting the TDS levels in each dilution to the
same TDS level as the 100% effluent using the
stock solution (prepared above). Each effluent
dilution is then tested individually for toxicity.
Comparable results for each effluent dilution
provides additional evidence for TDS toxicity.
5. Additional testing can be performed to identify the
TDS constituent(s) that are causing toxicity. The
toxicity of various cations and anions is well
known and a review of the literature (e.g., ENSR,
1998) can be helpful in indicating potential ions of
concern. The ions of concern can be evaluated by
spiking the ions into dilution water and measuring
the resulting toxicity. It should be noted that
toxicity may be caused by a combination of many
ions that exert their influence together. Therefore,
a single salt may not be solely responsible for the
observed toxicity.
TRE Example
Some of these procedures were used in a TIE at a
POTW in Georgia (Dawson et al., 1997) where
chronic effluent toxicity to C. dubia was observed.
TIE characterization tests conducted on the effluent
did not show a reduction in toxicity as a result of the
Phase I manipulations. Independent analyses of the
effluent indicated elevated chloride concentrations. A
mock effluent was prepared as described in step 3,
above, and the ion mixture was found to be as toxic to
C. dubia as the POTW effluent. Laboratory toxicity
data for sodium chloride (NaCl) were used to confirm
that the effluent chloride levels would impair
reproduction in C. dubia at the effect concentration.
Additional TIE studies were performed on the Georgia
POTW effluent using calcium addition and species
sensitivity tests. Calcium has been found to reduce
chloride toxicity in waters with similar ion
composition as the effluent and addition of calcium to
effluent samples reduced toxicity. Toxicity tests using
D. magna, which has been shown to be less sensitive
to chloride than C. dubia, also provided evidence for
chloride toxicity. Overall, the TIE results identified
chloride as a major contributor to effluent toxicity.
Ion Imbalance
Calcium and carbonate, in proper balance, with other
natural ions, are essential for the formation of new
exoskeleton for mysid shrimp and other crustaceans.
At low calcium carbonate levels (i.e., 15 mg/L CaCO3),
Ward (1989) observed 60% mortality in mysids
between the 48-hour and 72-hour exposure periods,
which corresponds well with the mysid molting cycle.
Low CaCO3 concentrations also appear to enhance
mysid sensitivity to other toxicants. Ward (1989)
observed a significant increase in the toxicity of Cd to
mysids when calcium carbonate levels were reduced.
The investigator should consider the potential effect of
ion balance as part of the TIE. Ion imbalance can
contribute to apparent toxicity in some marine
crustaceans when CaCO3 concentrations are 15 mg/L
or less.
Interpretation of Phase I Characterization Results
The following information on the interpretation of
Phase I characterization results is paraphrased from the
TIE manuals. The Phase I characterization provides
information on the types of toxicants in the POTW
effluent. In reviewing the Phase I data, whether for
acute or chronic toxicity characterization, caution is
needed to avoid making inaccurate conclusions about
the results. For example, as noted above, toxicity
removal by CIS SPE treatment does not necessarily
mean that non-polar organic toxicants are present.
Toxicity must be recovered in the methanol eluate test
to provide evidence for non-polar organic toxicants.
The following guidance is given by USEPA (1992a)
for interpreting Phase I data on various types of
toxicants. Note that the reduction or elimination of
toxicity is determined by comparing toxicity before
treatment, as measured by the baseline test, with
toxicity after treatment.
Non-Polar Organic Toxicants
Non-polar organic toxicants may be indicated if:
Toxicity in the post CIS SPE column test was
absent or reduced.
Toxicity is recovered in the methanol eluate test.
However, in those instances where methanol does
not recover toxicity from the CIS SPE column,
other solvents may be needed to elute the toxicants
(USEPA, 1993a).
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Toxicity is reduced by adding PBO to effluent
samples or methanol from the methanol eluate
test. PBO blocks the toxicity of metabolically
activated toxicants like organophosphate
insecticides (USEPA, 1992a).
Cationic Metals
Cationic metals may be indicated if:
Toxicity is removed or reduced in the EDTA
addition test.
Toxicity is removed or reduced in the post CIS
SPE column test.
Toxicity is removed or reduced in the filtration
test, especially when pH adjustments are coupled
with filtration.
Toxicity is removed or reduced in the sodium
thiosulfate addition test.
Erratic dose response curves are observed.
None of these characteristics is definitive, with the
possible exception of EDTA. la addition, toxicity may
be pH sensitive in the range at which the graduated pH
test is performed, but may become more or less toxic at
lower or higher pH depending on the particular metal
involved. This characteristic has not been
demonstrated for chronic toxicity to the extent it has
for the acute toxicity of several metals (USEPA,
1991a).
Surfactants
Surfactants may be indicated if:
Toxicity is reduced or removed in the filtration
test.
Toxicity is reduced or removed by the aeration
test. In some cases, toxicity may be recovered
from the walls of the aeration vessel using a
dilution water or methanol rinse.
Toxicity is reduced or removed in the post CIS
SPE column test. The toxicity may or may not be
recovered in the methanol eluate test. If a series of
methanol concentrations (e.g., 25, 50,75, 80, 85,
90, 95, and 100% in water) is used to elute the
column, toxicity may be observed in multiple
fractions.
Toxicity is reduced or removed in the post C18
SPE column test using unfiltered effluent.
Toxicity reduction/removal is similar to that
observed in the filtration test and toxicity may or
may not be recovered in the methanol eluate test
or by extraction from the glass fiber filter used in
the filtration test.
Toxicity degrades over time as the effluent sample
is held in cold storage (4ฐC). Degradation is
slower when the effluent sample is stored in glass
containers instead of plastic containers.
Ammonia
Ammonia may be indicated if:
Toxicity increases in the graduated pH test at
higher pH.
The effluent is more toxic to P. promelas than to
C. dubia.
Note: If the concentration of total ammonia (as
nitrogen) is 5 mg/L or more and chronic toxicity is
a concern, the potential for ammonia toxicity
should be evaluated.
Drawing conclusions about ammonia toxicity based
solely on observed concentrations can be misleading,
especially where chronic toxicity is a concern because
of the uncertainty about the chronic effects of
ammonia. Ammonia is an example of a toxicant that
acts independently of other toxicants in effluents.
Even though ammonia concentrations may appear to be
sufficient to cause all of the effluent toxicity, other
toxicants may be present and may contribute to toxicity
when ammonia is removed.
Oxidants
Oxidants may be indicated if:
Toxicity is removed or reduced in the sodium
thiosulfate addition test.
Toxicity is removed or reduced in the aeration
test.
The sample is less toxic over time when held at
4ฐC (and the type of container does not affect
toxicity).
C. dubia are more sensitive to the effluent than P.
promelas.
The presence of TRC in the effluent is not enough to
conclude that toxicity is due to an oxidant. However,
TRC concentrations of 0.05-0.1 mg/L or more in
100% effluent provides strong evidence for oxidant
36
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toxicity. Further evidence would be provided, if
dechlorination with sulfur dioxide (SO2) or another
dechlorinating agent removes or reduces toxicity
(USEPA, 1992a).
TDS
TDS may be indicated if:
pH adjustments do not remove or reduce toxicity
and a precipitate is not visible in the pH
adjustment test, pH adjustment and filtration test,
orpH adjustment and aeration test.
There is no loss of toxicity in the post CIS SPE
column tests, or a partial loss of toxicity, but no
change inconductivity measurements.
There is no change in toxicity with the EDTA
addition test, sodium thiosulfate addition test, or
the graduated pH test.
There is a greater sensitivity by C. dubia and D.
pulex compared to D. magna, together with high
conductivity readings.
A mock effluent prepared with the same ions as
the effluent exhibits similar toxicity as the effluent.
Toxicity is removed or reduced by ion exchange
resin.
Toxicity is not removed or reduced by passing the
effluent over activated carbon.
Appendices A, B, E, F, and G provide example Phase I
data and describe how results are used to select
additional TIE procedures for testing. The Phase n
and Phase m procedures (USEPA 1993a and 1993b)
are applicable to both acutely and chronically toxic
samples.
Acute and Chronic Toxicity Identification
(Phase II)
The Phase n guidance manual (USEPA, 1993a)
describes procedures that can be used to identify
specific toxicants such as non-polar organic
compounds, ammonia, cationic metals, chlorine, or
toxicants removed by filtration. The Phase n
procedures are applicable to both acute and chronic
toxicant identification. Phase n uses treatment and
toxicity testing techniques similar to Phase I and
incorporates chemical-specific analyses to identify the
toxicants. Examples of TIE Phase n studies are
provided in Appendices A, B, E, F, and G. Appendices
A, F, and G describe the use of Phase n techniques for
non-polar organic compounds, including high-
performance liquid chromatography (HPLC) for the
isolating toxicants. Appendices B and E describe the
application of Phase n procedures for identifying toxic
metals. Appendix G describes how Phase n
procedures were used to identify ammonia toxicity.
Acute and Chronic Toxicity Confirmation
(Phase III)
The toxicants identified in Phase EL may be confirmed
by a series of Phase HI steps, including correlating
toxicity and toxicant concentration from multiple
samples, observing test organism symptoms, evaluating
species sensitivity, spiking effluent samples with
suspected toxicants, and performing a mass balance to
account for all of the effluent toxicity. In many cases,
it will be appropriate for the Phase I, n, and m
evaluations to overlap because confirmation
information can be obtained during Phases I and n.
Examples of TIE confirmation testing are provided in
Appendices A, B, E, F, and G.
37
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Section 5
Toxicity Source Evaluation
Introduction
Once the effluent toxicants have been identified, a
follow-up evaluation can be conducted to locate the
sources of the toxicants. This evaluation may involve
a review of existing pretreatment program data or data
from the collection and analysis of additional samples
from industrial users. In some cases, the TIE may not
identify the specific compounds causing effluent
toxicity and, in the absence of data on toxicants, the
sources of toxicity must be tracked. Examples of
compounds that are not easily identified in the TIE
include surfactants and some non-polar organic
compounds (other than organophosphate insecticides).
Although the class of compounds may be indicated in
the TIE, it may not be possible to locate the sources
without information on the specific toxic compounds.
In these cases, a guidance is available to track the
sources of toxicity.
A toxicity source evaluation is conducted to locate the
sources of influent toxicity or toxicants that are
contributing to the POTW effluent toxicity. This
evaluation is performed in two tiers whether chemical-
specific or toxicity tracking is to be performed:
Tier Igenerally involves sampling and analysis
of wastewater samples collected from the main
POTW sewer lines.
Tier IIis performed using samples collected
from tributary sewer lines or point sources on the
main sewer lines found to be toxic in Tier I.
This tiered tracking approach can be used to identify
the sources of toxicity and/or toxicants through a
process of eliminating segments of the collection
system that prove to be non-toxic.
The flow diagram for the toxicity source evaluation is
presented in Figure 5-1. The choice of chemical-
specific analyses or toxicity tests for source tracking
will depend on the TIE data on the POTW effluent
toxicants. A chemical-specific investigation is
recommended in cases where the effluent toxicants
have been confirmed and can be traced to the
responsible sewer dischargers. If the sources of
toxicants are located, the TRE can then proceed to the
evaluation of local pretreatment limits as described in
Section 6. Toxicity tracking, using the refractory
toxicity assessment (RTA) approach described herein,
is required in situations where the TIE does not provide
conclusive data on the effluent toxicants. Prior to
toxicity analysis, sewer samples are subjected to the
same type of treatment as is provided by the POTW for
its influent wastewaters. This treatment step allows a
measurement of "refractory" wastewater toxicity,
which is the toxicity that passes through the POTW
and causes effluent toxicity. If toxicity tracking is
successful in locating the sources of toxicity,
pretreatment requirements can be set to reduce the
refractory toxicity contributed to the POTW.
In some cases, industrial users may modify or cease the
discharge of toxicity before specific sources are
identified. The abatement of effluent toxicity during
the course of TREs is not uncommon; however, efforts
to ensure ongoing compliance can be difficult when the
original sources of toxicity are not located. These
situations dramatize the importance of collecting
information on industrial pretreatment activities and
POTW operations in the early stages of the TRE. As
part of the toxicity source evaluation, POTW staff can
request industrial users to submit weekly or daily
reports of production and waste discharge activities
that can be used to indicate potential sources of
toxicity. This information also is helpful in subsequent
pretreatment control studies, if an industrial user is
identified as a source of toxicity (Botts et al., 1994).
38
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Toxicity Source Evaluation
Review of TRE Information
Results of Toxicity Identification Evaluation
Results of Pretreatment Program Review
1
Select Sampling Locations
Tier I
Chemical-Specific Investigation
of Sewer Lines or
Indirect Dischargers
Tier II
Tier I
Track Toxicity in Sewer Lines
Using RTA
Bench-Scale POTW Simulation
Using:
Sewer Wastewater Spiked
into POTW Influent
POTW Influent
TierH
Chemical-Specific Investigation
of Indirect Dischargers
1
Tier II
RTA of Sewer Line Tributaries
and Indirect Dischargers
POTW Simulation Tests
Inhibition Tests (Optional)
TIE Phase I Tests (Optional)
Review Tier I/II
Results and Repeat
Testing if Necessary
Additional
Information
Required?
TIE
Toxicity Control
Evaluation
Figure 5-1. Flow diagram for a toxicity source evaluation.
39
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Sampling Approach
The "Tier I" and "Tier H" designations refer to the
sampling approach to be taken in tracking the sources
of toxicity and/or toxicants.
Tier I- Toxicity Source Evaluation
Sampling locations for Tier I testing are established by
reviewing the pretreatment program data (Section 3)
and selecting appropriate sampling points on the main
sewer lines. In some cases, industrial users or tributary
sewer lines may be selected when substantial evidence
is available on potential sources of toxicity or
toxicants.
If the toxicants have been identified and chemical-
specific tracking is to be performed in Tier I, sampling
locations can include industrial users who have
manufacturing processes or use raw materials that are
known or suspected of containing the toxicants (e.g.,
metals from metal finishers). If the toxicant is
contributed by a large number of dischargers, sewer
line sampling is recommended in Tier I. For example,
sewer line sampling was conducted to determine the
sources of organophosphate insecticides in the City of
Fayetteville, North Carolina, sewer system and several
sewer systems in the San Francisco Bay Area in
California (see Appendix F). These studies indicated
that two insecticides, diazinon and chlorpyrifos, are
widely distributed in POTW collection systems.
If toxicity tracking is to be performed in Tier I, each
major sewer line should be sampled to ensure that all
possible sources in the collection system are
considered. Indirect discharger sampling is not
recommended in Tier I because of the large number of
sources that may need to be evaluated. Sewer line
testing may ultimately reduce the number of sampling
points by eliminating segments of the collection system
where toxicity is not observed (USEPA, 1983a).
In the RTA study conducted at Fayetteville, North
Carolina (Fillmore et al., 1990), sewer wastewater
samples were initially collected from manholes
throughout the collection system because of the large
number of potential sources of toxicity. Sources of
toxicity were subsequently identified by testing the
indirect dischargers located on the toxic sewer lines.
Tier II- Toxicity Source Evaluation
Results of Tier I are used to establish the sampling
locations for Tier II. The toxic sewer lines identified
by toxicity or toxicant tracking in Tier I can be further
evaluated in Tier n by sampling indirect dischargers or
tributary sewers on the toxic sewer lines.
Information on classes of toxicants such as surfactants
can be obtained by coupling the RTA protocol with
selected TEE procedures. For example, in the TRE at
the LRSA, New Jersey, sources of non-polar organic
toxicants were identified by passing RTA test samples
through CIS SPE columns (see Appendix G). Sources
of toxicity were indicated if toxicity was observed in
methanol eluates from the columns.
Sampling Conditions
Whether sampling of sewer lines or indirect
dischargers is conducted, 24-hour flow proportional
composite samples are recommended to characterize
daily variations in toxicant concentrations or toxicity.
In some cases, samples may be collected over less than
24 hours to observe the contribution of potential
intermittent sources of toxicants or toxicity.
Flow data must be gathered in order to determine the
relative contributions of toxicants or toxicity from the
sewer lines or indirect dischargers. Flow data can be
used to calculate the toxicant loadings, which will be
needed to develop local pretreatment limitations
(Section 6). Row data also will be needed to conduct
RTA testing, as described later in this section.
The sampling period for both sewer lines and indirect
dischargers should account for:
Discharge schedules for indirect dischargers
(i.e., intermittent versus continuous).
Temporary shut-down schedules for industry
maintenance.
Coordination with routine pretreatment program
monitoring, if possible.
For example, in the LRSA TRE (Appendix G), sources
of refractory toxicity were identified by sampling
during periods of normal industry activity and during
a period of temporary industry shut-down.
Other considerations for sampling are described in
Section 11. QA/QC sampling requirements are
discussed in Section 8.
Chemical-Specific Investigation
A chemical-specific approach can be used to trace the
influent sources of toxicants if definitive TIE data on
the causes of POTW effluent toxicity are available.
40
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This approach is not recommended in cases in which
the TIE data only indicate a broad class of compounds
(e.g., polar organic compounds), because the toxicants
may be contributed by a variety of sources that will be
difficult to pinpoint by chemical tracking.
Chemical-specific tracking should be conducted after
the effluent toxicants have been identified and
confirmed in TIE Phase n and in tests (USEPA 1993a
and 1993b).
The chemical-specific approach involves testing
indirect dischargers or sewer line samples for toxicants
using chemical analysis techniques. In some cases,
existing pretreatment program data maybe adequate to
identify the indirect dischargers that are contributing
the toxicants. It is likely, however, that further
sampling and analysis will be necessary, because
pretreatment program data generally do not include
information on toxicants typically identified in TIE
tests (e.g., compounds other than regulated pollutants).
Existing pretreatment program data may be used to
reduce the amount of sampling and analysis by
indicating which sources contribute toxic pollutants
that are similar to the effluent toxicants.
Chemical analysis methods for potential toxicants such
as ammonia, metals, and organic compounds are
described in several USEPA documents (USEPA
1979b, 1983b, 1985b) and Standard Methods for the
Examination of Water and Waste-water (APHA, 1995).
USEPA (1997) provides all of USEPA's methods for
analysis of water on a CD-ROM. USEPA's Phase H
TIE manual (1993a) also provides guidance for the
analysis of organophosphate insecticides, surfactants,
and metals. Analytical methods for organophosphate
insecticides have been improved to achieve the lower
detection limits necessary to assess insecticide toxicity
(USEPA, I993a; Durban etal., 1990). Enzyme-linked
immunosorbent assay (ELISA) procedures also are
available for selected organophosphate insecticides,
metals, and other compounds. ELISA methods offer
the advantage of low cost, rapid sample processing,
and field portability; however, these methods may not
be specifically approved by USEPA. Additional
analytical techniques can be found in American
Society for Testing and Materials (ASTM) manuals
and peer-reviewed journals such as the Analytical
Chemistry Journal. A qualified chemist should verify
the selected analytical method in the laboratory prior to
sampling and analysis.
A literature search also can be made to determine if the
toxicant could be a biodegradation product resulting
from POTW treatment. Where clear evidence is
available to show that the toxicant is a treatment by-
product, the sewer sample should be analyzed for the
precursor form(s) of the toxicant as well as the toxicant
itself.
In cases where chemical tracking is successful in
locating the sources of the POTW effluent toxicants,
the TRE can proceed to the selection and development
of toxicity control options such as local pretreatment
regulations (Section 6). Information on toxicant
distribution can be used in developing pretreatment
control options. For example, although a primary
contributor of ammonia was identified in the LRSA
TRE (Appendix G), system-wide pretreatment
limitations were adopted to address all non-domestic
sources of ammonia. In other situations, control
methods other than pretreatment limitations, such as
public education, may be needed to control the
discharge of a widely used toxicant. Public education
has been successfully used at a number of POTWs
(Appendix H) to control the use of organophosphate
insecticides, which can be contributed from many
domestic and commercial areas of the collection
system.
If the responsible indirect dischargers are not located,
the TIE results should be reviewed to confirm previous
conclusions. The chemical analysis results also should
be carefully reviewed to determine if errors or
waste water matrix effects may have caused inaccurate
results. In cases where the chemical-specific approach
is ultimately not successful, the source evaluation
testing should be repeated using toxicity tests in lieu of
chemical analyses, as described below.
Refractory Toxicity Assessment
Toxicity tracking may be required when the TIE
characterizes the toxicity as broad classes of toxicants
or identified toxicants cannot be confirmed. Toxicity
tracking also may be useful in situations in which there
are multiple effluent toxicants and the occurrence of
these toxicants in the POTW effluent is highly
variable. In such cases, toxicity testing may be more
cost-effective than chemical tracking.
The toxicity found in influent wastewaters is not
necessarily the same toxicity that is observed in the
41
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POTW effluent because the POTW is capable of
removing some toxic wastewater constituents. The
amount of sewer wastewater toxicity that could
potentially pass through the POTW must be estimated
by treating sewer samples in a simulation of the POTW
prior to toxicity analysis. This treatment step accounts
for the toxicity removal provided by the POTW.
A protocol has been developed for predicting the
potential for a sewer discharge to contribute to acute or
chronic toxicity in POTW effluents. This protocol,
referred to as the RTA procedure, has been
successfully used to track sources of acute and chronic
toxicity using both freshwater and estuarine/marine
species (Morris et al., 1990; Botts et al., 1992, 1993,
1994). Examples of RTA studies are presented in
Appendices C, D, and G.
The RTA protocol has been designed to simulate
conventional activated sludge processes, although it
has also been adapted to other POTW treatment
processes including single and two-stage nitrification
systems (Collins, et al. 1991), BNR processes
(Appendix D), and filtration treatment systems
(Appendices C and D). The RTA procedure described
herein involves treating sewer samples in a
bench-scale, batch simulation of a conventional
activated sludge process and measuring the resulting
toxicity. Batch simulations are appropriate for
plug-flow biological systems because batch processes
behave over time as plug-flow processes do with flow
time. Batch biological reactors have been used by
several researchers to screen wastewaters for activated
sludge inhibition (Grady, 1985; Adams et al., 1981;
Philbrook and Grady, 1985; andKangetal., 1983) and
non-biodegradable aquatic toxicity (Hagelstein and
Dauge, 1984; Lankford et al., 1987; and Sullivan et al.,
1987). Hagelstein and Dauge (1984) and Lankford et
al. (1987) have found that toxicity measurements
coupled with bioreactor tests can be a pragmatic way
to evaluate refractory wastewater toxicity.
The RTA protocol was developed in the USEPA TRE
research study at the City of Baltimore's Patapsco
POTW (Botts et al., 1987) to evaluate the potential for
indirect dischargers to contribute refractory toxicity.
Additional USEPA TRE research studies in Linden,
New Jersey; High Point, North Carolina; Fayetteville,
North Carolina; and Bergen County, New Jersey were
conducted to improve the RTA approach (Morris et al.,
1990; DiGiano, 1988; Fillmore et al., 1990; Collins et
al., 1991). The RTA procedure described herein is a
refined version of the method given in the municipal
TRE protocol (USEPA, 1989a).
The batch reactor used in RTA testing is designed to
simulate, as close as possible, the operating
characteristics of the POTW's activated sludge process
(e.g., MLSS concentration, DO level, and F/M ratio).
Two types of batch reactors are used, as shown in
Figure 5-2. One reactor serves as the control and
consists of the POTW influent and return activated
Wastewater and
Return Activated
Sludge
Plastic or Glass
Container
Air Supply
(Oil-Free)
Air Line
Tubing
Air Stone
Control Reactor
POTW Influent
(Control)
Spiked Reactor
Sewer/Industrial
Wastewater Spiked
into POTW Influent
Figure 5-2. Schematic of a refractory toxicity assessment test.
42
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sludge (RAS). The other reactor consists of sewer
waste water spiked into the mixture of POTW influent
and RAS. If the effluent toxicity of the reactor spiked
with sewer wastewater is increased relative to the
unspiked reactor, the sewer wastewater would be
considered a source of refractory toxicity. In the
spiked reactor, sewer wastewater is tested together
with the POTW influent in order to observe possible
interactive effects (e.g., additivity, antagonism) that
can occur when the wastewater and the total POTW
influent are combined and treated in the POTW.
A general description of the RTA procedure is
presented below. A step-by-step protocol for RTA
testing is provided in Appendix J. The basic steps in
the RTA approach are:
Conduct conventional pollutant analyses to
develop a profile for each wastewater to be tested
in the RTA.
Perform toxicity tests on the POTW's RAS
(filtrate) to determine its potential to cause an
interference in RTA testing.
Collect, characterize, and prepare wastewater
samples for RTA tests.
Calibrate the RTA batch reactors to achieve a
treatment level comparable to that of the POTW's
activated sludge process.
Calculate wastewater volumes to be used in RTA
tests.
Set-up and operate the RTA batch reactors.
Analyze batch effluent toxicity.
Evaluate the potential for the sewer waste waters to
inhibit activated sludge treatment (optional).
Conduct TIE Phase I tests to indicate the types of
refractory toxicants in the sewer wastewater
(optional).
Interpret the results.
It is important to emphasize that the RTA protocol
should be modified to address site-specific conditions.
For example, Appendix C describes an RTA study that
simulated a filtration treatment process in addition to a
nitrification treatment process. The following
summary of the RTA protocol is intended to be a
general guide to evaluating sources of toxicity using
simulations of suspended biological growth processes.
Best professional judgment is important in adapting the
procedures to treatment processes and conditions that
are unique to each facility.
POTW Wastewater Profile
The first step in the RTA is to characterize the POTW
influent (primary effluent), sewer wastewaters, and
RAS to be used in RTA testing. The POTW influent
wastewater should be collected from the effluent of the
POTW primary treatment process because primary
effluent is treated by the activated sludge process,
which is the main process to be simulated in the RTA.
RAS is recommended for use in batch testing because
it is in a concentrated form that can be easily diluted to
the target MLSS concentration. Mixed liquor from the
POTW's aeration basins can be used in lieu of RAS;
however, the activated sludge will need to be thickened
to the same suspended solids concentration as the RAS
before use.
Table 5-1 presents the analyses and information that
are needed to characterize the wastewaters to be used
in RTA testing. This information will be useful for
determining the following operating conditions for the
RTA batch reactors, including:
Determining the volume of sewer wastewater to
use in testing based on sewer line and indirect
discharger flow-rate data.
Determining whether nutrient addition is necessary
using information on the ratio of organic content
(BOD5 or COD) to nutrient concentrations (TKN
and TP).
Selecting a test period for the RTA reactors that is
based on the organic content of the sewer
wastewater. Some sewer wastewaters may have
substantially higher COD concentrations, which
will increase the initial COD level in the RTA
reactor. A longer treatment time may be needed to
ensure that the wastewater is treated to the same
level as the POTW influent.
Biotnass Toxicity Measurement
Sometimes the RAS used in testing can cause an
interference in the measurement of refractory toxicity.
In the Patapsco TRE, filtered samples of RAS were
found to be acutely toxic to C. dubia (Botts et al.,
1987). This toxicity was related to residual biosolids
that passed through the filter [10-micron (|im) pore
size]. The toxic biosolids caused the batch reactor
effluents in RTA tests to be acutely toxic and masked
the refractory toxicity of the wastewaters being tested.
This biomass interference reduced the effectiveness of
the RTA test for determining the sources of refractory
43
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Table 5-1. POTW Wastewater Profile Analyses for a Refractory Toxicity Assessment
Waste Stream
Information Required
RAS
TSS
VSS
NH3-N
pH
Primary effluent
BOD5
COD
TSS
TP
TKN
NH3-N
pH
Sewer line or indirect discharger wastewater
Location in collection system
Number/type of indirect dischargers
Flow, million gallons per day (mgd)
BOD5
COD
TSS
TKN
NH3-N
TP
pH
Other indirect discharger data
Type of discharger
Wastewater pretreatment system
Operations/production schedule
toxicity at the Patapsco WWTP. Additional tests
demonstrated that RAS toxicity could be removed by
filtration of the coarse filtrate through a 0.2 |im pore-
size filter or by centrifugation of the coarse filtrate at
10,000 times gravity (xg) for 20 minutes (Botts et al.,
1987).
Additional information obtained in the Linden Roselle,
New Jersey; Fayetteville, North Carolina; and Bergen
County, New Jersey USEPA TRE research studies
indicated that the POTW RAS filtrate was not acutely
toxic, and therefore did not cause an interference in
RTA testing (Morris et al., 1990; Fillmore et al., 1990;
Collins et al., 1991). The existing data on the toxicity
of sewage sludges are not sufficient, however, to
evaluate the occurrence of biomass toxicity at POTWs.
The following discussion provides information on how
to proceed, if POTW biomass toxicity is observed.
Prior to conducting the RTA, toxicity tests of the
POTW activated sludge should be performed to
determine if the biomass is toxic. This testing involves
toxicity measurement of two aliquots of RAS: coarse
RAS filtrate and coarse RAS filtrate subjected to
centrifugation to remove colloidal particles. The RAS
should first be filtered through a coarse glass fiber
filter (e.g., 10 (im pore size), which is the same type of
filter used for suspended solids analysis (APHA,
1995). Following coarse filtration, an aliquot of the
RAS filtrate should be further treated by centrifugation
at 10,000 xg for 10 to 15 minutes. Alternatively, the
coarse filtrate could be filtered through a 0.2 jam
membrane filter. However, tests should be conducted
to confirm that soluble toxicity is not removed by
sorption onto the filter.
Both the RAS filtrate and centrate should be tested for
acute or chronic toxicity using limited-scale tests
(USEPA 1993c, 1994a, 1994b). If results show that
centrifugation does not reduce biomass toxicity relative
to the coarse filtrate, then the RAS is not likely to
cause an interference in RTA testing. In this case, the
POTW biomass can be used directly in RTA testing
44
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and centrifugation of the RTA batch effluents will not
be required. Samples of RAS should be periodically
analyzed for toxicity during RTA testing to monitor for
possible biomass toxicity.
If the biomass coarse filtrate is observed to be more
toxic than the RAS centrate, the biomass toxicity may
interfere with RTA testing. Two options are available
in this case: removal of the toxic biosolids by fine
particle filtration (or centrifugation) of batch test
effluents, and use of an alternate biomass.
Biomass toxicity may be removed by applying the fine
particle filtration or centrifugation treatment steps to
the batch test effluents. In this case, the resulting RTA
effluent toxicity will only indicate soluble refractory
toxicity, not the total refractory toxicity (i.e., soluble
and paniculate).
Another approach to remove biomass toxicity is to use
a non-toxic biomass such as another POTW biomass or
a commercially available (freeze-dried) preparation. A
surrogate biomass will not be acclimated to the influent
wastewaters of the POTW being evaluated; therefore,
it may not treat the wastewaters as well as the POTW's
biomass. Nonetheless, an alternate biomass can
provide a level of treatment that will approximate the
refractory toxicity of the sewer wastewater. It may be
helpful to conduct a parallel series of RTA tests using
the toxic POTW biomass. The use of toxic POTW
biomass is suggested because it is acclimated to the
POTW influent wastewaters and will therefore provide
a level of batch treatment that is more similar to the
POTW treatment than that provided by the
unacclimated alternate biomass. In this case, fine
particle filtration or centrifugation is required to
remove the interfering biomass particles prior to
toxicity analysis. By performing RTA tests with
POTW biomass in parallel with RTA tests with
alternate biomass, both the soluble and total refractory
toxicity of the wastewater may be estimated.
An alternate biomass may be useful in cases where it is
necessary to simulate future modifications or additions
to the POTW activated sludge treatment process (e.g.,
conversion from conventional activated sludge to
nitrification). In these cases, a biomass that is
indicative of the future activated sludge, may not be
directly available at the POTW. An alternate biomass
can be obtained from another POTW that has a
biological treatment process similar to the treatment
process planned for the POTW. A TRE conducted by
the City of Durham, North Carolina, used this
approach to evaluate the toxicity reduction capability
of planned nutrient removal treatment systems
(Appendix D).
RTA Reactor Calibration Testing
Generally, ideal plug-flow conditions do not occur in
activated sludge processes; therefore, it will be
necessary to adjust the RTA batch treatment conditions
to account for the actual level of treatment achieved in
the POTW. One method of controlling the treatment
efficiency of activated sludge processes is to adjust the
biomass concentration, measured as MLVSS
concentration. Batch calibration tests can be
performed using a series of MLVSS concentrations
and the MLVSS concentration that most closely
simulates the POTW treatment efficiency can be
selected for RTA testing.
Prior to calibration testing, a target MLVSS
concentration can be estimated using mathematic
models. In the Fayetteville TRE, a steady state,
completely mixed, multi-stage model (Grady and Lim,
1980) was used to determine biokinetic coefficients
that best modeled the POTW treatment performance
(Fillmore et al., 1990). The biokinetic coefficients
were then used in a steady state plug-flow model
(Kornegay, 1970) to calculate a batch MLVSS
concentration that would theoretically simulate the
POTW treatment efficiency. The model results were
confirmed in bench-scale, batch reactor tests using a
range of MLVSS concentrations, including the
theoretical MLVSS concentration and several MLVSS
concentrations that bracketed the theoretical value. In
this case, the MLVSS concentration determined from
the calibration tests matched the theoretical MLVSS
value (Fillmore et al., 1990).
POTW primary effluent is typically used in RTA
calibration testing. The treatment efficiency of the
batch reactors can be evaluated by periodically
collecting and analyzing samples for COD and
toxicity. TKN, NH3-N, and TP may also be monitored
if the batch reactors are simulating BNR treatment
systems. Results of the batch reactor tests are then
compared to COD, nutrient, and toxicity data for the
POTW final effluent to indicate which batch reactor
achieved treatment comparable to the POTW. If there
are large differences between batch effluent results and
POTW effluent results, it may be necessary to evaluate
45
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different MLVSS concentrations in additional
calibration tests. In the Durham TRE, calibration tests
were used to define batch operating conditions for a
five-stage BNR process (Appendix D).
The RTA calibration study can also be used to
establish an appropriate test dilution series for the
toxicity tests of batch effluents. Where possible, the
dilution series for toxicity tests should bracket the
acute or chronic toxicity value (i.e., LC50 or ICp) as
closely as possible in order to reduce the span of the
95% confidence limits. Increased confidence in the
data is important because sources of refractory toxicity
are indicated based on a comparison between effluent
toxicity results for the sewer wastewater-spiked reactor
and the POTW influent (control) reactor (Figure 5-2).
The following example illustrates this point.
A wastewater from an industrial user is spiked into
POTW influent sample and tested using the RTA
procedure. The acute toxicity of the RTA effluent is
measured using two different dilution series: one test
series encompasses a wide range of samp?e
concentrations and the other series more closely
brackets the expected LC50. The dilution series and
resulting survival and LC50 values are shown in
Table 5-2.
Using dilution series #1, the LC50 for the industrial
wastewater test would be 35% sample with confidence
limits (95%) of 25% to 50% (based on binomial
model). Using series #2, the LC50 would also be 35%
sample, but the confidence limits (95%) would be
much tighter at 31 to 39% (based on probit method).
The results for the RTA control test together with the
industrial wastewater spiked test are shown in
Table 5-3.
In this example, if the control reactor LC50 had been
50% with confidence limits of 42 to 62%, the industrial
wastewater would have been indicated as a possible
source of toxicity based on the results of series #2,
because the 95% confidence limits do not overlap (i.e.,
31 to 39% versus 42 to 62%). However, if dilution
series #1 had been used, the industrial wastewater may
not have been judged to be a toxic source because the
confidence limits overlap (25 to 50% versus 42 to
62%). The partial mortality in the 35% concentration
in series #2 (Table 5-3) helps to more precisely define
the LC50. The narrow confidence limits in series #2
support the conclusion that the refractory toxicity of
the wastewater is significantly greater than the POTW
influent control (i.e., confidence limits do not overlap).
In the Reidsville and LRSA TREs (Appendices C and
G), the results of preliminary toxicity analyses were
used to adjust the dilution series to closely bracket the
expected IC25 and LC50 value of the batch effluent
samples. This approach allowed the identification of
sources of refractory toxicity that would not have been
indicated using a standard toxicity test dilution series.
Sample Collection
Wastewater and activated sludge samples should be
collected according to the procedures described in
Section 11. Sample volumes will be based on the
subsample volumes needed for periodic reactor
measurements and batch effluent toxicity testing.
Table 5-2. Example of Bracketing the LC50 Concentration in the RTA Sewer Wastewater Test
Test
Series #1
Series #2
Percent Survival in Sample Concentration
100
0
100
0
50
0
50
0
25
100
35
50
12.5
100
25
100
6.25
100
12.5
100
0
100
0
100
Source: Fillraore et al., 1990.
Table 5-3. Comparison of Control Test and Industrial Wastewater Spiked Test Results
Test
Series #1
Series #2
Control Test
LC50(CI)*
50% (42-62)
50% (42-62)
Industry Spiked Test 1
LCSO(CI) L
35% (25-50)
35% (31-39)
Potential Source
of Toxicity?
No
Yes
* Confidence intervals (95%) shown in parentheses.
46
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The volumes of wastewater and RAS to be used in
RTA testing will depend, in part, on whether acute or
chronic toxicity will be measured. Generally, a batch
reactor volume of 3 liters (L) is sufficient when
standard freshwater and marine/estuarine species (i.e.,
C. dubia, Daphnia sp., P. promelas, M. bahia) are to
be used for testing the acute toxicity of batch effluents.
A batch reactor volume of 10 L is adequate to measure
chronic batch effluent toxicity using the 7-day C. dubia
test.
Sample Characterization
Average characteristics of the sewer wastewater and
POTW primary effluent can be determined using the
wastewater profile data (Table 5-1). These data should
include historical results of BOD5, COD, TKN, TP,
TSS, NH3-N, and pH analyses. Analyses should also
be performed on the samples collected for RTA tests to
ensure that the wastewater characteristics are
consistent with historical data.
Preparation of RTA Test Mixtures
Two batch influent solutions are prepared for each test
of a sewer wastewater sample: sewer sample spiked
into POTW primary effluent, and primary effluent
alone. The sewer sample may be collected from a
sewer line or an industrial discharge. The amount of
sewer sample to be used in testing should reflect the
percent volume of sewer wastewater in the POTW
influent. In some cases, the wastewater toxicity from
small contributors may not be readily observed when
the wastewater is mixed by percent volume with
POTW influent. In these cases, it may be necessary to
use a greater volume of sewer wastewater than is
typically contributed to the POTW.
The volume of sewer wastewater (Vw) sample to be
added to the batch reactor is calculated as follows:
Vw (L) = -=- x (Vr - Vb) x Fw,
Qi
where: Qw is the sewer wastewater flow rate (mgd).
Qi is the average POTW influent flow rate
(mgd).
Vr is the total reactor volume (gal or L).
Vb is the volume of RAS biomass (gal or L).
Fw is the sewer wastewater flow concentra-
tion factor (e.g., 1, 2, 10 times the sewer
wastewater flow).
The selection of a flow concentration factor (Fw) will
depend on the percent flow of the sewer wastewater in
the POTW influent. A conservative, yet realistic,
approach would be to use a Fw that is based on the
maximum daily wastewater flow from the sewer
discharge in the past year. The Fw should not cause
the sewer wastewater to be 100% of the reactor
wastewater volume. For example, if the sewer
wastewater flow is greater than 20% of the POTW
influent flow, a Fw of less than 5 should be used. It is
necessary to test the mixture of sewer wastewater and
POTW primary effluent in order to evaluate the
interactive effects (e.g., additive or antagonistic) that
can realistically occur when these wastewaters are
combined at the POTW. All sewer samples should be
tested using the same Fw to allow a comparison of
batch effluent toxicity between the various sewer
wastewaters.
After determining the Vw, the volume of primary
effluent (Vpe) to be added to the batch reactors can be
calculated as:
Vpe = (Vr-Vb-Vw).
The batch reactor influents are prepared by mixing the
Vw and Vpe for the sewer wastewater spiked reactor
and measuring Vpe for the control reactor. In some
cases, it may be necessary to adjust the nutrient levels
or pH of the batch influents prior to testing as
described below.
The BOD5/TKN/TP ratio of the batch reactor influents
should be compared to the average BOD5/TKN/TP
ratio of the POTW influent, as determined from
historical or profile data. The sewer wastewater added
in the batch reactor influent may be deficient in
nutrients, especially if industrial wastewaters are used.
If necessary, nitrogen and/or phosphorus should be
added so that the BOD/TKN/TP ratios of the batch
reactor influent and POTW influent are similar.
Unless the BOD5 to nutrient ratios for the batch reactor
influent and POTW influent are clearly dissimilar,
nutrient addition is not recommended be'cause of the
potential for added nutrient salts to change the
sample's toxicity.
Using the profile data, BOD5 and nutrient (TKN, TP)
concentrations (C) in the batch reactor influent
(spiked) are calculated as follows:
47
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C (mg/L) = (vPexCPg)+(VwxCw)
(Vpe+Vw)
where: Vpe is the volume of primary effluent in
reactor (L).
Cpe is the BOD5 or nutrient concentration in
primary effluent (mg/L).
Vw is the volume of sewer wastewater in
reactor (L).
Cw is the BOD5 or nutrient concentration in
sewer wastewater (mg/L).
The typical BOD5/TKN/TP ratio for municipal sewage
is 100:5:1 (WEF/ASCE, 1992a). This ratio will ensure
that sufficient nutrients are available for consistent
batch treatment of the sewer wastewaters. If necessary,
phosphorus should be added in the form of three parts
monosodium phosphate (NaH2PO4) to four parts
disodium phosphate (NajHPOJ. Nitrogen should be
added as urea nitrogen, except in cases where ammonia
is suspected as a cause of effluent toxicity, because
urea nitrogen can be converted to ammonia during
biological treatment.
Following nutrient addition, the pH of the batch
influents may need to be adjusted to within the average
range of pH for the POTW influent. Typically, the
range of POTW influent pH values will be pH 6 to 9.
Hydrochloric acid and sodium hydroxide can be used
for pH adjustment.
Following nutrient addition and pH adjustment, the
batch influent toxicity should be measured to
determine if the added nutrients or pH adjustment
cause a change in sample toxicity. Substantial
differences between the initial toxicity and the adjusted
sample toxicity may indicate the presence of specific
types of toxicants. Use of pH adjustment for toxicity
characterization is discussed in the USEPA TIE Phase
I manuals (USEPA 1991a and 1992a).
The volume of RAS biomass (Vb) to be used in batch
testing should yield a batch MLVSS concentration that
is equal to the target MLVSS concentration determined
in calibration testing (see above). The amount of RAS
to be added to the total reactor volume (Vr) is
calculated as follows:
Vb(L) .Target MLVSS (mg/L)
RAS VSS (mg/L)
This equation also is used to determine the alternate
(non-toxic) biomass volume (Vnb), if required.
j
Synthetic Wastewater Testing (Optional)
In some cases it may be important to determine the
amount of refractory toxicity of the sewer wastewater
excluding the effects of other influent wastewaters. A
batch influent solution pontaining sewer sample spiked
into a synthetic wastewater can be used to determine
the individual refractory toxicity of the sewer sample.
The synthetic wastewater will provide a standard
substrate that will allow consistent treatment of the
sewer wastewaters.
