Whole Whole EffluentEffluent Toxicity Toxicity BasicsBasics

Betty Jane Boros-RussoBetty Jane Boros-RussoNJDEP, Office of Quality AssuranceNJDEP, Office of Quality Assurance

Christopher J. NallyChristopher J. NallyAmerican Aquatic TestingAmerican Aquatic Testing

HistoryHistory 16th century - scientists began testing 16th century - scientists began testing

the lethality of chemical compounds on the lethality of chemical compounds on animals prior to their use on humans for animals prior to their use on humans for therapeutic purposestherapeutic purposes

1930’s - some of the first uses of 1930’s - some of the first uses of aquatic organisms for testing to aquatic organisms for testing to determine the causes of observed fish determine the causes of observed fish killskills

1945 - some of the first methods for 1945 - some of the first methods for conducting toxicity tests were publishedconducting toxicity tests were published

Use of Toxicity Testing in Water Quality Based Toxics Control

To characterize and measure the To characterize and measure the aggregate toxicity of an effluent or aggregate toxicity of an effluent or ambient watersambient waters

To measure compliance with whole To measure compliance with whole effluent toxicity limitseffluent toxicity limits

As an investigative tool and to measure As an investigative tool and to measure progress in a toxicity reduction programprogress in a toxicity reduction program

As an ambient instream measure of As an ambient instream measure of toxicity to identify pollution sourcestoxicity to identify pollution sources

NJ WET Program NJ WET Program HistoryHistory

Early 1980’s - Acute monitoring and Early 1980’s - Acute monitoring and limits used on a routine basislimits used on a routine basis

1989 - Began use of chronic monitoring 1989 - Began use of chronic monitoring and chronic limitsand chronic limits

1993 - Group permit challenge on 1993 - Group permit challenge on chronic WETchronic WET

1996 - Settlement and initial chronic 1996 - Settlement and initial chronic WET program revisionsWET program revisions

1997 - Final program revisions adopted1997 - Final program revisions adopted

Test SpeciesTest Species

Species SelectionSpecies Selection

Sensitive species which are easily cultured Sensitive species which are easily cultured and readily available year roundand readily available year round

Must provide consistent and reproducible Must provide consistent and reproducible responseresponse

Also encourage ecologically, commercially Also encourage ecologically, commercially and or recreationally importantand or recreationally important

No one species is always the most sensitiveNo one species is always the most sensitive Species used is dependent upon salinity of Species used is dependent upon salinity of

receiving water and the state standardsreceiving water and the state standards

New Jersey Freshwater New Jersey Freshwater Acute Test SpeciesAcute Test Species

InvertebratesInvertebrates: (Daphnids): (Daphnids)– Ceriodaphnia dubiaCeriodaphnia dubia– Daphnia magnaDaphnia magna– Daphnia pulexDaphnia pulex

FishFish– Pimephales promelasPimephales promelas Fathead Fathead

MinnowMinnow– Oncorhynchus mykissOncorhynchus mykiss Rainbow Rainbow

TroutTrout– Salvelinus fontinalisSalvelinus fontinalis Brook TroutBrook Trout

Ceriodaphnia dubiaCeriodaphnia dubia

FemaleFemale approximately 2 approximately 2

mmmm

Photo compliments of Marinco Bioassay Laboratory

Pimephales promelasPimephales promelas

Photo by Karen McCabe from Animal Soup

New Jersey Saline Acute New Jersey Saline Acute Test SpeciesTest Species

InvertebratesInvertebrates: : – Mysidopsis bahia Mysidopsis bahia Opossum Opossum

ShrimpShrimp((Americamysis bahiaAmericamysis bahia))

FishFish– Cyprinodon variegatusCyprinodon variegatus Sheepshead MinnowSheepshead Minnow– Menidia beryllinaMenidia beryllina Inland SilversidesInland Silversides– Menidia peninsulaeMenidia peninsulae Tidewater Tidewater

SilversidesSilversides– Menidia menidiaMenidia menidia Atlantic SilversidesAtlantic Silversides

Mysidopsis bahiaMysidopsis bahia

Photo compliments of Marinco Bioassay Laboratory

Female approximately 6 mm in length

New Jersey Freshwater New Jersey Freshwater Chronic Test SpeciesChronic Test Species

InvertebratesInvertebrates: : – Ceriodaphnia dubiaCeriodaphnia dubia

FishFish– Pimephales promelasPimephales promelas Fathead Fathead

MinnowMinnow

AlgaeAlgae– Selenastrum capricornutumSelenastrum capricornutum

New Jersey Saline Chronic New Jersey Saline Chronic Test SpeciesTest Species

InvertebratesInvertebrates: : – Mysidopsis bahia Mysidopsis bahia Opossum ShrimpOpossum Shrimp

FishFish– Cyprinodon variegatusCyprinodon variegatus Sheepshead MinnowSheepshead Minnow– Menidia beryllinaMenidia beryllina Inland SilversidesInland Silversides– Menidia peninsulaeMenidia peninsulae Tidewater SilversidesTidewater Silversides– Menidia menidiaMenidia menidia Atlantic SilversidesAtlantic Silversides

Other Other – Arbacia punctulataArbacia punctulata Sea UrchinSea Urchin– Champia parvulaChampia parvula Red MacroalgaeRed Macroalgae

Test MethodsTest Methods

Rules for Conducting Rules for Conducting Toxicity TestsToxicity Tests

40 CFR 136.3 - Table 1A40 CFR 136.3 - Table 1A Effective November 15, 1995Effective November 15, 1995 Amended November 19, 2002 and Amended November 19, 2002 and

effective December 19, 2002 effective December 19, 2002 Methods must be followed as they Methods must be followed as they

are writtenare written

Incorporate by ReferenceIncorporate by Reference Methods for Measuring the Acute Toxicity of Effluents to Methods for Measuring the Acute Toxicity of Effluents to

Freshwater and Marine OrganismsFreshwater and Marine Organisms. 5th Edition, USEPA, . 5th Edition, USEPA, Office of Water, October 2002, EPA 821-R-02-012Office of Water, October 2002, EPA 821-R-02-012

Short-term Methods for Estimating the Chronic Toxicity Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater of Effluents and Receiving Waters to Freshwater OrganismsOrganisms. 4th Edition, USEPA, Office of Water, October . 4th Edition, USEPA, Office of Water, October 2002, October 2002, EPA 821-R-02-0132002, October 2002, EPA 821-R-02-013

Short-term Methods for Estimating the Chronic Toxicity Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and of Effluents and Receiving Waters to Marine and Estuarine OrganismsEstuarine Organisms. 3rd Edition. USEPA, Office of . 3rd Edition. USEPA, Office of Water, October 2002, EPA 821-R-02-014Water, October 2002, EPA 821-R-02-014

USEPA Methods USEPA Methods DocumentsDocuments

Health and safetyHealth and safety Quality assuranceQuality assurance Facilities, equipment and suppliesFacilities, equipment and supplies Test organisms and culture Test organisms and culture

methodsmethods Dilution waterDilution water

USEPA Methods USEPA Methods DocumentsDocuments

(cont.)(cont.)

Effluent sampling and handlingEffluent sampling and handling Endpoints and data analysisEndpoints and data analysis Individual test methodsIndividual test methods Report preparation and test reviewReport preparation and test review

Test TypesTest Types

Acute and Short-term Chronic Acute and Short-term Chronic TestsTests– Static non-renewalStatic non-renewal– Static renewalStatic renewal– Flow throughFlow through

Test Species dependentTest Species dependent Use dependentUse dependent

Test DesignTest Design

5 Concentrations + Control5 Concentrations + Control– Serial dilution’s of effluent and “control Serial dilution’s of effluent and “control

water” (also termed “dilution water”)water” (also termed “dilution water”)– Dilution series of 0.5 or greaterDilution series of 0.5 or greater– Single concentration testSingle concentration test

ReplicatesReplicates

Randomization (organisms/chambers)Randomization (organisms/chambers)

Perspective is EverythingPerspective is Everything

Test Conditions and Test Conditions and

Acceptability CriteriaAcceptability Criteria

Acute Toxicity TestsAcute Toxicity Tests

Test ProceduresTest Procedures– 96 hours or less (species specific)96 hours or less (species specific)– Mortality is the measured endpointMortality is the measured endpoint– For daphnia mortality determined by immobilizationFor daphnia mortality determined by immobilization

AdvantagesAdvantages– less expensive and time consuming than chronicless expensive and time consuming than chronic– endpoint is easy to quantifyendpoint is easy to quantify

DisadvantagesDisadvantages– indicates only lethal concentrationsindicates only lethal concentrations– only the effects of fast acting chemicals are only the effects of fast acting chemicals are

exhibitedexhibited

Acute Test Acceptability Acute Test Acceptability CriteriaCriteria

Minimum control survival at least 90%Minimum control survival at least 90%

Temperature maintained @ 20 +/- 1Temperature maintained @ 20 +/- 1oo C C

Maximum test organism age at start:Maximum test organism age at start:– 14 days for fish14 days for fish– 5 days for Mysid shrimp5 days for Mysid shrimp– 24 hours for daphnids 24 hours for daphnids