A synthetic wastewater should be prepared that has a
COD concentration that is equal to the average COD
concentration of the POTW primary effluent. The
volume of synthetic wastewater (Vsw) to be added to
the batch reactor is calculated using the same equation
that is used to calculate the volume of POTW primary
effluent. A synthetic wastewater has not been
developed that is consistently non-toxic (DiGiano,
1988). Prior to use in RTA testing, the synthetic
wastewater should be tested for toxicity to ensure that
it will not interfere with the measurement of refractory
toxicity.
Performance of RTA Tests
RTA testing is initiated when the batch influent
solutions are mixed with RAS and diffused air is
applied to the mixture. The aeration rate should be
adjusted to maintain a DO concentration equal to the
DO level observed in the POTW activated sludge
treatment process. Mechanical mixing using a
magnetic stirrer and teflon-coated stir bars may be
required to ensure complete mixing in the reactor. The
RTA tests must be performed in appropriate laboratory
fume hoods to prevent exposure of laboratory staff to
any toxic vapors stripped from the wastewater samples
(Section 9).
The organic loading to the batch reactors can vary
substantially depending on the type of sewer
wastewater being tested. For example, a wastewater
with a high COD concentration (e.g., >5,000 mg/L) is
likely to increase the COD loading to the RTA reactor.
The effect of this variation on batch treatment can be
minimized by adjusting the reactor treatment time to
achieve a constant "food-to-microorganism ratio" in
the batch reactor (F/Mb). F/Mb should be similar to
48
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the F/M of the POTW biological treatment process.
This adjustment will allow the biodegradable material
in the batch influent to be reduced to approximately
equal levels in all RTA tests. The required batch test
period (d) can be calculated as follows:
, _ Batch Influent COD(mg/L)
MLVSS (mg/L)xF/Mb
where: F/Mb is equal to the calculated F/M of the
primary effluent reactor (i.e., COD/MLVSS x
treatment period in days).
Both acute and chronic refractory toxicity can be
measured in RTA testing. In order to obtain
comparable toxicity results, RTA testing should utilize
the same species that was used for TIE tests or routine
compliance monitoring. Use of toxicity screening tests
such as bacterial bioluminescence tests (e.g.,
Microtoxฎ) in conjunction with the preferred test
species may provide additional information. These
screening tests are recommended when the waste
streams to be tested exert a high oxygen demand (i.e.,
high BOD concentration) which would otherwise
require aeration during testing and a possible loss of
toxicants. Standard procedures for toxicity
measurement are not practical due to the large number
of samples that will need to be processed in the RTA.
Instead, simplified acute toxicity test procedures, like
those presented in the USEPA TIE Phase I manuals
(USEPA 199 la, 1992a, 1996) are recommended.
Likewise, simplified procedures for short-term
measurement of chronic toxicity (USEPA 1992a,
1996) are recommended for chronic refractory toxicity
assessments. Oris et al. (1991) and Masters et al.
(1991) describe the use of an abbreviated version of
the 7-day chronic C. dubia test, referred to as the 4-day
test. However, the 7-day test has been the method of
choice for most RTA studies because the use of
younger test animals provides more consistent results.
Therefore, 7-day test data are better for discerning
differences between toxic and nontoxic sources.
The batch test mixtures are prepared for toxicity
analysis by allowing the mixed liquors to settle,
decanting the clarified supernatant, and filtering the
supernatant through a coarse glass fiber filter. The
coarse filtration step is used to more closely simulate
the POTW clarification process because solids settling
in bench-scale containers is not as efficient as the
POTW settling process. Note that this step may not be
required if the RTA includes a simulation of effluent
filtration processes at the POTW (see Appendix C). If
toxic biomass is used in the RTA tests, further
particulate removal is required to measure the soluble
refractory toxicity in the sewer wastewater. In this
case, the coarse filtrate can be filtered through a
0.2 nm pore-size glass filter or centrifuged at 10,000
xg for 10 to 15 minutes (American Society for
Microbiology, 1981) to remove colloidal size particles
from the wastewater. Membrane filters such as
cellulose nitrate filters may not be appropriate because
some soluble organic constituents may absorb onto the
filter. Prior to sample filtration, all filters should be
washed and filter blanks should be prepared using the
steps described in Section 8 and Appendix J.
Data Evaluation
Results of RTA testing are used to locate the sources
that are contributing refractory toxicity to the POTW.
A discussion of the evaluation of RTA results is
provided as follows.
Results of RTA Tests if POTW Biomass is Non-
toxic
Results for each sample analysis will consist of data on
two types of batch tests: tests of sewer sample spiked
into primary effluent, and a control test using primary
effluent alone. The batch test of the sewer sample/
primary effluent will indicate the toxicity that would
realistically occur upon mixture of the sewer
wastewater with POTW influent. Results of this test
are compared to results of the primary effluent control
test to determine if the addition of sewer wastewater
decreases the refractory toxicity (e.g., dilution or
antagonistic effect) or increases the refractory toxicity
(e.g., additive effect) of the primary effluent.
If the effluent toxicity of the sewer sample/primary
effluent test is greater than the effluent toxicity of the
primary effluent control test, the sewer wastewater
source may be a contributor of refractory toxicity.
POTW influent and sewer wastewater toxicity is
known to vary significantly over time; therefore, each
wastewater source should be tested several times over
an extended period (e.g., three times during both cold
and warm weather months) to determine the overall
potential for the discharge to cause POTW effluent
toxicity. Results of Tier I sewer line tracking can be
used to prepare a list of the toxic sewer lines. This list
can be compared to a sewer collection system map to
indicate tributary sewer lines or indirect dischargers to
be tested in Tier EL
49
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The TRE case study summaries in Appendices C and
G describe how RTA results were used to indicate
sources of refractory toxicity in the Reidsville and
LRSA TREs, respectively. These studies illustrate the
need to test several samples from each wastewater
source in order to account for the variability in
refractory toxicity over time.
Results of RTA Tests if POTW Biomass is
Toxic
In situations where the RAS coarse filtrate is found to
be more toxic than the RAS centrate, RTA tests may
use alternate (non-toxic) biomass in addition to tests
with the POTW biomass. The data for each sewer
sample analysis will consist of results of two batch
tests using alternate biomass (i.e., one test of sample/
primary effluent, and one test of primary effluent) and
results of two batch tests using toxic POTW biomass.
The results of tests that use alternate biomass will
provide an estimate of the total refractory wastewater
toxicity. The disadvantage of these tests is that the
alternate biomass is not acclimated to the POTW
influent wastewaters; therefore, it may not provide the
same level of treatment as the POTW acclimated
biomass.
Batch tests using toxic POTW biomass better reflect
the treatment efficiency of the activated sludge
process; however, manipulation of the batch effluent
(i.e., centrifugation or small particle filtering) removes
particles that normally are present in the POTW
effluent. Batch effluent treatment is necessary to
remove the interfering toxic biomass, but this treatment
may artificially change batch effluent toxicity. The
advantage of toxic biomass tests is that the soluble
refractory toxicity of source wastewaters can be
determined. The non-toxic biomass tests cannot
provide as good an estimate of soluble toxicity,
because alternate biomass is not acclimated to the
POTVV influent wastewaters. If both toxic biomass
and alternate nontoxic biomass are used in testing,
results are obtained on both the soluble and total
refractory toxicity of the sewer wastewater.
Inhibition Testing (Optional)
Inhibitory wastewater may upset the normal operation
of the POTW biological treatment process to the extent
that it causes toxicity pass-through. Biological
treatment inhibition may occur by three primary
mechanisms: competitive inhibition, non-competitive
inhibition, and substrate inhibition. The effect of
competitive inhibitors is most pronounced at low
substrate concentrations. Inhibition by non-
competitive inhibitors such as chromate or other heavy
metals is observed over a range of substrate
concentrations. The third mechanism of biological
inhibition, substrate inhibition, occurs at high substrate
concentrations.
Only substrate inhibition can be practically evaluated
in batch treatment tests. An example of the effects of
substrate inhibition on biological activity is shown in
Figure 5-3. This figure shows that substrate utilization
normally achieves a constant maximum rate as the
wastewater concentration is increased. If inhibitory
substances are present in the wastewater, the substrate
uptake rate would decrease as the wastewater
concentration is increased further.
Substrate inhibition can be assessed by monitoring
removal of substrate (e.g., BOD5, COD, TKN, andTP)
and oxygen uptake rates in the RTA batch reactors. A
series of dilutions of the sewer line or indirect
discharger wastewater is tested with POTW biomass:
one with 100% indirect discharger wastewater and at
least three consisting of serial dilutions (e.g., 50%,
25%, and 12.5%) of sewer wastewater. A range of
wastewater dilutions is necessary to compare organic,
nutrient, and oxygen removal rates over a range of
substrate concentrations. At high wastewater strengths
[e.g., 1 mg/L soluble COD (SCOD) to 4 mg/L
MLVSS], biomass activity will generally reach a
maximum rate (Figure 5-3). When wastewater
concentrations are increased, a decrease in COD,
nutrient, and oxygen removal rates would indicate the
presence of inhibitoiy materials.
SCOD, ammonia (SNH3-N), and phosphorus (SP)
removal can be used to calculate the specific substrate
utilization rate (SSUR). The SSUR is reported in units
of mg/L of soluble substrate per gram MLVSS per
minute (g MLVSS/min), and is calculated using the
equation:
SSUP, -
MLVSS (g/L) x Test Period (min)
where: Ci is the influent substrate concentration as
SCOD, SNH3-N, or SP.
Ce is the substrate concentration in periodic
samples collected from the batch reactor.
The POTW biomass used in batch testing contains
residual SCOD, SNH3-N and SP remaining from
50
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Non-inhibitory
wastewater
Inhibitory
wastewater
Figure 5-3. Theoretical results of inhibition testing.
biological treatment that must be accounted for when
calculating batch effluent concentrations. The
correction for biomass SCOD, SNH3-N, and SP is
calculated by the following equation:
SCOD = [(Vr) x (Ce, mg/L)] - [(Vb) x (Cb, mg/L)]
Vr
where: Cb is the concentration of SCOD, SNH3-N,
and SP in the RAS filtrate.
Vr is the total volume in the batch reactor (L).
Vb is the volume of RAS added to the reactor
(L).
Oxygen utilization can be measured as a specific
oxygen uptake rate (SOUR). SOUR is reported in
units of mg O2/L/g MLVSS/min and is calculated as
follows:
SOUR = -
Oxygen Consumed (mg/L)
MLVSS (g/L) x DO MeasurementPeriod (min)
The SSUR and SOUR data for the four wastewater
concentrations can be plotted as shown in Figure 5-3.
A reduction in the SSUR and SOUR rates for the full
strength sample test relative to the SSUR and SOUR
rates for the sample dilution tests would indicate the
presence of inhibitory material in the sewer wastewater
sample. The degree of inhibition can be inferred by the
amount of deviation in biomass activity rates between
the full strength sample test and the sample dilution
tests.
Phase I Toxicity Characterization (Optional)
TIE Phase I tests can be applied to the batch effluent of
the indirect discharger/primary effluent test to
determine the types of toxicants causing refractory
toxicity in the sewer wastewater. Results of the TIE
Phase I testing can be compared to TIE results for the
POTW effluent to determine if the sewer wastewater
contains the same types of refractory toxicants that
were observed in the POTW effluent. Sources that
discharge the same types of toxicants as those found in
the POTW effluent would be candidates for a
pretreatment control evaluation (Section 6). The TIE
Phase I procedure is described in Section 4.
Findings of the Toxicity Source Evaluation
The results of Tier I and Tier n testing should be
sufficient to confirm the sources of POTW effluent
toxicants or refractory toxicity. This information can
be used to evaluate and select pretreatment control
options (Section 6).
It is possible that the toxicity source evaluation results
will suggest that no single sewer line or indirect
discharger is a source of refractory toxicity. This case
may occur if the sources of toxicants or toxicity are
51
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widely dispersed throughout the collection system.
Examples of dispersed toxicants include organo-
phosphate insecticides (e.g., diazinon) and ammonia.
The inability to locate the toxicant or toxicity sources
may also indicate that the sewer sampling points did
not include all possible sources of the toxicants or
toxicity. In this case, it may be necessary to evaluate
additional sewer lines in the collection system.
In situations where the toxicity source evaluation
proves to be a prodigious task, the permittee may elect
to evaluate alternatives for in-plant toxicity control
(Section 6). The choice of pretreatment or in-plant
controls may be determined by assessing the best use
of the resources that are available for the TRE. In this
regard, POTW staff have the option to recover costs
associated with toxicity source evaluation through the
process of local limits development.
52
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Section 6
Toxicity Control Evaluation
Introduction
The goal of the TRE is to select and implement toxicity
control methods and technologies that will achieve
compliance with the permit limits for effluent toxicity.
Toxicity control evaluation involves assessing the
potential control options and selecting the best
option(s) for toxicity reduction based on technical and
cost considerations. Figure 6-1 illustrates the process
of evaluation and selection of toxicity control options.
Toxicity control may be accomplished either through
the implementation of pretreatment requirements or
POTW modifications. Examples of pretreatment
controls include local limits development and waste
minimization/pollution prevention requirements.
POTW modifications may include changes in
treatment chemical usage, enhanced operational
strategies, or addition of treatment processes.
Criteria for the selection of the preferred toxicity
control option(s) should be defined at the beginning of
the toxicity control evaluation. Recommended criteria
include:
Compliance with effluent toxicity limits
Compliance with other permits
Capital, operational, and maintenance costs
Ease of implementation
Reliability
Environmental impact.
Cost will be a primary selection criterion; however, the
selected control option must offer the best potential for
consistent, reliable toxicity reduction with the least
impact on other permit requirements. The selection
criteria should be used initially to screen all candidate
control options to determine which alternatives merit
further study. The preferred options can then undergo
an in-depth review in a pretreatment control evaluation
(e.g., local limits development) or in-plant control
evaluation (e.g., treatability studies). Information from
these evaluations will be used to select the most
feasible option(s) based on a more thorough
comparison of the criteria listed above. The final
selection process may require a quantitative
examination of the options using a scoring and ranking
system. Table 6-1 presents a matrix of in-plant toxicity
control options for the TRE case example provided in
Appendix G. Further discussion of the final selection
process is provided at the end of this section.
Identifying Toxicity Control Options
The TRE guidance is designed to identify possible
methods for toxicity reduction at the earliest possible
stage in the TRE process. As shown in the overall
schematic of the TRE process (Figure 1-1), sufficient
information may be available for toxicity control
evaluation at the completion of the POTW
performance evaluation conventional pollutant
treatability tests, TIE tests, and tbxicity/toxicant
tracking. Control options must be identified based on
ample data that clearly demonstrates the option's
technical feasibility.
POTW Performance Evaluation Treatability Tests
Treatability testing in the POTW performance
evaluation may identify options for improved
conventional pollutant treatment that also reduce
effluent toxicity to acceptable levels (Section 3). In
addition, the optional TIE Phase I tests may provide
information on the presence of in-plant toxicants such
as suspended solids or chlorine that is corroborated in
the operations and performance review. The treatment
steps in TIE Phase I also may provide information on
treatment options for control of the in-plant toxicants.
Potential control options may involve treatment
modifications or additions that are necessary to
improve conventional pollutant treatment and to reduce
or eliminate in-plant sources of identified toxicants.
Examples of these control options include dechlorin-
53
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Toxicity Control Evaluation
Develop Selection Criteria
Compliance with Effuent Toxicity Limits
Compliance with Other Permits
Capital, Operational, and Maintenance Costs
Ease of Implementation
Reliability
Environmental Impact
Select Control Alternatives
Based on TRE Results
i
Evaluation of
In-Plant Control Alternatives
Process Enhancement
POTW Modifications and Additions
Treatability Testing
1
Evaluation of
Pretreatment Control Alternatives
Allowable Headworks Loading
Analysis
Public Education
Local Limits
Industrial User Management
Case-by-Case Permitting
Information and
Data Review
Control Alternatives
Assessment
Select Toxicity
Control Alternatives
Implement Toxicity
Control Alternatives
Figure 6-1. Flow diagram for a toxicity control evaluation.
54
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Table 6-1. An Example of the Comparison of In-Plant Ammonia Treatment Alternatives (Ammonia
Concentrations of 90 mg/L NH3-N or Higher)
Treatment Technology
1 . Single-stage biological
nitrification
2. Two-stage biological
nitrification
3. Biological nutrient
removal with nitrification
4. Ammonia air stripping
5. Selective ion exchange
(including resin
regeneration)
6. Breakpoint chlorination
Capital Costs
Millions*
9.1
, 11.5
18.7
11.2
28.0
7.5
O&M
Costs
Millionst
1.5
2.4
2.8
1.3
6.2
6.8
Equivalent
Annual Cost
Millions*
3.4
5.0
6.4
3.3
12.7
11.5
Relative
Practicality!
Low
Impractical
Low
Very Low
Very Low
Very Low
Relative
Reliability*
Low
Low
Low
Low
Low
Low
* Approximate capital costs based in part on WPCF Nutrient Control Manual cost curves (WPCF, 1983). Values reflect conditions
of 17 mgd and 90 mg/L NH3-N. The values presented here have been modified from the cost curves to reflect engineering and
contingency costs at 25% and contractor's overhead and profit at 15%.
t Approximate overhead costs based on WPCF Nutrient Control Manual cost curves. Values reflect conditions of 17 mgd and
90mg/LNH3-N.
t Approximate equivalent costs amortized over 20 years, assuming an annual 5.00% increase in operation and maintenance costs
and an estimated annual interest rate of 8.86%.
ง Relative practicality based on typical technology applications, available land space, overall costs, and/or chemical usage
requirements.
# Relative reliability based on potential inhibition, temperature and pH sensitivity, and evidence that the technology is proven
reliable at 17 mgd and 90 mg/L NH3-N. Scores of "low" to "high" were used.
Source: LRSA (1991). Additional information on this TRE is presented in Appendix G. All costs shown are in 1991 dollars.
ation treatment to eliminate toxic levels of chlorine and
biological treatment optimization (e.g., increased
MCRT) to remove toxic ammonia concentrations.
TIE Tests
Results of TIE Phase I testing (Section 4) may indicate
the types of treatment that can be used to remove broad
classes of effluent toxicants (e.g., filterable material,
metals, organic compounds). For example, filterable
toxicants may be removed by granular media filtration.
The feasibility of options for removing classes of
toxicants can be evaluated in the POTW in-plant
control evaluation.
Alternatively, results of TIE Phases n and in may help
to identify and confirm the specific effluent toxicants
(Section 4). If the pretreatment program data are
adequate to determine the sources of the toxicants,
local limits can be developed and evaluated in the
pretreatment control evaluation. In this case,
pretreatment control would be preferred over in-plant
control because the costs of implementation are usually
lower. If pretreatment program data on the toxicants
are not available, chemical-specific testing will be
necessary to track the sources of the toxicants before
toxicity control selection can proceed.
Chemical-Specific Investigation
Chemical-specific tracking in the Tier I - toxicity
source evaluation may locate the sources of the POTW
effluent toxicants (Section 5). Once the sources have
been identified, pretreatment control options such as
local limits or waste minimization requirements can be
developed and evaluated.
Refractory Toxicity Assessment
Results of the Tier H RTA testing may identify the
indirect dischargers contributing refractory toxicity to
the POTW. Based on these results, POTW staff can
require the indirect discharger to limit the discharge of
wastewater toxicity even though the toxic wastewater
constituents have not been identified. In some cases,
55
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POTW staff may elect to perform optional TIE Phase I
analyses to provide information on the toxic
constituents in the indirect discharger wastewater.
This additional testing may be conducted so that
numerical pretreatment limits can be set.
Toxicity Control Screening Process
Using appropriate selection criteria, the preferred
toxicity control options are identified. Available
options can be compared using a ranking system (e.g.,
on a scale of 1 to 10). This screening process may be
relatively simple, although some estimate of costs (i.e.,
order of magnitude) will be useful in selecting the most
practical options. The selected options are then studied
in the pretreatment control evaluation and in-plant
control evaluation, as described below.
The example matrix in Table 6-1 compares in-plant
control options for ammonia toxicity. In this case,
costs and qualitative measures were used to rank the
various options. All of the in-plant control options
were found to be impractical or costly; therefore, the
sewerage authority investigated pretreatment controls.
The source of a majority of the ammonia loading was
an industry, which was considered to be controllable.
As a result, the sewerage authority required the
industry to implement ammonia control methods. The
cost to the authority was relatively low and involved a
headworks analysis for ammonia and reissuance of
discharge permits. Additional information on this TRE
is provided in Appendix G.
Pretreatment Control Evaluation
Pretreatment control options can be developed by
public works managers to prevent the pass-through of
toxicants, toxicity, and inhibitory material that have
been traced EO indirect dischargers. The primary
advantages of pretreatment control of toxicity are that
a smaller volume of waste can be managed by
addressing individual sources and the costs are usually
the responsibility of the industrial users. Pretreatment
requirements may involve a public education effort or
the implementation of narrative or numerical
limitations for POTW users.
The toxicants to be controlled may not be the same
parameters that are currently regulated under the
pretreatment program. Examples of these types of
toxicants include organophosphate insecticides, TDS,
biocides, and specialty chemicals used by industries.
In cases where current pretreatment regulations are
inadequate to address sources of toxicants or toxicity,
POTW staff should revise or adopt new permit
regulations or ordinances, as appropriate. In these
cases, it may be necessary to initiate the following
steps to control toxicants or toxicity:
Investigate public education approaches, if the
toxicant is widely used in the service area
(e.g., organophosphate insecticides).
Perform an allowable headworks loading analysis.
Decide whether to establish local limits or
implement a more directed approach, such as
industrial user management or case-by-case
requirements.
Develop a monitoring program to evaluate
compliance with the requirements.
These steps are described below.
Public education has been successfully used to control
toxicity at POTWs. Organophosphate insecticides
such as diazinon and malathion have been identified as
effluent toxicants at many POTWs, especially in the
southeast and southwest United States (Norberg-King
et al., 1989). Insecticides can be discharged by many
users in the POTW service area, including pest control
businesses, veterinarians, lawn care businesses,
apartment complexes, restaurants, hotels/motels, office
buildings, and homeowners. These users are usually
not included under pretreatment programs and it may
be impractical to control these sources by regulating
each discharge. Studies at POTWs in California
(Singhasemanon et ! al., 1997), Texas (City of
Greenville, 1991), Oklahoma (Engineering-Science,
Inc., 1992), and North Carolina (Fillmore et al., 1990)
have determined that public education is a viable
option for control of organophosphate insecticide
toxicity attributed to multiple sources. Recommended
steps in a successful public awareness program include
identifying the significant users of insecticides,
developing education materials targeted to users, and
distributing the materials on an ongoing basis during
periods of expected insecticide use. The City of
Greenville also enacted an ordinance to encourage the
environmentally sound use of insecticides and require
merchants to display public education materials where
insecticides are soH (City of Greenville, 1991).
Additional information on the identification and
control of organophosphate insecticides is presented in
Appendices F and H. ^Public education efforts may be
applied to control other effluent toxicants that are
widely used in POTW service areas and are not
56
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practical to regulate through local pretreatment
limitations.
POTW staff have successfully used revised or new
pretreatment regulations to reduce POTW effluent
toxicity (Appendices C, E, and G). Local pretreatment
limits can be developed to control sources of toxicants
or toxicity identified in the toxicity source evaluation.
USEPA's Guidance Manual on the Development and
Implementation of Local Discharge Limitations Under
the Pretreatment Program (USEPA, 1987b) describes
several approaches for developing local limits. These
approaches include:
Allowable Headworks Loading Method:
Numerical limits are defined based on the
maximum pollutant loadings that will allow
compliance with receiving water quality criteria,
sludge quality criteria, or protection against
treatment interferences.
* Industrial User Management Method: Based on
an in-depth review of indirect discharger practices,
POTW staff can set narrative limits for chemical
management practices (e.g., chemical substitution,
spill prevention, and slug loading control).
Case-by-Case Permitting: Technology-based
limits are established based on levels that can be
feasiblely and economically achieved by
industries.
Some of the local limits approaches can be adapted to
address effluent toxicants or toxicity. For example, the
allowable headworks loading method is well-suited for
developing limits to prevent the pass-through of
toxicants identified in POTW effluent TIE tests and
located by chemical-specific analyses in the toxicity
source evaluation. This method can be used to
establish the maximum level of the toxicant that can be
safely received by the POTW without exceeding the
effluent toxieity limit. The LRSA, New Jersey,
conducted an allowable headworks loading analysis to
address industrial sources of ammonia (see Appendix
G). The results of the analysis were used to develop
local limits for controllable sources in order to reduce
effluent toxicity caused by ammonia.
The industrial user management method provides a
framework for implementing chemical management
practices including slug discharge control. In cases in
which slug loadings contribute to POTW effluent
toxicity, spill prevention or load equalization can be
implemented at the industrial facility to moderate the
slug loadings. USEPA's Guidance Manual for
Control of Slug Loadings to POTWs (1988b and
1991c) describes methods for the development of slug
loading control programs.
The case-by-case permitting method can be used when
the POTW effluent toxicants cannot be identified, but
TIE information on the general classes of toxicants is
available or sources of toxicity have been located in the
toxicity source evaluation. Using TIE data, an
engineer may be able to select a pretreatment
technology that can remove general types of toxicants
(i.e., non-polar organic compounds). In cases where
the sources of toxicity have been identified, POTW
staff have the authority to require the indirect
discharger to take steps to limit the discharge of
refractory or inhibitory toxicity (USEPA, 1987b).
Although USEPA and the States with approved
pretreatment programs have overview authority, the
choice of which approach to use for local limits
development is the municipal government's decision.
The goal in developing local limits is to implement
pretreatment regulations that are technically and legally
defensible. Local limits can include provisions for
equitable recovery of costs associated with the toxicity
source evaluation and limits development.
In-PIant Control Evaluation
The objective of the in-plant control evaluation is to
select and evaluate feasible treatment options for the
reduction of effluent toxicity at the POTW.
Treatability testing may be conducted to determine the
toxicity removal effectiveness and operating
characteristics of the candidate treatment options.
These tests should use acute or chronic toxicity tests
and chemical analyses to evaluate the removal of
specific toxicants and/or toxicity. The resulting data
provide a basis for the final selection and conceptual
design of feasible POTW process modifications or
additions.
It is important to consider that major changes in
treatment plant facilities or operations may not be
practical due to the cost of new facilities or the
complexity of additional process operations. In these
situations, pretreatment control of toxicity may be
preferred over in-plant control. Wherever possible, the
in-plant control evaluation should be performed in
conjunction with the pretreatment control evaluation to
identify the most technically feasible and cost-effective
control option.
57
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Review Existing Information
The first step in the in-plant control evaluation is to
review the POTW performance evaluation data on the
POTW design (Section 3) to establish the physical
space available for new process additions and to
determine the idle facilities and equipment that could
be used for toxicity control. Operations and
maintenance information also should be reviewed to
determine if the POTW is capable of handling the
increased operational control that may be required with
process modifications or additions. In addition, POTW
performance evaluation information should be
reviewed to determine how the control options might
be integrated into the overall treatment system design.
TIE results on identified effluent toxicants can be used
to determine in-plant control options. Although
information on specific toxicants is well suited for the
application of pretreatment control limitations, POTW
staff may choose to evaluate in-plant control of these
toxicants. An example is the treatment of ammonia by
optimizing the POTW activated sludge process (e.g.,
increase MCRT) to achieve nitrification. In some
cases, TIE Phase I data on the classes of effluent
toxicant can be used to select options to be examined.
For example, if filterable material is the principal
effluent toxicant, possible options would include
improved solids clarification or granular media
filtration.
In-plant toxicity control may be achieved by
enhancement of the existing treatment system or by the
implementation of additional treatment processes. In-
plant control alternatives for different categories of
toxicants are summarized in Table 6-2. A description
of these control alternatives is provided as follows.
Process Enhancement
Biological Process Control
Biological process control is most easily applied to
suspended growth systems (e.g., conventional activated
sludge and BNR processes), although some
modifications to fixed film processes (e.g., trickling
filters and RBCs) may be feasible. The performance of
activated sludge and BNR systems is generally
controlled by adjusting several process parameters,
including MCRT, MLSS, DO levels, recycle ratio, and
F/M ratio. The treatment efficiency of the activated
sludge system is optimized by varying these
interrelated process parameters. A description of the
use of operational parameters for toxicant control is
provided as follows: "Removal of biodegradable toxic
compounds in suspended growth systems may be
improved by increasing the MCRT" (Adams et al.,
1981). MCRT can be increased by lowering the excess
sludge wasting rate. Longer MCRTs are necessary for
nitrification and can be beneficial for the
biodegradation of some types of organic compounds.
An example of this approach was practiced at a POTW
on the United States' east coast (Judkins and
Anderson, 1992). The facility was retrofitted to
achieve nitrification to reduce ammonia. Existing
treatment capacity, including aeration basins and
secondary clarification, was available to accommodate
the longer MCRTs and detention times needed to
accomplish nitrification and denitrification. The
retrofits involved increasing the air supply, changing
the air diffuser pattern, adding an anoxic zone in the
aeration basins, increasing the MCRT, and modifying
the return sludge flow. Usually, mixed liquor from the
aerobic zone of the biological treatment process is
recycled to the anoxic zone to accomplish
denitrification. However, it was possible in this case to
use the existing return sludge pumps to recycle the
secondary clarifier underflow to the anoxic zone. The
cost of the retrofit consisted of approximately
$100,000 in capital costs and an increase in annual
operating costs of about 25%.
High MLSS concentrations have been shown to
minimize the effects of inhibitory pollutants on
activated sludge treatment systems (WEF/ASCE,
Table 6-2. POTW In-PIant Control Technologies for Categories of Toxic Compounds
Biodegradable
Organic Compounds
and Ammonia*
Biological process control
Nutrient addition
Coagulation/precipitation
Non-Biodegradable
Organic Compounds
Filtration
Activated carbon
Coagulation/precipitation
Volatile Organic
Compounds |
Biological process control
Aeration
Heavy Metals and
Cationic Compounds
Filtration
Coagulation/precipitation
pH adjustment
* Air stripping, breakpoint chlorination, and ion exchange also may be considered for ammonia removal; however, the cost of these
technologies and the use of toxic additives such as chlorine often preclude their use.
58
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1992a). High MLSS concentrations increase the
potential for biodegradation and sorption of toxic
wastewater constituents and can help to protect the
treatment process from shock loadings. The maximum
MLVSS will often be limited by the available
secondary clarifier capacity. It is important to consider
the effect of increased MLVSS on secondary solids
separation and the TSS concentrations of the clarifier
effluent.
A decrease in F/M (based on BOD5) effectively
decreases the organic waste loading per unit of
biomass, which may improve the biodegradation of
some toxic compounds (Adams et al., 1981). The F/M
ratio is inversely related to MCRT.
Biological process control is not as easily
accomplished for fixed film processes, such as
trickling filters or RBCs. Some adjustments can be
made, however, such as varying the amount and point
of wastewater recirculation in a trickling filter to
potentially increase the removal of toxicants or
toxicity. In addition, secondary clarifier effluent can
be recirculated to dilute high-strength wastes prior to
treatment in a trickling filter or RBC. In some cases,
inhibitory pollutants may cause excessive sloughing of
the fixed film biomass. This problem may be rectified
by returning thickened secondary clarifier solids to the
fixed film process to help maintain a proper biomass
population.
Chemical Addition
The addition of chemicals or additives to waste streams
in existing POTW treatment processes may improve
toxicant or toxicity removal. Nutrients can be added to
influent wastewaters that have low nutrient levels
(relative to their organic strength) to improve
biological treatment. Lime or caustic chemicals can be
used to adjust wastewater pH for optimal biological
treatment or for coagulation and precipitation
treatment. Other chemical coagulants are used to aid
in removal of insoluble toxicants and to improve
sludge settling. Powdered activated carbon may be
applied in activated sludge systems to remove toxic
organic compounds. A description of each of these
treatment additives is provided as follows.
Addition of phosphorus, nitrogen, or sulfur may in
some cases improve biological treatment of industrial
wastewaters with low nutrient concentrations. The
optimal BOD/TKN/TP ratio for municipal activated
sludge treatment is 100:5:1. Lime and caustic
chemical addition may be used to increase influent
wastewater pH prior to primary sedimentation in order
toenhance the precipitation of heavy metals. Chemical
addition may also be appropriate for removal of metals
in sidestreams from sludge processing. Some metals,
however, such as iron and chromium will go into
solution rather than precipitate at alkaline pH. The
optimum pH range for metals precipitation varies for
each type of metal and the solubility/precipitation
equilibrium can be affected by other factors such as
dissolved solids concentrations in the wastewater.
Lime and caustic chemicals also provide additional
alkalinity, which is essential for biological treatment,
especially nitrification treatment, processes.
Polymers and inorganic coagulants such as alum and
ferric chloride can be introduced to POTW waste
streams to help remove insoluble pollutants.
Coagulants may be added to influent wastewater to
increase the sedimentation of toxic constituents in
primary treatment and thereby minimize the loading of
toxicants on the biological treatment process.
Coagulants also can be added after the activated sludge
aeration basins to control sludge bulking or reduce
effluent suspended solids, which may be associated
with effluent toxicity. The optimum conditions for
coagulation can be determined by conducting bench-
scale jar tests. These tests are used to establish the
optimum coagulant type and dose, the proper mixing
requirements, and the flocculent settling rates for
treatment (Adams et al., 1981).
Coagulants can adversely affect the characteristics of
sewage sludges, which could affect the sludge disposal
method. Coagulants may increase the toxicity of the
sludge (as measured by a TCLP) as a result of the
removal of toxic wastewater constituents or as a result
of the toxicity of the coagulant itself (e.g., metal salts).
Therefore, coagulants should be evaluated carefully
prior to use.
The addition of PAC to an activated sludge unit may
increase the removal of toxic organic chemicals.
Organic pollutants that are not biodegraded can be
removed by adsorption onto the surfaces of activated
carbon particles. Activated carbon also improves
sludge settleability by providing a substrate onto which
sludge floes can agglomerate. Although PAC
processes have been used in municipal wastewater
treatment, studies (Deeny et al., 1988) have shown that
PAC regeneration by wet-air oxidation breaks down
the activated carbon particles to carbon fines, which
59
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cany over the secondary clarifier weirs. In some cases,
periodic additions of PAC to an aeration basin can be
used to minimize the effects of toxic slug loadings,
thereby improving the stability of the activated sludge
system.
POTW Modifications and Additions
Where process enhancement is not feasible or will not
provide adequate toxicant removal, physical addition
to or modification of the POTW can be undertaken.
Additional treatment processes could include
equalization prior to treatment, instrumentation control,
BNR, and advanced wastewater treatment processes
such as coagulation/flocculation, granular media
filtration, and GAG treatment. Public works managers
also may consider enhancing effluent dilution through
the addition of an outfall diffuser or relocation of the
outfall to a larger water body.
Equalization
Equalization can be used prior to the biological
treatment process to dampen the effect of slug or
diurnal loadings of high-strength industrial wastes.
Equalization facilities can be provided to either
equalize wastewater flows or wastewater
concentrations. Flow equalization is partially provided
by existing primary sedimentation tanks and can be
enhanced by increasing the size of the primary tankage.
Concentration equalization requires mixing of the
wastewater to moderate intermittent pollutant loadings;
therefore, separate facilities must be provided.
Instrumentation Control
Instrumentation/monitoring can be used to help control
slug loadings of toxic constituents in the POTW
influent wastewater. For example, transient metals
loadings may be monitored by continuously measuring
the pH and conductivity of the influent wastewater. A
significant change in pH or an increase in conductivity
may indicate a slug loading of toxic material, which
can be manually or automatically diverted to a holding
basin. After equalization, the diverted wastewater can
be slowly added back to the influent waste stream to
dilute the material prior to treatment.
Outfall Diffuser/Relocation
Public works managers may choose to evaluate the
alternative of installing a diffuser or relocating the
outfall to achieve better dilution. For example, the
extension of a shoreline outfall to a submerged high-
rate diffuser in deeper water may promote rapid mixing
and achieve an acute dilution factor of 10 or more. If
allowed by applicable state water quality standards, the
effectiveness of outfall relocation or diffuser
installation can be evaluated along with other control
options. The reader is referred to USEPA's TSD
(1991b) for a discussion of the role of dilution in
permitting for whole effluent toxicity control and
details on mixing zone analyses and high-rate diffusers.
Advanced Wastewater Treatment
POTWs that only utilize primary and secondary
wastewater treatmetit may achieve toxicity reduction
by the addition of advanced wastewater treatment
processes such as coagulation/flocculation,
sedimentation, grariular media filtration, and granular
activated carbon. Each of these processes can provide
enhanced removal' of some toxicants and toxicity.
Treatability tests used to evaluate treatment process
additions are described below.
Treatability Testing
Bench-scale and pilot-scale treatability tests are
commonly used to evaluate treatment options that have
been selected for testing. Bench-scale or pilot-scale
tests offer several advantages compared to full-scale
testing, including a more manageable test unit size and
the ability to vary the operating conditions to evaluate
toxicity reduction. Treatability methods can range
from simple jar tests for testing coagulation/
flocculation options to flow-through bioreactors for
investigating thebipdegradation kinetics of wastewater
treatment.
j
During treatability testing, influent, effluent, and
sidestream waste waters of the treatment simulation are
tested for acute or chronic toxicity. Toxicity testing is
used to assess the effectiveness of the treatment option
in reducing wastewater toxicity and to determine the
fate of toxicity in the treatment process. Initial testing
should use the simplified toxicity test methods
described in the TIE manuals (USEPA 1991a, 1992a,
1996) because of the large number of samples that may
need to be tested. Toxicity screening tests such as
Microtoxฎ also may be used in conjunction with the
required test species to provide additional information.
These tests are recommended for waste streams with a
high oxygen demand (i.e., high BOD5 concentration),
which would otherwise require aeration when testing
with permit species. Aeration should be avoided
because it may remove volatile or oxidizable toxicants.
Definitive acute or chronic toxicity tests (USEPA
1993c, 1994a, 1994b, 1995) should be used at the
60
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completion of treatability testing to verify the option's
capability in meeting the NPDES permit limit.
Optional TIE Phase I analyses also may be performed
on treatability test samples to confirm toxicant removal
by the treatment option.
Activated Sludge/BNR Treatment
The basic parameters of interest in the design of
activated sludge/BNR systems include organic loading,
oxygen requirements, nutrient requirements, sludge
production, sludge settleability, and internal recycle
rates. Continuous flow systems are most useful for
evaluating activated sludge/BNR systems; however,
batch systems may provide sufficient treatability
information in some cases. An example of the use of
batch treatment tests in a TRE is provided in
Appendix D. This study determined that an upgrade of
a conventional activated sludge process to a five-stage
BNR process would achieve compliance with chronic
toxicity limits. Follow-up monitoring upon completion
of the upgrades confirmed the toxicity reduction.
Consideration should be given to evaluating design
specifications and operating conditions that are
expected to optimize the treatment of toxicants and
toxicity. These parameters may include relatively long
MCRTs and high MLVSS levels, which have been
shown to improve toxic pollutant removal and protect
the process from inhibitory wastes (Hagelstein and
Dauge, 1984; WEF/ASCE, 1992a).
Coagulation/FIocculation
The evaluation of coagulation and flocculation
treatment involves the use of bench-scale jar tests or
zeta potential tests to provide information on the
optimum coagulant type and dosage, mixing rates, and
flocculent settling rates for removal of solids and
flocculent suspensions (Adams et al., 1981). Results
of these tests are used to devise treatability tests to
evaluate the sedimentation of flocculent suspensions.
Sedimentation
Sedimentation involves the removal of suspended
solids or flocculent suspensions by gravity settling.
Sedimentation is evaluated by conducting a series of
settling column tests that measure the settling rates of
solids or flocculent suspensions (Adams et al., 1981).
Test results are used to calculate a settling profile that
can be used for clarifier design.
Granular Media Filtration
Filtration testing involves scaled-down models (usually
pilot-scale) of full-sized filters. The choice of filter
media and test-flow rates should correspond to the
intended design and operating criteria. Although the
process scale is reduced, the bed gradation and
thickness should be equivalent to anticipated full-scale
processes in order to predict actual treatment
performance (Adams et al., 1981).
Granular Activated Carbon
The carbon adsorption isotherm test is used to
determine the optimum type and dosage of activated
carbon for wastewater treatment (Adams et al., 1981).
Results of this test are used to prepare bench-scale or
pilot-scale carbon columns that can be used to evaluate
carbon exhaustion rates and the effect of carbon
regeneration on toxicity removal performance.
Toxicity Control Selection
The final process of toxicity control selection involves
an assessment of potential control options and selection
of the best option(s) for toxicity reduction based on
several criteria. In most cases, both a pretreatment
control evaluation and an in-plant control evaluation
will have been performed; therefore, the review
information should include the data developed in both
evaluations.
The choice of in-plant toxicity control or pretreatment
toxicity control will depend largely on the technical
and economic feasibility of POTW treatment
modifications and pretreatment controls. Pretreatment
control will be feasible in situations where the TIE data
and the toxicity source evaluation data are sufficient to
definitively identify the sources of toxicity. These data
should provide an indication of the variability of
toxicity and toxicants in the indirect discharge. If these
conditions are satisfied, POTW staff can set local
limits using the methods outlined above. In-plant
control will be preferred in cases where the
implementation of feasible treatment modifications or
additions is more practical than pretreatment control.
Data obtained in treatability studies should include
information on the variability of toxicity treatment
performance and the design criteria for implementing
the treatment option. In-plant options provide POTW
staff a direct method of controlling effluent toxicity;
however, in-plant modifications or additions may
substantially increase process operation requirements
and maintenance costs.
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Selection ofToxicity Control Options
Final selection of the preferred toxicity control
option(s) involves a comparison of the options using
appropriate criteria (see example in Table 6-1). It may
be necessary to select and implement more than one
control option to ensure compliance with effluent
toxicity requirements. The preceding evaluations
should provide sufficient information to document the
technical and cost considerations for each option.
Compliance with Effluent Toxicity Limits or
Requirements
Data gathered through the TRE should indicate that the
selected option will consistently achieve compliance
with the toxicity permit requirement. Sufficient
information should be provided to show that the option
will reduce effluent toxicity even during periods of
maximum occurrence of toxicity. If this information
includes bench and/or pilot-scale treatability data,
scale-up factors must be incorporated into estimates of
toxicity reduction to adjust for differences in treatment
efficiency between laboratory and full-scale treatment
systems. Likewise, safety factors should be included
in the calculation of local limits to allow for variation
in toxicant loadings to the POTW. It also may be
necessary to conduct a sensitivity analysis to evaluate
the effectiveness of the options under variable
conditions (e.g., variable toxicant loadings or treatment
performance).
A relative scoring system can be used to rate the
overall potential for the options to achieve permit
compliance. The scores can be entered into a matrix
table like that shown in Table 6-1.
Compliance with Other Permits
Steps taken to reduce effluent toxicity may have a
detrimental effect on other permitted activities such as
sludge disposal or air emissions. If toxicants are
expected to be transferred to sludge or air, the potential
effects on limitations specified in residuals and air
permits should be estimated. Each option should be
rated for its potential to comply with related permits.