Short-term Chronic Toxicity Short-term Chronic Toxicity TestsTests

Test ProceduresTest Procedures– typically 4-10 daystypically 4-10 days– Mortality, growth, fecundity, reproductionMortality, growth, fecundity, reproduction

AdvantagesAdvantages– more sensitive than acute, assess parameters more sensitive than acute, assess parameters

other than lethalityother than lethality– may better reflect real worldmay better reflect real world

LimitationsLimitations– more costly and time intensive than acutemore costly and time intensive than acute– more sensitive to low level contaminationmore sensitive to low level contamination

Chronic Test Acceptability Chronic Test Acceptability CriteriaCriteria

Minimum control survival 80%Minimum control survival 80%

Minimum control dry weight (average):Minimum control dry weight (average):– 0.25 mg for fish0.25 mg for fish– 0.20 mg for Mysid shrimp0.20 mg for Mysid shrimp

Minimum of 15 young (average) for control Minimum of 15 young (average) for control C. C. dubiadubia

Temperature maintained @ 25 +/- 1Temperature maintained @ 25 +/- 1oo C C

Maximum test organism age at start:Maximum test organism age at start:– 48 hours for fish48 hours for fish– 7 days for Mysid shrimp7 days for Mysid shrimp– 24 hours for daphnids24 hours for daphnids

Method Specific Test Method Specific Test ConditionsConditions

Test type and durationTest type and duration Temperature, light, DO, salinityTemperature, light, DO, salinity Chamber size and volumeChamber size and volume Species selection, age and feedingSpecies selection, age and feeding

Method Specific Test Method Specific Test Conditions (cont.)Conditions (cont.)

Dilution water Dilution water Dilution seriesDilution series SamplingSampling Test acceptability criteriaTest acceptability criteria Test measurementsTest measurements

Test MeasurementsTest Measurements

Dissolved oxygen cannot fall below 4 Dissolved oxygen cannot fall below 4 mg/l (initial and final)mg/l (initial and final)

pH (initial and final)pH (initial and final) conductivityconductivity total residual chlorinetotal residual chlorine total hardness and alkalinitytotal hardness and alkalinity salinitysalinity temperaturetemperature

Selection of Dilution WaterSelection of Dilution Water

May be either a standard laboratory May be either a standard laboratory water or the receiving waterwater or the receiving water

Choice of water is dependent on the Choice of water is dependent on the objectives of the testobjectives of the test– Absolute toxicity use standard waterAbsolute toxicity use standard water– Estimate of toxicity in uncontaminated Estimate of toxicity in uncontaminated

receiving water, use receiving waterreceiving water, use receiving water– Contaminated receiving water, use Contaminated receiving water, use

laboratory waterlaboratory water

Data and EndpointsData and Endpoints

Acute Test EndpointsAcute Test Endpoints

LC50LC50 - Concentration of effluent that is - Concentration of effluent that is lethal to 50 percent of the exposed lethal to 50 percent of the exposed organisms at a specific time of observation organisms at a specific time of observation (e.g. 96 hr LC50), (expressed as % effluent)(e.g. 96 hr LC50), (expressed as % effluent)

NOAECNOAEC - - No Observed Adverse Effect No Observed Adverse Effect ConcentrationConcentration– Lowest concentration at which survival is Lowest concentration at which survival is

not significantly different from the controlnot significantly different from the control– always set equal to 100% effluentalways set equal to 100% effluent

EC - Effect ConcentrationEC - Effect Concentration

Test DataTest Data

Typical dose response where mortality Typical dose response where mortality increases as the concentration of effluent in increases as the concentration of effluent in the mixture increases.the mixture increases.

LC50 would be somewhere between 25% LC50 would be somewhere between 25% effluent and 50% effluent.effluent and 50% effluent.

0% Mortality 0% mortality 20 % Mortality 40% Mortality 80% Mortality 100% Mortality

6.25 % EffluentControl

12.5 % Effluent

25.0% Effluent

50.0% Effluent

100.0% Effluent

Chronic Test EndpointsChronic Test Endpoints

IC25 - Inhibition Concentration IC25 - Inhibition Concentration - - Concentration of effluent which has an inhibitory Concentration of effluent which has an inhibitory effect on 25% of the test organisms for the effect on 25% of the test organisms for the monitored effect, as compared to the control monitored effect, as compared to the control (expressed as % effluent).(expressed as % effluent).

NOEC - No Observable Effect Concentration NOEC - No Observable Effect Concentration - Highest concentration of effluent tested which - Highest concentration of effluent tested which shows no statistically significant effect on the shows no statistically significant effect on the organisms as compared to the control organisms as compared to the control (expressed as % effluent).(expressed as % effluent).

Chronic Test DataChronic Test Data % % % % Average Average

EffluentEffluent MortalityMortality Dry weightDry weight % % w/Eggsw/Eggs

00 2.5 2.5 0.418 0.418 69.669.66.256.25 7.5 7.5 0.371 0.371 68.868.812.512.5 10.0 10.0 0.348 0.348 50.050.025.025.0 10.0 10.0 0.308 0.308 28.628.650.050.0 17.5 17.5 0.248 0.248 0.00.0100.0100.0 100.0 100.0 0.0 0.0 0.00.0

NOECNOEC 50.0% 50.0% 12.5% 12.5% 12.5% 12.5%IC25IC25 55.7% 55.7% 23.2% 23.2% 10.7% 10.7%

Toxicity ValuesToxicity Values

LC50, IC25, NOAEC: As a limit these values LC50, IC25, NOAEC: As a limit these values will INCREASE as the limit becomes more will INCREASE as the limit becomes more stringentstringent

– These are minimum limitsThese are minimum limits

LC50, IC25: When evaluating data, exhibit LC50, IC25: When evaluating data, exhibit more toxicity as the values decreasemore toxicity as the values decrease

Toxic Units: Maximum limitsToxic Units: Maximum limits

– As values increase as limits, they become less As values increase as limits, they become less stringentstringent

Toxic Units (TU’s)Toxic Units (TU’s)

Reciprocal of the fractional LC50, Reciprocal of the fractional LC50, NOEC, IC25 valueNOEC, IC25 value

Calculated by dividing the value Calculated by dividing the value into 100into 100– TUTUaa = 100/LC50 = 100/LC50

– TUTUcc = 100/IC25 = 100/IC25

Standard Reference Standard Reference

ToxicantToxicant

ProgramProgram

Standard Reference Standard Reference Toxicants (SRT’s)Toxicants (SRT’s)

PurposePurpose FrequencyFrequency Acceptability CriteriaAcceptability Criteria Control ChartsControl Charts

Control ChartsControl Charts

Quality Control Chart Ceriodaphnia dubiaAmerican Aquatic Testing, Inc.

Chart date: 05/21/03 Chart #: 75

0

100

200

300

400

500

600

700

Test Number

(ppm

KC

l)

IC25 Average Ave + 2 Std. Dev Ave - 2 Std Dev

Sample CollectionSample Collection

Subchapter 9Subchapter 9

N.J.A.C. 7:18 Subchapter 9 Sample N.J.A.C. 7:18 Subchapter 9 Sample RequirementsRequirements– Addresses collection, handling and Addresses collection, handling and

preservation of environmental preservation of environmental samplessamples

– Section 9.5 – Requirements for acute Section 9.5 – Requirements for acute toxicity testing samplestoxicity testing samples

Grab vs. CompositeGrab vs. Composite

Grab samples offer “snap shot” of effluentGrab samples offer “snap shot” of effluent

Composite samples offer “average view” of Composite samples offer “average view” of effluenteffluent

NJDEP requires sampling based on NJDEP requires sampling based on discharge typedischarge type– Continuous discharge – 24 hour composite Continuous discharge – 24 hour composite

samplesample– Intermittent discharge – grab or composite Intermittent discharge – grab or composite

each day that is representative of dischargeeach day that is representative of discharge

Frequency and HoldingFrequency and Holding

Daily for acute toxicity testing (single comp. Daily for acute toxicity testing (single comp. for daphnids)for daphnids)

Every 48 hours for chronic testingEvery 48 hours for chronic testing Minimum # of samples for 24 hour Minimum # of samples for 24 hour

composite - 48 (every 30 minutes)composite - 48 (every 30 minutes) Holding timesHolding times

– 24 hours to first use for acute and chronic 24 hours to first use for acute and chronic testingtesting

– 72 hours to use three times, chronic testing only72 hours to use three times, chronic testing only

Effluent Sampling Effluent Sampling ContainersContainers

Constructed of non-toxic materials:Constructed of non-toxic materials:– Glass – borosilicate, tempered or soda Glass – borosilicate, tempered or soda

limelime– 304 or 316 stainless steel304 or 316 stainless steel– Medical or food grade siliconeMedical or food grade silicone– Perfluorocarbons – Teflon, etc.Perfluorocarbons – Teflon, etc.– Plastics – polyethylene, polypropylene, Plastics – polyethylene, polypropylene,

polycarbonate,polystyrenepolycarbonate,polystyrene Containers rinsed with sample, used once Containers rinsed with sample, used once

and disposed of, or cleaned.and disposed of, or cleaned.