Capital, Operation, and Maintenance Costs
Sufficient detail on costs should be presented to allow
a straight-forward comparison of the control options.
Cost estimates should include the effort and materials
required for planning, implementation, operation, and
maintenance of the options. Cost information may be
obtained from equipment vendors, engineering
consultants, and existing data for comparable systems.
Costs for requisite environmental and construction
permits should be included.
In some cases, it may be possible to recover some of
the costs of implementation from responsible
dischargers. For example, municipalities may apply
surcharges to local limits or request in-kind funding for
POTW modifications or additions to recover the costs
of toxicity control. Anticipated cash returns should be
included in the final cost estimate.
Costs for all options can be ranked and a score can be
assigned and entered into a matrix table. Weighting
factors may be incorporated into the scoring if funding
of some options is uncertain.
Ease of Implementation
Factors such as land availability, permits, operability,
and maintenance will have a major influence on the
selection of options involving POTW modifications or
additions. Likewise, the economic impact and level of
community cooperation anticipated from new
pretreatment regulations will affect the selection of
pretreatment control options. Public works managers
should develop a list, of all potential constraints as well
as benefits of the candidate control methods. Benefits
should address items other than effluent toxicity
reduction such as improved treatment conditions or
better cooperation among POTW users. Each
constraint and benefit can be assigned a weighted
score, the individual values can be summed for each
option, and the total value entered into the matrix table.
Reliability
The selected option(s) must be dependable.
Pretreatment approaches or treatment processes that
tend to malfunction or fail because of difficulties in
executing complicated operational plans should be
avoided. Experiencein implementing similar projects
will be useful in defining the reliability of the options.
Public works managers should consider each option's
operational history, maintenance requirements, and
longevity.
Environmental Impact
Some options may require the evaluation of
environmental issues related to the protection of
wetlands, rare and endangered species, and cultural
resources. Although the costs of these evaluations are
included under the above cost criteria, other factors
will affect the decision-making process, including
public perception, time period for permit approval (if
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needed), and potential remediation issues. A score can
be developed based oh these factors and entered into
the matrix table.
Comparison of the Toxicity Control Options
Scores developed in the criteria evaluation are summed
for each option. These scores will incorporate all
necessary weighting factors; therefore, the total scores
for each option can be compared directly. The options
can be ranked according to their scores and the highest
ranked option(s) can be selected for implementation.
In some cases, it may be necessary to select more than
one toxicity control option to ensure that permit
compliance will be achieved. This approach is highly
recommended when the control options are relatively
inexpensive to implement, operate, and maintain.
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Section 7
Toxicity Control Implementation
Introduction
Once the evaluation and selection of toxicity control
options has been completed, the final steps in the TRE
are the implementation of the selected pretreatment
and in-plant control options and follow-up monitoring
to ensure permit compliance. The degree of effort in
the implementation step will depend on the severity of
the effluent toxicity and the complexity of the selected
control approaches. Depending on the findings of the
TRE, implementation may involve relatively minor
changes such as modifying POTW operating
procedures or more complex modifications such as
expanding the POTW's pretreatment program or
designing and constructing new treatment facilities.
Implementation
Using the results of the previous steps in the TRE, a
Toxics Control Implementation Plan (TCIP) should be
developed. The TCIP should summarize the results of
the TRE, results of the screening and selection of
toxicity control options, and justification for selecting
the preferred toxicity control option(s). For in-plant
control options, the TCIP should provide the basis of
design for the selected control options, including
capital and operating costs, and a schedule for design
and construction. For pretreatment control options, the
TCIP should specify the basis of selection and
technical justification for local limits and discharger
monitoring methods. In addition, the procedures for
implementing revised pretreatment regulations also
should be defined.
Follow-Up Monitoring
Once a control technology has been implemented, a
follow-up monitoring program should be developed
and implemented to ensure the effectiveness of the
selected control option(s). In most cases, the
conditions and frequency of monitoring will be set by
the regulatory agency. If source controls are
implemented, POTW staff should specify additional
monitoring requirements for indirect dischargers under
the pretreatment program. These requirements may
include verification of statements from industries that
the required reduction of toxicity has been made.
The POTW effluent monitoring program should be
designed to provide data to ensure that toxicity has
been reduced to acceptable levels and that the TRE
objectives have been met. This program may involve
more frequent monitoring than is required by the
NPDES permit, including monitoring to evaluate
daily, weekly, monthly, or seasonal variations in
effluent toxicity that were observed during the TRE.
Follow-up monitoring should utilize the test species
and methods specified in the discharge permit.
Additional tests, including surrogate methods applied
in the TRE, may be included to re-evaluate
correlations between test species that may have
changed as a result of the effluent toxicity reduction.
Any effluent toxicants that were determined to be
present prior to implementation of the control
technology should be monitored to ensure that
concentrations are below toxic levels. Approved
analytical methods! will generally be applied; however,
screening methods such as ELISA tests or other field
kits, which may not be specifically approved by
USEPA, can be used to evaluate trends and identify
potential problems for follow-up testing. As with
toxicity monitoring, the analytical program should re-
evaluate trends in toxicant concentrations observed
during the TRE. A discussion of an ongoing POTW
monitoring program for organophosphate pesticides is
described in Appendix F.
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Section 8
Quality Assurance/Quality Control
Introduction
A QA/QC program for the TRE should be developed
and implemented to ensure the reliability of the
collected data. The QA/QC program should include
addressing the monitoring of field sampling and
measurement activities, the review of laboratory
analysis procedures, and the documentation and
reporting of the analytical data. A QA/QC program
should be designed so that corrective action can be
quickly implemented to detect and eliminate erroneous
or questionable data without undue expense to the
project or major delays in the schedule.
The POTW laboratory manager should ensure that the
specific QA/QC requirements for TRE activities are
addressed by the facility's QA/QC plan. If a private
consultant is to be used for all or part of the TRE
testing, the POTW laboratory manager should request
a QA/QC plan from the consultant and review the
consultant's proposed QA/QC activities. Whether the
TRE is to be performed by the POTW laboratory or by
a consultant, it is essential that the project organization
include competent chemists, lexicologists, and
engineers who have adequate knowledge of TRE
methods.
The QA/QC plan should be prepared prior to the
initiation of the TRE and should contain the following
elements:
QA/QC objectives
Sample collection and preservation techniques
Chain of custody procedures
Analytical QA/QC
Laboratory equipment maintenance
QA/QC training requirements
Documentation and reporting procedures
Corrective action protocols.
Sample Collection and Preservation
To ensure quality control in sample collection
activities, the TRE sampling plan (Section 11) should
be strictly followed. In addition, the QA/QC plan
should state the minimum sample volumes, maximum
sampling holding times, and sample preservation
techniques for each analytical method. The sampling
requirements for conventional and priority pollutant
analyses are described in USEPA's Methods for
Chemical Analysis of Water and Wastes (USEPA,
1983b) and Standard Methods for the Examination of
Water and Wastewater (APHA, 1995). Sampling
requirements for acute toxicity tests are provided in
USEPA's Methods for Measuring the Acute Toxicity
of Effluents to Freshwater and Marine Organisms
(USEPA, 1993c) and Methods for Aquatic Toxicity
Identification Evaluations: Phase I, Toxicity
Characterization Procedures (USEPA, 1991a).
Sampling requirements for chronic toxicity tests are
provided in USEPA's Short-Term Methods for
Estimating the Chronic Toxicity of Effluents and
Receiving Waters to Freshwater Organisms (USEPA,
1994a), Short-Term Methods for Estimating the
Chronic Toxicity of Effluents and Receiving Waters to
Marine and Estuarine Organisms (USEPA, 1994b),
Short-Term Methods for Estimating the Chronic
Toxicity of Effluents and Receiving Waters to West
Coast Marine and Estuarine Organisms (USEPA,
1995), Toxicity Identification Evaluation:
Characterization of Chronically Toxic Effluents,
Phase I (USEPA, 1992a), and Marine Toxicity
Identification Evaluation (TIE) (USEPA, 1996).
It is important to routinely assess the effects of sample
holding times on wastewater toxicity to predict how
long samples can be kept before changes in toxicity
occur. For example, the acute TIE Phase I manual
(USEPA, 1991a) describes how testing the sample
toxicity on the day of collection and comparing this
initial toxicity to its baseline toxicity (tested 1 day
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later) can provide information on appropriate sampling
holding times for toxicity analysis. In chronic TIEs,
effluent manipulations are performed on the day the
sample is received so that the possible effects of any
toxicity degradation are minimized (USEPA, 1992a).
Other QA/QC considerations for TRE sample
collection include routine cleaning and inspection of
automatic sampling equipment, cleaning sample
containers according to the requirements for each
analytical method, and collecting duplicate samples
and field blanks. When preserving samples for
chemical analysis, only analytical grade preservatives
should be used to avoid contamination and
overestimation of analyte concentrations.
Unpreserved samples that are to be used for toxicity
and chemical analyses require sample containers that
are both lexicologically and analytically clean.
Equipment and containers used for toxicity test
samples require special cleaning procedures outlined
in USEPA manuals (1993c, 1994a, 1994b).
Chain-of-Custody
A chain-of-custody (COC) form should accompany all
samples to document the collection, preservation, and
handling of samples. The COC form should indicate
the sample identification number, sample type (i.e.,
composite or grab), date and time of collection, a brief
description of the sample, number of samples taken,
name of the person taking the sample and performing
field measurements, and sample characteristics such as
temperature, pH, total and free residual chlorine, and
conductivity. A field book also should be used to
record any field observations or conditions noted
during sampling along with other pertinent
information. Each laboratory should identify a sample
custodian to log in and store samples collected during
the TRE. The sample custodian should acknowledge
receipt of samples by signing the COC form and
noting the date and time of sample receipt, the sample
identification number, the laboratory assession code,
and sampling information such as temperature, pH,
and TRC. Upon receipt of the sample, a sample
tracking form should be used to record the date, time,
and volume of aliquots of the sample removed for
analysis, the analyst, and any changes in the nature of
the sample, including its toxicity, over time. All COC
and sample tracking forms should be maintained in a
permanent file so that information on specific samples
can be traced easily.
TRE Procedures
Analytical tests should provide data of an acceptable
quality for characterizing wastewater toxicity and for
evaluating methods and technologies for toxicity
reduction. Several test methods described in this
document are not standard procedures and require
careful attention to unique QA/QC procedures.
Special QA/QC procedures for each major TRE test
are discussed below. Whenever possible, these
procedures should be followed to ensure precise and
accurate results.
Toxicity Identification Evaluation
Special precautions for TIE tests are discussed in the
Phase I, E, and Hi! manuals (USEPA 1991a, 1992a,
1993a, 1993b, 1996). In general, strict adherence to
standard quality control practices is not required for
conducting Phase I analyses due to the large number
of toxicity tests to be performed and the tentative
nature of the toxicant characterization. Nonetheless,
system blanks and controls should be used whenever
possible to indicate toxicity artifacts caused by the
characterization procedures. In Phase n more
attention should be i paid to quality control in order to
identify interferences in toxicant characterization and
identification. Evert greater attention to quality control
is needed in Phase ;in. Sample manipulation should
be minimized in Phase El to prevent analytical
interferences and toxicity artifacts. Field replicates,
system blanks, controls, and calibration standards
should be used extensively to allow a precise and
accurate determination of the sample toxicants and
toxicity.
Specific precautions for characterization (Phase I) and
toxicity testing in TIE analyses are provided below.
Aeration
For air stripping or aeration tests, only a high quality
compressed air source should be used. Oil, water, and
dirt are undesirable contaminants in compressed air;
therefore, it is important to use equipment and filters
that generate dry, oil-free air. Oil-sealed air
compressors should not be used. Simple aeration
devices, such as those sold for use with aquariums, are
acceptable provided that the ambient laboratory air is
uncontaminated (USEPA, 1991a). Recommendations
for in-line filters, for air exchange systems in
laboratories are provided by USEPA (1993c).
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Filtration
High purity water, which has been adjusted to a
specified pH, should be used to rinse filters between
filtration steps (USEPA 1991a, 1992a). Filtration
equipment should be rinsed with 10% nitric acid
(HNO3), acetone, and high purity water between
sample aliquots. Filtration equipment should be made
of plastic to avoid leaching of metals or other toxicants
during acid washes. Toxicity can be checked by
testing filtered dilution water.
pH Adjustments
Concerns in the pH adjustment steps involve artificial
toxicity caused by excessive ion concentrations from
the addition of acid and base solutions, contamination
from impure acid and base solutions, and silver
contamination from some pH probes (USEPA 1991a,
1992a). The baseline toxicity test acts as a control for
indicating whether addition of acid and base solutions
increases effluent toxicity. Ultra-pure acids and bases
should be used to minimize artificial toxicity. During
pH measurement, toxic concentrations of silver can
leach from refillable calomel electrodes; therefore,
only solid state pH probes should be used.
Methanol/C18 SPE Column
HPLC grade methanol is required for CIS SPE
column preparation and extraction steps. A blank
toxicity test should be conducted for each methanol
reagent lot. In addition, a toxicity blank should be
performed on each CIS SPE column to check for
resin-related toxicity (USEPA 199 la, 1992a).
Sodium Thiosulfate Addition
The TIE manuals (USEPA 1991a, 1992a, 1996)
provide information on the toxicity of sodium
thiosulfate to several freshwater and marine species.
These manuals prescribe the amount of sodium
thiosulfate to use in testing. If alternative species are
to be used, the species tolerance should be evaluated
by adding increasing quantities of sodium thiosulfate
to aliquots of the sample, testing the resulting toxicity,
and comparing the toxicity to the sample's baseline
toxicity.
EDTA Addition
The TIE manuals (USEPA 1991a, 1992a, 1996) also
prescribe the concentration of EDTA ligand to be
added to samples. If alternative species are to be used
in the TIE, the same test approach noted above for
sodium thiosulfate can be applied.
Toxicity Tests
The organisms used to test the sample toxicity prior to
and following each characterization step should not be
subject to undue stresses such as contamination
(USEPA, 1991a). The test organisms should have had
no prior exposure to pollutants and their sensitivity
should be constant over time. To assess changes in the
sensitivity of the test organisms, a standard reference
toxicant test should be performed on a regular basis
and accompanying quality control charts should be
developed (USEPA 1993c, 1994a, 1994b). Reference
toxicant tests should be performed monthly. If test
organism cultures are not maintained in the laboratory,
reference toxicant tests should be performed with each
group of test organisms received, unless such
information is available from the vendor. Information
on obtaining and culturing species for toxicity testing
is provided in the acute and chronic toxicity test
manuals (USEPA 1993c, 1994a, 1994b).
The quality of the dilution water used in toxicity tests
will depend on the purpose of the TIE test and whether
the test is being performed for toxicant
characterization (Phase I), identification (Phase H), or
confirmation (Phase ID). Much of Phase I and parts of
Phase II rely on relative toxicity measurement;
therefore, water that is of consistent quality and will
support the growth and reproduction of the test species
is suitable for these phases of the TIE (USEPA 199 la,
1992a, 1996). The objective of Phase m, however, is
to confirm the true cause of toxicity; therefore,
artifacts are to be excluded and the choice of dilution
water should follow Phase HI guidance (USEPA,
1993b). Guidance for preparing the dilution waters is
described by USEPA (199 la, 1992a, 1996).
USEPA (199la) recommends feeding cladocerans
(i.e., C. dubia and Daphnia sp.) in the TIE test
solutions at the beginning of acute TIE toxicity tests.
Daily feeding is required in the chronic TIE tests
(USEPA, 1992a). Feeding requirements for selected
species are described in the acute and chronic toxicity
test manuals (USEPA 1993c, 1994a, 1994b, 1995).
Sample pH should be recorded at each sample
renewal. Additional pH measurements may be needed
during the test, especially if ammonia toxicity is a
concern.
DO measurements may be made at sample renewal or
at the end of the exposure period in the TIE. In cases
where low DO is a problem, DO adjustment should be
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performed at a rate that will not intentionally change
the sample toxicity.
Refractory Toxicity Assessment and Treatability
Tests
RTA and treatability tests are subject to a variety of
potential interferences due to the large number of
variables that must be accounted for and controlled
during testing. When performing RTA and treatability
analyses, it is important to hold all parameters
potentially affecting toxicity constant so that sample
toxicity is the sole variable. Important parameters to
be controlled in RTA testing include the test solution
temperature, DO level, and pH.
The QA/QC concerns for toxicity analysis in RTA and
treatability tests are the same as those noted above for
TIE tests. Selection and use of test species and dilution
water should follow procedures given in the USEPA
Phase I manual (USEPA 1991a, 1992a).
Potential sources of toxicity contamination should be
identified through the use of system blanks. As in TIE
testing, the filters used in RTA testing should be tested
to determine if toxicity is added during filtration.
Each of the solutions used in RTA testing, including
activated sludge, should be checked for toxicity. In
the Patapsco TRE, the RAS used in the RTA batch
tests was found to be acutely toxic to C. dubia (Botts
et al., 1987). Steps for addressing RAS toxicity are
described in Section 5. Similarly, the reagents used in
treatability testing such as chemical coagulants should
be screened for toxicity.
Held replicates, calibration standards, and analytical
replicates should be routinely performed during RTA
and treatability testing. Results of these quality
control analyses can be used to calculate the precision,
accuracy, and the sensitivity of each physical/chemical
analysis method used in these studies.
Chemical Analyses
Quality control for chemical analyses includes the use
of calibration standards, replicate analyses, spiked
sample analyses, and performance standards. The
detection limits and the recommended reagents for
method calibration and spiking are discussed in
USEPA's Methods for Chemical Analysis of Water
and Wastes (USEPA, 1983b) and Standard Methods
for Examination of Water and Waste-water (APHA,
1995). General information on laboratory quality
control for chemical analyses is provided in USEPA's
HandbookjbrAnalytical Quality Control in Water and
Wastewater Laboratories (USEPA, 1979a).
Equipment Maintenance
All facilities and equipment such as pH, DO, and
conductivity meters, spectrophotometers, GC/MS, and
HPLC instruments should be inspected and maintained
according to manufacturers' specifications. Standard
operating procedures (SOP) should be followed for
routine maintenance land calibration of each analytical
instrument. A maintenance log book also should be
kept for each major laboratory instrument.
The measurement of toxicity or trace compounds in
wastewater samples requires the use of carefully
cleaned instruments and glassware. Instruments that
involve flow-through analysis such as automated
spectrophotometers should be inspected to ensure that
flow-through parts ' (i.e., tubing) are periodically
replaced. New glassware may be contaminated with
trace amounts of metals; therefore, any glassware
being used in toxicity tests for the first time should be
soaked for three days' in 10% HNO3 (USEPA, 1991a).
For subsequent use in TIB and toxicity tests, the
glassware should be washed with phosphate-free
detergent, and sequentially rinsed with 10% HNO3,
acetone, and finally high-purity water (USEPA 1993c,
1994a, 1994b).
Documentation and Reporting of Data
Basic steps in a successful QA/QC program are the
documentation of the analytical data in meaningful,
exact terms, and reporting the analytical data in a
proper form for future interpretation and use. To
ensure the reliability of the data, its handling must be
periodically monitored and reviewed. This review
generally consists of three elements: an assessment of
laboratory record-keeping procedures, a review of the
data calculations, and a review of the final reported
data. On the basis: of these review steps and the
QA/QC analyses for precision and accuracy, the data
are accepted or rejected. This review process is
essential because some or all records may have to be
submitted for review by State or Federal regulatory
agencies.
Corrective Action
Procedures should be established to ensure that
QA/QC problems such as improper sampling
techniques, inadequate COC records, and poor
precision and accuracy results are promptly
investigated and corrected. When a QA/QC
deficiency is noted, the cause of the condition should
be determined and corrective action should be taken to
preclude repetition.
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Section 9
Health and Safety
Introduction
A health and safety (H&S) plan may be necessary to
establish policies and procedures to protect workers
from hazards posed by TRE sampling and analytical
activities. The general guidelines outlined in this
section should be integrated into existing H&S
programs even if a specific H&S plan is not required.
Whether a specific H&S plan is necessary or not will
depend on the conditions under which the TRE is
being conducted. For example, if the POTW operates
under an RCRA permit by rule, then H&S must be
addressed when collecting and analyzing hazardous
wastes.
Important considerations for H&S for TRE studies
include:
Identification of personnel responsible for H&S
matters
H&S information and training activities
Protective equipment required for TRE activities
Materials cleanup and disposal procedures
Emergency response contingencies.
Detailed information on the preparation and scope of
H&S plans is provided in the Occupational Safety and
Health Administration's (OSHA's) Safety and Health
Standards, General Industry (OSHA, 1976). The
following subsections discuss specific H&S
considerations for selected TRE activities.
Sample Collection and Handling
Working with waste streams, of unknown composition
is inherent to TREs. Samples of industrial sewer
discharges, municipal wastewater, and sewage sludge
can contain a variety of toxic and hazardous materials
(e.g., pathogens, carcinogens) at concentrations that
can be harmful to human health.
It is the responsibility of the laboratory sample
custodian to ensure that TRE samples are properly
stored, handled, and discarded after use (see
Section 8). Upon sample storage, the . sample
custodian should indicate the H&S considerations for
sample handling and disposal.
Exposure to toxic and hazardous sample constituents
should be minimized during sampling handling. The
principal routes of human exposure to toxics is via
inhalation, dermal absorption, and/or accidental
ingestion. Exposure can be minimized through the use
of proper laboratory safety equipment such as gloves,
laboratory aprons or coats, safety glasses, respirators,
and laboratory hoods. Laboratory hoods are especially
important when testing wastewaters containing toxic
volatile substances such as volatile priority pollutant
compounds, hydrogen sulfide, or hydrogen cyanide.
Proper dermal protection such as using neoprene
gloves for solvent-containing wastes also is important.
Laboratory managers should consult the
manufacturers' specifications in selecting appropriate
clothing materials for protection against specific
chemicals.
Residual wastewater samples and wastes generated
during TRE studies should be disposed of properly.
Residual municipal wastewater and other non-
hazardous wastes can be disposed directly into the sink
drain if the TRE is being conducted at the POTW.
Residual industrial samples and other wastes that may
contain hazardous materials should be decontaminated
and/or disposed of in accordance with hazardous waste
regulations (NIOSH, 1977).
TRE Methods
Specific precautions to be, followed for selected TRE
techniques are described below.
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Toxicity Identification Procedures (TIE)
USEPA's TIE Phase I manuals (USEPA 1991a,
1992a, 1996) address the general H&S concerns
involved in performing TIE testing. Ventilation is a
specific concern when performing the Phase I aeration
procedure. The aeration test should be performed in
laboratory hoods to prevent exposure to toxic volatile
compounds or pathogens resulting from aeration.
H&S considerations for aquatic toxicity testing are
addressed in USEPA's toxicity test manuals (USEPA
1993c, 1994a, 1994b, 1995). Special precautions need
to be taken for on-site mobile laboratories in the
handling and transportation of chemicals, supply of
adequate ventilation and safe electrical power, and
disposal of waste materials.
Refractory Toxicity Assessment and Treatdbility
Tests
Proper ventilation also is important when conducting
RTAs and treatability tests in the laboratory. Hoods
should be used to capture and vent potential volatile
compounds that are stripped from the wastewater
during biological treatment tests.
Physical/chemical treatability testing may involve the
use of hazardous reagents such as acids or caustics.
Caution should be taken in the handling and disposal
of these chemicals.
Chemical Analyses
Several reagents used for chemical-specific analyses
(e.g., priority pollutants, COD, etc.) are toxic or
hazardous substances. Analysts should be familiar
with safe handling procedures for all reagents used in
testing, including the practice of proper chemical
storage to avoid storing incompatible chemicals
together (NIOSH, 19J77;OSHA, 1976). After use, the
waste chemicals should be converted into a less
hazardous form in the laboratory before disposal or
disposed of by a commercial disposal specialist.
General Precautions
USEPA (1977) and the American Chemistry Society
(1979) describe additional laboratory safety
procedures that can be used in TRE studies, including:
Use of safety arid protective equipment such as
eye protection (safety goggles, eye wash), fire
hazard protection (smoke and fire detectors, fire
extinguishers), and electrical shock protection
(ground-fault interrupters for wet laboratories).
Protocols for emergency response and materials
cleanup.
Personnel training in H&S procedures.
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Section 10
Facilities and Equipment
Introduction
Laboratories should be equipped with all the basic and
specialized laboratory equipment required to conduct
the TRE, and laboratory personnel should be skilled
and experienced in operating this equipment. The
facilities and equipment needed to perform a TRE will
be different for each POTW and will depend on the
type of testing to be performed in the TRE. In general,
the minimum facilities and equipment for initiating a
TRE will include the equipment needed for toxicity
and Tffi testing (USEPA 1991a, 1992a, 1993c, 1994a,
1994b, 1996). As additional information becomes
necessary, facility and equipment needs will depend
on the physical/chemical characteristics of the
causative toxicants aiid the toxicity control approaches
to be evaluated. For example, the selection of bench-
scale equipment and/or pilot plant facilities for
treatability studies will be dictated by the control
options to be tested (e.g., physical/chemical processes
such as filtration or biological processes).
The choice of whether to work on-site or off-site will
depend on the stage of the TRE, the approach for
tracking sources of toxicants or toxicity, and the
requirements for treatability testing. In general, the
equipment and time required for conducting TEE tests
makes on-site testing less feasible. If the loss of
sample toxicity over time is minimal, TIE samples can
be shipped and tested off-site, usually at much less
cost than on-site testing. If toxicity tracking using
RTA tests is required, on-site testing is recommended
for the treatment phase of the RTA, because fresh
samples of the POTW RAS biomass must be used.
Treatability tests that require continuous supplies of
POTW influent or process wastewaters and/or
activated sludge (i.e., flow-through bioreactor tests)
also may be more efficiently conducted in on-site
facilities. Some treatability evaluations require unique
or sophisticated equipment (e.g., ultra-filtration
apparatus) that is not readily available for on-site
work. In these situations, the equipment vendor may
be able to conduct the required tests at their facility.
The general equipment requirements for each of the
main TRE methods are summarized below. H&S
equipment is discussed in Section 9.
Toxicity Identification Evaluations
Laboratories should be equipped with the equipment
and materials needed to conduct the TEE, including
filtration and air-stripping equipment, pH and DO
meters, CIS SPE columns, fluid metering pumps,
required reagents for the TEE manipulations, and
facilities for organism handling, water preparation,
sample holding, and glassware cleaning. Equipment
requirements for culturing standard test species are
described in USEPA's acute and chronic toxicity test
manuals (USEPA 1993c, 1994a, 1994b, 1995).
More sophisticated analytical equipment is required
for the TEE Phase n toxicant identification and TEE
Phase in toxicant confirmation procedures. The
choice of analytical instruments for these procedures
will depend on the compound to be measured.
Equipment may include an analytical balance, a
GC/MS, an HPLC, an atomic absorption (AA)
spectrometer, an inductively coupled plasma (ICP)
spectrometer, an ultraviolet-visible spectrophotometer
(UV-VIS), an ion chromatograph, ion selective
electrodes, a pH meter, a conductivity meter, and a
refractometer. Use of inert materials such as
perfluorocarbon plastics for TEE Phases n and HI are
recommended to protect against toxicity artifacts
(USEPA, 1991a).
Refractory Toxicity Assessment and
Treatability Tests
Laboratories should be equipped with the basic
equipment for setting up and operating the RTA batch
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reactors, including an air supply, electrical supply, and
a laboratory hood. Instruments for monitoring the
batch reactors include respirometer and/or oxygen
meter, pH meter, ion selective electrode meter and
probes, total TOC analyzer, spectrophotometer for
COD and nutrient (e.g., ammonia and nitrate)
analyses. A drying oven, muffle furnace, and
analytical balance will be needed for TSS and VSS
measurements.
The equipment for toxicity testing will depend on the
choice of toxicity screening tests. Depending on the
species to be used, it may be more economical to
culture the test organisms than to purchase them. In
some cases, rapid screening tests such as a bacterial
bioluminescence test (e.g., Microtoxฎ) may be used as
a surrogate method for toxicity testing (see the
Billerica, Massachusetts, case history in Appendix A).
General Analytical Laboratory Equipment
General laboratory equipment such as refrigerators, a
water purification' system, and commonly used
reagents are neede'd to support the TIE and RTA
analyses. The type of water purification system
needed for testing !is described by USEPA (1993c,
1994a, 1994b, 1995).
72
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Section 11
Sample Collection and Handling
Introduction
The most important criterion in sampling is to obtain
a sample that is representative of the discharge.
Several samples will need to be collected to ensure
that the samples represent the typical lexicological and
chemical quality of the wastewater. Guidelines for
sample collection and handling are presented in the
acute and chronic toxicity test manuals (USEPA
1993c, 1994a, 1994b, 1995) and the Phase I TEE
documents (USEPA 1991a, 1992a, 1996). The WEF
also has published a useful guide to sampling at
POTWs (WEF, 1996).
A sampling plan should be prepared to document the
procedures to be followed in TRE sampling. This plan
should include:
A description of sampling locations
Sampling equipment and methodology
ซ Sample deli very requirements.
These elements are discussed in the following
subsections. QA/QC procedures for sampling are
addressed in Section 8. Procedures include preparing
COC forms, maintaining sampling equipment, and
identifying the minimum volume requirements,
holding times, and preservation techniques for
samples.
Sampling Location
Sampling locations should be established where
representative samples can be readily obtained. When
sampling waste streams within the POTW, care should
be taken to exclude unwanted waste streams and select
a sampling point that is most representative of the
discharge (e.g., the common discharge channel for
secondary clarifiers). The sampling location for the
POTW effluent should correspond with the
biomonitoring sampling point stated in the NPDES
permit. If the permit does not specify whether the
effluent sample is to be collected prior to or following
the chlorination/dechlorination treatment process, the
choice of a sampling location will depend on the
toxicants of concern. Generally, sampling at the point
of final discharge is the best option; however,
sampling both before and after chlorination/
dechlorination may help to determine if toxicity is
caused by chlorination (i.e., TRC) or dechlorination.
If samples are collected following the chlorination
process, free chlorine and TRC should be measured
when sampling is completed and upon initiation of
toxicity tests. These results will provide information
on the potential for chlorine toxicity.
Wastewater sampling for toxicity source evaluations
requires knowledge of sewer discharge locations.
Sampling may be conducted at the point of sewer
discharge or within the sewers in the municipal sewer
collection system. The choice of sampling points for
sewer line tracking may be based on existing
pretreatment program data. If these data are not
available, a sampling scheme can be devised to locate
sources of toxicity by testing and eliminating segments
of the collection system that prove to be non-toxic. In
some cases, indirect dischargers may have multiple
sewer discharges that need to be included when
sampling.
RTA testing requires samples of the POTW influent
(primary effluent) and activated sludge. Primary
effluent samples should be collected at the overflow
weirs of the primary sedimentation tanks. Activated
sludge samples can be collected from the aeration
basin effluent weirs or the RAS pipelines.
POTW Sampling
The choice of grab or composite samples of POTW
waste streams (i.e., effluent and influent wastewater
and process waste streams) will depend on the
physical/chemical characteristics and variability of the
73
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toxicants. Initial effluent toxicity characterization
(TEE Phase I) should utilize 24-hour composite
samples in order to ascertain the daily, weekly, or
seasonal variability of the causative agents. If effluent
toxicity is not easily observed in 24-hour time
composites, flow proportional composite or grab
samples may be used to observe possible flow-related
peaks of toxicity. In the latter phases of the TEE, grab
samples may be used to determine the variability in the
type and concentration of effluent toxicants (USEPA
1991a, 1992a). A discussion of the use of grab versus
composite sampling for toxicity tests is provided by
USEPA (USEPA 1993c, 1994a, 1994b, 1995). The
choice of sampling techniques for chemical-specific
analyses is dependent on the type of compounds to be
measured (e.g., grab sampling for volatile organic
compounds).
When evaluating the treatment efficiency of the
POTW or its unit processes, collection of the influent
and effluent wastewaters should be lagged by the
hydraulic retention time (HRT) of the treatment
process in order to obtain comparable samples. For
example, if the HRT of the treatment plant is 20 hours,
the effluent sampler should be timed to start 20 hours
after influent sampling is initiated. Likewise,
sampling of wastewater from industries or sewers
should account for the travel time in the collection
system (i.e., POTW influent sample collection should
lag industry sample collection).
Samples also should be collected during representative
discharge periods. An evaluation of the POTW
operations and performance at the time of sampling
can be made by comparing the effluent sample
concentrations of BOD5, TSS, and other pollutants to
long-term historical averages and/or permitted values
for those parameters.
Effluent samples are often collected, shipped, and
stored in plastic containers. However, some toxicants
such as surfactants may adsorb to plastic. A simple
way to check for this characteristic is to collect and
ship samples in both glass and plastic containers, then
test the samples for toxicity (USEPA, 1991a). A
greater loss of toxicity in plastic containers as
compared to glass containers may indicate the
presence of toxic surfactants.
The sample volume requirements for TIE Phase I tests
are provided by USEPA (1991a, 1992a, 1996).
Volume requirements for POTW samples that are used
in RTA tests are given in Section 5.
If TEE or physical/chemical treatability testing is being
conducted off-site, samples should be shipped on ice
to maintain the sample temperature at 4ฐC. RTA and
some biological treatability tests require fresh or
continuous samples of POTW waste streams, which
requires testing to be conducted on-site. Samples of
RAS and activated sludge should be delivered to the
on-site laboratory and used immediately in testing to
prevent changes in the biomass that can occur during
long-term storage. Biomass samples should be
vigorously aerated for a minimum of 15 minutes
before use in the RTA or treatability tests. POTW
influent and process wastewater samples required for
on-site RTA or treatability studies should be used on
the day of sample collection.
Sewer Discharge Sampling
The choice of grab or composite samples of indirect
discharges will depend on the physical/chemical
characteristics and variability of the toxicants. The
sample type also will be dictated by the stage of the
toxicity source evaluation. In Tier I testing, 24-hour
flow proportional composite samples are
recommended to characterize daily variability while
accounting for variations in flow. Flow proportional
sampling should be scheduled to coincide with
production schedules for industrial discharges, the
frequency of intermittent inputs for RCRA discharges,
and the schedule of remedial activities for CERCLA
discharges. This information is usually available in
the POTW's pretreatment program reports.
Sampling techniques'for flow proportional composites
should account for the potential loss of volatile
compounds. For samples collected for chemical
analysis or refractory toxicity testing, zero headspace
sampling methods can be used to minimize volatile
losses. In some cases, grab sampling may be used in
lieu of zero headspace methods to reduce sampling
costs; however, care should be exercised in collecting
samples that are representative of the discharge.
In Tier n, grab sampling can be used in addition to
composite sampling; to assess the variability of the
toxicants. This type of sampling requires in-depth
knowledge of the production schedules and the
pretreatment operations of the discharger.
74
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Section 12
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Quality-Based Permitting for Toxic Pollutants.
Office of Water Enforcement and Permits,
Washington, D.C.
USEPA. 1987b. Guidance Manual on the
Development and Implementation of Local
Discharge Limitations Under the Pretreatment
Program. Office of Water Enforcement and
Permits, Washington, D.C.
USEPA. 1988a. Methods for Aquatic Toxicity
Identification Evaluations: Phase I Toxicity
Characterization Procedures. EPA/600/3-88/034.
National Effluent Toxicity Assessment Center,
Duluth, Minnesota.
USEPA. 1988b. Development of Slug Loading
Control Programs for Publicly Owned Treatment
Works. Office; of Water Enforcement and
Permits, Washington, D.C.
USEPA. 1989a. Toxicity Reduction Evaluation
Protocol for Municipal Wastewater Treatment
Plants. EPA/600/2-88/062. Office of Research
and Development, Risk Reduction Engineering
Laboratory, Cincinnati, Ohio.
USEPA. 1989b. Generalized Methodology for
Conducting Industrial Toxicity Reduction
Evaluations. EPA/600/2-88/070. Water
Engineering Research Laboratory, Cincinnati,
Ohio.
USEPA. 1989c. Handbook: Retrofitting POTWs.
EPA/625/6-89/020. Center for Environmental
Research Information, Cincinnati, Ohio.
USEPA. 1990. User Documentation: POTW Expert,
Version 1.0. EPA/625/11-90/001. Office of
Research and Development, Cincinnati, Ohio.
USEPA. 1991a. Methods for Aquatic Toxicity
Identification Evaluations: Phase I, Toxicity
Characterization Procedures. Second Edition.
EPA/600/6-91-003. National Effluent Toxicity
Assessment Center, Duluth, Minnesota.
USEPA. 1991b. Technical Support Document for
Water Quality-Based Toxics Control. EPA/505-2-
90-001. Office ;of Water Enforcement and
Permits, Washington, D.C.
USEPA. 1991c. Guidance Manual for Control of
Slug Loadings to 'POTWs. 21W-4001. Office of
Water Enforcement and Permits, Washington,
D.C.
USEPA. 1991d. Methods for Determination of
Metals in Environmental Samples. EPA 600/4-
91/010. Office of Research and Development,
Washington, D.C.;
USEPA. 1992a. Toxicity Identification Evaluations:
Characterization of Chronically Toxic Effluents,
Phase I. EPA/600/6-91-005F. National Effluent
Toxicity Assessment Center, Duluth, Minnesota.
USEPA. 1993a. Methods for Aquatic Toxicity
Identification Evaluations: Phase II Toxicity
Identification Procedures for Samples Exhibiting
80
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Acute and Chronic Toxicity. EPA/600/R-92-
080. National Effluent Toxicity Assessment
Center, Duluth, Minnesota.
USEPA. 1993b. Methods for Aquatic Toxicity
Identification Evaluations. Phase HI Toxicity
Confirmation Procedures for Samples Exhibiting
Acute and Chronic Toxicity. EPA/600/R-92-081.
National Effluent Toxicity Assessment Center,
Duluth, Minnesota.
USEPA. 1993c. Methods for Measuring the Acute
Toxicity of Effluents and Receiving Waters to
Freshwater and Marine Organisms. Fourth
Edition. EPA/600/4-90-027F. Environmental
Monitoring Systems Laboratory, Cincinnati, Ohio.
USEPA. 1993d. Treatability Data Base, Version 5.
EPA/600/C-93/003a. Risk Reduction
Engineering Laboratory, Cincinnati, Ohio.
USEPA. 1994a. Short-Term Methods for Estimating
the Chronic Toxicity of Effluents and Receiving
Waters to Freshwater Organisms. Third Edition.
EPA/600/4-91/002. Environmental Monitoring
and Support Laboratory, Cincinnati, Ohio.
USEPA. 1994b. Short-Term Methods for Estimating
the Chronic Toxicity of Effluents and Receiving
Waters to Marine and Estuarine Organisms.
Second Edition. EPA/600/4-91/003.
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio.
USEPA. 1994c. Methods for Determination of
Metals in Environment Samples Supplement I.
EPA 600/R-94/111. Office of Research and
Development, Washington, D.C.
USEPA. 1995. Short-Term Methods for Estimating
the Chronic Toxicity of Effluents and Receiving
Waters to West Coast Marine and Estuarine
Organisms. EPA/600/R-95-136. National
Exposure Research Laboratory, Cincinnati, Ohio.
USEPA. 1996. Marine Toxicity Identification
Evaluation (TIE) Guidance Document, Phase I.
EPA/600/R-96/054. Office of Research and
Development, Washington, D.C.
USEPA. 1997. Methods and Guidance for Analysis
of Water CD ROM Version 1.0. Office of Water,
EPA-821-97-001. Washington, D.C.
Walsh, G.E., and R.L. Garnas. 1983. Determination
of Bioactivity of Chemical Fractions of Liquid
Wastes Using Freshwater and Saltwater Algae and
Crustaceans. Environ. Sci. Tech. 17: 180-82.
Ward, S.H. 1989. The Requirements for a Balanced
Medium in Toxicological Experiments using
Mysidopsis bahia with Special Reference to
Calcium Carbonate. Environmental Toxiocology
and Hazard Assessment: 12th Volume, ASTMSTP
No. 1023: 402-12. U.M. Cowgill and L.R.
Williams, eds. American Society for Testing and
Materials, Philadelphia, Pennsylvania. 402-12.
Water Environmental Federation. 1996. Wastewater
Sampling for Process and Quality Control - MOP
OM-1. Water Environment Federation,
Alexandria, Virginia.
Water Environment Federation and American Society
of Civil Engineers (WEF/ASCE). 1992a. Design
of Municipal Wastewater Treatment Plants,
Volume I, Chapters 1-13. WEF Manual of
Practice No. 8 andASCE Manual and Report on
Engineering Practice No. 76. Water Environment
Federation, Alexandria, Virginia, and American
Society of Civil Engineers, New York, New York.
Water Environment Federation and American Society
of Civil Engineers (WEF/ASCE). 1992b. Design
of Municipal Wastewater Treatment Plants,
Volume II, Chapters 13-20. WEF Manual of
Practice No. 8 and ASCE Manual and Report on
Engineering Practice No. 76. Water Environment
Federation, Alexandria, Virginia and American
Society of Civil Engineers, New York, New York.
Water Pollution Control Federation (WPCF). 1983.
Nutrient Control. Manual of Practice FD-7,
Facilities Design, Alexandria, Virginia.
Water Research Commission (WRC). 1984. Theory,
Design and Operation of Nutrient Removal
Activated Sludge Processes. Wiechers HNS, ed.
Water Research Commission, Pretoria, South
Africa.
Weis, P., J.S. Weis, C-M Chen, and A. Greenberg.
1992. Treated Municipal Wastewaters: Effects of
Organic Fractions on Development and Growth of
Fishes. Environ. Toxicol. Chem. 11: 1451-59.
81
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Section 13
Bibliography
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Ahel, M., and W. Giger. 1985. Determination of
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Ahel, M., and W. Giger 1985. Determination of
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Jirka, A.M., and M.J. Carter. 1975. HACH COD
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Marcomini, A., and W. Giger, 1987. Simultaneous
Determination of Linear Alkylbenzenesulfonates,
Alkylphenol Polyethoxylates and Nonylphenol by
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USEPA. 1983. Treatability ManwaZ. EPA/600/2-82-
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83
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Appendix A
Original Case Histories: Lessons Learned
Since the USEPA research studies in the 1980s and the
first TREs performed to meet permit requirements,
there have been significant advances in the
development and refinement of TRE procedures.
These advancements become apparent upon review of
the original case histories published in the first edition
of the TRE manual. The case histories have been
revisited in this manual to note the lessons learned and
new approaches that can be taken to conduct TREs.
Many lessons have been learned in applying Tffi/TRE
procedures to different types of effluents using a
variety of freshwater and saltwater test species.
Perhaps the most significant improvements in the
methods since the original case histories were
performed have been the development and application
of methods to:
Identify causes of short-term chronic toxicity to
both freshwater and estuarine/marine species.
Track sources of chronic toxicity that can not be
readily characterized in the TIE.
Characterize, identify, and confirm organophos-
phate insecticide toxicity.
Characterize toxic metals using improved EDTA
and sodium thiosulfate tests.