Sampling LocationSampling Location

NJPDES sample location must be used NJPDES sample location must be used for toxicity test sampling for toxicity test sampling

– This is generally the same sampling location This is generally the same sampling location required for all other parametersrequired for all other parameters

Prechlorination sampling may be requiredPrechlorination sampling may be required Post dechlorination sampling may be requiredPost dechlorination sampling may be required

– Location should always be specified in the Location should always be specified in the permitpermit

PreservationPreservation

For toxicity testing only For toxicity testing only temperature preservation temperature preservation permittedpermitted

Refrigeration during sampling Refrigeration during sampling optionaloptional

Refrigeration or icing immediately Refrigeration or icing immediately upon collection requiredupon collection required

SAMPLING DOCUMENTATIONSAMPLING DOCUMENTATION

Chain of CustodyChain of Custody– Facility informationFacility information– Date, time, sample ID, sampler ID, sample Date, time, sample ID, sampler ID, sample

location informationlocation information– Signatures for custody transferSignatures for custody transfer

Signatures are important!!Signatures are important!!– Avoids confusionAvoids confusion– Prevents sampling from occurring when Prevents sampling from occurring when

plant not operating normallyplant not operating normally

New Jersey Toxicity Testing New Jersey Toxicity Testing ProgramProgram

Whole Effluent Toxicity Approach to Water Quality Based Toxics Control

WET is used as an effluent parameter to WET is used as an effluent parameter to measure the aggregate toxic effect of the measure the aggregate toxic effect of the discharge of toxic pollutants to surface discharge of toxic pollutants to surface waterswaters

Goal is to protect aquatic biota and achieve Goal is to protect aquatic biota and achieve surface water quality standardssurface water quality standards

Limits are set to be met at the “End of the Limits are set to be met at the “End of the pipe” to satisfy the “No toxics in toxic pipe” to satisfy the “No toxics in toxic amounts” narrative water quality standardamounts” narrative water quality standard

The Whole Effluent ApproachThe Whole Effluent ApproachCapabilitiesCapabilities

Toxicity of all effluent constituents are Toxicity of all effluent constituents are measured and the toxic effect can be measured and the toxic effect can be regulated with one parameterregulated with one parameter

Implements the national policy of no Implements the national policy of no toxics in toxic amountstoxics in toxic amounts

Chemical interactions are assessedChemical interactions are assessed Unknown toxicants are addressedUnknown toxicants are addressed Bioavailability of toxic constituents is Bioavailability of toxic constituents is

assessed and the interactions of assessed and the interactions of constituents accounted forconstituents accounted for

The Whole Effluent The Whole Effluent ApproachApproach

LimitationsLimitations

No direct human health protectionNo direct human health protection Carcinogenicity, mutagenicity and Carcinogenicity, mutagenicity and

bioaccumulations are not assessedbioaccumulations are not assessed No direct treatmentNo direct treatment Predictivity of results should be carefully Predictivity of results should be carefully

assessedassessed No persistency on sediment coverageNo persistency on sediment coverage Incomplete knowledge of a causative Incomplete knowledge of a causative

toxicanttoxicant

Program StructureProgram Structure Laboratory Certification Program for Laboratory Certification Program for

Acute and Chronic ToxicityAcute and Chronic Toxicity

Permit ProgramPermit Program– Whole Effluent Toxicity LimitsWhole Effluent Toxicity Limits– Toxicity Testing Monitoring RequirementsToxicity Testing Monitoring Requirements– Toxicity Reduction EvaluationsToxicity Reduction Evaluations

Compliance Testing Program Compliance Testing Program (Enforcement)(Enforcement)

Laboratory Certification Laboratory Certification ProgramProgram

Regulations Governing the Regulations Governing the Certification of Laboratories and Certification of Laboratories and Environmental Measurements Environmental Measurements (N.J.A.C. 7:18)(N.J.A.C. 7:18)– Subchapter 7 contains test methodsSubchapter 7 contains test methods– Subchapter 9 contains the procedures Subchapter 9 contains the procedures

governing sample collection and governing sample collection and handlinghandling

– Formal certification programFormal certification program– National and state programsNational and state programs

WET Certification WET Certification ComponentsComponents

Personnel Personnel qualificationsqualifications

Laboratory facilities Laboratory facilities and safetyand safety

Equipment and Equipment and instrumentationinstrumentation

Sample collection, Sample collection, handling and handling and preservationpreservation

Test MethodologyTest Methodology General lab General lab

practicespractices Quality controlQuality control Reference toxicant Reference toxicant

datadata Records and data Records and data

reportingreporting Test acceptability Test acceptability

criteriacriteria

Acute Toxicity Acute Toxicity MethodsMethods

Methods contained in rule at N.J.A.C. Methods contained in rule at N.J.A.C. 7:187:18– 5 concentrations + control5 concentrations + control– replicatesreplicates

96 hours or less (species specific)96 hours or less (species specific) Mortality or immobilizationMortality or immobilization Receiving water for dilution preferredReceiving water for dilution preferred Test species Test species

Chronic Toxicity MethodsChronic Toxicity Methods

Incorporated by reference in N.J.A.C. Incorporated by reference in N.J.A.C. 7:18-7.1(a)7:18-7.1(a)

Part V includes additional requirementsPart V includes additional requirements USEPA Methods (USEPA Methods (40 CFR 136)40 CFR 136) Certified laboratoriesCertified laboratories Same test species as acute testing Same test species as acute testing

PermittingPermitting

Limit CalculationLimit Calculation

N.J.A.C. 7:14A-13 - Effluent Limitations N.J.A.C. 7:14A-13 - Effluent Limitations for DSW Permits for DSW Permits

USEPA’s Technical Support Document USEPA’s Technical Support Document – Acute and Chronic WQBELsAcute and Chronic WQBELs– Values of 0.3 and 1.0 used to interpret Values of 0.3 and 1.0 used to interpret

narrative “no toxics” criterianarrative “no toxics” criteria– Reasonable Potential determinations based Reasonable Potential determinations based

on site specific data on site specific data Effluent toxicity standard at N.J.A.C. 7:9-Effluent toxicity standard at N.J.A.C. 7:9-

5.7(a) is an LC505.7(a) is an LC50>>50% effluent50% effluent

Who Gets What??Who Gets What??

What gets imposed - acute / chronic, What gets imposed - acute / chronic, limits or monitoring only, is highly limits or monitoring only, is highly dependent on what type of data is dependent on what type of data is availableavailable

Generally, if no data exists a limit will Generally, if no data exists a limit will not be imposed right away, unless the not be imposed right away, unless the discharge is the result of a cleanupdischarge is the result of a cleanup

Final limits are affected significantly by Final limits are affected significantly by available dilution and the acute:chronic available dilution and the acute:chronic ratioratio

Limits - The Bottom LineLimits - The Bottom Line

Variability of data affects the Variability of data affects the final limit outcomefinal limit outcome

The more data the betterThe more data the better Ensure input values are Ensure input values are

appropriateappropriate

Permit RequirementsPermit Requirements Limit and testing frequencyLimit and testing frequency Test species and methodTest species and method Reporting requirements (endpoints)Reporting requirements (endpoints)

– Repeat testing requirementsRepeat testing requirements Characterization requirementsCharacterization requirements

– Split samplesSplit samples Toxicity Reduction RequirementsToxicity Reduction Requirements Compliance schedule vs. triggerCompliance schedule vs. trigger

– Interim vs. final limitsInterim vs. final limits– 3 or 5 years3 or 5 years

Toxicity Reduction Toxicity Reduction Evaluations (TRE)Evaluations (TRE)

Specific TRE language is included at Specific TRE language is included at N.J.A.C. 7:14A-13.17(a)N.J.A.C. 7:14A-13.17(a)

Language to exclude test results not Language to exclude test results not considered representative included at considered representative included at N.J.A.C. 7:14A-13.14(a)2N.J.A.C. 7:14A-13.14(a)2

Permittees responsibilityPermittees responsibility NJDEP oversight roleNJDEP oversight role Series of stepped requirementsSeries of stepped requirements Apply whether limit in effect or notApply whether limit in effect or not

Regulatory IssuesRegulatory Issues

Recent DevelopmentsRecent Developments

USEPA Support for WETUSEPA Support for WET

1984 - EPA National Policy for WQBEL 1984 - EPA National Policy for WQBEL development for Toxic Pollutantsdevelopment for Toxic Pollutants

1989 - 40 CFR 122.44 Revised for 1989 - 40 CFR 122.44 Revised for WQBELsWQBELs

1991 - Technical Support Document for 1991 - Technical Support Document for Water Quality-based Toxics ControlWater Quality-based Toxics Control