Characterize surfactant toxicity using multiple TIE
manipulations.
Confirm toxicants by the correlation approach.
The use of some of these updated methods is described
below using the original case histories as examples.
The following summaries are intended to show how
similar TREs can be performed more quickly, cost-
effectively, and accurately using the current
procedures. These summaries also portray the steps
taken over the last 10 years to improve the TRE
procedures.
Baltimore, Maryland
In January 1986, USEPA, in cooperation with the City
of Baltimore, began the first research study to develop
a pragmatic approach and methods for conducting
TREs at WWTPs {Bolts et al., 1987). The City's
Patapsco WWTP was selected for this study because of
evidence of acute and chronic effluent toxicity. In
addition, USEPA wfas interested in conducting a TRE
at an urban WWTP, like the Patapsco WWTP, which
receives its influent from a wide range of industrial
discharges. The objectives of the TRE were to
characterize the WWTP's capability for treatment of
toxicity, evaluate techniques to identify the specific
components of toxicity, and assess methods to trace
toxicity to its source(s).
The study results showed that the WWTP influent had
significant acute and chronic toxicity as measured by
C. dubia [(mean 48-hour LC50=2.6% and mean 7-day
chronic value (ChV)=1.2%], M. bahia (mean 96-hour
LC50=23%), and Microtoxฎ (EC50=8%). Although
significant toxicity reduction occurred through
treatment, substantial toxicity remained. The 48-hour
LC50 for C. dubia averaged 6.3% effluent. An
evaluation of the WWTP operations indicated that
treatment performance was not the major cause of
effluent toxicity.
Results of the TIE showed that acute effluent toxicity
was removed by passing effluent samples through a
C18 SPE column. Recovery of toxicity in the 75 to
95% methanol/water eluates from the C18 column
suggested that the toxicants were non-polar organic
compounds with relatively high octanol-to-water
partition coefficients. However, GC/MS analysis of
the toxic non-polar organic fractions was not
successful in identifying the specific nonpolar organic
84
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toxicants. Additional testing showed that the toxicants
sorbed onto suspended solids in the effluent. Solids
greater than 0.2 |im were found to be the major toxic
fraction.
TIE Procedure Update
Since this study, USEPA developed procedures for
identifying non-polar organic toxicants (1993a). If
non-polar organic toxicity is indicated in the Phase I of
the TIE, the toxicant(s) can be isolated and
concentrated to improve the chances of identification
using GC/MS analysis. This approach has been
helpful in identifying organophosphate insecticides as
causes of effluent toxicity at some POTWs (see
examples below and Appendix F).
An evaluation of wastewa.ter samples from selected
candidate industries was performed to determine the
major contributors of refractory toxicity to the WWTP.
An important goal of this study was to develop and
evaluate methods for tracking sources of toxicity in
POTWs. A protocol was designed to measure the
toxicity remaining after treatment at the WWTP, which
is the toxicity that passes through in the final effluent.
The residual or "refractory" toxicity of five major
industrial users of the WWTP was evaluated by
treating wastewater samples in a bench-scale batch
simulation of the WWTP activated sludge process.
Microtoxฎ results indicated that two of the five
industries were contributing refractory toxicity to the
WWTP. Results of C. dubia tests were inconclusive
due to an interference in the treatment simulation. This
interference appeared to be caused by residual toxicity
in the RAS used in testing.
RTA Procedure Update
Biomass toxicity may be reduced by washing the
RAS with buffer solutions or laboratory water.
Alternatively, a surrogate biomass from a POTW with
a similar type of biological treatment process may
be obtained for testing. Details are presented in
Section 5.
Hollywood, California
In the late 1980s ;and early 1990s, the USEPA
laboratory in Duluth, Minnesota, tested several POTW
effluents in the process of developing TIE procedures.
One of these effluents was the City of Hollywood
POTW, which exhibited acute toxicity to C. dubia
(Amato et al., 1992).
TIE Phase I tests showed that treatment with a CIS
SPE column was the only step that reduced effluent
toxicity. Acute toxicity was recovered from the CIS
column by eluting the column with methanol.
Additional CIS SPE column tests performed on 16
effluent samples showed that toxicity was consistently
eluted in the 80 and 85% methanol fractions, which
suggested that the cause of toxicity was the same
among the various samples. These results provided
evidence that the toxicant(s) was a non-polar organic
compound(s). Further concentration and separation of
the toxic fractions was done, followed by confirmation
GC/MS analyses of the fractions. Analysis of selected
80 and 85% methanol fractions by GC/MS found
sufficient concentrations of the insecticide diazinon to
account for the observed acute toxicity to C. dubia.
TIE Procedure Update
In recent TIE guidance, USEPA (1991 and 1993a)
recommends adding a metabolic blocker, PBO, to
toxic effluent samples or methanol eluates as a
subsequent test for the presence of metabolically
activated toxicants like organophosphate insecticides.
PBO has been shown to block the acute toxicity of
diazinon, parathion, methyl parathion, and malathion
to cladocerans, but does not affect acute sensitivity to
dichlorvos, chlorfenvinphos, and mevinphos (Ankley
et al., 1991). A reduction in acute or chronic toxicity
by the PBO addition together with toxicity removal by
the CIS SPE column and concentration data can
provide strong evidence for the presence of selected
organophosphate insecticides.
In the confirmation step (USEPA, 1989b), three Phase
in confirmation steps were used to confirm diazinon as
a cause of effluent toxicity: toxicant correlation, mass
balance, and additional species testing.
Toxicant correlation was evaluated by plotting effluent
diazinon concentrations and effluent LC50 values as
shown in Figure A-l. The correlation coefficient (r
value) was significant and confirmed that, from sample
to sample, diazinon was consistently the cause of acute
effluent toxicity. Also, the intercept of the regression
line at 100% effluent (0.325) was near the diazinon
LC50 of 0.35 ng/L, which indicated that diazinon
accounted for nearly all of the observed acute effluent
toxicity.
85
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10
I i:
10-'
10
LC50 (%)
102
Figure A-l. Acute LC50 of Hollywood effluent versus
diazinon concentration (actual correlation shown by solid
line; predicted 1:1 correlation by dashed line) (Source:
USEPA, 1988).
TEE Procedure Update
USEPA (1993b) recommends a straight-forward
correlation approach to determine if a consistent
relationship exists between the concentration of the
toxicant(s) and effluent toxicity. This approach
involves comparing the toxic units of the toxicant to
whole effluent toxic units. Toxicant concentrations are
converted to toxic units (i.e., measured concentration
divided by the toxicant's acute or chronic endpoint)
and the resulting values are plotted versus whole
effluent toxic units. Since this study, additional acute
toxicity data for diazinon and other organophosphate
insecticides have become available for calculating
toxic units for these toxicants (Ankley et al., 1991;
Amato et al., 1992; and Bailey et al., 1997). The
correlation approach is useful for determining the
extent to which the identified toxicants contribute to
effluent toxicity. Using the above example, diazinon
would be confirmed as the primary toxicant if the slope
is 1 and the intercept is 0 for a plot of diazinon toxic
units versus effluent toxic units. In some cases,
additional toxicants may be indicated using this
technique (see the City of Largo, Florida, example
below).
The mass balance confirmation approach involved
passing samples through a CIS SPE column, eluting
the column with a series of eight methanol
concentrations, and testing the toxicity of the methanol
fractions. The combined toxic, combined nontoxic,
and all fractions were combined and tested at whole
effluent concentrations. The results showed that the
toxicity of the combined toxic fractions was similar to
the toxicity of all fractions together and the toxicity of
the original effluent samples. These results provided
further confirmation that effluent toxicity was
associated with non-polar organic toxicants.
The final confirmation step involved testing effluent
samples with P. promelas, which are at least 100 times
less acutely sensitive to diazinon than C. dubia
(USEPA 1987,1988). Test results showed only slight
acute toxicity to the minnows as compared to the
average acute LC50 of about 60% for C. dubia. Acute
toxicity to P. promelas was interpreted as evidence that
a toxicant other than diazinon was present in the
samples. However, this additional toxicant(s) was not
a significant contributor to toxicity and its identity was
not evaluated. In! summary, the Phase HI testing
confirmed that diazinon was the principal effluent
toxicant.
Largo, Florida
The USEPA Duluth Laboratory also evaluated effluent
samples from the City of Largo POTW. A TIE was
performed to identify the causes of acute effluent
toxicity (USEPA, 1987).
TIE Phase I tests showed that CIS SPE column
treatment removed acute effluent toxicity. Toxicity
was not reduced by the other Phase I treatments,
including filtration, EDTA addition, or sodium
thiosulfate addition.
An additional 18 effluent samples were passed through
CIS SPE columns in Phase H Elution of the columns
with methanol showed that acute toxicity was
consistently isolated in the 75 and 80% methanol
concentrations, although occasional toxicity was also
observed in the 70 and 85% methanol concentrations.
GC/MS analysis of the toxic fractions identified
diazinon as a cause of acute effluent toxicity.
In Phase HI, five confirmation steps were used to verify
that diazinon was: the cause of effluent toxicity:
toxicant correlation; toxicant spiking, mass balance,
additional species testing, and test species symptoms.
Acute effluent toxicity and diazinon concentrations
were converted to TUs and were plotted to determine
86
-------
the toxicant correlation to whole effluent toxicity
(USEPA, 1989b). As shown in Figure A-2, more acute
toxicity was present than would be explained by
diazinon alone; the slope of the linear regression was
less than 1 and all of the plotted data points are below
the expected 1:1 relationship for diazinon and effluent
toxicity. Spiking experiments also showed that
doubling the concentration of diazinon in effluent
samples did not result in a doubling of effluent toxicity.
These results suggested that diazinon was not the sole
cause of acute effluent toxicity.
5.00
4.00 -
I
Expected Regression
Observed Regression
3.00 -
2.00 -
1.00-
0.00
0.00 1.00 2.00 3.00 4.00 5.00
Whole Effluent TUs
Figure A-2. Correlation of diazinon TUs versus whole
effluent TUs (Source: USEPA, 1988).
TIE Procedure Update
The current approach (USEPA, 1993b) is to plot
effluent TUs on the Y-axis (dependent variable) and
toxicant TUs on the x-axis (independent variable).
See Figure A-3.
Follow-up GC/MS analyses identified chlorfenvinphos
(CVP) and malathion in effluent samples. Malathion
did not appear in concentrations high enough to cause
acute toxicity to C. dubia, although CVP
concentrations were sufficient to contribute to effluent
toxicity (48-hour LCSOs of 1.4 and 0.35 ug/L,
respectively, according to D. Mount, personal
communication, USEPA, Duluth, Minnesota, 1989).
The correlation analysis was repeated using the
summed toxic units for both diazinon and CVP versus
whole effluent toxic units (USEPA, 1993b). As shown
in Figure A-3, the slope of the regression line was
close to 1, the y-intercept was nearly zero, and the
r-value indicated a good correlation (r = 0.73). These
results show that diazinon and CVP accounted for
nearly all of the acute effluent toxicity.
5-
t Observed
Theoretical
= 0.73
slope = 0.82
y-intercept = 0.46
0123456
TUs of Suspect Toxicant(s)
Figure A-3. Correlation of diazinon and CVP TUs versus
whole effluent TUs (Source: USEPA, 1993b).
Additional confirmation testing involved analyzing 13
effluent samples using the CIS SPE column mass
balance approach. As shown in Table A-l, in 12 of the
13 tests, the toxicity of all methanol fractions
combined was slightly greater than the toxic fractions
combined. Various mixtures of the three identified
insecticides were tested to determine if interactive
effects (i.e., antagonistic or synergistic) could account
for the difference in toxicity. These tests showed that
the toxicity of the insecticides was strictly additive.
These results indicated that the higher toxicity of "all
fractions" compared to the toxicity of the "toxic
fractions" may be due to another unidentified toxicant,
rather than interaction among the identified toxicants.
The additional toxicity observed in the "all fraction"
test was attributed to 70% methanol/water fraction,
which exhibited slight and intermittent toxicity. This
fraction was initially included in the "nontoxic
fraction" test; however, the mass balance approach
87
-------
Table A-l. Comparison of Whole Effluent TUs
and Methanol Fraction TUs
Sample
A
B
C
D*
E
F*
G
H
I
J
K
L
Main
Acute Toxic Units (TUa)
Whole
Effluent
1.18
2.00
1.93
<1.00
2.00
1.15
1.33
3.70
2.86
2.27
2.27
2.27
2.13
All-
Fractions
1.64
2.94
2.86
1.15
1.75
1.06
1.52
3.03
2.86
1.72
2.04
1.67
2.18
Toxic-
Fractions
1.43
3.13
2.53
<1.00
1.64
<1.00
1.13
2.86
2.44
1.64
2.00
1.59
2.00
* Values excluded from mean calculations due to less-than values.
indicated it to be a slightly toxic fraction. When the
toxic units of the 70% fraction are added to the "toxic
fraction" result, nearly all of the toxicity is accounted
for. Due to the intermittent toxicity of this fraction,
additional testing to identify the toxicant was not
performed.
Additional species testing with P. promelas provided
further evidence that the toxicants werp
organophosphate insecticides. No acute toxicity was
observed with P. promelas, which are known to be
orders of magnitude less sensitive to diazinon than
C. dubia (USEPA 1987,1988).
As a final confirmation step, the same symptoms to
C. dubia were observed after exposure to effluent
samples, toxic methanol fractions, and laboratory water
spiked with Bear lethal levels of diazinon, CVP, and
malathion. Similar symptoms were observed for all
test solutions, which suggested that the same toxicant
was responsible in each case.
Lawton, Oklahoma
The City of Lawton was required by USEPA Region 6
to initiate a TRE study in 1991, based on evidence of
chronic effluent toxicity at its POTW (Engineering
Science, Inc., 1991). The permit limit of no chronic
lethality at the critical instream dilution of 96%
(i.e., NOEC >96% effluent) was exceeded. Toxicity
test results showed that the effluent was toxic to C.
dubia, but not P. promelas.
TIE Phase I tests were conducted in 1991 to
characterize the chronic effluent toxicants
(Engineering Science, Inc., 1991). The permit limit
was based on lethality to C. dubia and P. promelas in
chronic toxicity tests; therefore, the TEE tests focused
on lethality instead of reproduction or growth effects.
The Phase I tests evaluated percent survival of C.
dubia, the most sensitive organism, over 5 to 7 days in
100% effluent. In addition, acute lethality results (48-
to 72-hour exposure) also were collected to assist in the
evaluation.
The results indicated a consistent reduction in effluent
toxicity by passing \ samples through the C18 SPE
column. Chronic lethality data showed that no other
treatment consistently removed toxicity. Toxicity was
recovered by eluting the C18 SPE column with
methanol, which indicated the presence of nonpolar
organic toxicants. Sample adjustment to pH 3 and pH
11 also reduced toxicity in all but two samples, which
suggested that the toxicants could be denatured under
acidic or basic conditions.
TIE Procedure Update
As noted above, PBO, a metabolic blocker, can be
added to toxic effluent samples, CIS SPE fractions, or
HPLC fractions to test for the presence of meta-
bolically activated toxicants such as organophosphate
insecticides. j
Reproduction data for C. dubia, although not required
as part of compliance testing for the Lawton POTW,
may have been useful in characterizing the effluent
toxicants. These data may provide a more sensitive
endpoint than survival in 100% effluent when
comparing the effects of the various TIE treatments.
TIE Phase n tests were performed on three samples
evaluated in the Phase I characterization and involved
the following steps as described by USEPA (1989a):
C18 SPE columns were eluted with a series of
increasing methanol concentrations (25, 50, 75,
80, 85, 90, 95, and 100%) to isolate the toxicants.
The acute toxicity of each eluted fraction was
determined and ithe fractions found to be toxic
were combined. The combined toxic fractions
were then reconcentrated using a second C18 SPE
88
-------
column. Acute toxicity tests were used instead of
chronic toxicity tests because the methanol elution
concentrated the toxicants to acutely toxic levels.
The concentrated sample was separated into 30
fractions using HPLC and the. toxicity of each
fraction was measured. Again, the toxic fractions
were combined and reconcentrated on another CIS
SPE column.
The combined toxic sample was then analyzed by
GC/MS.
As shown in Table A-2, toxicity was consistently
isolated in the 75 and 80% methanol fractions,
although toxicity was also recovered in the 50%
methanol fraction of one sample. Further separation of
the toxicants by HPLC recovered toxicity in a
relatively narrow band of fractions (fractions 22 to 28).
Table A-2. Summary of TIE Phase II Results
Sample
Sample Collection Data (1992)
4/28
6/11
7/16
C. dubia percent survival in 100% sample
Original effluent
Post CIS SPE
SPE eluate
(Ix effluent)
50
100
0
0
100
0
0
80
0
Toxic methanol fractions (>20% mortality)
Methanol/water
(Ix effluent cone.)
HPLC fraction no.
(Ix effluent cone.)
50%
75%
80%
15
22-25
30
75%
80%
25
28 .
75%
80%
22
24
Organophosphate insecticides in effluent (ng/L)
Diazinon
Diazinon oxon
0.22
0.1
0.42
<0.1
0.71
1.45
GC/MS analysis of the toxic HPLC fractions identified
several potentially toxic compounds, including the
organophosphate insecticide, diazinon, and its
metabolite, diazinon oxon (Table A-2). The 48-hour
LC50 of diazinon to C. dubia is reported to range from
0.35 to 0.61 ug/L (Amato et al., 1992; Ankley et al.
1991). Based on the low end of this range, the
diazinon concentrations in the Lawton effluent were
high enough to cause acute toxicity to C. dubia in two
of the three samples tested (0.42 and 0.71 |ig/L for the
June and July samples, respectively).
C. dubia acute toxicity tests were conducted to
evaluate the potential contribution of diazinon oxon to
effluent toxicity. The 48-hour LC50 for diazinon oxon
was determined to be 1 |J.g/L. These data indicate that
the diazinon oxon concentration in the July effluent
sample (1.45 |ig/L) was high enough to contribute to
the observed acute toxicity.
TIE Procedure Update
Data on the chronic toxicity of organophosphate
insecticides is limited. Unpublished data (TRAC
Laboratories, 1992) suggest that C. dubia may be
chronically sensitive to 0.12 to 0.38 jag/L diazinon (see
also Section 2). Chronic data would have been useful
in defining the potential for diazinon to contribute to
chronic toxicity at the Lawton POTW.
Further testing focused on confirming the contribution
of diazinon and diazinon oxon to effluent toxicity. A
partial Phase HI confirmation was performed using the
following steps (USEPA, 1989b):
Assessing diazinon's physical/chemical properties
in relation to the TIE results.
Determining the contribution of diazinon and
diazinon oxon to whole effluent toxicity based on
measured effluent concentrations.
Reviewing effluent toxicity data for a 3-year
period to determine if the occurrence of effluent
toxicity matched seasonal insecticide use
(Engineering Science, Inc., 1992).
Diazinon matches the general toxicant profile
developed as part of the TIB. Removal of diazinon on
the CIS SPE column and its elution at high methanol
concentrations is consistent with diazinon's
characteristic as an organic chemical of low polarity.
The observed reduction in toxicity by pH adjustment
also is indicative of diazinon's tendency to break down
under acidic and alkaline conditions.
Concentrations of diazinon and diazinon oxon were
measured in 13 effluent samples collected from April
1 through August 21, 1992. Chronic toxicity data for
the insecticides were not available at the time;
therefore, it was not possible to apply the correlation
approach. However, in seven cases, diazinon exceeded
the 0.35 [ig/L acute toxicity value for C. dubia. In two
of these cases, diazinon oxon concentrations also
exceeded the acute toxicity value of 1.45 (ig/L- These
89
-------
data suggested that diazinon and diazinon oxon were
likely to cause mortality equal to or greater than that
found in the effluent samples.
A review of effluent toxicity data from 1989 to 1992
indicated a greater incidence of toxicity in the spring
and summer of each year when insecticides are most
often used. Effluent toxicity decreased in late summer
and fall and generally disappeared in the winter
months. These data support the evidence that toxicity
is associated with insecticides.
TEE Procedure Update
Confirmation of the role of diazinon and other
toxicants would have been more definitive if the
current Phase HI procedures (USEPA, 1993b) for
chronic toxicants had been applied. Useful procedures
for confirming organophosphate insecticide toxicity
include the correlation, mass balance, and species
symptoms approaches. An example of the use of these
procedures is presented in Appendix F.
Based on previous studies (City of Greenville, 1991; C.
Kubula, personal communication, City of Greenville,
Texas, Public Works Department, 1992), the City of
Lawton decided to implement a public awareness
program in 1993 to control the discharge of
insecticides to the POTW. Information on the proper
use and disposal of insecticides was printed in
newspaper articles and on monthly water bills
(Engineering-Science, Inc., 1993). An electronic
message sign with insecticide information was also
located at major intersections. Since August 1993, the
POTW effluent has met the toxicity permit limit
(NOEC >96% effluent) with the exception of 2 months
in 1994 and several months in 1995 (as of September
1997). Although diazinon was not confirmed as ;an
effluent toxicant, the City's ongoing insecticide control
effort appears to have been successful in achieving
compliance with the chronic toxicity limit.
Akron, Ohio
A survey of six Ohio municipal wastewater treatment
plants was conducted to determine the level of toxicity
reduction that can occur in POTWs (Neiheisel et al.,
1988). Of the six WWTPs, the City of Akron's
Botzum WWTP received the most toxic influent
wastewater. Significant toxicity reduction was
achieved through treatment; however, the effluent had
an impact on the Cuyahoga River. A bioassessment
study of the river in 1984 revealed a severe impairment
to aquatic communities downstream of the WWTP
discharge. A review of the WWTP's operating records
showed a history ,of intermittent bypasses of raw
wastewater during storm events.
Based on the survey results, the Botzum WWTP was
selected by USEPA as a site for a TRE research study.
The research study focused on conducting toxicity tests
of the effluent and the bypassed wastewater and
characterization of the variability and sources of the
impairment to the receiving water (Mosure et al.,
1987). In addition, TIE tests were performed to try to
identify the effluenjt toxicants.
Toxicity test results indicated that although CSOs may
contribute intermittently to poor river quality, the
continuous effluent discharge was probably the major
cause of the observed impact (Mount and Norberg-
King, 1985).
The TIE testing isolated toxicity on the C18 SPE
column and the toxicity was eluted in the 85%
methanol/water fraction (Mosure et al., 1989). These
results suggested that non-polar organic compounds
were a principal cause of effluent toxicity. Metals also
were implicated as effluent toxicants. However, before
toxicant identification and confirmation could be
performed, effluent toxicity abated.
The cause of this abatement is not known, although the
following events may have contributed to the improved
effluent quality. These events include:
Increasing MLSS concentrations in the WWTP
aeration basins.
The shutdown of a large chemical manufacturing
plant that discharged to the WWTP.
Overall improvements in WWTP operation and
the pretreatment program (Mosure et al., 1987).
Biological surveys of the Cuyahoga River in 1986
continued to show poor water quality despite the
decrease in effluent toxicity (Mosure et al., 1987). It is
possible that other dischargers to the river were
contributing to the impairment or the recovery rate of
the river was slower than anticipated.
Billerica, Massachusetts
A study was conducted at the City of Billerica's
WWTP to evaluate sources of toxicity in the facility's
90
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Toxicity Control Evaluation Update
Abatement of effluent toxicity during the course of
TREs is not uncommon. However, efforts to ensure
ongoing compliance can be difficult when the original
causes and sources of toxicity are not known. These
situations dramatize the importance of documenting
industrial pretreatment activities and POTW operations
in the early stages of the TRE. Weekly or daily reports
of production and waste discharge activities by
industrial users can provide a useful history of events
that can be used to indicate potential sources. This
information is also helpful in subsequent pretreatment
control studies if an industrial user is identified as a
source of toxicity (Botts et al., 1994).
collection system (Durkin et al., 1987). A purpose of
the study was to evaluate the usefulness of Microtoxฎ
as a tool for tracing sources of toxicity.
The Billerica study was conducted in five stages:
Screening for WWTP influent toxicity.
Testing samples from pump stations in the
collection system.
In-depth testing to determine the time of day when
toxicity was observed at the pump stations.
Testing of the main sewer lines above the pump
stations where toxicity was indicated.
Final testing of tributary sewers.
Of the 11 pump stations tested, 2 were found to have
highly toxic wastewaters. In one of these pump
stations, high levels of toxicity occurred only during
the 8 a.m: to 2 p.m. time period. Further investigation
of the intermittently toxic pump station provided
evidence that the principal source of toxicity was an
industrial park.
References
Amato, J.R., D.I. Mount, EJ. Durban, M.L.
Lukasewycz, G.T. Ankley, and E.D. Robert.
1992. An Example of the Identification of
Diazinon as a Primary Toxicant in an Effluent.
Environ. Toxicol. Chem. 11: 209-16.
Ankley, G.T., J.R. Dierkes, D.A. Jensen, and G.S.
Peterson. 1991. Piperonyl Butoxide as a Tool
in Aquatic Toxicological Research with
Organophosphate Insecticides. Ecotoxicol.
Environ. Saf. 21: 266-74.
RTA Procedure Update
Toxicity screening tools such as Microtoxฎ have been
used to identify sources of toxicity in POTW collection
systems. It is necessary to first determine if a
correlation exists between the compliance test and the
screening test to ensure that the toxicity measured by
the surrogate tool is the same toxicity indicated by the
species used for compliance testing. This correlation
can be performed using POTW effluent; however, it is
important to note that correlation results may be
different for individual industrial discharges. As a
result, the screening test may yield false positive or
false negative results.
The advantage of screening tests is that a large number
of samples can be processed at relatively low cost. As
an alternative to these tools, POTW staff may consider
using the permit test species in an abbreviated test
procedure such as that used in the TIE (USEPA1991).
The cost of these tests can be comparable to
commercially available screening tests if the number of
replicates or sample concentrations is reduced or the
exposure time is decreased.
Although this study indicated a potential source of
toxicity, a final determination of the source(s) of
toxicity would require first treating the sewer samples
in a simulation of the POTW to provide an accurate
estimate of the refractory toxicity of the waste stream.
Otherwise, as discussed in Section 5, the toxicity
results may overestimate the toxicity of the discharge
because some toxicity removal generally occurs in the
POTW. A description of the updated RTA protocol is
given in Section 5.
Bailey, H.C., J.L. Miller, M. J. Miller, L.C. Wiborg, L.
Deanovic, and T. Shed. 1997. Joint Toxicity of
Diazinon and Chlorpyrifos Under Conditions of
Acute Exposure to Ceriodaphnia dubia. Environ.
Toxicol Chem. 16: 2304-08.
Botts, J.A., J.W. Braswell, E.G. Sullivan, W.C.
Goodfellow, B.D. Sklar, and A.G. McDearmon.
1987. Toxicity Reduction Evaluation at the
Patapsco Wastewater Treatment Plant. Water
Engineering Laboratory, Cincinnati, Ohio.
Botts, J.A., T. Schmitt, E. Wilson, M. Collins, D.
Waddell, R. Diehl, and L. Ehrlich. 1994.
Refractory Toxicity Assessment: An Alternative
Approach for Chronic Toxicity Reduction
Evaluations. Technical paper presented at the
Annual Conference and Exposition of the Water
91
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Environment Federation, Chicago, Illinois. Paper
#AC944404.
City of Greenville. 1991. TRE Phase B Final Report.
Prepared for the City of Greenville, Texas.
Durkin, PP., C.R. Ott, D.S. Pottle, and G.M. Szal.
1987. The Use of the Beckman Microtoxฎ
Bioassay System to Trace Toxic Pollutants Back
to Their Source in Municipal Sewerage Systems.
Proceedings of the Water Pollution Control
Federation Association Annual Meeting, New
Hampshire.
Engineering-Science, Inc. 1991. Toxicity Reduction
Evaluation Phase I Report. Submitted to the City
of Lawton, Oklahoma.
Engineering-Science, Inc. 1992. Toxicity Reduction
Evaluation: Toxicant Identification and
Confirmation. Submitted to the City of Lawton,
Oklahoma.
Engineering-Science, Inc. 1993. Review ofDiazinon
Control Options. Submitted to the City of
Lawton, Oklahoma.
EXTOXNET. 1990. Pesticide Information Profile for
Diazinon, Extension Toxicology Network.
Oregon State University, Corvalles, Oregon.
Microbics Corporation. 1982. Microtoxฎ System
Operating Manual. Carlsbad, California.
Mosure, T.E., J.P. Pierko, J.M. Schmidt, R.A.
Monteith, and J.S. Mosser. 1987. Toxicity
Reduction Evaluation at the Akron Water
Pollution Control Station, Draft Report.
Cooperative agreement #CR813681010. USEPA
Office of Research and Development Risk
Reduction Engineering Laboratory, Cincinnati,
Ohio.
Mount, D.I., and T.J. Norberg-King. 1985. Toxicity
Reduction Evaluation: Akron STP - Part I.
USEPA Environmental Research Laboratory,
Duluth, Minnesota.
Neiheisel, T.W., W.B. Horning, B.M. Austern, D.F.
Bishop, T.L. Reed, and J. F. Estemik. 1988.
Toxicity Evaluation at Municipal Wastewater
TreatmentPlants.7oซrwa/o/W2/erPo//H/. Control
Fed. 60(1): 57-67.
TRAC Laboratories, Inc., 1992. Unpublished data.
Prepared by TRAC Laboratories, Inc. Denton,
Texas. '
USEPA. 1987. Toxicity Identification Evaluation of
the Largo, Florida POTW Effluent. J.R. Amato,
D.I. Mount, M. Lukasewycz, E. Durham, and E.
Robert. National Effluent Toxicity Assessment
Center, Duluth, Minnesota.
USEPA. 1988. Memorandum from Donald Mount to
USEPA Region IX. June 25, 1988.
USEPA. 1989a.} Methods for Aquatic Toxicity
Identification Evaluations: Phase II: Toxicity
Identification Procedures. EPA/600/3-88/035.
Office of Research & Development, Duluth,
Minnesota. :
USEPA. 1989b. Methods for Aquatic Toxicity
Identification Evaluations, Phase HI. Toxicity
Confirmation ] Procedures. EPA/600/3-88/036.
Office of Research & Development, Duluth,
Minnesota. ;
USEPA. 1991. Methods for Aquatic Toxicity
Identification Evaluations: Phase I Toxicity
Characterization Procedures. Second Edition.
EPA/600/6-91-003. National Effluent Toxicity
Assessment Center, Duluth, Minnesota.
USEPA. 1992. Toxicity Identification Evaluations:
Characterization of Chronically Toxic Effluents,
Phase I. EPA/600/6-91-005F. National Effluent
Toxicity Assessment Center, Duluth, Minnesota.
USEPA. 1993a. ; Methods for Aquatic Toxicity
Identification Evaluations: Phase II Toxicity
Identification Procedures for Samples Exhibiting
Acute and Chronic Toxicity. EPA/600/R-92-080.
National Effluent Toxicity Assessment Center,
Duluth, Minnesota.
USEPA. 1993b. Methods for Aquatic Toxicity
Identification Evaluations. Phase III Toxicity
Confirmation Procedures for Samples Exhibiting
Acute and Chronic Toxicity. EPA/600/R-92-081.
National Effluent Toxicity Assessment Center,
Duluth, Minnesota.
92
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Appendix B
TRE Case Study:
Central Contra Costa Sanitary District, Martinez, California
Abstract
TRE Goal:
Test Organisms:
TRE Elements:
Toxicant Identified:
Toxicity Controls:
NOEC;>10%
Echinoderms (S. pufpuratus
and D. excentricus)
TIE
Copper
Pretreatment requirements
Summary
Chronic toxicity was detected in a municipal effluent
with the echinoderm fertilization assay. D. excentricus
(sand dollar) appeared more sensitive to the effluent
than did S. purpuratus (purple urchin). A Phase I TEE
was conducted using procedures described by USEPA
(1988a) that were adapted to the echinoderm
fertilization toxicity test. The Phase I TIE implicated
cationic metals as the cause of chronic toxicity, and
follow-up investigations suggested that Cu was the
primary cation responsible. As part of the TIE, toxicity
tests were conducted on ammonia and several cations.
No observable effect concentrations for D. excentricus
were >13.4 jag/L silver (Ag), >9.4 |ag/L Cd, 3.8 to 13.1
(ig/L Cu, >0.7 |ag/L mercury (Hg), and 10 mg/L
nitrogen as total ammonia. The data also suggested
that inter-specific differences in sensitivity to Cu and
ammonia exist between D. excentricus and S.
purpuratus.
Key Elements
1. TIE procedures for freshwater organisms can be
successfully modified to apply with the
echinoderm fertilization toxicity test.
2. This study demonstrated that Cu could have
accounted for the intermittent effluent toxicity
observed.
3. Echinoderms exhibited comparatively high
sensitivity to Cu with EC50s for both species of
approximately 25 ug/L.
4. Source control measures were successful in
reducing Cu concentrations by approximately
25%.
Introduction
Permit Requirements
The Central Contra Costa Sanitary District (CCCSD,
Martinez, California) was required by the State Water
Quality Control Board, San Francisco Bay Region, to
conduct a TRE to identify the chemical constituents in
their final effluent that were responsible for observed
chronic toxicity in the echinoderm fertilization toxicity
tests. Results of monthly compliance tests showed
frequent exceedance of the discharge permit limit
(NOEC ;> 10% effluent).
Description of the Treatment Plant
The CCCSD WWTP provides secondary level
treatment for combined domestic, commercial, and
industrial wastewater from a 126-square mile area with
a population of approximately 400,000. The treatment
plant has an average dry weather design capacity of 45
mgd and currently discharges an annual average flow
of 38.7 mgd into upper San Francisco Bay. Treatment
facilities consist of screening, primary sedimentation,
activated sludge, and secondary clarification followed
by chlorination in contact basins. In the treatment
process, waste-activated sludge is thickened via
flotation thickeners, and lime is added to assist in
dewatering with centrifuges. The combined primary
and waste-activated sludge is dewatered and
incinerated in multiple-hearth furnaces. The effluent
TSS and BOD concentrations average <10 mg/L. Total
ammonia concentrations range from 10-35 mg/L with
an average of 25 mg/L.
93
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Toxicity Identification Evaluation
General Procedures
The echinoderm fertilization toxicity tests weire
conducted on the final effluent according to published
procedures (Dinnel, et al., 1982, as modified by S.
Anderson, 1989) using the West Coast species S.
purpuratus and D. excentricus. The purpose of the test
is to determine the concentration of a test substance
that reduces egg fertilization by exposed sperm relative
to fertilization in a control solution. Two species were
used in this test because the echinoderms are obtained
from feral populations which are gravid at different
times during the year. Effluent samples were 24-hour
flow-proportional composites. Samples were screened
for toxicity within 36 hours of collection. The effluent
salinity was adjusted to 30% using hypersaline brine
(90%), and the pH was adjusted to 8.0 ฑ 0.05.
Phase I TIE Studies
The results of this TIE have been published elsewhere
(Bailey, et al., 1995). The Phase I TIE included the
procedures described by USEPA (1988a). After
completing the TIE manipulations, the effluent was
salinity and pH adjusted as previously noted.
Table B-l. Summary of Results of Phase I TIE Conducted
on Two Effluent Samples with D. excentricus
Treatment
PH3
pHll
Filtration
Aeration
EDTA
Sodium thiosulfate
Post CIS SPE
column
Mcthanol cluate
add-back
Sample 1
No effect on toxicity
Eliminated toxicity
No effect on toxicity
No effect on toxicity
Eliminated toxicity
Eliminated toxicity
No effect on toxicity
No toxicity
Sample 2
Increased toxicity
No effect on toxicity
No effect on toxicity
No effect on toxicity
Eliminated toxicity
Eliminated toxicity
No effect on toxicity
No toxicity
Phase I TIEs were conducted on two effluent samples.
The data for both samples (Table B-l) suggested that
EDTA and sodium thiosulfate were consistently the
most effective treatments in reducing toxicity.
Extraction of the sample with SPE columns did not
reduce toxicity, suggesting that non-polar organics and
weak organic acids and bases were not causes of
toxicity. This conclusion is supported by the fact that
clution of the columns with methanol did not yield
toxicity. The effectiveness of EDTA in eliminating
toxicity suggested that a divalent cation(s) was
responsible for toxicity in the samples tested. The
concurrent effectiveness of sodium thiosulfate in
reducing toxicity suggested that the potential suite of
cations was limited to Cd, Cu, and Hg (USEPA, 1991).
In one case, toxicity also appeared to be increased by
temporarily reducing the sample pH to 3; greater
toxicity at lower pHs has been associated with Cu
(Schubauer-Berigan et al., 1993).
Because the effluent samples contained moderate
levels of ammonia (2025 mg/L total ammonia), the
potential contribution of ammonia to effluent toxicity
was determined by comparison with ammonia toxicity
tests. This approach was taken because the TIE
guidelines evaluate ammonia toxicity by adjusting the
pH of the test solution and preliminary data indicated
that these pH adjustments adversely affected
fertilization success.
Contribution of Ammonia to Toxicity
Ammonia toxicity tests were conducted in natural
seawater spiked with ammonia chloride; fertilization
success was evaluated using logarithmically spaced
concentrations across a range of 1.0 to 100.0 mg/L N
as total ammonia. Test solutions were adjusted to pH
8.0 ฑ 0.05 prior to exposure.
The NOECs for D. excentricus and S. purpuratus were
both 10 mg/L N as total ammonia. Based on the
unionized fraction, the NOECs were Q.21 and
0.17 mg/L N for D. excentricus and S. purpuratus,
respectively (calculated per USEPA, 1988a). However,
large differences existed between the response of the
two species at concentrations higher than the NOEC.
For S. purpuratus, the IC25 was greater than 100 mg/L
N as total ammonia (1.69 mg/L N as unionized
ammonia) compared with an IC25 estimate of 16.5
mg/L N (0.34 mg/L N as unionized ammonia) for D.
excentricus. Because the upper limit of ammonia
concentrations in the effluent was 25 mg/L N as total
ammonia, these results suggested that ammonia alone
could not account for NOECs that were ^33% effluent,
a concentration that would correspond to a maximum
of 8.25 mg/L N as total ammonia.
Identification and Confirmation of the Role of
Cationic Metals
Sensitivity of echinoderms to cationic metals
Once it appeared that a divalent cation was responsible
for the effluent's toxicity, candidate metal ions (Cd, Cu,
and Hg) and Ag were evaluated for toxicity with
94
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D. excentricus and S. purpuratus. Metal solutions
were prepared in moderately hard freshwater (USEPA,
1991) using reagent grade salts of Cu, Cd, and Hg.
The CCCSD also was concerned about the potential
for Ag to contribute to effluent toxicity; therefore, tests
were performed with silver salts. Stock concentrations
of metals were confirmed by either graphite furnace
(Ag, Cu, and Cd) or cold vapor (Hg) AA spectroscopy
(APHA, 1989). Hypersaline brine was then added (1/3
brine:2/3 metal solution) to bring the salinity to 30%,
and the pHs of the solutions were adjusted to 8.0 ฑ
0.05 prior to exposure. This procedure was analogous
to the preparation of the effluent samples prior to
testing. Serial dilutions that incorporated a 50%
dilution factor were made from the stock solutions to
achieve exposure concentrations that bracketed those
found in the effluent. The NOECs from multiple
toxicity tests on Ag, Cd, Cu, and Hg with D.
excentricus and S. purpuratus are summarized in Table
B-2. Side-by-side comparisons between the two
species are shown by the paired values in the table.
Table B-2. NOECs Obtained for/), excentricus and
S. purpuratus Exposed to Different Metals*
Metal
Ag
Cd
Cu
Hg
'. NOECS (ug/L)
D. excentricus
>13.4
>9.4
>67.0
10.0
13.1
5.4
3.8
8.0
>0.7
>2.2
S. purpuratus
>13.4
Not tested
>67.0
20.0
19.7
Not tested
Not tested
Not tested
>0.7
Not tested
* When seasonally available, concurrent tests were conducted
with both species. Values given as jig metal/L (Bailey et al.,
1995).
In some cases, seasonal spawning constraints
precluded conducting concurrent tests with S.
purpuratus. One comparison was conducted with Ag;
the NOECs for both species were >13.4 ng/L. Two
tests were conducted with Cd; in both cases the highest
concentrations tested (9.4 and 67.0 (ig/L) failed to
produce any measurable effects on fertilization
success. Five tests were performed on Cu with D.
excentricus. The NOECs ranged between 3.8 and 13.1
ug/L with an average of 8.1 pg/L. In two of three
concurrent tests with S. purpuratus, the NOECs were
1.5 to 2 times greater than those obtained with D.
excentricus. In two tests with Hg, no effects on
fertilization success were found at concentrations up to
0.7 and 2.2 ug/L, respectively.
Comparison of toxic concentrations of metals
with concentrations found in the effluent
The NOECs for each of the metals were compared
with the discharger's analytical records to determine
which metals were present individually in the effluent
at concentrations high enough to inhibit fertilization
success. Toxicity ratios were calculated for each metal
[metal concentration in effluent (|ig/L) -=- NOEC
(ug/L)]. A ratio greater than 1 suggested that the
metal(s) was present in the effluent at concentrations
high enough to produce toxicity. Conversely, a ratio of
1, or less, suggested that the concentration of metal
was 13.4
>9.4; >67
3.8-13.1
>0.7; >2.2
Ratio
<;0.3
sO.2
0.4-5.3
<;0.6
* Values given as 7.5 (80.0 ฑ 2.0)ug metal/L.
Confirmation of the role of Cu in effluent toxicity
The next confirmation step compared fertilization
success in an effluent sample against that in seawater
spiked with copper sulfate (CuSO4) to the same
concentration found in the effluent. These exposures
were conducted simultaneously using the same
gametes fromD. excentricus. Fertilization success also
95
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was evaluated in an effluent sample spiked with
different concentrations of Cu, such that subsamples of
the effluent contained 1, 2, and 3 times the amount of
Cu (measured concentrations) as the original sample.
Serial dilutions, which incorporated a 50% dilution
factor, were then prepared from the unspiked and 2x
spiked samples and fertilization success evaluated with
ฃ>. excentricus. Depending on the results, it could be
determined whether Cu was responsible for toxicity in
the effluent. The reasoning was if Cu was the primary
factor controlling toxicity, then the LOECs and
NOECs obtained for the spiked and unspiked samples
should be the same, based on Cu concentration.
Similarly, based on percent effluent, the NOEC and
LOEC associated with the spiked sample should be one
dose level lower than in the unspiked sample.
The results of parallel toxicity tests with D. excentricus
on effluent and seawater spiked with Cu at
concentrations found in the effluent are summarized in
Table B-4. Based on Cu concentration, the NOECs
and LOECs were the same between the effluent sample
and the concurrent toxicity test with seawater spiked
with Cu. Furthermore, the percent fertilization was
similar at corresponding Cu concentrations in both
toxicity tests. These data suggested that Cu accounted
for the reduction in fertilization success associated with
this effluent sample. Fertilization success in an
effluent sample and the same sample spiked with Cu is
shown in Table B-5.
Discussion
The data demonstrated that procedures for conducting
TIEs with freshwater organisms can be successfully
applied to the echinoderm fertilization toxicity test.
Table B-4. Comparison of NOECs, LOECs, and Percent
Fertilization Obtained withD. excentricus Exposed to Effluent
and Seawater Spiked with Cu
Treatment
Effluent
Seawater Cu
spike
: NOEC* .
3.8 (89.3 ฑ 3.0)
: 7.5 (80.0 ฑ2.0)
:'LOEC*;:''
7.5 (73.3 ฑ6.1)
7.5 (80.0 ฑ 2.0)
* Percent fertilization given in parentheses (mean ฑ SD).
The results of this study suggest that Cu could have
accounted for the intermittent toxicity demonstrated by
the echinoderm fertilization test. Of the four metals
identified in the Phase TIE, Cu was the only one that
occurred in the effluent at concentrations that
overlapped the toxic range. Confirmatory studies
conducted with two different effluent samples also
showed that Cu could account for the adverse effects
observed with the whole effluent. Paired tests also
suggested that Cu exhibited greater toxicity to
D. excentricus than to S. purpuratus. This is important
because S. purpuratus generally exhibited less
sensitivity to the effluent.
Source control measures implemented by the CCCSD
successfully reduced Cu concentrations in the effluent
by 25%. This reduction made it difficult to obtain
samples with sufficient toxicity to fully complete the
confirmatory phase of the TEE. In fact, nearly all the
samples tested at the end of the TIE failed to produce
a measurable response with S. purpuratus.
Acknowledgments
This work was supported wholly or in part by the
CCCSD, Martinez, California. Bart Brandenburg,
Table B-5. Percent Fertilization Obtained with D. excentricus Exposed to Effluent and Effluent Spiked with Cu*
Unspiked effluent
Effluent (%)
0.0
8.4
16.8
33.5
67.0 (IxCu)
ug/LCu
0.0
0.8
1.6
3.3
6.6
Fertility (%)
96.0 ฑ 2.5 ,
96.7 ฑ3.1,
97.3 ฑ 1.2
91.3 ฑ1.2
82.0 ฑ 4.7f
Effluent spiked with Cu
Effluent (%)
0.0
8.4
16.8
33.5 (IxCu)
67.0 (2xCu)
67.0 (3xCu)
ug/LCu
0.0
1.6
3.3
''. 6.6
13.2
19.8
Fertility (%).'
96.0 ฑ 2.5
90.7 ฑ 2.3
90.3 ฑ 2.3
83.3 ฑ 2.7|
74.8 ฑ 2.2f
71.7 ฑ 12.9f
* Fertilization data are the means and standard deviations of three replicates.
t Significantly less than controls; p < 0.05.
96
-------
Bhupinder Dhaliwal, and Jim Kelly of the CCCSD
managed the various aspects of this project. The TRE
studies were conducted at AQUA-Science, Davis,
California, under the direction of Jeffrey L. Miller,
Ph.D., and Michael J. Miller.
References
American Public Health Association (APHA),
American Water Works Association, and Water
Pollution Control Federation. 1989. Standard
Methods for the Examination of Water and
Wastewater. 17th ed. American Public Health
Association, Washington, D.C.
Anderson, S.L. 1989. Effluent Toxicity
Characterization Program. Report to the San
Francisco Bay Regional Water Quality Control
Board. Oakland, California.
Bailey, H.C., J.L. Miller, M.J. Miller, and B.S.
Dhaliwal. 1995. Application of Toxicity
Identification Procedures to the Echinoderm
Fertilization Assay to Identify Toxicity in a
Municipal Effluent. Environ. Toxicol. Chem. 14:
2181-86.
Dinnel, P.A., Q.J. Stober, S.C. Crumley, and R.E.
Nakatani. 1982. Development Of A Sperm Cell
Toxicity Test For Marine Waters. In J.G. Pearson,
R.B. Foster, and W.E. Bishop, eds. Aquatic
Toxicology and Hazard Assessment: Fifth
Conference. STP 766. American Society for
Testing and Materials, Philadelphia, Pennsylvania.
Schubauer-Berigan, M.K., J.R. Dierkes, P.D. Monson,
and G.T. Ankley. 1993. pH Dependent Toxicity
of Cd, Cu, Ni, Pb, and Zn to Ceriodaphnia dubia,
Pimephales promelas, Hyalella azteca, and
Lumbriculus variegatus. Environ. Toxicol. Chem.
11: 1261-66.
USEPA. 1988a. Methods For Aquatic Toxicity
Identification Evaluations: Phase I Toxicity
Characterization Procedures. EPA/600/3-88/034.
National Effluent Toxicity Assessment Center,
Duluth, Minnesota.
USEPA. 1988b. Methods For Aquatic Toxicity
Identification Evaluations: Phase II Toxicity
Identification Procedures. EPA/600/3-88/035.
National Effluent Toxicity Assessment Center,
Duluth, Minnesota.
USEPA. 1988c Methods For Aquatic Toxicity
Identification Evaluations: Phase III Toxicity
Confirmation Procedures. EPA/600/3-88/036.
National Effluent Toxicity Assessment Center,
Duluth, Minnesota.
USEPA. 1991. Methods for Aquatic Toxicity
Identification Evaluations: Phase I, Toxicity
Characterization Procedures. Second Edition.
EPA/600/6-91-003. National Effluent Toxicity
Assessment Center, Duluth, Minnesota.
USEPA. 1995. Short-Term Methods for Estimating
the Chronic Toxicity of Effluents and Receiving
Waters to West Coast Marine and Estuarine
Organisms. EPA/600/R-95/136. National
Exposure Research Laboratory, Cincinnati, Ohio.
97
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Appendix C
! i
TRE Case Study:
City of Reidsville, North Carolina
Abstract
TRE Goal:
Test Organism:
TRE Elements:
Toxicant Identified:
Toxicity Controls:
NOEC >90%
C. dubia
TIE and Toxicity Tracking
Assessment (RTA)
Surfactants
Pretreatment requirements
Summary
The TRE study used a novel approach to identify the
sources of POTW effluent toxicity. Subsequent
modifications in chemical usage by industrial
contributors successfully reduced effluent toxicity to
the NOEC limit in 1994. Further studies are in
progress to ensure consistent compliance with the
toxicity limit.
Key Elements
1. Other TRE procedures can be used if the TIE
cannot identify the effluent toxicants. One such
procedure uses a toxicity-based tracking approach
to locate the sources of toxicity in municipal
collection systems.
2. The toxicity-based tracking approach, referred to
as the RTA procedure, can be adapted to fit the
site-specific conditions at each POTW.
3. Once identified, the toxic contributors can be
required through the industrial pretreatment
program to reduce the discharge of toxicity.
Practical control techniques are available to
industries, including substitution of toxic
chemicals, waste minimization, and pollution
prevention.
Introduction
The City of Reidsville was required by the North
Carolina Division of Environmental Management
(NCDEM) to conduct a TRE based on evidence of
chronic effluent toxicity at its POTW. Monthly
NOECs for C. dubia have averaged about 35% effluent
since 1992. These values show that chronic effluent
toxicity has consistently exceeded the discharge permit
NOEC limit of 90% effluent.
Background
In 1992, the City submitted a TRE plan and initiated
TIE studies to determine the cause(s) of the effluent
toxicity. Chronic TIE Phase I (Tier I) tests indicated
that surfactants were the principal toxicant group. This
evidence was based on toxicity reduction by filtration,
aeration, and CIS SPE in the Phase I tests. TIE Phase
n tests were performed to try to identify the toxic
surfactant compounds; however, the results were
inconclusive because of the difficulty in isolating the
toxicants and the lack of conventional analytical
techniques for surfactant compounds. The toxicants
removed by the CIS SPE column were recovered by
eluting the column with methanol, but toxic
compounds could not be identified in the column
extract (Burlington Research Inc., 1993).
In cases where the TIE is not successful in identifying
the effluent toxicants, other TRE steps can be used to
gather information on the nature and sources of
effluent toxicity. USEPA and several municipalities
have worked together in USEPA funded studies to
develop the RTA method, which can be used to assess
the potential toxicity contribution from indirect
dischargers in sewerage collection systems (USEPA,
1989a; Botts et al., 1987; Morris et al, 1991; Fillmore
et al., 1990; Collins et al., 1991). The RTA procedure
involves treating industrial wastewater samples in a
bench-scale, batch simulation of the POTW, and
measuring the resulting toxicity. The toxicity
98
-------
remaining after batch treatment, referred to as
refractory toxicity, represents the toxicity that passes
through the POTW and is discharged in the effluent.
Several municipalities have successfully used the RTA
procedure to identify industrial sources of toxicity
(Botts et al., 1992; Morris et al., 1991; and
Engineering-Science, Inc., 1992).
Description of Treatment Plant
The major treatment processes at the Reidsville POTW
are extended activated sludge treatment and filtration.
Influent wastewater, which averages 2.8 mgd, is
initially screened and then treated in two activated
sludge aeration basins equipped with mechanical
surface aerators. Both carbonaceous BOD and
ammonia are removed in this single-stage aeration
system. After 48 hours contact time, the basin effluent
flows to the final clarifiers for solids clarification. The
clarified effluent is then passed through sand filters to
remove remaining suspended solids that may
contribute to effluent BOD. The filter effluent is
disinfected with chlorine gas and dechlorinated and
aerated prior to discharge. Waste activated sludge is
thickened and aerobically digested for land application.
Refractory Toxicity Assessment Procedure
Selection of Industries for Testing
Acute and chronic toxicity tests were performed on
raw wastewater from the seven permitted significant
industrial users in the Reidsville collection system.
The industrial wastewater samples were tested at
concentrations that reflected the average flow
contribution of the industries to the POTW (dilutions
were made with reconstituted lab water).
The results showed that five of the seven industries
were contributing chronic toxicity to the POTW (Table
C-l). It is possible that at least some of the raw
wastewater toxicity would be removed by treatment at
the POTW; therefore, the five toxic industrial users
were selected for further evaluation by RTA testing. A
description of the industries evaluated in the RTA is
provided in Table C-2.
Test Procedure
A step-by-step description of the RTA procedure is
given in Section 5 and Appendix J. The generic
procedure must be adapted to simulate the treatment
processes and operating conditions at each POTW.
Several types of treatment processes can be simulated,
including conventional activated sludge systems (Botts
et al., 1987; Morris et al., 1991; and Fillmore et al.,
Table C-l. Chronic Toxicity of Raw Industrial
Wastewaters
Industry
A
B
C
D
E
F
G
C. dubia Chronic Pass/Fail Result*
May
1992
Fail
Fail
Fail
Fail
Pass
Pass
Pass
June
1992
Fail
NTf
Fail
NT
Pass
Pass
Pass
July
1992
Fail
Fail
Fail
Fail
Fail
Pass
Pass
April
1993
Fail
Fail
Fail
Fail
Fail
NT
NT
* Tests were conducted using industrial wastewater diluted
according to its percent contribution to the total POTW
influent.
t NT = Not tested.
Table C-2. Description of Industries Evaluated in the
RTA
Industry
A
B
C
D
E
Domestic
Type
Textile
Tobacco
Products
Can
Making
Food
Processing
Metal
Finishing
Flow
(mgd)
1.072
0.308
0.085
0.189
0.031
%Flow* to
POTW
65
28
10
12
2
38
* Based on maximum industrial flow and minimum
POTW influent flow, except for domestic, which is
based on average flow and minimum POTW influent
flow.
1990), single and two-stage nitrification processes
(Collins et al., 1991), and BNR systems (Botts et al.,
1992).
The RTA simulated the two main treatment processes
at the Reidsville POTW: the activated sludge and sand
filtration processes. Wastewater samples were first
treated in biological reactors and then the clarified
effluents were passed through a bench-scale sand filter
column.
99
-------
Two types of simulations were tested as shown in
Figure 5-2 (see Section 5). A control simulated the
existing treatment conditions and treated only the
POTW influent. The second simulation evaluated the
addition of die industrial discharge to the POTW and
treated the industrial wastewater spiked into the POTW
influent.
The amount of industrial wastewater spike represented
the conservative condition of maximum industrial flow
and minimum total influent flow at the POTW. The
operating conditions for the simulations are described
in Table C-3.
Table C-3. Comparison of Operating Conditions for the
City of Reidsville POTW Processes and RTA Simulation
Tests
POTW Process
Specifications
Treatment
Plant
RTA
Simulation
Activated Sludge Process
Mixed liquor solids
(mg/L)
DO (mg/L)
Treatment period
(hours)
2,200-2,500
>2
48
2,240-2,740
2.4-9.2
48
Sand Filter Process
Filtration rate
(gpm/sf)
Total filter area (sf)
Sand particle size
(mm)
Sand depth (inches)
Water depth on
filter (ft)
Backwash rate
(gpm/sf)
0.8
2,520
0.45
10
0-7
12
0.8
0.09
0.45
10
0.1-2.5
5 (estimated)
The results of the control and spiked simulations are
compared to determine whether addition of the
industrial wastewater increases effluent toxicity. Ah
industry would be considered a source of toxicity if the
effluent of the spiked simulation is more toxic than the
control effluent.
Sampling
Three rounds of RTA tests were performed over a
4-month period. Twenty-four hour composite samples
of the industrial wastewaters, POTW influent,
domestic wastewater, and POTW effluent were
collected for testing. In addition, a grab sample of the
POTW RAS was collected on the day of testing.
Domestic wastewater was tested because TRE studies
at other municipalities have shown that domestic
sources can contribute to effluent toxicity (Botts et al.,
1990). The POTW effluent served as a baseline for
comparison with the: RTA control to determine if the
treatment performance of the simulations and the
POTW were similar)
Toxicity Monitoring
Following biological treatment, the clarified reactor
effluents were passed through the sand filter column
and the resulting filtrates were tested for chronic
toxicity using C. dubia, the test species specified in the
NPDES permit. Each RTA effluent sample was used
for both test initiation and renewals on days 3 and 5 of
the toxicity test (USEPA, 1989b).
Results ;
Source Characterization
Two rounds of RTA tests were used to characterize the
sources of toxicity. As shown in Figure C-l, the
effluent TUc for the two control simulation tests in
Round 1 were 3.8 and 3.1. These values compare well
with the POTW effluent (TUc =3.6). The control
simulation effluents in Round 2 also exhibited similar
toxicity (TUc =3.0 and 2.9) as the POTW effluent
(TUc =3.4). These results indicate that the RTA test
accurately simulated the POTW with respect to toxicity
treatment. !
As shown in Figure C-l, the effluent from the
simulation spiked with Industry A wastewater was
about twice as toxic (TUc=6.7) as the control effluents
in both rounds of tests. Effluent TUc values for the
simulations spiked with other industrial wastewaters
were similar to or less than the effluent TUc for the
controls.
The results of both rounds of testing indicate a
potential for Industry A to contribute toxicity to the
POTW. The results for the simulations spiked with the
other industrial wastewaters suggest that Industries B,
C, D, and E do not contribute measurable toxicity to
the POTW.
Toxicity Confirmation
A recent study for a New Jersey municipality found
that an industry was contributing toxicity in amounts
100
-------
Round 1 Toxicity Results
6_
u 5
O A
I i
H 3 -
o
1 2
a
v
ซ *
_g.
%flil
5SO.l1
2^i**s : ^i^i
l^ ^ ง
^ ง: '
Control Control POTW A B C D
Simulation Tests
Note: Industry E was not tested in round 1.
Round 2 Toxicity Results
6_
u
i
O A -
^ 4
I ,
H 3
'I
r\
^
%
^ f ?,
^&.v v
1 C*
E
i
Domestic
*"j p
^ s 5
Control Control POTW A B C D
Simulation Tests
1
E
^ %
;c5
i
i
Domestic
1010P-10
Figure C-l. Results of RTA (rounds 1 and 2).
101
-------
high enough to mask other smaller sources of toxicity
(Morris et al., 1991). It was necessary to remove the
larger source of toxicity from the RTA test regime
before other significant sources could be identified.
The City of Reids ville decided to conduct a third round
of tests to determine if a similar situation was
occurring at their POTW.
Round 3 involved using a mock influent that did not
contain Industry A wastewater. The mock influent was
used in lieu of the POTW influent for the controls and
the spiked simulations. The mock influent consisted of
samples collected from each major sewer line with the
exception of the sewer receiving Industry A
wastewater.
Toxicity results for the RTA simulation effluents are
presented in Figure C-2. A comparison of results
shows that the effluent of the Industry A spiked
simulation was several times more toxic (TUc=6.8)
than the control effluent (TUc =1.2). These results
provide further evidence that Industry A is a source of
toxicity. The simulations spiked with Industry C and
D wastewater had similar effluent toxicity (TUc=1.3
for both) compared to the control. These data indicate
that Industries C and D are not contributing significant
toxicity to the POTW.
The simulation spiked with domestic wastewater had
greater effluent toxicity (TUc=2.3) than the control
(TUc=1.2). These results suggest that this waste
stream may be a source of toxicity; however, results of
Round 1 and 2 indicate that domestic wastewater
collected from other areas of the collection system is
not a problem. Further studies are planned to evaluate
the potential toxicity contribution from domestic
sources throughout the collection system.
Discussion ,
The results of this study indicate that Industry A is a
major contributor to chronic effluent toxicity at the
Reidsville POTW. None of the other industries (B, C,
D, and E) were found to discharge measurable toxicity
even after the potential toxicity interference from
Industry A was removed.
In January 1994, the City of Reidsville implemented a
program to minimize or eliminate the discharge of
industrial chemicals that may contribute to the POTW
effluent toxicity. Although the RTA results indicated
Round 3 Toxicity Results
Control Control POTW
A B C
Simulation Tests
Domestic
Note: A replicate control and POTW effluent were not tested in round 3. Industries B and E were not indicated to be sources
of toxicity in rounds 1 and 2; therefore, these industries were not tested in round 3.
1010P-11
Figure C-2. Results of RTA (round 3).
102
-------
that Industry A is the major contributor of chronic
toxicity, all of the City's eight permitted industrial
users were requested to participate. The program
involved:
An evaluation of current chemical usage and the
selection of alternative materials of low toxicity,
low inhibition potential, and high biodegradability.
An on-site evaluation of waste-minimization
practices by the North Carolina Office of Waste
Reduction.
Particular attention was given to surfactant products or
chemicals with surfactant constituents because the TIE
had indicated surfactants to be the principal toxicant in
the POTW effluent. Industries were requested to
maintain chronological records of changes in chemical
usage, production, and housekeeping practices. These
records were used to compare the timeline of industry
modifications to results of chronic toxicity monitoring
at the POTW.
Follow-up monitoring results showed a substantial
reduction in effluent toxicity. Beginning in March
1994, the IC25 values (an endpoint that approximates
the NOEC) for 7 of 10 monthly C. dubia toxicity tests
were 2:90%. A review of the industries' chronological
records established a correlation between toxicity
reduction and chemical optimization practices,
especially those implemented at Industry A.
However, in 1995 occasional chronic effluent toxicity
was again observed., Since early 1997, the effluent has
exhibited consistent chronic toxicity
(NOEC=30-45%). Current studies are focusing on
treatment with polymer, which has shown to reduce
toxicity in bench-scale tests. The City is also working
with the industries to implement additional chemical
optimization and waste minimization practices. In
addition, construction is underway to extend the outfall
from a small creek to a river, which will afford greater
dilution. In 1998, the City will need to meet a revised
chronic toxicity limit of an NOEC of approximately
61%.
Summary
The RTA protocol was initially developed as part of
TRE research studies funded by the USEPA Risk
Reduction Engineering Laboratory in Cincinnati, Ohio.
The procedure was intended to be used by
municipalities as a tool for tracking sources of toxicity
in sewer collection systems; however, the RTA
approach has evolved to suit other purposes. In
addition to toxicity tracking (Collins et al., 1991), the
RTA protocol has been used to determine the
compatibility of planned discharges to POTWs
(Engineering-Science, Inc., 1992, 1993) and to
establish compliance with toxicity-based pretreatment
limits (Morris et al., 1991).
Acknowledgments
Burlington Research, Inc. (Burlington, North Carolina)
and Engineering-Science, Inc. (Fairfax, Virginia),
conducted the TRE study. Burlington Research, Inc.,
performed the TIE tests, with assistance by EA
Engineering, Science and Technology, Inc. (Sparks,
Maryland), and the evaluation of industrial chemical
usage. Engineering-Science, Inc., performed the RTA
study. Burlington Research, Inc., and Engineering-
Science, Inc., acknowledges Mr. Jerry Rothrock
(Director of Public Works, City of Reidsville), and
Donald Waddell, LisaHaynes, Mitzy Webb, and James
Fain (Hydro Management Services) for their
assistance.
The material presented in this appendix includes
copyrighted data presented in a technical paper for the
67th Annual Water Environment Federation Conference
(Botts et al., 1994). WEF has granted permission to
include the data in this document.
References
Botts, J.A., J.W. Braswell, E.G. Sullivan, W.L.
Goodfellow, B.D. Sklar, and A.G. McDearmon.
1987. Toxicity Reduction Evaluation at the
Patapsco Wastewater Treatment Plant. Risk
Reduction Engineering Laboratory, Cincinnati,
Ohio. Cooperative Agreement No. CR812790-01-
1. NTIS # PB 88-220 488/AS.
Botts, J.A., L.B. Fillmore, E.J. Durban, W.A.
Goodfellow, T. Pereira, and D.F. Bishop. 1990.
Evaluation of the Role of Diazinon in the Toxicity
of a Municipal Wastewater Treatment Plant
Effluent Proceedings of the Third National
Pesticide Conference, November, 1990,
Richmond, Virginia.
Botts, J.A., T.L. Morris, J.E. Rumbo, and C.H.
Victoria-Rueda. 1992. "Case Studies - Munici-
palities." In Toxicity Reduction: Evaluation and
Control. D.L. Ford, ed. Technomic Publishing
Co., Lancaster, Pennsylvania.
103
-------
Bolts, J.A., T. Schmitt, E. Wilson, M. Collins, D.
Waddell, R. Diehl, and L. Ehrlich. 1994.
Refractory Toxicity Assessment: An Alternative
Approach for Chronic Toxicity Reduction
Evaluations. Technical paper presented at the
Annual Conference and Exposition of the Water
Environment Federation, Chicago, Illinois. Paper
#AC944404.
Burlington Research, Inc. 1993. Toxicity Reduction
Evaluation: CityofReidsville, Phase I/II Report.
Burlington, North Carolina.
Collins, M.A., T.L. Morris, J.A. Botts, T. Norberg-
King, J. Thompson, and D.I. Mount. 1991.
Chronic Toxicity Reduction Evaluation Study at
the Bergen County Utilities Authority Wastewater
Treatment Plant. Draft Report, 1991. USEPA,
Risk Reduction Engineering Laboratory,
Cincinnati, Ohio. USEPA Contract No.
68-03-3431.
Engineering-Science, Inc. 1992. Results of the
Refractory Toxicity Assessment for an Auto Parts
Manufacturer. Prepared by Engineering-Science,
Fairfax, Virginia.
Engineering-Science, Inc. 1993. Results of the
Refractory Toxicity Assessment for a Store
Fixtures Manufacturer. Prepared by Engineering-
Science, Fairfax, Virginia.
Welch, and J.A
Fillmore, L.B., T.L. Morris, T.L. Champlin, M.C.
.Botts. 1990. Toxicity Reduction
Evaluation at the City of Fayetteville Cross Creek
Wastewater Treatment Plant. Draft Report, 1990.
USEPA, Risk Reduction Engineering Laboratory,
Cincinnati, Ohio. USEPA Contract No. 68-03-
3431. :
Morris, T.L., G. Fare, and J. Spadone. 1991. Toxicity
Reduction Evaluation at the Linden Rosette
Sewerage Authority Wastewater Treatment Plant.
Presented at the 64th Water Pollution Control
Federation Conference, Toronto, Ontario, October
7-10, 1991. #AC91-055-001.
USEPA. 1989a. ; Toxicity Reduction Evaluation
Protocol for Municipal Wastewater Treatment
Plants. EPA/600/2-88/062. Risk Reduction
Engineering Laboratory, Cincinnati, Ohio.
USEPA. 1989b. Short-term Methods for Estimating
the Chronic Toxicity of Effluents and Receiving
Waters to Freshwater Organisms. EPA/600/4-
89/001. Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio.
USEPA. 1991b. technical Support Document for
Water Quality-Based Toxics Control.
EPA/505/2-90/001. Office of Water, Washington,
D.c. ;
104
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Appendix D
TRE Case Study:
City of Durham, North Carolina
Abstract
TRE Goal:
Test Organism:
TRE Elements:
Toxicant Identified:
Toxicity Controls:
NOEC = 100%
C. dubia
Toxicity treatability
evaluation
TEE not performed
Proceeded with planned
POTW upgrades
Summary
The City of Durham evaluated the expected toxicity
reduction to be achieved by planned upgrades of their
POTWs. Chronic toxicity reduction was evaluated
through the use of bench-scale simulations of the
upgraded POTWs. Results indicated that the new
POTWs would reduce chronic toxicity to compliance
levels. Based on this evidence, the TRE was waived
until the new POTWs were online and effluent toxicity
reduction could be confirmed. The upgraded POTWs
became operational in late 1994 and effluent
monitoring results have shown no chronic toxicity after
consistent treatment performance was achieved.
Key Elements
The TRE study used a unique approach to evaluate
chronic toxicity reduction. This approach may be
useful to other .municipalities that have TRE
requirements, yet are planning upgrades of their
POTWs. The key elements of interest in the City of
Durham study include the following:
1. In cases where POTW staff are planning to
upgrade their POTWs, it may be more practical to
evaluate the toxicity reduction to be achieved by
the upgrade than to conduct TIE tests on the
existing POTW effluent. The treatability approach
is recommended when the upgrade is expected to
improve toxicity reduction, such as nitrification
treatment for ammonia removal; however,
additional evidence is needed to confirm the
expectation.
2. A bench-scale simulation of the upgraded
treatment system can be used to generate an
effluent that is similar to the effluent expected for
the new POTW. Calibration tests should be
performed to ensure that the quality of the
simulation effluent is similar to that of the planned
POTW effluent.
3. The treatability approach should be thoroughly
described in the TRE plan and the regulatory
authority should accept the plan prior to testing.
Introduction
Permit Requirements
Since 1987, NCDEM has required the City of Durham
to monitor the effluents of its four POTWs for chronic
toxicity using the North Carolina pass/fail test. The
pass/fail test consists of 10 replicates of the effluent at
the critical instream waste concentration (IWC) and a
control. The effluent test concentrations corresponding
to the IWC were 63.8% for the Eno River POTW,
100% for Lick Creek POTW, 98.7% for Farrington
Road POTW, and 100% for Northside POTW. The
test results indicated unacceptable levels of chronic
effluent toxicity for each of the four POTWs. In each
case, a statistically lower number of C. dubia young
were observed in the effluent concentration as
compared to the control.
Based on the effluent toxicity monitoring results,
NCDEM required the City of Durham to initiate a TRE
in January 1990. The goal of the TRE was to identify
methods for reducing chronic effluent toxicity to
acceptable levels at each of the treatment plants by
105
-------
January 1991. The City of Durham submitted a plan
within 60 days that described a unique approach for
implementing the TRE program.
Instead of the traditional TRE approach of testing the
existing effluents, the City proposed to evaluate the
expected chronic toxicity reduction to be achieved by
planned upgrades to the POTWs. Toxicity reduction
would be evaluated through the use of bench-scale
simulations of the upgraded POTWs. This approach
was favored over conventional TRE methods, such as
TIE tests, because it was anticipated that the degree
and nature of the effluent toxicity would change upon
startup of the new treatment plants.
Description of the Treatment Plants
In 1990, the City of Durham, North Carolina, had four
POTWs: Eno River (2.5 mgd), Farrington Road (13
mgd), Lick Creek (1.5 mgd), and Northside (10 mgd).
In anticipation of the need for additional treatment
capacity, the City decided to close the Eno River and
Lick Creek treatment plants and divert the flow to an
expanded Northside plant. At the same time, NCDEM
established draft permit limits for several parameters,
including phosphorus. The new permit limits would
require advanced wastewater treatment; therefore, in
addition to the Northside plant expansion, the City of
Durham decided to upgrade the Northside and
Farrington Road POTWs plants to include BNR
treatment.
During the TRE, the Northside POTW comprised
primary treatment followed by trickling filters, a
single-stage nitrification process, secondary
clarification, and chlorine disinfection. The Northside
POTW upgrade involved building a new treatment
system in parallel with the existing system, which
would treat the flow diverted from the former Eno
River and Lick Creek plants. The new treatment
system was planned to consist of primary clarifiers and
a five-stage BNR process designed to remove nitrogen
and phosphorus. Effluents from the new and existing
treatment systems will be combined, treated with
aluminum sulfate (alum), passed through a filtration
process, and disinfected by UV light prior to discharge
to Ellerbe Creek.
The Farrington Road POTW was planned to be
converted from a two-stage nitrification process to a
five-stage BNR process similar in design to that
planned for the Northside plant. Final effluent
treatment, like the Northside plant, will involve alum
treatment, filtration, and UV disinfection.
Wastewater Treatment Plant Simulations
The new treatment processes for the Northside and
Farrington Road POTWs were planned to be similar;
therefore, the simulation designs were nearly identical.
A batch mode of operation instead of a continuous
flow mode was selected to reduce study costs. Both
simulations, as shown in Figure D-l, comprised a BNR
process, followed by alum flocculation, settling, and
effluent filtration. Phosphorus and nitrogen removal
was achieved in the; BNR process, which involved
treating the influent wastewater with activated sludge
in five consecutive stages (anaerobic, anoxic, aerobic,
anoxic, and aerobic).; The BNR process effluent was
then treated with aliim and passed through a dual
media filter column to remove additional phosphorus.
Chronic toxicity tests using C. dubia (USEPA, 1989)
were performed on the final simulation effluents to
evaluate the expected effluent quality of the full-scale
treatment systems. '
Simulation of the Northside POTW involved treating
the combined influents of the three POTWs scheduled
for consolidation: the Eno River, Lick Creek, and
Northside plants. The influents were combined in
proportion to their respective flow rates. The
Farrington Road POTW influent was used directly in
the simulation tests of the Farrington Road facility.
Each simulation influent was settled for approximately
2 hours to simulate primary sedimentation.
The activated sludge used in the simulations was
collected from a municipal treatment plant that had a
BNR process similar to the system planned for the City
of Durham POTWs.; RAS was collected from the
plant's clarifier return line and mixed liquor solids
were collected from the aeration basins. RAS was
mixed with the simulated primary effluent in the first
BNR simulation stage (anaerobic). Phosphorus
removal was enhanced in the subsequent BNR stages
by replacing a portion of the RAS with nitrate rich,
aeration basin sludge. The nitrate was an essential
source of oxygen for phosphorus removing bacteria in
the BNR anoxic stage.
Following biological treatment, the activated sludge
was settled and the clarified effluent was withdrawn
and treated with alum. Alum treatment involved flash
mixing and settling. The clarified supernatant was then
106
-------
RAS
Primary Effluent
15-Gallon
Vessel
Replace RAS
with Nitrate
Rich Sludge
Air
Anaerobic
Stage
1st Anoxic
Stage
Aerobic
Stage
2nd Anoxic
Stage
Air
Alum
Supernatant
2nd Aerobic
Stage
Clarification
Stage
Alum Treatment
Stage
4-Inch Diameter
Column
18 Inches
Anthracite
12 Inches Sand
Final
Effluent
Filtration Stage
Figure D-l. Flow diagram for waste-water treatment simulations.
passed through an anthracite/sand filter column, which
was operated in a constant headless mode. Prior to
testing, the anthracite and sand in the filter columns
was distributed by backwashing the columns in the
upflow direction using tap water. The filter columns
were then rinsed with deionized water in the
operational (downflow) mode.
The general operating conditions for the treatment
simulations are shown in Table D-l. Some of the
operating procedures for the simulations were modified
during calibration testing to achieve the desired
treatment performance.
Calibration of the Treatment Simulations
Prior to the toxicity evaluation, calibration tests were
performed to match the simulation performance to
expected performance for the upgraded POTWs. Also,
several toxicity tests were performed during the
calibration testing to verify that the simulation
materials and additives (i.e., activated sludge, alum)
would not introduce unexpected toxicity. The toxicity
tests followed USEPA procedures (1989) for C. dubia,
the test organism specified in the City's discharge
permits.
The calibration testing involved varying the operation
of the simulations and monitoring the resulting effluent
quality. The objective was to achieve a reduction in
influent concentrations of BOD5, COD, TKN, NH3-N,
NO3-N, TP, PO4-P, and TSS to levels approximating
those expected in the effluents of the planned treatment
plants. Treatment performance was evaluated by
varying the treatment times for each step.
The treatment times evaluated during the calibration
testing were 90,100, and 110% of the design HRT. A
summary of the conventional pollutant results for the
calibration study is shown in Tables D-2 and D-3.
Also shown are the monthly average permit limitations
and the design effluent characteristics for the planned
facilities.
Biological Treatment
All. BNR process simulations successfully achieved
carbonaceous BOD5 removal and nitrification. As
shown in Table D-2, the batch biological process
removed BOD5, COD, and ammonia concentrations to
well below design effluent levels. TKN concentrations
in the simulation effluents also met the design levels.
107
-------
Table P-l. Farrington Road and Northside Simulation Operating Conditions
Parameter
Farrihgton Road
POTW
Design*
Simulation
Northside
POTW
Design*
Simulation
Biological Treatment Step
Primary effluent volume
Eno River
Lick Creek
Northside
Average MLSS
Minimum DO
Anaerobic
1st Anoxic
1st Aerobic
2nd Anoxic
2nd Aerobic
Temperature (ฐC)
24.5 mgd
3,000 mg/L
Omg/L
Omg/L
2 mg/L
Omg/L
4 mg/L
10-26
100%
3,508 mg/L
<0.2 mg/L
<0.2 mg/L
2mg/L
<0.2 mg/L
4 mg/L
20-25
3.00 mgd
6.94 mgd
1:1.53 mgd
3,000 mg/L
!Omg/L
0 mg/L
2 mg/L
Omg/L
4 mg/L
12-29
14.0%f
32.3%f
53.7%t
3,481 mg/L
<0.2 mg/L
<0.2 mg/L
2 mg/L
<0.2 mg/L
4 mg/L
20-25
Alum/Filtration Treatment Steps
Alum dose after biological
treatment
Depth of anthracite/sand in filter
Constant headless level in filter
Average filtration rate
10 mg/L
878"
4ft
2.4 gpm/ft2
20 mg/L
878"
4ft
2.4 gpm/ft2 $
.5 mg/L
18712"
2-8 ft
4 gpm/ft2
10 mg/L ง
18712"
4ft
4.1 gpm/ft2 #
* Source: Hazen and Sawyer; R.L. Taylor, personal communication to J.A. Botts, Design Information for the Treatment
Plant Expansions. December 10,1990, Raleigh, North Carolina. ;
t Percent of total simulation influent volume. '
$ Filtration rate was 4.2 gpm/ft2 for April 4-5 simulation. ;
ง Alum dosage increased to 20 mg/L for April 10-11 simulation.
# Filtration rate was 7.1 gpm/ft2 for April 45 simulation.
The BNR simulations did not consistently achieve the
effluent permit levels for phosphorus (Table D-2). No
phosphorus removal was observed in the April 4-5 test.
For subsequent tests, the percentage of aeration basin
sludge added to the anoxic stage was increased to
stimulate phosphorus removal. This modification!
resulted in a decrease in phosphorus to near design
levels in the April 10-11 test. As shown in Table D-3,
phosphorus was initially released by the bacteria in the
anaerobic stage, which is common in BNR systems.
However, unlike the April 4-5 test, the phosphorus
was re-assimilated in the anoxic and aerobic stages as
would be expected. These results demonstrated that
phosphorus removal can be achieved in the batch
simulation tests. The lack of phosphorus removal in
the April 18-19 test appeared to be related to the poor
quality of the activated sludge on the day of testing.
The BNR simulations also did not achieve consistent
denitrification (Table D-2). The Northside simulation
test on April 10-11 reduced nitrate to a level
(1.7 mg/L) close to the design effluent concentration
(1.0 mg/L). All other simulation tests achieved only
slight nitrate removal. The lack of nitrate removal in
108
-------
Table D-2. Comparison of Calibration Test Results to Permit Limitations and Design Criteria (mg/L)
Parameter
Monthly Average*
Effluent Permit
Limits
Designf Effluent
Characteristics
Calibration Results
Apr 4-5
Apr 10-11
Apr 18-19
Northside POTW
BODS
COD
TSS
TKN
NH3-N
NO3-N
TP
24.0/12.0 $
NA*
30
NA
16.0/8.0 ง
NA
2
5
51
10
1.5
0.5
4.75
0.5
1
21
0
1.5
0.2
5.9
6
1
17
5
1.5
0.1
1.7
0.8
1
26
0
0.9
0.05
12.4
6
Farrington Road POTW
BOD5
COD
TSS
TKN
NH3-N
NO3-N
TP
10.0/7.0 $
NA
30
NA
4.0/2.0 $
NA
2
5
45
10
1.5
0.5
1
0.5
1
23
1
1.9
0.1
7.1
7.4
1
26
5
1
0.1
6.5
0.6
1
23
2
0.8
0.1
14.7
7.1
* Values are interim limits for the period beginning January 1, 1991, and lasting until 3 months after construction completion.
t Source: Hazen and Sawyer, R.L. Taylor, personal communication, to J.A. Bolts, Design Information for the Treatment
Plant Expansions December 10,1990, Raleigh, North Carolina.
$ Winter and Summer limits, respectively.
ง No limit established in permit.
the Farrington Road simulation may have been due to
the short anoxic treatment time (approximately 3
hours) as compared to the Northside simulation (more
than 4 hours). The simulation procedure was modified
to increase the anoxic treatment time for the Farrington
Road simulation to attempt to achieve denitrification
during the effluent toxicity evaluation.
The toxicity test results indicated that the RAS
supernatant used in simulation testing was not acutely
toxic (LC50 z 100%). Therefore, the activated sludge
was not expected to cause an acute toxicity
interference in the simulation tests.
Alum Treatment
As shown in Table D-3, only a slight removal of
phosphorus was observed in the alum treatment step.
Solids flocculation did not occur at the designed alum
dosages (10 mg/L for Farrington Road POTW and
5 mg/L for Northside POTW). Alum dosages were
Table D-3. Total Phosphorus Results (mg/L) for the
Calibration Tests Conducted on April 10-11,1990
Wastewater/Sludge
Influent
RAS
Basin sludge
Biological treatment
Anaerobic effluent
1st aerobic effluent
2nd aerobic effluent
(Clarifier effluent)
Alum treatment supernatant
Farrington
Road
Simulation
5.49
13.5
4.13
32.2
2.33
1.48
1.06
Northside
Simulation
3.95
13.5
4.13
20.7
3.05
1.78
1.55
109
-------
increased two-fold; however, no additional phosphorus
removal was achieved.
The effect of alum on effluent toxicity was evaluated
by comparing the toxicity of the wastewater before and
after alum treatment. The results show that the alum
did not add acute toxicity to the wastewater (i.e., LC50
>100% before and after alum addition).
Filtration Treatment
The filter columns were very efficient in removing
suspended solids (Table D-2). As a result, nutrients
and COD associated with the solids were further
reduced. Total phosphorus concentrations decreased
by nearly half after filtration (Table D-3).
The deionized water rinsates from the filter columns
were analyzed for toxicity prior to testing. The results
indicated that the filter media would not add acute
toxicity to the simulation effluent (rinsate LC50
Discussion of Calibration Results
The calibration results indicated that bench-scale tests
could effectively simulate the effluent quality expected
for the new POTWs. Pollutant removal was similar
whether the simulations were tested at 90, 100, or
110% of the design HRT. BOD5, COD, TKN,
ammonia, and TSS were consistently reduced to levels
expected to be achieved by the planned facilities.
Although nitrate and phosphorus were not treated to
design effluent levels, no adverse effects on toxicity
treatment in the simulations were anticipated. The
calibration results also indicated that the simulation
materials would not contribute artifactual toxicity.
Toxicity Treatment Evaluation
Tests of the calibrated simulations were performed to
determine if the new POTWs would eliminate chronic
toxicity. The operating parameters for the simulations
were based on the design HRT treatment condition
(100%). An exception was the treatment time for the
second anoxic treatment stage of the Farrington Road
simulation, which was increased to stimulate
denitrification. In addition, the alum dosages for both
simulations were increased to enhance the flocculation
necessary for phosphorus removal.
The treatment plant simulations were implemented on
two occasions. Performance criteria were applied to
ensure that the effluent quality was sufficient for
toxicity evaluation. These criteria, shown in
Table D-4, were based on the treatment performance
that was consistently achieved in the calibration tests.
Treatment Performance Results
A summary of the conventional pollutant results for the
simulation effluents is shown in Table D-4. The
results show that the simulations consistently achieved
the design effluent concentrations for BOD5, COD,
TSS, and ammonia. Effluent TKN concentrations
were within the simulation performance criterion of
5 mg/L. The effluent concentrations of total
phosphorus and nitrate also were within the simulation
performance criteria levels. Overall, the simulation
effluents were judged to be suitable for toxicity
analysis based on the simulation performance criteria.
Toxicity Evaluation Results
Results of toxicity tests, presented in Table D-5, show
that the simulation effluents were not acutely toxic to
C. dubia (48-hour LCSOs 100% effluent). Chronic
toxicity results show that the simulation effluents did
not inhibit C. dubia reproduction (NOEC of 100%
effluent). Only the effluent of the Farrington Road
simulation on May 29-30,1990, adversely affected C.
dubia survival (NOEC = 75% effluent). The chronic
toxicity of this effluent was due to significant mortality
in the 100% effluent concentration.
Sulfide was detected in the May 29-30 Farrington
Road simulation effluent at a concentration that may be
chronically toxic to C. dubia (1.6 mg/L). The sulfide
NOEC for D. magna at pH 7.6-7.8 is reported to be
1.0 mg/L (USEPA, 1990). Although the toxicity of
sulfide to C. dubia is unknown, the sensitivities of D.
magna and C. dubia to many classes of toxicants are
similar (Mount and Norberg, 1984). The pH values of
the Farrington Road simulation effluent and the value
used for the reported test also were similar (i.e., 7.85
versus 7.6 to 7.8); therefore, the potential toxicity of
sulfide in the simulation sample should be comparable
to that of the reported test (Note: the concentration of
hydrogen sulfide, the most toxic form of sulfide,
increases when pH decreases). Based on this evidence,
the chronic toxicity observed in the May 29-30
Farrington Road simulation effluent may be related to
sulfide. i
Discussion
The TRE study was completed within the 1-year time
frame specified by NCDEM. The results of this study
indicated that the addition of new BNR and filtration
treatment processes at the City of Durham POTWs
110
-------
- Table D-4. Comparison of Simulation Test Results to Performance Criteria
Parameter
Simulation
Performance Criteria
'(mg/L)*
Simulation Effluent Results (mg/L)
May 29-30
June 6-7
Northside POTW
BOD5
COD
TSS
TKN
NH3-N
NO3-N
TP
5
51
10
5
0.5
15
8
1
22
3
2
0.1
5.5
1.2
1
21
2
- NAf
0.1
11.3
3.2
Farrington Road POTW
BOD5
COD
TSS
TKN
NH3-N
NO3-N
TP
5
45
10
5
0.5
15
8
1
22
4
2.3
0.1
5.2
1.5
1
22
1
NA
0.1
9.3
3.8
* Simulation performance criteria based on calibration results and design effluent levels (Hazen and Sawyer; R.L. Taylor,
personal communication, to J.A. Botts, Design Information for the Treatment Plant Expansions. December 10,1990,
Raleigh, North Carolina).
f NA = not available.