1994 - WET Control Policy Updated1994 - WET Control Policy Updated 1995 - Incorporation of WET methods in 1995 - Incorporation of WET methods in

40 CFR 13640 CFR 136

October 26, 1995October 26, 1995

40 CFR 136.3 revised to establish standard 40 CFR 136.3 revised to establish standard protocols for conducting WET testsprotocols for conducting WET tests

Incorporates acute and chronic test Incorporates acute and chronic test method manuals by referencemethod manuals by reference

Supplemental Information Document Supplemental Information Document provides responses to comments raisedprovides responses to comments raised

Revisions to Part V to reference 40 CFR 136Revisions to Part V to reference 40 CFR 136

Settlement Agreement Settlement Agreement RequirementsRequirements

July 24, 1998July 24, 1998 Variability Guidance DocumentVariability Guidance Document Method Guidance DocumentMethod Guidance Document Interlaboratory Variability StudyInterlaboratory Variability Study Rulemaking actionsRulemaking actions

ResultsResults

8 of 10 methods had test completion rates 8 of 10 methods had test completion rates >90%>90%

Test completion rate of 82% for Test completion rate of 82% for CeriodaphniaCeriodaphnia

Successful test completion rate of Successful test completion rate of approximately 64% for Selenastrumapproximately 64% for Selenastrum

7 of 10 test with no false positives7 of 10 test with no false positives

9 of 10 methods had false positives<5%9 of 10 methods had false positives<5%

ConclusionsConclusions

WET Variability Study results WET Variability Study results confirmed EPAs conclusions that WET confirmed EPAs conclusions that WET methods provide sufficient precision methods provide sufficient precision and can be reliably used in permitsand can be reliably used in permits

In September 2001, EPA proposed to In September 2001, EPA proposed to ratify its previous approval of the ratify its previous approval of the methods evaluated in the studymethods evaluated in the study

Technical Corrections Technical Corrections NoticeNotice

February 2, 1999 (64 FR 4975)February 2, 1999 (64 FR 4975) Incorporated into the WET final rule Incorporated into the WET final rule

an errata document an errata document corrects minor errors and omissionscorrects minor errors and omissions provides clarificationprovides clarification established consistency among the established consistency among the

methods manuals and the final rulemethods manuals and the final rule

Variability Guidance Variability Guidance DocumentDocument

July 18, 2000 (65 FR 44528)July 18, 2000 (65 FR 44528) Guidance to regulatory authorities, Guidance to regulatory authorities,

permittees, and testing labs on permittees, and testing labs on measurement variability in WET testing measurement variability in WET testing

Explains the toxicity test protocol, Explains the toxicity test protocol, organisms, chemical and physical organisms, chemical and physical conditions, renewals, dilution series, conditions, renewals, dilution series, test design, measurements (mortality test design, measurements (mortality reproduction) data analysis and test reproduction) data analysis and test endpointsendpoints

Method Guidance Method Guidance DocumentDocument

July 28, 2000 (65 FR 46457)July 28, 2000 (65 FR 46457) % Minimum Significant Difference% Minimum Significant Difference Confidence intervals Confidence intervals Concentration response Concentration response

relationshiprelationship Dilution series selectionDilution series selection Dilution water selectionDilution water selection

Laboratory Errors Laboratory Errors

Errors in the analysis and reporting Errors in the analysis and reporting of WET test results were prevalent.of WET test results were prevalent.

Errors ranged from single data Errors ranged from single data entry or rounding errors to errors entry or rounding errors to errors in statistical method selection for in statistical method selection for use.use.

Most errors had minor effects on Most errors had minor effects on test results.test results.

Guidance to Testing Guidance to Testing LaboratoriesLaboratories

Maintain QC control charts for IC25 PMSD.Maintain QC control charts for IC25 PMSD. Routinely plot average treatment responses Routinely plot average treatment responses

and replicate data to identify anomalies and and replicate data to identify anomalies and excessive variability.excessive variability.

Ensure that the upper PMSD is not exceeded.Ensure that the upper PMSD is not exceeded. Use at least four replicates for minnow tests.Use at least four replicates for minnow tests. Additional topics: lab quality control, Additional topics: lab quality control,

standardizing reference toxicants, standardizing reference toxicants, acceptance limits for ref-tox test results.acceptance limits for ref-tox test results.