Table D-5. Toxicity of Simulation Effluents to C. dubia*
Date
May 29-30, 1990
June 6-7, 1990
Simulation
Farrington Road
Northside
Farrington Road
Northside
48-hour LC50
(%Effluent)
100
>100
>100 .
>100
NOECf ' '
(%Effluent)
75 ง
100
100
100
LOECI
(%Effluent)
100 ง
>100
>100
>100
* 7-day chronic toxicity test (USEPA Method 1002.0) according to USEPA (1989).
t NOEC for Northside is based on survival and reproduction. Results for Farrington R'oad are based on survival.
t LOEC for Northside is based on survival and reproduction. Results for Farrington Road are based on survival.
ง Denotes statistically significant inhibition of survival.
would reduce chronic effluent toxicity to levels
required under the North Carolina discharge permit.
Sulfide, a potential effluent toxicant, was not expected
to be a problem because the final effluents of the new
treatment plants are aerated to meet instream DO
standards. The sulfide should be volatilized or
oxidized in this aeration step.
The POTW upgrades were implemented beginning in
November 1994. Results of effluent monitoring
through the second quarter of 1997 show that the
POTWs are in compliance with the chronic toxicity
limits. The limits were revised to NOECs 2:90% for
both plants. One test failure was observed in January
1995; however, this result may have been related to the
111
-------
start-up of the new treatment processes. Since then,
the City has passed all quarterly tests at both POTWs.
Bench-scale batch tests were successfully used to
simulate the treatment processes planned for the new
POTWs, including the BNR treatment process. In
addition to carbon removal and nitrification, the;
simulations achieved phosphorus removal to near
permit levels. Although nitrate was not reduced to
permit levels, the observed concentrations did not
cause chronic toxicity.
The study findings suggest an alternative TRE
approach is appropriate in cases where POTW staff is
planning upgrades or improvements to their WWTPs.
Toxicity reduction can be evaluated by conducting
bench-scale batch simulations of the planned upgrades.
This testing can be used to determine the potential for
compliance with discharge limits for toxicity. If non-
compliance is anticipated, further testing can be
performed to evaluate the additional improvements
necessary for toxicity reduction. In cases where the
conclusions of a bench-scale toxicity evaluation are
uncertain, pilot-scale tests may be warranted.
i
Acknowledgments
The City of Durham TRE study was conducted by
Engineering-Science, Inc., under contract to Hazen and
Sawyer, P.C., and the City of Durham. The assistance
of A.T. Rolan, W.W. Sun, Vicki Westbrook, and
Susan Turbak (City of Durham); Linda Forehand,
Tim Morris, Jim McReynolds, and Tory Champlin
(Engineering-Science, Inc.); and William L.
Goodfellow (EA Engineering, Science and
Technology, Inc.) are acknowledged.
The material presented in this appendix includes
copyrighted information published in text by
Technomic Publishing Company, Lancaster,
Pennsylvania (Botts et al, 1992). Technomic
Publishing Company has granted permission to include
the information in this document.
References
Botts, J.A., T.L. Morris, J.E. Rumbo, and C.H.
Victoria-Rueda. 1992. Case Histories-Munci-
palities. In Toxicity Reduction: Evaluation and
Control. D.L. Ford, ed. Technomic Publishing
Co., Lancaster, Pennsylvania.
Engineering-Science, Inc. 1989. Test Plan for the
City of Durham. Toxicity Reduction Evaluation
Project. Prepared for the City of Durham, North
Carolina.
Engineering-Science, Inc. 1990. Results of the Devel-
opment and Implementation of Waste-water
Treatment Plant Simulations for the City of
Durham TRE. Prepared for the City of Durham,
North Carolina. .
Mount, D.I., and TJ. Norberg. 1984. A Seven-Day
Life Cycle Cladoceran Test. Environ. Toxicol.
Chem. 3:425-34.
USEPA. 1989. Short-Term Methods for Estimating
the Chronic Toxicity of Effluents and Receiving
Water to Freshwater Organisms. Second Edition.
EPA/600/4-89/001. Office of Research and
Development, Cincinnati, Ohio.
USEPA. 1990. Aquatic Information Retrieval Toxi-
city Data Base, Office of Research and
Development, National Health and Environmental
Effects Research Laboratory, Mid-Continent
Ecology Division, Duluth, Minnesota.
112
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Appendix E
TRE Case Study:
Michigan City Sanitary District, Indiana
Abstract
TRE Goal: LC50 ;> 100%, NOEC ;>62%
Test Organisms: C. dubia and P. promelas
TRE Elements: TIE
Toxicant Identified: Metals
Toxicity Controls: Pretreatment requirements
Summary
Acute and chronic TIE studies indicated that metals
were the primary cause of effluent toxicity. An
industrial user was identified as a major source of
metals loadings to the POTW. The POTW staff
required the industrial user to discontinue a cadmium
plating operation and, as a result, the POTW effluent
has achieved compliance with the acute and chronic
toxicity limits (MCSD, 1993).
Key Elements
1. Less expensive acute TIE procedures can be used
in lieu of chronic TIE procedures to help
characterize the causes of chronic effluent toxicity.
However, chronic TIE testing is needed to confirm
the acute TIE results.
2. CIS SPE can remove toxicity caused by
compounds other than non-polar organic
compounds. In this study, CIS SPE treatment
removed toxicity caused by metals. These results
demonstrate the importance of needing to recover
toxicity from the CIS SPE column before
concluding that non-polar organic compounds are
causing effluent toxicity.
3. TIE Phase I data may provide sufficient
information to proceed to the selection of
pretreatment controls for toxicity reduction.
Although specific toxic metals were not identified
in this study, evidence of metals toxicity was
successfully used to set pretreatment requirements.
Introduction
Permit Requirements
The NPDES permit for the Michigan City Wastewater
Treatment Plant (MCWTP) requires acute and chronic
toxicity monitoring using C. dubia and P. promelas.
The permit specifies that the effluent must not
demonstrate chronic effluent toxicity at effluent
concentrations of 62% or less (< 1.6 TUc) and that the
effluent must not be acutely toxic (e.g., LC50 ^ 100%,
< 1.0 TUa). Based on evidence of unacceptable acute
and chronic toxicity, Michigan City was required to
perform a TRE. The Michigan City Sanitary District
submitted a TRE plan and initiated TIE testing. The
objective of the TIE was to characterize, identify, and
confirm the causes of acute and chronic effluent
toxicity so that an appropriate toxicity reduction
strategy could be developed and implemented.
Description of Treatment Plant
The MCWTP comprises an activated sludge process
with single-stage nitrification and advanced waste
treatment of the secondary effluent. The facility is
designed for an average wastewater flow of 12-million
gallons per day (mgd) and 96.7% removal of BOD5
and 96% removal of suspended solids. Monthly
average effluent limits for ammonia are 2 mg/L in
summer and 6 mg/L in the winter. Influent phosphorus
is reduced with an iron salt added at the aeration tanks.
Additional phosphorus and suspended solids removal
is accomplished by sand filtration of the secondary
effluent. Total phosphorus is reduced by 80%, which
results in effluent concentrations of less than 1 mg/L.
Post aeration equipment is provided to increase the
effluent DO concentration prior to discharge to Trail
Creek. During the months of June through September
(which coincides with the seeding of Trail Creek with
..smoltSia'ndilater.fishiDnignation up;Trail Creek), a:pure
113
-------
oxygen system supersaturates the plant effluent to a
DO concentration in excess of 13.0 mg/L.
Toxicity Identification Evaluation
Initial Toxicity Characterization
When both acute and chronic toxicity requirements
must be met, POTW staff must decide whether to use
acute or chronic TEE procedures to determine the
effluent toxicants. Acute TIE procedures can be used
to provide information about the causes of chronic
toxicity and may be preferred because they are
simpler and less costly than chronic TIE tests. Follow-
up confirmation tests can be performed using chronic
TIE procedures to determine if additional toxicants are
contributing to chronic toxicity. If an effluent exhibits
marginal and intermittent acute toxicity, it may not be
possible to identify the causes of effluent toxicity using
acute TIE procedures. In this case, chronic TIE
procedures should be used.
The initial TIE work at the MCWTP focused on
characterizing the causes of acute effluent toxicity
because previous testing indicated that the effluent
exhibited consistent acute toxicity. C. dubia were used
as the test organism based on previous tests showing it
to be more sensitive to the MCWTP effluent than P.
promelas.
The toxicity characterization tests conducted during the
first quarter of the TIE program included the following
effluent manipulations:
Pressure filtration (1.0 um filter).
Submicron filtration (0.22 um filter) following
pressure filtration (performed on one sample)
Aeration.
C18 SPE following filtration.
Cation resin treatment following filtration/CIS
SPE treatment.
Anion resin treatment following filtration/CIS
SPE treatment. :
As shown in Table E-l, the four effluent samples
characterized from April through June 1991 were
consistently toxic and the magnitude of toxicity was
similar in each sample (1.5 to 2.5 TUa). Slight
reductions in toxicity occurred following filtration and
aeration and acute toxicity was completely removed by
the CIS SPE column. Toxicity removal by the cation
and anion resins could not be determined because the
sample was first passed through the CIS SPE column,
which removed all of the toxicity. In retrospect, it
would have been preferable to treat the samples with
the ion exchange resins following filtration rather than
after CIS SPE treatment. Relatively nonpolar organic
compounds are preferentially adsorbed onto the CIS
SPE column; therefore, toxicity removal by the CIS
Table E-l. Acute Toxicity Characterization Test Results from April 1991 Through June 1991
Characterization Test
Baseline (whole effluent)
Filtration
Aeration co
Post CIS SPE t
Cation exchange T
Anion exchange ฃ
C dubia LC50 (TUa)*
4/18/91
42 (2.4)
51 (2.0) f
40 (2.5)
>100 (0.0)
>100 (0.0)
>100 (0.0)
5/16/91
40 (2.5)
79 (1.3) t
62(1.6)
>100 (0.0)
>100 (0.4)
>100 (0.0)
6/5/91
46 (2.2)
54(1.9)f
51 (2.0)
>100 (0.0)
>100 (0.0)
>100 (0.2)
6/19/91
67 (1.5)
ง
ง
>100 (0.0) #
ง
ง
* C. dubia 48-hour LC50 values expressed as percent effluent with acute TUs (100/LC50) in parentheses.
t Effluent first pressure filtered through a Gelman A/E glass fiber filter (1.0 um).
t Effluent first pressure filtered through a Gelman A/E glass fiber filter (1.0 um), followed by filtration through a Micro
Separation, Inc., 0.22 um nylon filter.
ง Characterization manipulation not conducted.
# Fine stream of air bubbles passed through an effluent sample placed in a graduated cylinder.
o> Effluent sequentially pressure filtered (1.0 um) and passed over a CIS SPE column.
i Effluent passed directly over a CIS SPE column. '
t Effluent passed over a Bio-Rex MSZ 50 cation resin after pressure filtration and CIS SPE treatment.
Effluent passed over a Bio-Rex MSZ 1 anion resin after pressure filtration and CIS SPE treatment.
114
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SPE treatment during the initial characterization tests
suggested that non-polar or semi-polar organic
compounds were causes of effluent toxicity.
Evaluation of Toxicity Removed by CIS SPE
The CIS SPE column can remove toxicants other
than non-polar organic compounds, including
organometallic complexes, certain metal ions,
surfactants, and some high molecular weight organic
compounds. Accordingly, additional tests were
performed from July through October 1991 to obtain
information about the types of compounds removed by
the CIS SPE treatment. In an attempt to recover
toxicity from the CIS SPE column, sequential elutions
were performed with methanol, methylene chloride, 3N
hydrochloric acid, and 9N sodium hydroxide. Metals
were evaluated as possible causes of toxicity
concurrently with the CIS SPE tests. Metals toxicity
was investigated by adding EDTA to whole effluent
samples and testing for acute toxicity. EDTA forms
complexes with many toxic metals and, when added at
appropriate concentrations, can render metals non-
toxic.
Results of the CIS SPE column and EDTA tests are
summarized in Table E-2. In contrast to previous tests,
the acute toxicity of the whole effluent from August
through October 1991 was variable and intermittent
(Table E-2). Four of the seven effluent samples were
not acutely toxic. The three acutely toxic samples were
rendered non-toxic by the CIS SPE treatment;
however, toxicity was not recovered by eluting the CIS
SPE columns with methanol, methylene chloride, 3N'
hydrochloric acid, or 9N sodium hydroxide. Toxicity
could not be successfully eluted from CIS SPE
columns using conventional organic extraction
techniques; therefore, it was concluded that the toxicity
removed by the column was not caused by typical non-
polar or semi-polar organic compounds.
Addition of EDTA to the three acutely toxic samples
eliminated acute toxicity, suggesting that toxicity was
caused by metals. The EDTA results provide evidence
that the toxicity removed by the CIS SPE column was
not caused by non-polar or semi-polar organic
compounds. Instead, it indicated that metals or
organometallic complexes were removed in the CIS
SPE column tests. These results demonstrate the
importance of needing to recover toxicity from the C18
SPE column before concluding that non-polar organic
compounds are a cause of effluent toxicity.
Evaluation of Metal Toxicity
Additional testing was performed to evaluate metals as
a cause of chronic effluent toxicity to C. dubia.
Chronic tests were used to help avoid problems
associated with the intermittent acute toxicity;
however, acute toxicity endpoints (e.g., 48-hour LC50)
were also obtained from the chronic tests. During
October 1991 through January 1992, 7-day static
renewal C. dubia survival and reproduction tests were
performed on whole effluent samples and whole
Table E-2. Toxicity Characterization Test Results from July 1991 Through October 9,1991
1 <
Sample Date
7/10/91
7/24/91
8/07/91
8/22/91
9/11/91
9/25/91
10/09/91
C,dซWaLC50(TUa)*
Baseline
(Final Effluent)
>100 (0.2)
>100 (0.0)
61 (1.6)
52 (1.9)
>100 (0.4)
>100 (0.2)
<100 (>1) #
Post CIS SPE t
ง
>100 (0.2)
>100 (0.0)
>100 (0.4)
ง
>100 (0.0)
ง
EDTA*
ง
>100 (0.0)
>100 (0.0)
>100 (0.0)
ง
>100 (0.0)
>100 (0.0) co
* C. dubia 48-hour LC50 values expressed as percent with TUs (100/LC50) in parentheses.
t Effluent passed over a C18 SPE column.
$ EDTA was added to the final effluent at a concentration of 186 mg/L.
ง Characterization manipulation was not conducted.
# Test conducted only in 100% effluent; as a result, LC50 and TUa values could not be calculated.
co EDTA concentration in the 10/09/91 sample was 18.6 mg/L.
115
-------
effluent samples with EDTA added. As shown in
Table E-3, three of the five samples exhibited acute
toxicity and four of the five were chronically toxic.
The 48-hour LC50 values for all of the EDTA treated
samples were greater than 100% effluent. EDTA
addition also eliminated chronic toxicity in two
samples and reduced chronic toxicity in a third sample.
These results provided additional evidence that metals
cause acute effluent toxicity, and also suggested that
metals were a primary cause of chronic effluent
toxicity.
The correlation approach and spiking approach
described by USEPA (1989a) were used to confirm
that metals were causing effluent toxicity. The
correlation approach is intended to evaluate the
relationship between the concentration of suspected
toxicants and effluent toxicity. Toxicity and metals
data (aluminum, Cd, Cu, Ni, and Zn) for six effluent
samples were compared by correlation analysis. All
metals were measured as total metals.
Linear regression analysis indicated a good correlation
(regression coefficient of 0.72) between effluent
toxicity and effluent Cd concentrations. However,
when data from May 1991 through December 1992
were pooled with the data set, the correlation between
effluent toxicity and effluent Cd concentrations was
not statistically significant. A comparison of the mean
Cd concentrations from samples collected during a
toxic period (May 1991 to December 1991), and those
taken during a non-toxic period (May 1992 to
December 1992) indicated a trend. The mean Cd
concentration was 4.1 |jg/L during the toxic period and
0.47 ug/L during the non-toxic period. These data
provide evidence that Cd was contributing to effluent
toxicity. No significant correlation was observed
between effluent toxicity and the concentration of the
other metals or the sum of all the metals.
The objective of the spiking approach was to determine
whether an increase in the concentration of a suspected
toxicant would cause a proportional increase in
toxicity. Chronic C. dubia toxicity tests were
performed on three chronically toxic effluent samples
both with and without added Cd, Cu, Ni, and Zn. The
metals were added in nominal concentrations
approximating those typically found in the MCWTP
effluent. The results indicated that effluent toxicity did
not consistently increase when the metals were spiked
individually or in combination. Therefore, the results
of the spiking tests did not confirm that Cd or other
metals were contributing to effluent toxicity.
Toxicity Control Evaluation and
Implementation
Although the TIE did not conclusively identify the
specific causes of effluent toxicity, the weight of
evidence indicated that effluent toxicity was caused by
metals. As a result, Michigan City investigated
possible sources of metals in the collection system.
Pretreatment program data indicated that a cadmium
plating facility in the MCWTP service area was
consistently out of compliance with pretreatment
limitations for metals. Based on the persistent
pretreatment permit violations, the cadmium plating
Table E-3. Acute and Chronic Toxicity of MCWTP's Effluent (with and without added EDTA) from October 1991 Through
January 1992
Sample Date
10/30/91
11/14/91
12/04/91
12/18/91
01/08/92
Final Effluent
Acute
LC50 (TUa) t
73 (1.4)
>100 (0.0)
>100 (0.0)
84 (1.2)
60 (1.7)
Chronic
NOECt
50
62
100
<50
<50
Final Effluent with EDTA Added *
Acute
LC50 (TUa) t
>100 (0.0)
>100 (0.0)
>100 (0.0)
>100 (0.0)
>100 (0.0)
Chronic
NOEC $ . . ; v:
100
62
100
100
50
* EDTA concentration in the 10/30/91 and 11/14/91 tests was 5 mg/L. EDTA concentration in the 12/04/91,12/18/91 and
01/08/92 tests was 10 mg/L. :
t C. dubia 48-hour LC50 values expressed as percent effluent with acute TUas (100/LC50) in parentheses.
t Reproduction NOEC values expressed as percent effluent calculated from 7-day static-renewal chronic tests with C. dubia.
116
-------
company was issued a consent decree to terminate their
cadmium plating operation. The cadmium plating
operation was shut down in April 1992.
The impact of the shutdown on effluent toxicity was
evaluated by performing 4-day modified chronic C.
dubia tests on whole effluent samples at approximately
2-week intervals from May through September 1992
(total of nine tests). The 4-day modified chronic tests
consisted of four concentrations and a control, five
replicate test chambers per concentration, and the tests
were initiated with 3-day old C. dubia. This modified
approach has been demonstrated to produce results that
are comparable to the 7-day test (Masters et al., 1991).
The results of these tests showed that acute and chronic
effluent toxicity to C. dubia had been eliminated.
Discussion
Subsequent chronic testing with C. dubia and P.
promelas using compliance monitoring procedures
(USEPA, 1989b) confirmed the reduction in effluent
toxicity following shutdown of the cadmium plating
operation. The acute and chronic toxicity of the
MCWTP effluent from inception of the TRE through
December 1992 is summarized in Figure E-l. The
correlation between the cadmium plating operation
shutdown and improved effluent toxicity is clearly
evident. Based on the improved effluent toxicity, the
TRE was terminated and semiannual acute and chronic
toxicity compliance monitoring was initiated.
However, starting in August 1996 significant
reproductive effects were observed in 100% effluent as
compared to the test control. Subsequent TIE testing
was inconclusive because effluent samples were
nontoxic. Michigan City has submitted a letter to the
Indiana Department of Environmental Management
(IDEM) requesting changes in the effluent monitoring
program. The requested changes include the use of
reconstituted laboratory water as dilution water in lieu
of receiving water to minimize potential contamination
and reducing the frequency of monitoring if no toxicity
is observed in three consecutive tests. As of October
1997, a decision from IDEM was still pending.
Acknowledgments
This TIE effort was conducted by Great Lakes
Environmental Center, Traverse City, Michigan, under
contract to the Michigan City Sanitary District.
References
Masters, J.A., M.A. Lewis, D.H. Davidson, and R.D.
3.0 T
Chronic Toxicity Acute Toxicity
Cadmium plating operation discontinued
Toxicity Test Date
Note 1. Recorded toxicity is an artifact of the statistical analysis.
Note 2. TUa's and chronic TUs are based on 48-hour LC50 and NOEC values for C.dubia, respectively.
Figure E-l. Acute and chronic effluent toxicity: 1991 through 1992.
117
-------
Bruce. 1991. "Validation of a Four-Day C. dubia
Toxicity Test and Statistical Considerations
in Data Analysis" Environ. Toxicol. Chem. 10:
47-55.
Michigan City Sanitary District (MCSD). 1993. Draft
Final Report on Toxicity Identification/Reduction
Evaluation for Michigan City Waste-water
Treatment Plant. Prepared by Great Lakes
Environmental Center, Traverse City, Michigan.
USEPA. 1989a. Methods for Aquatic Toxicity
Identification Evaluations: Phase III Toxicity
Confirmation Procedures. EPA/600/3-88/036.
Office of Research and Development, Duluth,
Minnesota. ;
USEPA. 1989b. Short-Term Methods for Estimating
the Chronic Toxicity of Effluents and Receiving
Waters to Freshwater Organisms. Second
Edition. EPA/600/4-89/001. Office of Research
and Development, Cincinnati, Ohio.
118
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Appendix F
TRE Case Study:
Central Contra Cost Sanitary District, Martinez, California,
and Other San Francisco Bay Area POTWs
Abstract
TRE Goal:
Test Organism:
TRE Elements:
Toxicants Identified:
Toxicity Controls:
No significant acute toxicity
at 100% effluent
C. dubia
TIE and source identifi-
cation
Diazinon and chlorpyrifos
Multi-faceted public aware-
ness program; ongoing
program to identify and
control sources; ongoing
effort to identify POTW
processes and operations
that effectively remove
organophosphate
insecticides.
Summary
Acute toxicity to C. dubia was consistently detected in
a POTW effluent. Application of Phase I, H, and m
TIE procedures showed that the toxicity was caused by
diazinon and one or more additional organophosphate
insecticides. Follow-up studies, which required
development of more sensitive analytical methods,
showed that chlorpyrifos was present at levels that
exceeded the NOEC in all effluent samples that were
toxic to C. dubia. Influent and effluent monitoring
studies of San Francisco Bay Area POTWs identified
large differences in both influent loading and removal
of the two insecticides between the POTWs. All the
POTWs sampled achieved substantial removal of
diazinon and chlorpyrifos from influent wastewater.
Higher removal of both insecticides were generally
associated with POTWs that had filtration treatment,
extended mean cell residence times, chlorine contact
times, and/or long retention in ponds. Source
identification studies showed that the majority of the
influent mass loading of the two insecticides was from
residential sources. A multi-faceted outreach program
was initiated within the POTW service area.
Monitoring of effluent toxicity and insecticide
concentrations to assess the effectiveness of the public
outreach program is on-going.
Key Elements
1. The organophosphate insecticides, diazinon, and
more recently, chlorpyrifos, have been implicated
as causes of toxicity to C. dubia in POTW
effluents.
2. Published TIE procedures are available to identify
organophosphate insecticide toxicity (USEPA
1991,1992,1993a, 1993b, 1996). Application of
new methods and procedures assisted in providing
a more quantitative assessment of the role of
diazinon and chlorpyrifos in effluent toxicity.
3. Source identification studies at the CCCSD
demonstrated that the majority of the diazinon and
chlorpyrifos influent loading was from residential
sources.
4. Regional influent and effluent monitoring studies
demonstrated patterns in influent diazinon and
chlorpyrifos loadings at the CCCSD, which
suggest there were demographic differences in use
and disposal practices for organophosphate
insecticides.
5. A multi-faceted public outreach program was
implemented in the POTW service area. The
effectiveness of the program is being assessed by
frequent measurements of influent and effluent
levels of diazinon and chlorpyrifos and effluent
toxicity tests.
119
-------
6. Monitoring studies showed that San Francisco Bay
Area POTWs achieve substantial removal of both
diazinon and chlorpyrifos. The highest levels of
removal are associated with systems that have
filtration systems, extended MCRTs, and/or longer
chlorine contact times.
Introduction
Permit Requirements
During 1990-1991, the CCCSD conducted an effluent
toxicity characterization program in which 18 acute
toxicity tests were performed. The effluent produced
detectable acute toxicity to C. dubia in 12 of the 18 test
events. The CCCSD's NPDES permit requires no
significant acute toxicity at 100% effluent; therefore, a
TRE study was required by the California State Water
Quality Control Board, San Francisco Bay Region, to
determine the causes and sources of the acute toxicity.
This study was performed in addition to the TRE study
that addressed effluent toxicity caused by Cu (see
Appendix B). The CCCSD was required to meet
permit limits based on toxicity testing using both C.
dubia and echinoderms.
Description of the Treatment Plant
A description of the treatment plant is presented in
Appendix B.
Facility Performance Evaluation
As part of the TRE study, the CCCSD conducted an
internal facility performance evaluation to determine if
the treatment system was operating at design
performance specifications. A review of all relevant
operating parameters indicated that there were no
obvious performance deficiencies. During this period,
monthly effluent tests showed intermittent acute
toxicity to C. dubia, but no toxicity was detected to
juvenile P. promelas (15- to 60-day-old).
Toxicity Identification Evaluation
USEPA TIE methods were used as guidance in
conducting the Phase I (1988a), Phase H (1988b) and
Phase IE TEE studies (1988c).
Phase I TIE - Toxicity Characterization
A total of five Phase I TIE studies were conducted with
the CCCSD final effluent to characterize the class of
the toxicant(s) responsible for the acute toxicity to C.
dubia. Tests were 48-72 hours in duration and TIE
treatments were not renewed during the tests. TIE
treatments were conducted on 100% effluent. The
results, shown in Table F-l, indicated that the toxicity
was consistently reduced by treatment with CIS SPE
columns at pHz (initial pH of the sample) and PBO
addition. Treatments that produced a partial decrease
in toxicity in two or more samples included adjustment
to pH 3 and aeration. Treatments that consistently did
not decrease toxicity included pH adjustments, sodium
thiosulfate, EDTA, or graduated pH treatment.
The results of the Phase I TIE studies showed that
acute toxicity was consistently reduced by the C18
SPE column treatment, which removes non-polar
organic chemicals. The methanol eluates from the
C18 SPE column were toxic when added to dilution
water at a concentration equivalent to 1.5 times (1.5X)
the concentration in the effluent sample. It is important
to note that the 1.5X calculation assumes that the
toxicity was completely removed from the effluent
sample by the C18 SPE column and further, that the
toxicity was completely recovered from the column in
the methanol eluate.
PBO was effective in preventing acute toxicity to C.
dubia in all five samples. PBO blocks the metabolic
activation and subsequent toxicity of organophosphate
insecticides, which require metabolic activation to
exhibit toxicity (Ankley et al., 1991). The
ineffectiveness of sodium thiosulfate and EDTA
suggest that oxidants and/or cationic metals were not
implicated in the toxicity. The results of the graduated
pH test also suggested that ammonia did not contribute
to toxicity. Overall, the Phase I TIE results indicated
that the effluent toxicity was due to non-polar organic
toxicant(s), specifically one or more organophosphate
insecticides, which require metabolic activation to
produce toxicity. Diazinon, a metabolically activated
organophosphate insecticide, has been reported to
cause toxicity in municipal effluents (Norberg-King et
al., 1989; Amato et al., 1992); therefore, subsequent
Phase EC studies focused on identifying organo-
phosphate insecticides. Effluent and diazinon-spiked
laboratory water were used to determine if the TIE
treatments produced similar effects.
Phase II- Toxicity Identification
A total of four effluent samples were processed in
Phase n. PBO completely prevented toxicity in all
four effluent samples, suggesting that metabolically
activated organophosphate insecticides were
responsible for the acute toxicity. The Phase I TIE
showed that the toxicity could be both removed by and
120
-------
Table F-l. Matrix of Results of Phase I TIE Conducted on Five Effluent Samples with C. dubia
Treatment
Reduces Toxicity Due To
Samples with Substantially Reduced Toxicity
CIS SPE column (pffi)*
CIS eluate toxic'
PBO addition
Filtration
Aeration
Adjustment to pH 3
Adjustment to pH 11
Thiosulfate addition
EDTA addition
Graduated pH test
Non-polar organics, metals
Confirms non-polar organics
Organophosphate insecticides
Filterable toxicants
Volatile/oxidizable toxicants
Acid hydrolyzable toxicants
Base hydrolyzable toxicants
Oxidants, some metals
Cationic metals
Ammonia, metals
* pHz = initial pH.
recovered fromCIS SPE columns; therefore, the Phase
n TIE procedures focused on the use of the columns to
fractionate the sample for further characterization.
Aliquots of the samples were concentrated on CIS SPE
columns and the columns were eluted with a series of
methanol:water mixtures (USEPA, 1993a). Acute
toxicity tests were then conducted on each fraction at
1.5X the original effluent concentration.
The 75% fraction from all the effluent samples was
acutely toxic. In some samples, adjacent fractions
(e.g., 70, 80, and 85%) also exhibited acute toxicity.
The toxic fractions were combined, concentrated, and
sequentially fractionated using HPLC. For
comparison, an analytical standard of diazinon was run
immediately prior to each effluent sample HPLC run.
A total of 30 fractions were collected during the HPLC
linear gradient (30-100% methanol:water for 25
minutes with 5 minutes at 100% methanol). Each
fraction was assayed at 1.5X the original effluent
concentration with C. dubia, and toxic fractions were
treated with PBO to ascertain the presence of
organophosphate insecticides. This procedure was
similar to that described by USEPA (1993a). The
results are summarized in Table F-2.
The diazinon standard consistently produced acute
toxicity in one fraction (19), and in one HPLC run,
toxicity also was observed in another fraction (18). All
four effluent samples also produced acute toxicity in
fraction 19 and occasionally in adjacent fractions (18
and 20).
As shown in Table F-2, in all cases, PBO provided
protection against acute toxicity in the HPLC fractions
in which toxicity occurred (18-20). However, PBO
did not protect against the toxicity of fractions 12 and
13. The results of the PBO treatment of the toxic
fractions suggested that one or more metabolically
activated organophosphate insecticides, such as
diazinon, had a role in the toxicity of all four effluent
Table F-2. Summary of TIE Phase II Results
Sample
Diazinon (Runs 1-4)
Effluent 1
Effluent 2
Effluent 3
Effluent 4
Toxic Fractions
18*+, 19*+
18*+, 19*+
12-f, 19*+
18*+, 19*+, 20$
13f-, 19*+,
* PBO provided full protection against toxicity.
t PBO provided no substantial protection against toxicity.
t PBO provided partial protection against toxicity.
121
-------
samples. Diazinon consistently eluted in the same
fractions that were identified in the effluent samples;
therefore, further studies focused on confirming the
presence of diazinon in the HPLC fractions and
refining procedures for the accurate determination of
diazinon in effluent samples. This latter aspect was
challenging because diazinon is toxic to C. dubia at
low concentrations (LC50=0.26-0.58 ug/L) (USEPA,
1991; Ankley et al., 1991; Bailey et al., 1997), and the
CIS SPE column extracts of the effluent samples
contained numerous interferences which made analysis
by gas chromatography (GC) problematic. Diazinon
analysis generally followed procedures described by
USEPA (1993a). Diazinon was quantitatedby GC/MS
using selected ion monitoring. The detection limit for
this procedure in the CCCSD effluent matrix was
0.010 ug/L of diazinon.
Phase IH- Toxicity Confirmation
The role of diazinon in the CCCSD's effluent toxicity
was assessed using the correlation approach (USEPA,
1988c). Hie purpose of the correlation approach is to
determine whether there is a consistent relationship
between the concentration of the suspected toxicant
and the degree of effluent toxicity. If the correlation is
not robust, the role of the suspect toxicant in the
effluent toxicity should be re-examined.
A total of seven CCCSD effluent samples collected
during July and August 1992 were evaluated by
comparing the expected toxicity based on diazinon
(48-hour LC50=0.38 ug/L) with the measured effluent
toxicity. The 48-hour toxicity of the effluent samples
ranged from 1.25-2.17 TUa. Diazinon concentrations
in these samples ranged fromO. 120-0.280 ug/L, which
corresponds to 0.32-0.74 TUa based on the 48-hour
LC50 for diazinon (i.e., 0.12 ug/L + 0.38 ug/L and
0.28 ug/L -T- 0.38 ug/L). The oxygen analog of
diazinon (diazinon oxon) was not detected
(<0.010 Ug/L) in any of the effluent samples analyzed.
Treatment of the toxic samples with PBO resulted in
full reduction of toxicity in five samples, partial
reduction in one sample, and no reduction in one
sample. The effluent TUa and diazinon TUa values for
the seven toxic samples are plotted in Figure F-l along
with the theoretical regression line, which depicts the
case where all of the toxicity measured in the sample is
due to diazinon (diazinon TUa = effluent TUa).
The linear regression of effluent TUa versus diazinon
TUa had an R2 value of 0.75 (p<;0.01), which indicates
that diazinon concentrations can account for 75% of
2.5
2-
1.5 H
1 -
0.5
Effluent Regression Line
r2 = 0.87
slope =1.69 /
y-intercept = 0.74 S
Theoretical Regression Line
r
0.5:
1
i
1.5
2.5
Diazinon TUa
Figure F-l. Effluent TUs versus diazinon TUs in the CCCSD
effluent samples.
the variability in the toxicity of the effluent samples.
However, the regression is above the theoretical
regression line, which suggests that either the
analytical procedure for diazinon was consistently
detecting less than the actual effluent concentration,
and/or there were one or more additional toxicants
present in the effluent samples. Further studies were
undertaken to assess both possibilities.
Analytical procedures were reviewed by the CCCSD
and were found to have acceptable levels of precision
and accuracy. In an effort to identify the missing
toxicant(s), more rigorous extraction procedures were
applied to additional samples of effluent that were
toxic to C. dubia. The effluent samples were
exhaustively extracted with methylene chloride,
evaporated to dryness, and resolubilized in hexane.
Analysis of the extracts by GC/MS revealed the
presence of chlorpyrifos, a metabolically activated
organophosphate insecticide, in all the toxic effluent
samples at concentrations greater than the NOEC of
0.030 ug/L (AQUA-Science, 1992; Bailey et al.,
1997).
Follow-Up TIE Studies
Before further Phase El studies were initiated, a series
of studies were conducted to validate the Phase I and
n TIE findings for diazinon and to determine why the
Phase n TIE process failed to identify chlorpyrifos as
a toxicant in the CCCSD effluent. The results of these
studies are summarized in Table F-3.
122
-------
Table F-3. Summary of Follow-Up TEE Studies
TIE Treatment
pH adjustment
PBO addition
CIS SPE
HPLC fractionation
Sample stability
studies
Effect on Organophosphate Insecticides
Diazinon is degraded rapidly at pH 3, but is relatively stable at pH 1 1
PBO at 100-700 ug/L effectively protects against three times LC50 concentration of
diazinon and chlorpyrifos (1.6 and 0.24 ug/L, respectively). Effectiveness of PBO is
affected by the matrix; therefore, use a range of PBO additions (USEPA, 1991a, 1993a).
Diazinon is well recovered (80-100%) from CIS SPE columns
Diazinon elutes sharply in specific methanol/water fractions: 75-80% methanol fractions
for CIS SPE columns
Chlorpyrifos is poorly recovered from CIS SPE columns (40-50% recovery)
Chlorpyrifos tends to elute in broad bands: 80-95% methanol fractions for CIS SPE
columns
Diazinon is well recovered in specific fractions from C18 HPLC columns
Recovery of chlorpyrifos from C18 HPLC columns is highly variable (20-60% recovery)
Significant amounts (20-40%) of diazonin and chlorpyrifos are lost from influent and
effluent samples stored in either glass or plastic containers for 48 hours
Effluent samples should be analyzed or extracted within hours of collection
The follow-up studies provided additional insight into
the initial Phase I and II TIE results. The instability of
diazinon at pH 3 is consistent with the reduction in
effluent toxicity after pH 3 treatment. Diazinon is well
recovered through the Phase n concentration and
fractionation steps (Bailey etal, 1996); therefore, toxic
fractions corresponding to those produced by diazinon
standards should be present in all toxic effluent
samples, as was demonstrated in the TIE.
On the other hand, the low overall recovery of
chlorpyrifos from C18 SPE columns would explain the
failure to detect chlorpyrifos toxicity in the effluent
C18 SPE and HPLC fractions. For example, using the
values in Table F-3, the recovery of chlorpyrifos in
HPLC fractions could be as low as 8% (i.e., 40%
recovery from 3 mL SPE column x 40% recovery from
1 mL SPE column x 50% recovery from HPLC
column). This level of recovery would require an add-
back of more than 12X to ensure that concentrations of
chlorpyrifos in the HPLC fractions and the effluent
samples were comparable. This study indicated that
add-backs of fractions at levels substantially greater
than 1.5X should be avoided because of the potential
to amplify the toxicity due to toxicants that are below
the toxic threshold in the effluent, but are well
conserved through the TIE process. This could lead to
erroneous identification of chemicals that do not have
a causal role in the effluent toxicity.
A critical issue facing the investigator is how to
identify toxicants that are not well recovered through
the TIE process. Recently, procedures have been
developed to selectively remove diazinon and
chlorpyrifos from effluent samples using antibody-
mediated processes (Miller et al., 1996; Miller et al.,
1997). This process involves treating the effluent
sample with the chemical-specific antibody preparation
that selectively removes up to 95% of the target
chemical (either diazinon or chlorpyrifos). By
conducting effluent toxicity tests before and after the
antibody treatment, the exact contribution of the target
chemical to the overall toxicity can be determined. In
addition, use of sequential antibody treatments to
remove both diazinon and chlorpyrifos from the
effluent matrix can indicate the extent to which
toxicity is not due to either compound. The residual
toxicity can be further characterized through the TIE.
Alternative Analytical Procedures
A major limitation of the TIE study was obtaining
accurate and timely analytical information on levels of
insecticides in effluent samples and TIE treatments.
The GC/MS methods that were available involved
tedious extractions, clean-up, and the use of expensive
analytical equipment that was fully scheduled for
compliance-related purposes. ELIS A procedures were
evaluated as an alternative analytical method for the
analysis of diazinon and chlorpyrifos in subsequent
Phase in TIE and source identification studies.
123
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Commercially available ELISA kits (Beacon
Analytical, Scaresborough, Connecticut) have some
distinct advantages over GC or GC/MS methods,
including cost ($40-70 versus $250-500 per sample),
sample volumes (100 uL versus liters), sample turn-
around (hours versus days or weeks), and equipment
costs ($3,000 versus >$50,000). The detection limit
for ELISA kits for diazinon and chlorpyrifos
(0.030 ug/L) is also comparable to that for GC/MS.
An interlaboratory study involving 6 laboratories and
a total of 19 influent samples was conducted to
compare the performance of ELISA, GC, and GC/MS
procedures for diazinon and chlorpyrifos. The study
showed that ELISA values for both insecticides were
highly correlated (R2 >0.95) with GC and GC/MS
results for those laboratories (Singhasemanon et al.,
1997). The results were comparable over a wide range
of concentrations (i.e., 0.030 to 31.5 ug/L for diazinon
and 0.030 to 9.8 ug/L for chlorpyrifos).
Based on the excellent performance of the ELISA
procedures in the interlaboratory study, ELISA
procedures were used to monitor diazinon and
chlorpyrifos concentrations in the CCCSD influent and
effluent samples during follow-up studies, including
source identification, POTW influent removal studies,
and monitoring the effectiveness of public outreach
programs.
Source Identification Studies
Source Study 1
A reconnaissance study was conducted in August 1995
to identify potential sources of diazinon and
chlorpyrifos in wastewater from selected residential
and commercial sources within the CCCSD collection
system. A total of 36 24-hour composite samples of
influent were analyzed for the two insecticides by
ELISA. The samples included daily and/or hourly
composite samples collected from a residential
community, and from selected businesses within the
CCCSD collection system, including self-service pet
grooming facilities, operations centers for pest control
operators, and kennels.
The measured levels of diazinon and chlorpyrifos were
coupled with estimated flows from the various sources
to provide estimates of overall contribution of the two
insecticides to the CCCSD's influent. The results are
shown in Table F-4.
Diazmon and chlorpyrifos concentrations in the
wastewater from the residential sources were highly
variable (0.050-0.720 ug/L and <0.050-0.520 ug/L,
respectively). Peak concentrations of both insecticides
in the residential samples were measured in the
samples collected on Saturday afternoon. The cause of
the spikes of the insecticides in the residential
wastewater is under further study and may be related to
home use and/or improper disposal of these chemicals
during weekend activities (e.g., lawn care operations
for diazinon and pet flea control for chlorpyrifos).
Diazinon and chlorpyrifos levels in wastewater
samples collected from commercial sources also were
highly variable (<0.030-16.0 ug/L and 0.040-5.4
ug/L, respectively). The highest concentrations of both
insecticides were measured in wastewater samples
from a commercial kennel.
[
Overall, the reconnaissance study showed that although
high levels of diazmon and chlorpyrifos were detected
in some of the wastewater samples from commercial
sources, the vast majority of the loading of the
insecticides into CCCSD influent during the sampling
period was from residential sources. This finding
agrees with an earlier study of sources of diazinon in
Fayetteville, NC (Fillmore et al., 1990).
Table F-4. Diazinon and Chlorpyrifos Concentrations in Wastewater Samples from Selected Residential and Commercial
Sources in the CCCSD
Source
Residential
Commercial: Pest control operators
Pet groomers
Kennels
Diazinon ;
ug/L
0.050-0.720
<0.03-1.10
<0.03-0.10
0.070-16.00
% of Total
Influent Loading
101
3
<1
2
Chlorpyrifos
ug/L
<0.05-0.52
0.060-1.80
0.04-7.00
3.10-5.40
% of Total
Influent Loading
94
4
2
1
124
-------
Source Study 2
Results of the reconnaissance study were used by the
CCCSD and the California Department of Pesticide
Registration (CADPR) to develop a plan for a more
definitive study that was conducted from June to
September 1996 (Singhasemanon et al., 1997). m this
study, over 200 flow-proportional 24-hour composite
samples were collected from each of 5 residential areas
and 12 businesses (pet groomers, pest control
operators, and kennels) within the CCCSD collection
system. How measurements were made at selected
sampling points in order to calculate mass loadings of
diazinon and chlorpyrifos. The measured flows in
residential areas were compared with modeled flow
data obtained from a computer program [Sewer
Network Analysis Program (SNAP) 1989, developed
by the CCCSD]. The SNAP program applies modeled
land use, groundwater infiltration, and CCCSD plant
influent data to determine flow rates from the sampled
areas. Concentrations of the insecticides were
measured using ELISA, GC, and/or GC/MS
procedures. The loading of diazinon and chlorpyrifos
in the CCCSD influent from residential sources was
estimated by multiplying the mean insecticide
concentrations measured from the residential sites by
the SNAP flow rates from the sampled sources. The
commercial loading was estimated by multiplying the
mean insecticide concentrations measured at each
business by the measured flows and the number of
similar businesses in the sewer service area. The data
were analyzed using a computer program (SASฎ, SAS
Institute, Inc, 1994, Version 6.1, Gary, North
Carolina), which calculated the Uniformly Minimum
Variance Unbiased Estimator (UMVUE) for the mean
influent loading concentrations for the insecticide
(Singhasemanon et al., 1997). The mean UMVUE
influent concentrations and associated loading for
diazinon was 0.230 [ig/L and 34.7 g/day, respectively.
Corresponding values for chlorpyrifos were 0.145 |ig/L
and 15.0 g/day. The percentage of the total loading
contributed by residential, commercial and unknown
sources is shown in Figure F-2.
The CADPR study concluded that:
Levels of diazinon and chlorpyrifos were highly
variable in wastewater samples from both
residential and commercial sources.
Residential neighborhoods contributed the
majority of diazinon and chlorpyrifos to the
CCCSD's influent.
Although relatively high concentrations of both
insecticides were found at commercial sources,
low flows from these sources resulted in relatively
small mass loadings.
A mass balance showed that a significant mass of
chlorpyrifos and, particularly, diazinon was
unaccounted for. Uninvestigated sources such as
restaurants, nurseries, and industrial facilities
should be sampled in future studies.
Future source reduction strategies should focus on
residential customers to identify and correct
behaviors that contribute to disposal of
organophosphate insecticides to the sewer system.
Diazinon
Unknown
(42%)
Commercial
(6%)
Residential
(52%)
Chlorpyrifos
Unknown
(25%)
Residential
(60%)
Commercial
(15%)
Figure F-2. Percent mass contribution of sources to the CCCSD influent.
125
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As a result of the source identification studies, the
CCCSD:
Initiated a multi-faceted public outreach program
targeting residential costumers to increase public
awareness of the proper use of disposal of
insecticides. The initial program included point of
sale information sheets, newspaper articles,
television ads, and billboards. A program to
enhance public awareness of proper insecticide use
by promoting integrated pest management
. practices is on-going.
Shared study information with interested POTWs
and State and Federal regulatory agencies.
Initiated frequent effluent monitoring of diazinon
and chlorpyrifos coupled with an effluent toxicity
program to monitor the success of the public
outreach program.
Planned further studies to identify homeowner
practices that contributed to the discharge of
insecticides to the collection system.
Reviewed disposal practices with pest control
operators, pet care businesses, and kennels within
the District.
Conducted a study to identify the toxicity of
alternative products for pet flea control.
Loading and Removal of Diazinon and
Chlorpyrifos
Study 1
As an ancillary part of the CADPR source
identification study, diazinon and chlorpyrifos were
measured in seven consecutive daily samples of
influent and effluent from CCCSD and two nearby
POTWs [Union Sanitary District (USD), Fremont,
California, and the Regional Water Quality Control
Plant (RWQCP), Palo Alto, California]. The purpose
of the study was to assess differences in loading and
removal efficiencies for the POTWs. The three
POTWs had similar influent flows (25-38 mgd),
aeration detention times (3.8-5.6 hours), and clarifier
detention times (2.0-4.2 hours). However, the CCCSD
and the USD had shorter MCRTs (1.6-1.8 days versus
11.6 days) and shorter chlorine contact time (30-50
minutes versus 90 minutes) when compared to the
RWQCP. In addition, the RWQCP treatment process
incorporates two-stage aeration and dual media
filtration to optimize particulate removal. The results
of the study are shown in Figure F-3.
Daily concentrations of both diazinon and chlorpyrifos
in the three POTWs varied widely during the sampling
period. The CCCSD consistently had the highest
influent and effluent concentrations of both
insecticides, followed by the USD and the RWQCP.
The CCCSD and the USD, which have similar
treatment processes, had similar removal efficiencies
for diazinon (32 and 24%, respectively), and
chlorpyrifos (53 and 49%, respectively). The
RWQCP, which has longer chlorine contact time, two-
stage aeration, and dual media filtration had the highest
removal efficiencies for diazinon (82%) and
chlorpyrifos (71%). The effect of these parameters on
the removal and/pr degradation of diazinon and
chlorpyrifos in municipal influent was further
evaluated in a subsequent study.
Study 2
A larger scale study was conducted to confirm the
findings of the CADPR study, which suggested that
there may be demographic and/or microclimatic
differences in influent loadings of diazinon and
chlorpyrifos to POTWs within the same region and
moreover, there may be differences in removal
efficiencies of the two insecticides in POTWs using
different treatment systems. Seven daily 24-hour
composite samples of influent and effluent were
collected from 9 Bay Area POTWs during August
1997. The POTWs included the CCCSD and the cities
of Fairfield-Suisun, Hayward, Palo Alto, Petaluma,
San Francisco, San Jose, Union City, and Vallejo.
Samples were analyzed for diazinon and chlorpyrifos
within 24 hours of collection using ELISA (AQUA-
Science, 1997). The results for diazinon and
chlorpyrifos are shown in Figure F-4. Information on
the characteristics of each POTW treatment system is
shown in Attachment 1.
The results of this study confirmed and extended the
findings of the previous study. A summary is provided
below.
Mean influent concentrations for both diazinon
and chlorpyrifos were highly variable and ranged
from 0.278-1.211 |ig/L and 0.030-0.176 ug/L,
respectively. These results suggest-that there are
regional demographic, and possibly, climatic
differences in use and disposal practices for the
insecticides.
All the POTWs achieved substantial removal of
the two insecticides from influent (up to 98% for
diazinon and up to 86% for chlorpyrifos). These
removal rates are generally higher for both
insecticides than were observed in the previous
study. The highest levels of removal were
126
-------
+0.474 +0.26
CCCSD USD
Influent and Effluent Samples
| Diazinon Influent 03 Diazinon Effluent
RWQCP
Chlorpyrifos Influent l~l Chlorpyrifos Effluent
Figure F-3. Mean diazinon and Chlorpyrifos concentrations (ฑstd) in influent and effluent from three Bay Area POTWs.
associated with POTWs that had filtration, longer
MCRTs and chlorine contact times, and long
retention in ponds.
Mean effluent concentrations for diazinon and
Chlorpyrifos ranged from <0.030-0.241 ug/L and
<0.030-0.085 |ig/L, respectively. The combined
mean effluent concentrations for both insecticides
exceeded 1.0 TUa in only three of the nine
POTWs sampled (including the CCCSD).
Overall, the results showed that all the POTWs
sampled during this period had potentially toxic
levels of diazinon and Chlorpyrifos in their
influents. However, all the POTWs achieved
substantial removal of both insecticides.
Another round of sampling was scheduled for February
1998 to assess seasonal effects on influent levels and
removal rates from the POTWs.
Alternative Pet Flea Control Products
Toxicity source investigations by the CCCSD
suggested that pet flea control products were a major
source of Chlorpyrifos in the influent (AQUA-Science
1995a and 1995b). Before the CCCSD could
recommend alternative products, it was necessary to
conduct studies to determine the toxicity of several
commonly used pet flea dips and shampoos. The acute
toxicity of six flea shampoos and four dips was
evaluated with C. dubia (AQUA-Science, 1995a;
Miller et al., 1994). Although the products tested
varied widely in toxicity, shampoos were generally less
toxic than the dips. The most toxic products tested
contained Chlorpyrifos (IC25s of 0.800 to 2.30 ug/L as
product), which were 2,500-7,000 times more toxic
than the least toxic product tested, which contained
D-limonene (IC25 of 5.687 ug/L). The products
containing pyrethrins and permethrin had intermediate
levels of toxicity (IC25s of 0.149-4.683 ug/L).
Calculations (with the associated assumptions on use
rate, system losses, and dilution) indicated that only
flea dip products containing Chlorpyrifos were
sufficiently toxic to produce measurable effluent
toxicity to C. dubia.
Effects of Household Bleach on Aqueous
Concentrations of Diazinon and Chlorpyrifos
A study was conducted to determine if household
bleach could be recommended to residential customers
127
-------
+0.099
+0.001
CCCSD Fairfield Hay ward Palo Alto Petaluma San , San Jose Union
Francisco
+1.15
Vallejo
0.14
CCCSD Fairfield Hayward Palo Alto Petaluma San San Jose Union Vallejo
Francisco
Influent and Effluent Samples
I Influent [J Effluent
Figure F-4. Mean chlorpyrifos and diazinon concentrations (ฑstd) in influent and effluent from mine Bay Area POTWs during
August 1997.
128
-------
as a measure to degrade diazinon in spray container
rinsate and chlorpyrifos from pet flea washes prior to
disposal into the sewer. Samples of tap water were
spiked with high concentrations of diazinon
(60.0 fig/L) and chlorpyrifos (10.0 |J.g/L) and treated
with either 0.005 or 5% solutions of household bleach
for 24 hours. After neutralization, concentrations of
the insecticides were measured by ELISA (AQUA-
Science, 1995a). Both bleach concentrations reduced
concentrations of the insecticides by 86-92%. The
study suggested that household bleach may be a
effective pretreatment for waste solutions of diazinon
and chlorpyrifos prior to disposal. Additional studies
are planned to further define bleach exposure times and
concentrations under actual use conditions, and to
characterize the chemical oxidation products produced
by the chlorine treatment.
Diazinon and Chlorpyrifos Concentrations in
Water Samples from Restaurant Grease Traps
The CADPR source identification study recommended
follow-up studies to determine concentrations of
diazinon and chlorpyrifos in wastewater from
restaurants. Water samples were collected from the
grease traps of eight restaurants in the CCCSD service
area (AQUA-Science, 1997). ELISA was used to
measure concentrations of the two insecticides.
Diazinon and chlorpyrifos concentrations ranged from
0.192-4.197 jig/Land 0.265-4.313 ng/L, respectively.
The highest concentrations of both insecticides were
found in wastewater from the same restaurant. The
uses that contributed to these insecticide residues in the
wastewater are currently being investigated by the
CCCSD.
Regulatory Activities
Chlorpyrifos-Related
In January 1997, Dow-Elanco, as part of an agreement
with USEPA, announced the following actions
associated with the registered uses of chlorpyrifos (L.
Goldman, USEPA Assistant Administrator for
Prevention, Pesticides and Toxic Substances. Press
Release on January 16, 1997):
Withdrawal of chlorpyrifos from indoor broadcast
and fogger flea control markets.
Withdrawal of chlorpyrifos from direct application
pet-care uses (shampoos, dips, and sprays).
Increase marketing of ready-to-use products to
replace concentrated formulas.
Increase training and supervision of pest control
operators.
Revise chlorpyrifos labels to limit retreatment
intervals.
If the chlorpyrifos in POTW influent loading is due to
indoor and pet-care uses and/or misapplications by pest
control operators, these actions should substantially
reduce influent loadings of this chemical.
Diazinon-Related
In 1996, Novartis Crop Protection, Inc., the major U.S.
registrant of diazinon, submitted voluntary label ,
changes to USEPA to warn users not to dispose of this
product into sanitary or storm drains. Novartis also
developed educational materials with this message and
provided the materials to selected cities in Texas and
California. In 1997, Novartis completed a 4-year study
with several POTWs in USEPA Region VI on diazinon
occurrence and treatability (Novartis, 1997). Afollow-
up study is on-going with a California POTW to
identify treatment processes that consistently optimize
removal of diazinon (D. Tierney, personal
communication, Novartis Crop Protection, 1997).
Discussion
In this case study, USEPA TIE procedures were used
to identify organophosphate insecticide toxicity in a
POTW effluent. Phase I and n TIE procedures
identified diazinon as a candidate toxicant. Phase HI
TIE studies determined that effluent diazinon
concentrations were significantly correlated with the
extent of the effluent toxicity, but diazinon only
accounted for approximately half of the effluent's
toxicity. The follow-up TIE studies identified
chlorpyrifos at potentially toxic concentrations in the
toxic effluent samples. ELISA procedures were shown
to provide sensitive and accurate measurements of the
two insecticides in samples of POTW influent and
effluent, and these procedures were used extensively in
follow-up TIEs and source identification studies.
Additional TIE experiments found chlorpyrifos to be
poorly recovered through the Phase I and n TIE
processes, which may explain why it has not been
identified as a toxicant in other effluent TIEs.
The source identification studies at the CCCSD and
other Bay Area POTWs showed that the influents
contained highly variable, and often potentially toxic,
levels of diazinon and chlorpyrifos, which appearto be
originating primarily from residential rather than
commercial sources. However, only a relatively small
number of commercial sources have been sampled to
date. Thus, it is possible that certain business types
129
-------
(e.g., restaurants and nurseries) may be significant
contributors of the two insecticides into wastewater.
All of the POTWs that were sampled to date have
demonstrated substantial removal of both insecticides
from their influents. This was surprising because it
was generally believed that these insecticides were
poorly treated by POTWs (J.L. Miller, personal
communication, Aqua-Science, Inc., Davis, California,
April 1998). The available data suggest that there were
substantial differences in influent loadings of diazinon
and chlorpyrifos between POTWs within the San
Francisco Bay region. Further studies are planned to
explore the demographic basis for these differences to
evaluate patterns of insecticide use. Seasonal trends in
insecticide removal efficiencies are currently being
monitored in nine Bay Area POTWs. Public outreach
programs, supported, in part, by the manufacturers of
diazinon and chlorpyrifos, have been implemented by
the CCCSD and other POTWs across the country to
increase awareness of the proper use and disposal of
insecticides. Recent regulatory actions have resulted
in the withdrawal of chlorpyrifos from the pet flea
control market, and this action, coupled with the
enhanced training of applicators and the increased use
of prediluted insecticide products, may eventually
reduce the influent loadings. Monitoring studies are in
place at the CCCSD and elsewhere to determine if
these programs will result in reduced influent loadings
and decreased incidences of insecticide-related effluent
toxicity.
Acknowledgments
This work was supported wholly or in part by the
CCCSD, Martinez, California. Bart Brandenburg,
Bhupinder Dhaliwal, and Jim Kelly of the CCCSD
managed the various aspects of this project. The TRE
studies were conducted at AQUA-Science, Davis,
California, under the direction of J.L. Miller and MJ.
Miller.
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Attachment I
Summary of POTW Treatment System Characteristics
CCCSD
39mgd
Primary sedimentation
Air-activated sludge (MART 1.6 days)
Secondary clarification
UV disinfection
Fab-field
13 mgd
Primary sedimentation
Oxidation towers with clarification
Air-activated sludge (MART 12-14 days)
Secondary clarification
Tertiary filtration with dual media
Chlorine disinfection (90-120 minutes)
Hayward
12 mgd
Valuators
Primary sedimentation
Fixed film reactors (sludge age n/a)
Anaerobic digester
Final clarifiers
Chlorine disinfection (~100 minutes)
Palo Alto
26 mgd
Primary sedimentation
Fixed film reactor to mixed aeration basins with
activated sludge (MART 11.6 days)
Secondary clarifiers
Mixed media filtration
Chlorine disinfection (90 minutes)
Petaluma
6 mgd
Primary clarification
41% to activated sludge
32% to trickling filter
27% bypasses to ponds where retention time is about
100 days :
San Francisco
17 mgd
Primary sedimentation
Air-activated sludge (MART ~ 0.86 days)
Secondary clarification
Sodium hypochlorite disinfection
San Jose
137 mgd
Primary sedimentation
Air-activated sludge (MART ~ 4 days)
Secondary clarification
Nitrification and clarification (MART -11 days)
Tertiary filtration with backwash to clarification (for
flow equilibrium)
Chlorine disinfection (40-60 minutes)
Union
31 mgd
Primary sedimentation
Air-activated sludge (MART ~ 1.75 days)
Secondary clarifiers
Chlorine disinfection (30 minutes)
I
Vallejo
12 mgd
Primary sedimentation
Biological filters
Aeration basins (MART ~ 3 days)
Clarification
UV disinfection and sodium hypochlorite contact
(8 minutes)
132
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Appendix G
TRE Case Study:
Linden Roselle Sewerage Authority, New Jersey
Abstract
TRE Goal:
Test Organism:
TRE Elements:
Toxicants Identified:
Toxicity Controls:
96-hour LC50;> 50%
Interim goal of LC50 ^30%
M. bahia
Facility performance
evaluation, TIE, toxicity
source evaluation
Ammonia, non-polar
organic compounds,
surfactants
Pretreatment limits
Summary
Ammonia was confirmed as the primary cause of
toxicity, and pretreatment limits were developed to
reduce effluent ammonia concentrations. Secondary
causes of toxicity were complex and highly variable.
Toxicity-based procedures were used to identify
industrial sources of toxicity and develop pretreatment
limits to control secondary causes of toxicity.
In 1997, a major source of ammonia was eliminated.
An acute toxicity test performed since then showed a
reduction in effluent toxicity (LC50 = 72%) to
compliance levels (i.e., LC50 >50%). Additional tests
are planned to confirm this initial result.
Key Elements
1. TIE procedures may need to be modified to
evaluate multiple causes of effluent toxicity. In
this study, it was necessary to remove toxic
effluent concentrations of ammonia in the TIE
before other causes of toxicity could be identified
and confirmed.
2. If TIE analyses are successful in confirming
causes of effluent toxicity (e.g., ammonia),
chemical-specific analyses can be used to identify
4.
sources and pretreatment limits can be developed
for controllable toxicants.
If the TEE is inconclusive or the causes of toxicity
are variable and complex, the RTA approach can
be used to track the industrial sources of toxicity in
the collection system. Once identified, the toxic
dischargers can be required to meet pretreatment
limits for toxicity.
If effluent toxicity is contributed by controllable
industrial sources, pretreatment controls are more
practical than in-plant controls.
Introduction
Permit Requirements
The LRSA New Jersey Pollutant Discharge
Elimination System (NJPDES) permit contains an
acute whole effluent toxicity limit of LC50 >50%
effluent. A 96-hour static renewal M. bahia (mysid)
test is used to monitor compliance with the limit.
Based on observed toxicity to mysids, the NJPDES
permit was amended to include a requirement to
perform a TRE. In July 1992, the LRSA entered into
an administrative consent order (AGO) with the New
Jersey Department of Environmental Protection
(NJDEP) to establish a compliance schedule for
reducing acute effluent toxicity. The AGO established
a compliance date of October 31,1996, if pretreatment
controls are implemented and a compliance date of
December 31, 1997, if in-plant controls are
implemented. The AGO also includes TRE milestones
and an interim whole effluent toxicity limitation of an
LC50 of 30%. The acute effluent toxicity limit of an
LC50 of 50% becomes effective on May 1, 2000.
Description of the Treatment Plant
The LRSA POTW serves a 13-square-mile area in
northeastern New Jersey. The POTW has a design
133
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flow of 17 mgd and is presently treating a wastewater
flow of about 13 mgd. Approximately 20% of the
influent flow is contributed by 40 industrial users.
Primary treatment consists of screening and degritting
followed by primary sedimentation. The primary
effluent is then treated by roughing (trickling) filters
and conventional activated sludge treatment.
Following secondary clarification, the effluent is
disinfected with chlorine and then discharged to the
Arthur Kill estuary. The NJPDES permit specifies that
samples for toxicity testing be collected prior to
chlorination.
Plant Performance Evaluation
A limited POTW performance evaluation was
conducted during a USEPA TRE research study to
determine if POTW operations or performance was
contributing to the observed acute toxicity. The
POTW performance evaluation findings showed that
industrial wastewater contributions have a significant
effect on the variability and concentration of influent
constituents. For example, in 1987, influent BOD5
varied from 292 to 636 mg/L, oil and grease ranged
from 11 to 132 mg/L, and ammonia-nitrogen varied
from 17 to 119 mg/L (Morris et al., 1990). The
influent variability requires the LRSA to make
significant modifications to plant operations, such as
operating one or two aeration basins, to maintain
optimum treatment. Despite this variability, the LRSA
has consistently met NJPDES permit effluent limits for
conventional pollutants.
Overall, the POTW performance evaluation indicated
that the operation and performance of the LRSA
POTW was satisfactory and the treatment processes
did not appear to be contributing to effluent toxicity
(Morris et al., 1990). The POTW performance
evaluation also indicated that the ammonia
concentrations observed in the effluent warranted
further evaluation as a cause of effluent toxicity.
Pretreatment Program Review
Monthly average influent ammonia concentrations at
the LRSA have been as high as 150 mg/L. A review of
the influent ammonia data indicated consistently lower
ammonia levels in July of each year (LRSA, 1990a).
The decreased ammonia concentrations were related to
the temporary shutdown of a manufacturing process at
a major industrial contributor.
Toxicity Identification Evaluation
An objective of the LRSA TRE was to identify the
causes of effluent toxicity in order to select controls for
reducing toxicity. Initial TIE Phase I and Phase n
testing was performed in 1989 using C. dubia as a
surrogate test species. C. dubia were used because
little information was available at the time for using
mysids as a TEE test organism. Subsequent TIE testing
in 1991 was performed using mysids to confirm that
the causes of toxicity identified using C. dubia were
also causes of toxicity to mysids.
TIE Phase I
During the USEPA study, three effluent samples were
tested using the TIE Phase I procedures (USEPA,
1988). The Phase I results and ammonia data indicated
that ammonia was a primary cause of effluent toxicity.
Toxicity reduction by CIS SPE suggested that non-
polar organic compounds were also contributing to
effluent toxicity (Morris et al., 1990).
TIE Phases II and III
TIE Phase H (USEPA, 1989b) and Phase HI (USEPA,
1989c) analyses were performed using C. dubia and
mysids to identify arid confirm ammonia and non-polar
organic toxicants as causes of effluent toxicity (LRSA
1990b, 1991; Morris et al., 1992). It was necessary to
remove ammonia toxicity in the TIE before other
toxicants could be evaluated. A serial treatment
approach was used, to evaluate the contribution of
non-polar organic toxicants to acute effluent toxicity.
Effluent samples were first treated with zeolite to
remove ammonia and then non-polar organic toxicity
was evaluated using CIS SPE column treatment and
GC/MS analyses. A separate CIS SPE column test
was performed using whole effluent to determine if
zeolite treatment had removed non-polar organic
toxicity.
Results of the non-polar organic toxicant confirmation
tests, presented in Table G-l, show that filtration, CIS
SPE column treatment, and zeolite treatment reduced
toxicity to both mysids and C. dubia. The combined
treatment steps removed all of the acute toxicity to both
species. Following filtration, zeolite treatment
removed 1.3 to 2.0 TUa, while the CIS SPE column
removed 1.5 to 4.3 TUa. Acute toxicity to both species
was recovered in the 80 to 100% methanol/water
fractions from the C18 SPE column. Although only
0.3 TUa were recovered from the column, previous
tests had shown greater recovery (>2 TUa). The lower
recovery of non-polar organic toxicity in this sample
may be due to the presence of toxicants that are
difficult to elute from the C18 SPE column (e.g.,
surfactants were indicated as a possible toxicant based
134
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Table G-l. TEE Phase III Results; Non-Polar Organic Compound Confirmation (LRSA POTW)
Sample Description*
Baseline toxic units
Post-filtration treatment
Aliquot No. 1
Post-filtration and CIS SPE column treatment (original pH)
Combined toxic methanol/water
CIS SPE column fractions*
Aliquot No. 2 '
Post-zeolite treatment
Post-zeolite and CIS SPE column treatment
Combined toxic methanol/water fractions from zeolite/
CIS SPE column treatment*
TUa (100/LC50)
C. dubiaf
4.3
2.8
100 ง
0.3
1.5
<1.0
0.3
M, bahia$
8.5
6.3
100 ง
0.3
4.3
<1.0
0.3
* Effluents of serial treatment steps.
t 48-hour C. dubia acute toxicity test.
t 96-hour M bahia acute toxicity test.
ง Percent mortality in 100% sample after 48 and 96 hours for C. dubia and M. bahia, respectively.
# Methanol/water fractions were evaluated at 5 times and 2.5 times whole effluent concentration for C. dubia and M. bahia,
respectively.
on the toxicity removed by filtration). Overall, the
results showed that mysids were sensitive to the same
non-polar organic toxicity as C. dubia. These tests
confirmed non-polar organic toxicants as a cause of
effluent toxicity to mysids.
Difficulties were encountered in trying to identify and
confirm the specific non-polar organic toxicants. TIE
Phase H procedures (USEPA, 1989b), which included
HPLC separation and GC/MS analyses, tentatively
identified more than 20 non-polar organic compounds
as potential causes of toxicity. In addition, many
potentially toxic unknown compounds were detected.
The results suggested that the majority of the
compounds were related to industrial sources because
the compounds are not typically found in domestic
wastewater. Further work was not performed to
identify the toxic non-polar organic compounds
because:
Little or no toxicity data were available for most of
the non-polar organic compounds identified in the
effluent (e.g., no LC50 values for the specific non-
polar organic compounds); therefore, it was not
possible to determine if the concentrations present
in the effluent were acutely toxic.
The non-polar organic toxicants varied from
sample to sample, which made it difficult to
determine consistent causes of non-polar organic
toxicity.
Many of the compounds detected were unknowns.
The TIE results indicated that, in addition to ammonia,
non-polar organic toxicity may need to be controlled to
achieve compliance with the acute toxicity limit. Due
to the difficulty in determining the non-polar organic
toxicants, the LRSA decided to use a toxicity-based
approach to identify the sources of non-polar organic
toxicity and other non-ammonia effluent toxicity.
Toxicity Source Evaluation
The available information indicated that both ammonia
and non-ammonia (e.g., non-polar organic) toxicity
was being contributed by controllable industrial
sources. Therefore, pretreatment controls were
deemed to be feasible and source evaluation studies
were performed to identify the sources of ammonia and
non-ammonia toxicity. Sources of ammonia were
identified by a chemical-specific approach and sources
of non-ammonia toxicity were identified by a
toxicity-based approach. The resulting information
was used to develop appropriate pretreatment limits.
135
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Chemical-Specific Source Evaluation
The LRSA conducted studies to locate the major
sources of ammonia in the collection system. Key
manholes and industrial discharges were sampled and
tested for total ammonia from 1990 through 1992. the
results indicated one major industrial source of
ammonia in the collection system. Based on the survey
results, the LRSA developed and implemented
pretreatment limits to reduce effluent ammonia
concentrations (LRSA, 1993a).
Toxicity-Based Source Evaluations
The toxicity-based approach used RTA procedures that
involved treating industrial wastewater samples in
bench-scale, batch simulations of the POTW activated
sludge process and measuring the resulting toxicity
(USEPA, 1989a). The toxicity remaining after batch
treatment, referred to as "refractory" toxicity,
represented the toxicity that passes through the POTW
and causes effluent toxicity. As shown in Figure 5-2
(Section 5), two types of batch reactors are tested. A
control reactor simulated the treatment plant and
treated only the POTW influent. The second reactor
evaluated the addition of the industrial discharge to the
POTW by treating industrial wastewater spiked into
the POTW influent. An industrial discharge would be
considered a source of toxicity if effluent from the
spiked reactor was more toxic than the control reactor
effluent.
Initial RTA tests conducted during the USEPA study
indicated that refractory toxicity was limited to an
industrialized area of the collection system. Following
the USEPA study, ammonia was confirmed as the
primary cause of effluent toxicity and the major source
of ammonia was identified. Accordingly, subsequent
RTA tests focused on identifying sources of
non-ammonia toxicity. In 1992, RTA testing was
performed to evaluate sources of non-polar organic
toxicity because non-polar organic compounds had
been identified as a major cause of non-ammonia
toxicity.
The procedure for measuring non-polar organic
toxicity involved passing the RTA batch effluent
samples through a CIS SPE column, eluting the
column with methanol, and performing a toxicity test
on the methanol elution (LRSA, 1992a). This
procedure provided a direct means of measuring non-
polar organic toxicity and it eliminated interferences
associated with toxic ammonia concentrations
(i.e., ammonia was not captured by or eluted from the
CIS SPE column).
The toxicity source evaluation identified two industrial
dischargers of non-polar organic toxicity (LRSA,
1992b). Nonpolar organic toxicity tests performed on
the effluent during this period suggested that non-polar
organic toxicity was variable and that there may be
other causes of non-ammonia toxicity. Therefore,
further RTA testing was conducted in 1993 to identify
sources of non-ammonia toxicity that may be caused by
non-polar organic compounds and other unidentified
compounds.
The ammonia pretreatment limits were not to become
effective until after July 1995; therefore, the LRSA
influent and effluent ammonia concentrations remained
high during 1993. It was necessary to remove
ammonia toxicity in RTA testing in order to identify
sources of non-ammonia toxicity (LRSA, 1993b).
Zeolite treatment of the batch effluent samples to
remove ammonia was considered, but previous studies
indicated that zeolite also may remove non-ammonia
toxicity. Therefore, two alternative approaches were
used to remove ammonia toxicity in the RTA. First,
testing was conducted during periods of low influent
ammonia concentrations, which occurred during the
annual summer shutdown of the ammonia-contributing
industrial process. During this period, ammonia
concentrations werb not acutely toxic; therefore, RTA
testing would provide a direct measure of the
non-ammonia toxicity contributed to the POTW. The
second approach was used when the ammonia
contributing process was fully operational and involved
using a simulated plant influent (SPI). The SPI
consisted of sewer wastewater collected from all major
trunk lines except the sewer line serving the ammonia
discharger. It was also necessary to wash the RAS
used in the RTA to reduce the ammonia concentrations
associated with the RAS (LRSA, 1993c).
The 1993 RTA testing was intended to identify those
industries that would be required to meet pretreatment
requirements to control non-ammonia toxicity.
Thirty-two of the 40 industrial users were evaluated
either directly or indirectly by testing sewer wastewater
samples collected from key manholes. Previous RTA
results and information obtained in an industrial user
waste survey were used to select the industries to be
tested.
136
-------
The results of RTA tests performed in July and
October 1993 are presented in Table G-2. If the
effluent toxicity of the sewer wastewater spiked reactor
was greater than that of the control reactor on two
occasions, the discharge was considered a source of
toxicity. Industries A, B, E, and F were indicated as
sources of non-ammonia toxicity based on the results
of direct testing of their industrial discharges. These
results support the findings of the USEPA study, which
identified industries A, B, and E as sources of toxicity,
and the 1992 study, which identified industries B and
E as sources of non-polar organic toxicity. Six other
industries were identified as suspected sources based
on the results obtained for key manholes 9 and 12.
LRSA plans to test these suspected sources directly to
determine which industries are contributing toxicity.
Table G-2. Results of Refractory Toxicity Assessment, July and October 1993*
RTA Reactor
Effluent
Control Reactor
Spiked Reactors
Industry A
Industry B
Industry C
Industry D
Industry E
Industry E 5x f
Industry F
Industry G
Industry H
Industry I
Industry J
Key manhole 1
Key manhole 3
Key manhole 4
Key manhole 7A
Key manhole 9 ง
Key manhole 10
Key manhole 12 #
Key manhole 14
Key manhole 15
Roselle flume
96-Hour Mysid TUa (100/LC50)
JullS
<1.0
<1.0
1.45
<1.0
NT
NT
NT
NT
NT
NT
NT
NT
<1.0
NT
NT
<1.0
1.1
NT
1.33
NT
NT
NT
Jull6
<1.0
NT
NT
NT
<1.0
<1.0
NT
<1.0
NT
NT
NT
NT
NT
NT
<1.0
NT
NT
NT
NT
NT
<1.0
<1.0
Jul22
1.63
1.92
1.89
NT
NT
NT
NT
NT
<1.0
NT
NT
NT
NT
NT
NT
NT
NT
1.33
1.81
1.33
NT
NT
Jul23
1.05
NT
NT
NT
NT
1.19
4.0
2.18
NT
NT
NT
NT
NT
1.12
NT
<1.0
<1.0
NT
NT
NT
NT
NT
Octl9
2.0
3.39
NT
NT
NT
NT
NT
1.55
NT
2.28
NT
NT
NT
t
NT
NT
6.1
NT
1.71
NT
NT
NT
Oct20
1.75
1.22
NT
NT
NT
1.75
NT
1.86
NT
NT
1.29
1.81
NT
NT
NT
NT
NT
NT
1.63
NT
NT
NT
Source of Refractory
Toxicity?0
n/a
YES
YES
NO
NO
YES
YES
YES
NO
NO
NO
NO
NO
NO
NO
NO
YES
NO
YES
NO
NO
NO
* Spiked reactor results shown in bold indicate greater TUa than the control. Increased toxicity in the spiked reactor
effluent compared to the control indicates a source of refractory toxicity.
t Tested at five times the normal flow contribution to evaluate anticipated increase in flow.
t Toxicity test was invalid based on unacceptable control survival.
ง Key manhole 9 receives wastewater from three industries.
# Key manhole 12 receives wastewater from three industries.
w If a spiked reactor result was greater than that of the control on two occasions then the discharge was considered a source
of refractory toxicity.
NT Not tested.
137
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Toxicity Control Evaluation
The LRSA evaluated control options for ammonia and
non-ammonia toxicants. The objective was to identify
and assess the available options and to determine the
most cost effective and pragmatic approaches for
reducing effluent toxicity to acceptable levels.
Ammonia Toxicity Control Evaluation
A modified acute toxicity test procedure was
developed by the LRSA and approved by the NJDEP
to control pH drift in the toxicity test. The pH in
previous LRSA compliance tests typically drifted up to
8.0 to 8.5, which resulted in an overestimation of
ammonia toxicity (i.e., unionized ammonia concen-
trations increase as pH increases). The modified test
procedure maintains pH in the toxicity test at the
receiving system pH of 7.4. This modification
provides a more accurate measurement of instream
ammonia toxicity.
Using ammonia toxicity values for mysids published
by USEPA (1989d), a linear regression model was
prepared to predict the concentration of ammonia in
the effluent which, in the absence of other toxicants,
should result in compliance with the acute toxicity
limit. The ammonia value generated by the model
accounts for toxicity test conditions that affect the
concentration of unionized ammonia (e.g., pH,
temperature, and salinity). The model determined that
the acute toxicity limit could be met with an effluent
ammonia concentration of 35 mg/L (LRSA, 1991).
Several options forin-plant treatment of ammonia were
evaluated to achieve the ammonia target level. As
shown in Table 6-1 (Section 6), none of the six options
evaluated was practical based on technical and cost
considerations. In addition, significant inhibition of
nitrification was observed during treatability tests,
indicating that inhibitory compounds would need to be
controlled if nitrification was selected as a control
option (LRSA, 1991). Based on these results and the
results of the ammonia source evaluation, chemical-
specific pretreatment limits were selected as the best
approach for controlling toxicity caused by ammonia
(LRSA, 1993a). \
i
Non-Ammonia Toxicity Control Evaluation
The TIE indicated that the causes of non-ammonia
toxicity were complex and highly variable and the
specific compounds causing non-ammonia toxicity
could not be identified and confirmed. Consequently,
the necessary information was not available to develop
chemical-specific pretreatment limits.
As an alternative to pretreatment limits, activated
carbon treatment at the POTW was evaluated based on
its effectiveness in reducing effluent toxicity caused by
a variety of compounds including non-polar organic
toxicants. Both PAC and GAC treatment were
considered and found to be cost prohibitive (T.L.
Morris, Technical Memorandum to LRSA, Evaluation
of Granular Activated Carbon at LRSA, January 19,
1993). It also was determined that the use of PAC
treatment would result in unacceptable sludge quality.
The LRSA elected to implement pretreatment controls
because controllable industrial sources of non-
ammonia toxicity had been identified and practical
in-plant treatment options were not available. It was
determined that the pretreatment limits must be
toxicity-based because of the lack of specific
information on the causes of non-ammonia toxicity.
The proposed pretreatment approach involved RTA
testing to determine which industries should be issued
limits and which industries should be monitored to
assess the need for future limits (LRSA, 1993c).
Implementation Of Toxicity Controls
Ammonia Pretreatment Limits
The approach used to develop pretreatment limits for
ammonia was relatively straightforward. As required
by the AGO, the LRSA submitted a work plan for
developing ammonia pretreatment limits to the NJDEP
in April 1992 and the plan was approved in May 1992
(LRSA, 1992c). Using the target ammonia level of 35
mg/L and the ammonia survey data, an allowable
headworks loading approach (USEPA, 1987) was
followed to develop draft pretreatment limits. The
LRSA published the draft limits for public notice and
comments were received and reviewed. In January
1993, the proposed ammonia pretreatment limits and
the LRSA's response to public comments were
submitted to the NJDEP. The limits were approved in
March 1993 and industrial users were to comply with
the limits by July 1995 (LRSA, 1993a).
Toxicity-Based Pretreatment Limits for
Non-Ammonia Toxicity
The LRSA is one of the first municipalities to develop
toxicity-based pretreatment limits to control non-
ammonia toxicity. At the time of this study, toxicity-
based pretreatment limits had not been applied
138
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elsewhere and. there was no specific guidance on
developing such limits. The selected approach was
based on the available TRE information and involved
several aspects of various pretreatment approaches
recommended by USEPA (1987).
The LRSA submitted a work plan for development of
the limits to the NJDEP in June 1993 (LRSA, 1993b).
The proposed approach was designed to address both
major and minor sources of non-ammonia toxicity
(LRSA, 1993c) and to ensure compliance without
unnecessary controls. The proposed limits will consist
of the following components referred to collectively as
a toxicity management program (TMP):
Narrative local pretreatment limit of "no discharge
of refractory toxicity."
Pass/fail toxicity-based limit using the RTA
procedure as a compliance test (i.e., the effluent
LC50 of the industrial user spiked reactor may not
be less than the LC50 of the control reactor
effluent).
Industrial user (if toxicity is found) may be
required to implement a toxicity reduction
program comprising requirements to identify
causes and sources of toxicity, implement
industrial user managementpractices, and evaluate
and establish other controls to ensure compliance
with the toxicity-based limits.
RTA monitoring requirements and decision
criteria for determining if an industrial user needs
to continue with the TMP.
Provisions to allow industries to be relieved from
the TMP requirements if toxicity requirements are
met.
Compliance schedule including milestones and
progress reports.
Reopener clause stating that the pretreatment
permit will be modified to include chemical-
specific limits if the causes of toxicity are
identified.
The proposed pretreatment limit approach falls under
the case-by-case/best professional judgment approach
described by USEPA (1987), but also includes
toxicity-based requirements, industrial user
management practice, and chemical-specific
components. The TMP approach is consistent with
USEPA recommendations for monitoring and
controlling effluent toxicity through the NPDES.
The RTA procedures had not been used for compliance
monitoring purposes in New Jersey. Therefore, a
site-specific RTA protocol (LRSA, 1994) was
submitted to the NJDEP for review and approval prior
to development of the draft pretreatment limits. The
RTA protocol was approved by the NJDEP in June
1996. Pretreatment program permits for several
industries were modified to include the TMP
provisions. These industries are currently required to
conduct quarterly monitoring using the RTA protocol.
Discussion
Chemical-specific pretreatment limits are being
implemented to control toxicity caused by ammonia
and toxicity-based pretreatment limits are in place to
control non-ammonia toxicity. The major source of
ammonia ceased its discharge of the ammonia-laden
waste stream in 1997. As a result, effluent ammonia
concentrations at the LRSA treatment plant decreased
to about 30 mg/L!_A_co.mpliance test performed after
the ammonia source was eliminated showed improved
effluent quality (i.e.-, LC50 = 72%). Additional tests
are planned to confirm this initial result.
It is possible that the ammonia pretreatment limits
alone will achieve compliance with the acute effluent
toxicity limit. However, due to the complex and
variable nature of the non-ammonia toxicity, it is not
possible to accurately predict if the ammonia reduction
will achieve consistent-compliance with the permit
limit LC50 ;>50%). The LRSA has established
pretreatment requirements for non-ammonia toxicity to
ensure full and timely compliance with the toxicity
limit. The need for industrial users to control
non-ammonia toxicity is ultimately tied to compliance
with the acute effluent toxicity limit. If necessary,
industrial users may request relief from these
requirements if the effluent consistently complies with
the acute effluent toxicity limit.
Acknowledgments
The USEPA research study was funded by the Office
of Research and Development, Risk Reduction
Engineering Laboratory in Cincinnati, Ohio. The
LRSA assisted USEPA and its technical consultant,
Engineering-Science, Inc. (Fairfax, Virginia), in
conducting this study. TIE and toxicity analyses for
the USEPA study were performed by EA Engineering,
Science and Technology, Inc. (Sparks, Maryland), and
the USEPA's National Effluent Toxicity Assessment
139
-------
Center (NETAC). Subsequent TRE work was
performed by Engineering-Science, Inc., in association
with EA. Engineering-Science, Inc., and the LRSA
acknowledge the assistance of Gary Fare and Judy
Spadone (LRSA), the LRSA Board members, William
Goodfellow (EA), and John Botts, Mark Collins, Tim
Morris, and Tim Schmitt (Engineering-Science, Inc.).
The material presented in this appendix includes
copyrighted information published in text by
Technomic Publishing Company, Lancaster, PA (Botts
et al., 1992). Technomic Publishing Company has
granted permission to include the information in this
document.
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Developing Secondary Toxicant Pretreatment
Limits. Prepared by Engineering-Science, Inc.,
Fairfax, Virginia.
Linden Roselle Sewerage Authority. 1993c. LRSA
Toxicity Reduction Program 1993: Toxicity
Source Evaluation Final Report. Prepared by
Engineering-Science, Inc., Fairfax, Virginia.
Linden Roselle Sewerage Authority. 1994. Draft
Protocol for Conducting Refractory Toxicity
Assessment Testing Under the Linden Roselle
Sewerage Authority Industrial Pretreatment
Program. Prepared by Engineering-Science, Inc.,
Fairfax, Virginia.
Morris, T.L., J.A. Botts, J.W. Braswell, M.C. Welch,
and W.L. Goodfellow. 1990. Toxicity Reduction
Evaluation at the Linden Roselle Sewerage
Authority Wastewater Treatment Plant. Draft
Report. Prepared for the USEPA, Office of
Research and Development, Risk Reduction
Engineering Laboratory, Cincinnati, Ohio.
USEPA Contract No. 68-03-3431.
Morris, T.L., G. Fare, and J. Spadone. 1992. Toxicity
Reduction Evaluation at the Linden Roselle
140
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Sewerage Authority Wastewater Treatment Plant.
Water, Environment, and Technology, June: 8-16.
USEPA. 1987. Guidance Manual on the Development
and Implementation of Local Discharge
Limitations Under the Pretreatment Program.
EPA Office of Water Enforcement and Permits,
Washington, D.C.
USEPA. 1988. Methods for Aquatic Toxicity
Identification Evaluations: Phase I Toxicity
Characterization Procedures. EPA/600/3-88/034.
National Effluent Toxicity Assessment Center,
Duluth, Minnesota.
USEPA. 1989a. Toxicity Reduction Evaluation
Protocol for Municipal Wastewater Treatment
Plants. EPA/600/2-88/062. Office of Research
and Development, Risk Reduction Engineering
Laboratory, Cincinnati, Ohio.
USEPA. 1989b. Methods for Aquatic Toxicity
Identification Evaluations: Phase II Toxicity
Identification Procedures. EPA/600/3-88/035.
National Effluent Toxicity Assessment Center,
Duluth, Minnesota.
USEPA. 1989c. Methods for Aquatic Toxicity
Identification Evaluations: Phase III Toxicity
Confirmation Procedures. EPA/600/3-88/036.
National Effluent Toxicity Assessment Center,
Duluth, Minnesota.
USEPA. 1989d. Ambient Water Quality Criteria for
Ammonia (Saltwater). EPA/440/5-88/004. Office
of Water Regulations and Standards, Criteria and
Standards Division, Washington, D.C.
141
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Appendix H
Toxicity Control Options for Organophosphate Insecticides
Organophosphate insecticides, including diazinon,
chlorpyrifos, malathion, and chlorfenvinphos, have
been found to cause effluent toxicity at POTWs
throughout the United States (Norberg-King et al.,
1989; Amato et al., 1992; USEPA, 1987; Botts et al.,
1992; Kllmore et al., 1990). A case study of the
occurrence of Organophosphate insecticide toxicity at
POTWs in the San Francisco Bay area is presented in
Appendix F. Although procedures are available for
identifying Organophosphate toxicants, less is known
about how to control Organophosphate insecticides in
POTW effluents. This section describes approaches
for Organophosphate toxicity control that have been
successfully implemented at POTWs. Information is
also presented on ongoing research into POTW
operational improvements that may reduce effluent
concentrations of Organophosphate toxicants.
A review of the literature suggests that two approaches
may be successful in reducing Organophosphate
compQunds at POTWs:
Public education to limit the discharge of
Organophosphate compounds to the POTW.
POTW modifications, particularly involving
enhancements to the biological treatment and
chlorine disinfection processes.
The latter approach has been the subject of a research
study being funded by the two principal manufacturers
of Organophosphate compounds in North America:
Novartis Crop Protection, Inc., and Makhteshim-Agan
of North America, Inc.
Public Education Approach
Organophosphate insecticides are used widely for pest
control by homeowners, restaurants, veterinarians, and
other commercial businesses. These sources are not
readily controlled by pretreatment program regulations.
Alternative efforts to minimize the use or disposal of
Organophosphate insecticides must have broad appeal
to the public at large.
Organophosphate insecticide control measures that
have been considered by POTW staff include public
outreach and education programs and approaches to
restrict the use of Organophosphate compound
applications. Efforts to ban or restrict the use of
Organophosphate insecticides have not been successful,
largely because of concern about legal issues and the
difficulty in controlling the sale of Organophosphate
compounds outside of the community.
Restrictions on Organophosphate Insecticide
Use '
In 1990, the City of Largo, Florida, evaluated the
feasibility of banning the use of diazinon and other
Organophosphate insecticides (malathion and
chlorfenvinphos) to control effluent toxicity (C.
Kubula, personal communication, City of Largo,
Florida, 1992). It was determined that a diazinon ban
would likely increase the use of other, equally toxic,
insecticides. For example, Dursbanฎ, a likely
alternative insecticide, contains chlorpyrifos, which
has been found to be more toxic than diazinon. Also,
restrictions on diazinon use would apply only to new
supplies, not to insecticides already in stock at stores.
The City of Largo estimated that the stockpiled
diazinon would last for more than a year. An effective
control program would also require the cooperation of
neighboring communities in limiting the purchase of
diazinon outside of the community. In addition, the
local banning of federally approved insecticides would
be controversial. It was anticipated that insecticide
manufacturers and distributors would challenge the
City's authority to implement such controls. Based on
142
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this analysis, the City of Largo determined that banning
diazinon would not be a practical control option.
Public Education Campaigns
Based on the impracticality of insecticide bans, the
City of Largo elected to pursue a public awareness
approach to control diazinon toxicity. The City of
Greenville, Texas, also implemented a public
education program in 1990 (City of Greenville, 1991).
The first year of the program focused on determining
significant users of the insecticide and developing
educational materials. The following years have
involved distributing the materials and conducting
other informational activities.
The City of Greenville initially identified nine groups
of diazinon users: pest control businesses, lawn care
businesses, veterinarians, animal shelters, janitorial
services, apartment complexes, restaurants, hotels, and
retail stores (City of Greenville, 1991). The residential
population also was added as a target user group. The
City service area was divided into sections, and a
telephone survey was conducted. Information was
gathered on diazinon use, including existing supplies
and application and waste disposal practices, and
business owners and homeowners were notified of the
importance of controlling diazinon wastes. The
program involved the following public education
activities:
Brochures and handouts
Pest control fact sheets describing integrated pest
management methods, which focused on
minimizing insecticide usage
Mass mailings
Newspaper articles
ซ Public service announcements
Occasional talk shows on local radio stations
Biweekly presentations to schools and business
groups
A telephone information line.
The City of Greenville also enacted an ordinance to
encourage environmentally sound use of insecticides.
The ordinance requires retail vendors, pest control
services, and apartment managers to distribute
educational material to customers and to periodically
report insecticide applications to the City.
The results of the Greenville education campaign are
encouraging. Beginning in December 1993, the
treatment plant effluent was not toxic to C. dubia for 3
consecutive months. The public awareness effort is
continuing and the City will monitor its effect on
toxicity reduction.
The City of Largo initiated a public education
campaign in 1992. An information brochure was
prepared and distributed in 1993. Effluent toxicity
decreased; however, it was not known if the reduction
is related to the public education program. A strong
emphasis has not been placed on the program because
the City has opted for a land irrigation treatment
system in lieu of continued effluent discharge.
As noted in Appendix A of this manual, diazinon and
its toxic metabolite diazoxon were tentatively
identified as effluent toxicants at the City of Lawton
POTW. The City decided to implement a public
awareness program in 1993 to control the discharge of
insecticides to the POTW (Engineering Science, 1993).
Information on the proper use and disposal of
insecticides was printed in newspaper articles and on
monthly water bills. An electronic message sign with
insecticide information also was located at major
intersections. Since August 1993, the POTW effluent
has met the toxicity permit limit (NOEC >96%
effluent) with the exception of 2 months in 1994 and
several months in 1995 (as of September 1997).
Although diazinon was not confirmed as an effluent
toxicant, the City's ongoing insecticide control effort
appears to have been successful in achieving
compliance with the chronic toxicity limit.
POTW Operational Improvements
Diazinon Treatment
In 1992, Novartis Crop Protection, Inc., in cooperation
with Makhteshim-Agan of North America, Inc.,
initiated a study on diazinon and its relationship to
effluent toxicity at POTWs (Novartis, 1997). A
principal objective of the study was to determine the
treatability of diazinon and assess its fate in POTWs.
Research on this subject included a survey of POTWs
in which organophosphate insecticide toxicity was
observed and bench-scale treatability tests were
conducted to evaluate diazinon removal by various
treatment methods and operating conditions.
Two types of POTW biological treatment processes
were investigated in the Novartis study: fixed film
(trickling filter and RBC) and activated sludge.
Influent and effluent concentrations at several POTWs
in the southwestern United States were compared to
determine removals of diazinon and chlorpyrifos.
143
-------
Overall, the data indicated that diazinon reduction
could be achieved in conventional POTW treatment
processes. A statistical analysis of the data showed
that the fixed film process had a significantly lower
percent removal (p=0.95) for diazinon than the
activated sludge process or a combined fixed film/
activated sludge process. A similar trend was observed
for chlorpyrifos, although no significant differences
were found between the process types.
Bench-scale treatability testing was conducted to
further evaluate the fate of diazinon in typical POTW
processes. These tests considered the effect of design
and operating conditions for biological treatment
processes on diazinon removal and effluent toxicity.
Additional tests were performed to investigate the
effect of physical/chemical processes, including
chemical precipitation, chlorination/dechlorination, and
post aeration on diazinon concentrations and toxicity.
As shown in Figure H-l, a correlation was found to
exist between diazinon removal and sludge retention
time (SRT), HRT, and MLSS concentration in
activated sludge treatment tests. The primary removal
mechanism in the activated sludge tests was adsorption
onto the biological solids. These results suggest that
diazinon removal may be improved by increasing the
SRT, HRT, and/or MLSS concentration of the
treatment process.
Auxiliary process studies provided additional
information on treatment of diazinon (Novartis, 1997).
Chemical precipitation using ferric chloride and
polymer only slightly reduced diazinon levels. No
major change in diazinon concentrations was observed
whether the coagulants were added to primary
wastewater or secondary treated wastewater prior to
clarification. Chlorination treatment was effective in
reducing diazinon from secondary clarifier effluent;
however, chronic toxicity was unchanged. Qualitative
results suggest that the chlorine oxidized diazinon to
diazoxon, a by-product that exhibits similar toxic
effects as diazinon. Post aeration of secondary clarifier
effluent also reduced diazinon levels; however, once
again, chronic toxicity was not significantly changed.
Again, it was assumed that diazinon was oxidized to
diazoxon. !
Additional tests evaluated the fate of diazinon in
POTWs (Novartis, 1997). Anecdotal evidence from
other studies (Fillmore et al., 1990) and the treatability
studies suggested that adsorption onto solids was the
dominant removal mechanism. Therefore, the tests
focused on partitioning of diazinon and chlorpyrifos
onto primary and mixed liquor solids. These tests
showed that about 30% of the diazinon and 85 to 90%
of the chlorpyrifos present in POTW primary influent
samples is adsorbed onto primary influent solids.
Mixed liquor adsorption results revealed that
approximately 65 to 75% of the diazinon added to the
mixed liquor adsorbed onto the biomass. Diazinon
adsorption was greater for a 30-day SRT biomass than
for a 15-day biomass. Chlorpyrifos strongly adsorbed
to the biomass; 100% was removed.
Summary
Studies have shown that organophosphate compounds
can be effectively controlled through public education
(City of Greenville, 1991; Engineering Science, Inc.,
1993). This effort may vary from the distribution of
educational materials to the enactment of ordinances
that require strict accounting of insecticide use. The
studies conducted to date indicate that characterization
of the sources of organophosphate compounds is key
to the development of a successful toxicity control
program.
Recent information shows that relatively simple
enhancements to POTWs may help to reduce
organophosphate compounds. Factors affecting
diazinon and chlorpyrifos removal include the SRT,
HRT, and MLSS concentrations in activated sludge
processes, chlorination/dechlorination, and post
aeration. Further studies are in progress to better
define the operating conditions that will promote
organophosphate compound removal (D. Tierney,
personal communication, Novartis Crop Protection,
Inc., 1997).
144
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70 -r
_ 65
ฃ 60--
1 55 --
ts 50
S 45 +
ฃ 40 +
35 --
30
I
ง
I
70 T
30
1,000
70 -
65 -
60 -
55 -
50 --
45 -
40
35 --
30 --
R2 = 0.543
10
12
HRT(hrs)
1,500
2,000
MLSS (mgfl)
2,500
3,000
R2 = 0.9956
10
15
20
SRT (days)
Figure H-1. Diazinon removal as a function of SRT, HRT, and MLSS concentration (reprinted with the permission
of Novartis Crop Protection, Inc.) (Source: Novartis, 1997).
145
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References
Amato, J.R., D.I. Mount, EJ. Durban, M.L,
Lukasewycz, G.T. Ankley, and E.D. Robert!
1992. An Example of the Identification of
Diazinon as a Primary Toxicant in an Effluent;
Environ. Toxicol. Chem. 11: 209-16. ;
Bolts, J.A., T.L. Morris, J.E. Rumbo, and C.H.
Victoria-Rueda. 1992. Case Histories - Munici-
palities. Toxicity Reduction: Evaluation and
Control. D.L. Ford, ed. Lancaster, PA:
Technomic Publishing Co.
City of Greenville. 1991. TRE Phase B Final Report.
City of Greenville, North Carolina.
|
Engineering-Science, Inc. 1991. Toxicity Reduction
Evaluation Phase I Report. Submitted to the City
of Lawton, Oklahoma, September 1991. Parsons
Engineering Science, Inc., Fairfax, Virginia.
j
Engineering-Science, Inc. 1992. Toxicity Reduction
Evaluation: Toxicant Identification and Confirma-
tion. Submitted to the City of Lawton, Oklahoma,
Parsons Engineering Science, Inc., Fairfax,
Virginia.
Engineering-Science, Inc. 1993. Review of Diazinon
Control Options. Submitted to the City of Lawton,
Oklahoma, April 1993. Parsons Engineering
Science, Inc., Fairfax, Virginia.
Fillmore, L.B., T.L. Morris, T.L. Champlin, M.C.
Welch, and J.A. Bolts. Draft 1990. Toxicity
Reduction Evaluation at the City of Fayetteville
Cross Creek Wastewater Treatment Plant. Draft
Report. USEPA Risk Reduction Engineering
Laboratory, Cincinnati, Ohio.
Norberg-King, T.J., M. Lukasewycz, and J. Jensen.
1989. Results of Diazinon Levels in POTW
Effluents in the United States, Technical Report
14-89. USEPA, National Effluent Toxicity
Assessment Center, Dululh, Minnesola.
Novartis Crop Protection, Inc., and Makhteshim-Agan
of North America, Inc. 1997. Investigation of
Diazinon Occurrence, Toxicity, and Treatability in
Southern United States Publicly Owned Treatment
Works. Technical Report 3-7, Environmental
Affairs Departmenl, Greensboro, North Carolina.
USEPA. 1987. Toxicity Identification Evaluation of
the Largo, Florida POTW Effluent. J.R. Amalo,
D.I. Mounl, M. Lukasewycz, E. Durham, and E.
Robert. National Effluent Toxicity Assessment
Center, Duluth, Minnesola.
USEPA. 1989a. Methods for Aquatic Toxicity
Identification Evaluations: Phase II: Toxicity
Identification Procedures. EPA/600/3-88/035.
Office of Research and Development Duluth,
Minnesola.
USEPA. 1989b. Methods for Aquatic Toxicity
Identification Evaluations, Phase HI.
EPA/600/3-88/036. Toxicily Confirmalion
Procedures. Office of Research and Development,
Dululh, Minnesola.
USEPA. 1992. Toxicity Identification Evaluation:
Characterization of Chronically Toxic Effluents,
Phase I. EPA/600/6-91-005F. National Effluenl
Toxicily Assessmenl Center, Dululh, Minnesota
USEPA. 1993a. Methods for Aquatic Toxicity
Identification Evaluations: Phase II Toxicity
Identification Procedures for Samples Exhibiting
Acute and Chronic Toxicity. EPA/600/R-92-080.
National Effluent Toxicity Assessment Center,
Duluth, Minnesota.
USEPA. 1993b. Methods for Aquatic Toxicity
Identification Evaluations: Phase III Toxicity
Confirmation Procedures for Samples Exhibiting
Acute and Chronic Toxicity. EPA/600/R-92-081.
National Effluent Toxicity Assessment Center,
Duluth, Minnesota.
146
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Appendix I
Pretreatment Program Chemical Review
Introduction
It may be possible in limited cases to identify the toxic
influent sources by comparing pretreatment program
data on suspected sources to chemical-specific and
toxicity data on the POTW effluent. The objective of
the PPCR is to determine the sources of toxicity by
comparing chemical data on industrial dischargers to
toxicity data reported in the literature. The
pretreatment program information should include flow
and chemical monitoring data on the industrial users,
descriptions and schedules of industrial production
campaigns, and inventories of chemicals used in
production. The final outcome of this review should
be an improved understanding of the industries'
processes and chemical usage, and the possible
identification of sources of toxicity. Source
identification through the PPCR approach has been
successful in reducing effluent toxicity at POTWs with
a limited number and type of industrial inputs (Diehl
and Moore, 1987).
General Procedure
The main steps in a PPCR are to:
Gather the pertinent pretreatment program data
Compare the data to POTW effluent toxicity
results and/or TIE data
Identify potential influent source(s) of toxicity
Evaluate and recommend a toxicity control
option(s).
A brief description of each of these steps follows.
Collect Data on Industrial Users
Data on all categorical, significant non-categorical and
other potential toxic dischargers (e.g., industrial users
with local limits and RCRA and CERCLA inputs)
should be collected. A list of pertinent information
that should be considered in a PPCR is presented in
Table 2-3. The data collection effort should include a
survey of each industrial user, using the example
checklist shown in Table 1-1.
Information on chemicals that may be used in
manufacturing processes can be obtained from the
Encyclopedia of Chemical Technology (Kirk-Othmer,
1982). Although OSHA regulations require that
information on hazardous chemicals is to be made
available to the public on MSDSs, information on
various "specialty" chemicals can be difficult to obtain.
When data on a "specialty" chemical are not disclosed,
a literature review can be performed to determine the
chemical's acute toxicity and biodegradability. This
information allows assumptions to be made concerning
the biodegradability of the chemical at the POTW and
the potential for the chemical to.cause effluent toxicity.
An initial indication of the possible toxic pollutants
causing effluent toxicity can be made by comparing
expected or actual effluent concentrations to toxicity
values provided in the literature.
Compare PPCR Data to POTW Effluent Toxicity
Results
Information on the magnitude, variability, and nature
of the POTW effluent toxicity can be compared with
the PPCR data to determine the soufces(s) of possible
problem chemicals. This comparison can be made
using statistical analyses to determine if the variability
in the source characteristics can be related to the
variability in the POTW effluent toxicity. A
description of data analysis techniques for comparing
POTW and industry pretreatment data follows.
Two types of statistical analyses can be used to
compare the pretreatment program chemical data and
POTW effluent toxicity data: linear regression (Draper
and Smith, 1966) and cluster analysis (Pielou, 1984;
Romesburg, 1984).
147
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Table M. PPCR Data Sheet
Industry Name
Notes:
Address
Notes:
Industrial Category (SIC Code)
Notes:
TRE Objectives
Notes:
Manufactured Products
Notes:
Chemicals Used
Notes:
Amounts (write on MSDS)
Notes:
MSDS
Process in which chemical is used
(write on each MSDS)
Notes:
Aquatic toxicity/biodegradability information
on all chemicals used. Review MSDS,
supplier information, and literature
Notes:
Engineering drawings of facility
Notes:
Production flowchart and line schematic
Notes:
All floor and process drains with schematic
Notes:
Wastewater pretreatment system schematic
Notes:
Facility records
Notes:
Water usage, water bills
Notes:
DMRs for 24 months
Notes:
Pretreatment system operations data
Notes:
Pretreatment system operator interview
Notes:
Spilt prevention control plan
Notes:
RCRA reports, hazardous waste manifests
Notes:
All Attached
None
_ Available
. Available
. Available
. Available
. Available
. Available
Available
Available
Available
Partial Available
Some
No
No
No
No
No
No
No
No
No
148
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Linear regression analysis is used to find correlations
among the variables in the data base and to relate
changes in POTW effluent toxicity to the variables. A
cluster analysis using pattern recognition software can
weigh and evaluate the significance of toxics/toxicity
correlations. The determination of concentration
/response relationships through statistical analysis
should not be considered as a definitive answer to
toxicity tracking because of the complexity of the
factors contributing to toxicity in POTW effluents.
The following example illustrates how a stepwise
linear regression technique can be used in a PPCR
assessment. The technique is used to identify how
changes in several variables can impact the presence
and variability of effluent toxicity. Table 1-2 presents
an example data sheet for a POTW serving one
manufacturing plant. In this example, only a few
POTW effluent industry variables were used in the
linear regression analysis; however, additional
variables also could be added in the regression
analysis.
The following variables are the "X" variables:
Industry variables:
LBS is the manufactured product per month
(millions of pounds).
INFLOW is the discharge flow based on water
usage (mgd).
Table 1-2. Data Sheet for Regression Analysis
POTW effluent variables:
OELOW is the recorded effluent flow (mgd).
COD is the chemical oxygen demand concentra-
tion (mg/L).
BOD5 is the biochemical oxygen demand concen-
tration (mg/L).
Cu is the copper concentration (mg/L).
Cr is the chromium concentration (mg/L).
Zn is the zinc concentration (mg/L).
The following variable is the "Y" variable:
LC50 is the acute LC50 as percent effluent.
By applying standard stepwise linear regression, the
variables OELOW, BOD5, Cr, and Cu were eliminated
because they were insignificant to toxicity. Stepwise
linear regression showed that the remaining (X)
variables were significant as regressed versus (Y)
LC50. This analysis indicated that Zn, COD, LBS, and
INFLOW were correlated with POTW effluent
toxicity.
Identify Source(s) of Toxicity
Based on the data analysis, a list of the possible
contributors to effluent toxicity at the POTW can be
developed. Sources of suspected toxicants should be
selected based on toxicant loading calculations.
Industrial users who contribute potentially toxic
Month
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Parameter
LBS
0.80
1.01
1.20
1.25
1.16
0.90
0.90
1.20
1.30
1.27
1.10
0.90
INFLOW
1.2
1.5
1.7
1.7
1.6
1.2
1.2
1.6
1.8
1.7
1.6
1.2
OFLOW
1.0
'1.2
1.4
1.5
1.4
1.0
0.9
1.4
1.6
1.4
1.4
1.0
COD
30
33
41
39
30
28
25
23
25
26
30
40
BOD5
10
11
15
14
12
11
10
9
15
18
17
21
Cu
0.73
0.61
0.78
0.65
0.66
0.68
0.71
0.72
0.69
0.72
0.71
0.75
Cr
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
Zn
1.6
1.9
2.0
1.6
1.5
1.4
1.8
1.9
2.0
2.1
1.9
2.0
LC50
20
20
18
18
22
30
40
38
40
33
28
22
149
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loadings of suspected toxicants would be candidates
for a toxicity control evaluation.
Recommend Toxicity Control Option(s)
Of the potential toxicity control options, toxic chemical
substitution or elimination is usually the most
pragmatic approach. Thus, a follow-up interview with
the toxic discharger(s) should be conducted to develop
information concerning techniques for the preferred
use of problem chemicals. A list of useful interview
questions is shown in Table 1-3. These questions may
enable the industry to identify problem areas and
possible corrective actions in the use of toxic
chemicals in manufacturing. Source control may
include substitution or elimination of problem
chemicals, flow reduction, equalization, spill control,
and manufacturing process changes.
Table 1-3. Summary of the PPCR Chemical Optimization Procedure
1. Objectives
a. Optimize chemical usage amounts in production and water treatment processes.
b. Optimize chemical structures in process chemicals ensuring biodegraclability or detoxification is possible.
c. Establish process controls over incoming raw materials, measuring possible toxic components. Example:
corrosion-resistant finish put on steel by manufacturer that must be removed prior to part fabrication.
2. Strategy
a. Determine the role of each chemical in the process. This is done by supplier interviews and review of data
gathered during the initial survey. Ask the questions:
Can less of this chemical be used?
Has the optimum amount been determined for each process? ;
Do other suppliers offer compounds that will perform as well at lesser concentrations?
Is the compound in reality a part of the manufacturer's water treatment system and independent of product
production?
OBJECTIVE: Use less chemicals per pound of product produced.
b. Discover the biodegradability and toxicity of the process chemical. This is done by supplier interview, review
of MSDS information, and literature search. Suppliers may not want to supply exact chemical formulations.
In this case, ask industry to request supplier to perform tests to develop needed data. Questions to ask:
What are the components in the product?
What is its aquatic toxicity?
Is the product biodegradable? :
What is the rate of biodegradation or half-life?
Are there other component chemicals on the market that meet manufacturing requirements, but are low in
toxicity and highly biodegradable?
OBJECTIVE: Use chemicals that will not create or contribute to toxicity problems.
c. Establish process controls over incoming raw materials. Many raw materials have chemicals used in their
manufacturing that are removed in the production of the final product. Many raw materials may have trace
contaminants that may cause toxic problems. Questions to ask:
What chemicals are used in the manufacturing of the raw material?
What are the residual amounts of these raw material contaminants or by-products?
Are there quality-control procedures that measure the amounts of these chemicals?
What are the statistical process measures used in the monitoring of these chemicals in the raw materials?
150
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Table 1-3. Summary of the PPCR Chemical Optimization Procedure (continued)
If these chemicals are required to be removed before the raw materials can be used in manufacturing the
final product, what purpose do the chemicals serve in raw material manufacturing?
Can they be eliminated?
Can they be made less toxic or more biodegradable?
OBJECTIVE: Understand all raw materials being used and encourage development of QA procedures
to monitor toxic chemicals removed during processing.
3. Outcome of Investigations
a. There will be a list of all chemicals used in processing and manufacturing of products. Included will be the
amounts used, why the chemicals are used, and if optimization is being practiced.
b. MSDS sheets for all chemicals used will be on file.
c. A list of chemicals applied or used in the manufacturing of all raw materials will be on file under that raw
material with the residual amounts noted if possible.
d. There will be a list of all chemicals and raw materials purchased on a monthly basis and the amount of
product produced.
OBJECTIVE: Hard information to be used in data analysis.
4. Use of opportunities available due to past experience
a. With experience in various industries, certain chemicals will become "known" as typically used in some
process of manufacturing.
b. These known compounds can be categorized and toxicity determinations made. Once found toxic, the first
information the industry must supply to the POTW staff conducting the TRE is whether these chemicals
are used in its manufacturing process, in raw materials, or in water treatment processes.
c. Letters also are sent to raw material suppliers asking if these compounds are used in raw material
production. If they are, the supplier is asked to submit prototype alternative raw materials that do not
contain these compounds.
d. This can be done at the beginning of the TRE for known problem chemicals. Indeed, control regulations
also usually involve establishing limits for selected known toxics in industrial operations.
e. What is accomplished by this process can be remarkable. First, the supplier is alerted that these compounds
can cause his or her customers problems, resulting in a search for an alternative raw material source that
is free of these objectionable chemicals. A successful market search reduces the market demand for
contaminated or objectionable raw material.
5. Tests to help assess toxicity/biodegradability on speciality formulated chemicals and mixtures and to help
evaluate competitive products
a. BOD?,BOD20.
b. BOD5, BOD20 performed at LC50 concentration with toxicity test performed on settled effluent from test.
c. COD before and after BOD5, BOD20 at LC50, EC50 concentrations.
d. Estimate biodegradability by using BOD5 and COD tests and the calculation (BOD5 - COD)/COD x 100
of 10 or 20 mg/L solutions of chemical; this can be repeated at a 20-day BOD.
e. Biomass inhibition tests (see detailed procedures given in Section 5).
f. LC50 on products; screening dilutions 1-10,000 ppm.
OBJECTIVE: Help industry determine relative biodegradability and toxicity of various raw materials,
products, and by-products.
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References
Diehl, R., and S. Moore. 1987. Case History: A
North Carolina Municipal TRE. Toxicity Iden-
tification/Reduction Evaluation Workshop, Water
Pollution Control Federation Conference,
Philadelphia, Pennsylvania.
Draper and Smith. 1966. Step-wise Multiple Regres-
sion: Applied Regression Analysis. New York,
New York: John Wiley and Sons.
Kirk-Othmer. 1982. Encyclopedia of Chemical
Technology. New York, New York: Wiley
Interscience.
Pielou. 1984. Cluster Analysis Techniques: The
Interpretation of Ecological Data. New York:
Wiley Interscience.
Romesburg, H.C. 1984. Cluster Analysis for
Researchers. Lifetime Learning Publications.
Gelmont, California.
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Appendix J
Refractory Toxicity Assessment Protocol:
Step-by-Step Procedures
The following protocol provides step-by-step
procedures for designing and executing RTA studies to
track sources of acute and/or chronic toxicity in POTW
collection systems. This protocol describes the
following steps:
Using characterization data to evaluate waste
streams of concern.
Accounting for toxicity in the activated sludge
biomass to be used in testing. .
Adapting and calibrating the protocol to site-
specific conditions.
Collecting and analyzing samples to be used in
testing.
Preparing RTA test mixtures.
Performing RTA tests.
Evaluating the inhibitory potential of waste
streams.
Performing TIE Phase I tests on RTA effluents
(optional).
The RTA protocol was first developed in the USEPA
TRE research study at the City of Baltimore's Patapsco
POTW (Botts et al., 1987) to evaluate the potential for
indirect dischargers to contribute refractory toxicity.
Additional USEPA TRE research studies in Linden,
New Jersey; High Point, North Carolina; Fayetteville,
North Carolina; and Bergen County, New Jersey were
conducted to improve the RTA approach (Morris et al.,
1990; DiGiano, 1988; Fillmore et al., 1990; Collins et
al., 1991). The RTA protocol described below is a
refined version of the method given in the first edition
of the Municipal TRE Protocol (USEPA, 1989).
The RTA procedure has been used to track sources of
acute and chronic toxicity using both freshwater and
estuarine/marine species (Morris et al., 1990; Botts et
al., 1992, 1993, 1994). Examples of RTA studies are
presented in Appendices C, D, and G. The RTA
protocol has been designed to simulate conventional
activated sludge processes, although it has also been
adapted to other POTW treatment processes including
single and two-stage nitrification systems (Collins, et
al. 1991), BNR processes (Appendix D), and filtration
treatment systems (Appendices C and D).
A. POTW Wastewater Profile
Characterization data are generated for each waste
stream to be tested in the RTA.
1. Collect grab samples of RAS and 24-hour
composite samples of POTW primary effluent
and selected sewer wastewaters (i.e., sewer
line wastewater or indirect discharges).
2. Analyze RAS samples (filtrate) for TSS, VSS,
NH3-N, and pH.
3. Analyze primary effluent and sewer
wastewater samples for BOD5, COD, TSS,
TKN, TP, NH3-N, and pH.
4. Determine the type of unit processes, type of
discharge (e.g., continuous versus
intermittent), operations schedule, and flow
rate for the discharge points selected for
evaluation (see Section 5).
5. Repeat above steps on several samples to
characterize variability over time.
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B. Biomass Toxicity Measurement
Biomass toxicity is measured to evaluate the potential
for toxicity interferences in the RTA.
1. Collect 5 liters of fresh RAS and aerate
vigorously for 15 minutes.
2. Prepare glass fiber filter [same type used for
TSS analysis (APHA, 1995) by rinsing two 50
ml volumes of high purity water through the
filter.
I
3. Rlter sufficient volume of RAS for two acute
or chronic toxicity tests.1
I
4. Centrifuge a portion of the RAS filtrate at
10,000 xg for 10 to 15 minutes. Alternatively,
filter RAS filtrate through a 0.2 um pore-size
filter if blank tests show that the filter does not
remove soluble toxicity or add artificial
toxicity (see Section 5).1
5. Test RAS filtrate and RAS centrate/fine
filtrate for acute toxicity using procedures
described by USEPA (1991a, 1991b) or for
chronic toxicity using limited-scale methods
provided by USEPA (1992a, 1992b, 1992c,
1995,1996).
6. Repeat above steps on several RAS samples
to characterize variability over time.
7. If RAS filtrate is more toxic than the RAS
centrate/fine filtrate, obtain non-toxic biomass
(e.g., another POTW biomass or a
freeze-dried preparation) (see Section 5).
C. RTA Reactor Calibration Testing2
Calibration tests are performed to select the RTA test
operating conditions that most closely simulate the
POTW operation and performance.
1 Positive pressure filtering is recommended. Chronic
toxicity measurement will require larger filtrate
volumes than acute tests.
2 RTA calibration is recommended. If resources are
limited, POTW staff may select test conditions that
reflect POTW operating conditions. However, RTA
reactor performance should be compared to POTW
performance to ensure that the RTA procedure
effectively simulates the POTW processes.
1. As described in Section 5, estimate MLVSS
concentration for RTA batch tests using
mathematical models (Grady and Lim, 1980;
Kornegay, 1970). Alternatively, use the
average MLVSS concentration for the POTW.
2. Select a series of MLVSS concentrations
(e.g., four) that includes the model MLVSS
concentration. Calculate the volumes of RAS
(Vr) needed to yield the MVLSS
concentrations in the batch reactors. If the
RAS was found to be toxic (i.e., RAS filtrate
is more toxic than RAS centrate in step B-5
above), also select appropriate volumes of
non-toxic biomass (Vnb). An equation for
calculating Vb and Vnb is:
Vb orVnb(L)= TargetMLVSS (mg/L)
RASVSS(mg/L)
where: Vr is the reactor test volume.
3. Add each RAS volume (Vb and Vnb, if
needed) to pre-cleaned glass or clear plastic
containers. Add diffused air using air stones
and gently aerate. Note that it may be
necessary to filter the air supply to prevent
contamination (e.g., compressor oil) of the
reactor mixed liquors.
4. Add primary effluent (Vpe) to each reactor
containing Vb and Vnb. Vpe can be
calculated using the following equation:
Vpe = (Vr-Vb).
5. Adjust aeration rate to maintain DO at
concentrations typically observed in POTW
activated sludge process. Mechanical mixing
using a magnetic stirrer and teflon-coated stir
bars may be required to ensure complete
mixing. Periodically check and adjust DO
level.
6. Periodically check the batch reactor pH.
Adjust pH to 6-9 range, if necessary.
7. Periodically collect 50-100 ml samples of
batch reactor mixed liquor from each reactor
(e.g., 1- to 2-hour intervals).
8. Allow mixed liquor samples to settle for 15
minutes. Rinse glass fiber filters as stated in
154
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step B-2 above. Filter each mixed liquor
supernatant using separate filters.
9. Stop aeration after the required reaction
period, allow the Vb (and Vnb) to settle for 15
minutes, and filter the clarified batch effluents
as described in step C-8.
10. Analyze filtered batch mixed liquor and
effluent samples to determine COD removal
over time.
11. Decant additional clarified batch effluent for
toxicity analysis. Filter each batch effluent
using rinsed filters.1 Wash filter apparatus
between each sample filtration using high-
purity water.
12. Batch filtrates that were treated with toxic
biomass (Vb) must be centrifuged at 10,000
xg for 10 to 15 minutes to remove colloidal
size particles. Viscous mixtures may require
faster or longer centrifugation (ASM, 1981).
Alternatively, the batch filtrates may be
filtered through a 0.2 |im pore-size filter if the
filter does not remove soluble toxicity (see
Section 5).3 Filter blank analyses should be
performed for each filter type using high-
purity water.
13. Analyze the batch effluent filtrates, centrates,
and filter blanks for acute or chronic toxicity
using the procedures referenced in step B-5.
14. Calibration test results can be used to select a
batch MLVSS concentration that achieves a
level of COD and toxicity removal similar to
that provided by the POTW activated sludge
process (see Section 5).
D. Sample Collection
Representative samples are collected from each waste
stream to be tested in the RTA.
1. Upon completion of the RTA calibration, tests
can be conducted to evaluate the refractory
toxicity of sewer wastewaters.
2. Obtain 24-hour, flow-proportioned composite
samples of sewer wastewater (i.e., sewer line
wastewater or indirect discharger effluent)
and POTW primary effluent. If possible, lag
collection of the primary effluent sample by
the estimated travel time of the sewer
wastewater to the POTW.
3. Collect 10 liters of RAS (and non-toxic
biomass, if needed) on day of test.
E. Sample Characterization (performed on
day of sample collection)
Sample characterization data are collected to set the
operating conditions for the RTA.
1. Analyze sewer wastewater for BOD5, COD,
TSS, TKN, TP, NH3-N, andpH.
2. Prepare glass fiber filter as stated in step B-2.
Filter RAS and test filtrate for acute or
chronic toxicity using the procedures
referenced in step B-5.4
3. Determine percent volume of sewer
wastewater in POTW influent based on flow
data gathered in the wastewater profile (step
A above).
F. Preparation of RTA Test Mixtures
Two types of batch reactors are prepared: one
consisting of the POTW influent (primary effluent) and
RAS, which serves as a control, and another consisting
of the sewer wastewater spiked into the POTW influent
and RAS.
1. Calculate the volume of sewer wastewater
(Vw) based on the sewer wastewater flow and
the desired flow concentration factor (Fw).
Information on selecting an appropriate Fw is
presented in Section 5. Vw can be calculated
using the following equation:
Vw(L)=-ฐ^-x(Vr-Vb)xFw,
Qi
Positive pressure filtering is recommended. Also,
chronic toxicity measurement will require larger
filtrate volumes than acute toxicity tests.
Positive pressure filtering is recommended. Also,
chronic toxicity measurement will require larger
filtrate volumes than toxicity tests.
155
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where: Qw is the sewer wastewater flow rate
(mgd).
Qi is the average POTW influent
flow rate (mgd).
Fw is the sewer wastewater flow
concentration factor (e.g., 1, 2, 10
times the sewer wastewater flow).
2. Calculate the volume of primary effluent
(Vpe) using the following equation:
Vpe=(Vr-Vb-Vw).
3. Prepare spiked batch reactor influent by
mixing Vw with Vpe and measure Vpe for
control batch reactor influent.
4. If necessary, add nutrients to adjust the
BOD/TKN/TP ratio of the spiked batch
influent to equal the average BOD5/TKN/TP
ratio of the POTW influent (or 100:5:1). An
equation for calculating BOD5, TKN, and TP
concentrations in the spiked batch influent is:
BOD5,TKN.orTP(C,mg/L) =
5
(Vpe+Vw)
where: Cpe is the BOD5 or nutrient concen-
tration in primary effluent (mg/L).
Cw is the BOD5 or nutrient concen-
tration in sewer wastewater (mg/L).
5. If necessary, adjust pH of batch influents to
pH range for POTW influent.
6. Test sample toxicity (using methods
referenced in step B-5) after nutrient addition
and pH adjustment to determine if the batch
influent toxicity is changed by these steps.
7. Select volume of RAS (Vb) to yield the
MVLSS concentration determined in
calibration testing (step C above). If RAS is
toxic (i.e., RAS filtrate is more toxic than
RAS centrate), also select appropriate volume
of non-toxic biomass (Vnb). The equation for
calculating Vb and Vnb is provided in step
C-2.
8. Add each RAS volume (Vb and Vnb, if
needed) to pre-cleaned glass or clear plastic
containers.
9. Add spiked batch influent and control batch
influent to reactors containing Vb (and
reactors containing Vnb, if needed).
G. Performance of RTA Tests
The spiked batch reactor influent and control batch
reactor influent are treated and the resulting effluents
are tested for toxicity.
1. Add diffused air to reactors using air stones
and gently aerate. Note that it may be
necessary to filter the air supply to prevent
contamination (e.g., compressor oil) of the
reactor mixed liquors.
2. Adjust aeration rate to maintain DO at
concentrations typically observed in the
POTW activated sludge process. Mechanical
mixing may be required to ensure complete
mixing. Periodically check and adjust the DO
level.
3. Periodically check the batch reactor pH and
adjust to pH 6-9 range, if necessary.
4. The treatment period for the control reactor
should be equal to the average HRT of the
POTW aeration system. For the spiked
reactor, calculate the required reaction period
necessary to achieve a batch F/M ratio (F/Mb)
equal to the nominal F/M ratio determined in
calibration testing (step C above). F/Mb can
be calculated using the following equation:
TestPeri0d(dayS)^BatchInfluentCOD(mg/L),
(MLVSS (mg/L) x F/Mb
where: F/Mb is equal to the calculated F/M
of the control (primary effluent)
reactor.
F/Mb = CODpe/(MLVSS x test
period, days).
156
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5. Stop aeration after the required reaction
period and allow the Vb (and Vnb) to settle
for 1 hour. Decant the clarified batch
supernatant for toxicity analysis. Filter each
batch supernatant using rinsed filters.5 Wash
filter apparatus between each sample filtration
using high-purity water.
6. Batch filtrates that were treated with toxic
biomass (Vb) must be centrifuged at 10,000
xg for 10 to 15 minutes to remove colloidal
size particles (ASM, 1981). Alternatively, the
batch filtrates may be filtered through a 0.2
)im pore size filter if the filter does not
remove soluble toxicity (see Section 5).1
Filter blank analyses should be performed for
each filter type using high-purity water.
7. Analyze the batch filtrates, centrates, and
filter blanks for acute or chronic toxicity using
the procedures referenced in step B-5 above.
H. Synthetic Wastewater Testing (Optional)
Synthetic wastewater can be used in lieu of POTW
influent (primary effluent) in the RTA to determine the
toxicity of the sewer wastewater.
1. Select non-toxic synthetic wastewater.
Confirm that the synthetic wastewater is
non-toxic using toxicity test procedures
referenced in step B-5 above.
2. Prepare synthetic wastewater solution with
SCOD concentration equal to the average
SCOD of the POTW primary effluent.
3. Prepare volume of synthetic wastewater
(Vsw) equal to the volume of primary effluent
(Vpe) used above for the sewer
wastewater/primary effluent batch test.
4. Add Vw and Vsw to a reactor containing Vb
(and a reactor containing Vnb, if needed).
5. After batch treatment, analyze batch effluent
toxicity as described in step G above.
5 Positive pressure filtering is recommended. Also,
chronic toxicity measurement will require larger
filtrate volumes than acute toxicity tests.
I. Inhibition Testing (Optional)
The RTA protocol can be used to evaluate the
inhibitory potential of the sewer wastewater.
1. Add equal volumes of Vb to four reactors.
Add diffused air and gently aerate.
2. Prepare a series of four sewer wastewater
concentrations (e.g., 100, 50, 25 and 12.5%
wastewater) by adding sewer wastewater to
toxicity test dilution water (freshwater).
3. If necessary, add nutrients to adjust batch
influent BOD/TKN/TP ratio.
4. Add sewer wastewater volumes (e.g., Vw 100,
Vw50, Vw25 and Vwl2.5) to the reactors.
5. Adjust aeration rate to maintain DO at
concentrations typically observed in the
POTW activated sludge process. Mechanical
mixing may be necessary to ensure complete
mixing. Periodically check and adjust DO
level.
6. Periodically check the batch reactor pH and
adjust to pH 6-9 range, if necessary.
7. Subsample 300 ml from each reactor at 30
minutes and every 2 hours following test
initiation. Immediately measure oxygen
utilization using the BOD bottle method
(APHA, 1995). Return the subsamples to the
reactors immediately following oxygen
utilization measurement. Alternatively,
oxygen utilization can be measured using
respirometric techniques.
8. Subsample 50 ml from each reactor at 5
minutes and every 2 hours following test
initiation, and at completion of the test. Also,
subsample 50 ml of the original undiluted
RAS. Filter the subsamples through a 0.45
jam pore-size filter and measure the SCOD of
the filtrates.
9. Calculate oxygen and COD utilization rates,-
as described in Section 5 of this manual, and
plot rates versus sewer wastewater
concentration. Lower oxygen and COD
removal rates with increasing wastewater
concentration may indicate inhibition.
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J. Phase I Toxicity Characterization (Optional)
1. TIE Phase I tests may be conducted on RTA
test effluents using indirect discharger
wastewatef spiked into primary effluent.
Additional volumes are required for TIE
Phase I testing; therefore, the batch reactor
volume will need to be increased accordingly
(USEPA 199 la, 1992a, 1996).
2. TIE Phase I tests should be performed on
effluent filtrates from RTA tests that use
non-toxic POTW biomass.
References
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