Guidance to NPDES Guidance to NPDES PermitteesPermittees

Use one laboratoryUse one laboratory Review your laboratory’s control Review your laboratory’s control

chartscharts Check test acceptability criteriaCheck test acceptability criteria Check sample holding times and Chain Check sample holding times and Chain

of custody’sof custody’s Obtain at leas 10 data points over >1 Obtain at leas 10 data points over >1

year to characterize effluent variabilityyear to characterize effluent variability

Guidance to RegulatorsGuidance to Regulators

Review the test reportsReview the test reports Evaluate PMSD as well as TACEvaluate PMSD as well as TAC Conduct routine lab auditsConduct routine lab audits Review SRT control chartsReview SRT control charts

Proposed Rule Proposed Rule Amendments Amendments

September 28, 2001 (66 FR 49794)September 28, 2001 (66 FR 49794) Specific revisions to the test Specific revisions to the test

methods and proposed to ratify its methods and proposed to ratify its previous approval of the methodsprevious approval of the methods

Comment period scheduled to end Comment period scheduled to end on November 27, 2001, extended on November 27, 2001, extended to January 11, 2002to January 11, 2002

Final RuleFinal Rule

Issued November 19, 2002Issued November 19, 2002 Vol. 67. No. 223, 40 CFR 136Vol. 67. No. 223, 40 CFR 136 Effective December 19, 2002Effective December 19, 2002 Ratified most of the previously Ratified most of the previously

adopted methodsadopted methods Amended the table containing the Amended the table containing the

toxicity methodstoxicity methods

Ratification of Ten Ratification of Ten MethodsMethods

Methods are repeatable and reproducibleMethods are repeatable and reproducible Available and applicableAvailable and applicable RepresentativeRepresentative Variability study showed high rate of Variability study showed high rate of

successful completionsuccessful completion Do not often produce false positive Do not often produce false positive

results results Exhibit precision comparable to chemical Exhibit precision comparable to chemical

methods approved at 40 CFR 136methods approved at 40 CFR 136

Withdrawal of Two Withdrawal of Two MethodsMethods

Holmesimysis costataHolmesimysis costata Acute Test Acute Test– west coast test organismwest coast test organism

Champia parvulaChampia parvula Reproduction Reproduction TestTest

Methods can still be usedMethods can still be used

Amendment to 40 CFR Amendment to 40 CFR 136.3 Table 1A136.3 Table 1A

Clarified mysid test method does not Clarified mysid test method does not apply to apply to Holmesmysis costataHolmesmysis costata

Added method numbers to acute testsAdded method numbers to acute tests Modified footnotes and references to Modified footnotes and references to

cite the updated version of the method cite the updated version of the method manualsmanuals

Revise the parameter measured in Revise the parameter measured in marine tests to refer to organisms “of marine tests to refer to organisms “of the Atlantic Ocean and Gulf of Mexico”the Atlantic Ocean and Gulf of Mexico”

Impact of the AdoptionImpact of the Adoption Blocking by parentageBlocking by parentage CeriodaphniaCeriodaphnia test test

endpointendpoint pH driftpH drift Dilution seriesDilution series Dilution waterDilution water Pathogen interferencePathogen interference Variability criteriaVariability criteria Minimum number of Minimum number of

replicatesreplicates Test requirements / Test requirements /

recommendationsrecommendations

Reference toxicant Reference toxicant testingtesting

Sample collection and Sample collection and holding timesholding times

Sampling holding Sampling holding temperaturetemperature

BiomassBiomass Total residual chlorineTotal residual chlorine Additional minor Additional minor

correctionscorrections

Ceriodaphnia dubiaCeriodaphnia dubia Chronic Toxicity TestChronic Toxicity Test

Mandated use of a very specific Mandated use of a very specific procedure of “Blocking by Known procedure of “Blocking by Known Parentage” with at least six neonatesParentage” with at least six neonates

Neonates from a single parent may Neonates from a single parent may be used to initiate more than one testbe used to initiate more than one test

Elimination of use of fourth brood Elimination of use of fourth brood organismsorganisms

pH DriftpH Drift

Changes between proposal and Changes between proposal and adoptionadoption

Permitted in chronic methods onlyPermitted in chronic methods only Specific procedures to Specific procedures to

demonstrate need for pH control demonstrate need for pH control Specific procedures for pH control Specific procedures for pH control

during testingduring testing

PMSDPMSD

Percent Minimum Significant Percent Minimum Significant DifferenceDifference

Test Method: Endpoint 10th PMSD 90th PMSD

Fathead Minnow Growth 12 30C. dubia Reproduction 13 47Sheepshead minnow Growth (6.3) (23)Inland Silverside Growth 11 28Mysid Growth 11 37

PMSD values calculated with Dunnett’s test must be between within the range established by the 10th and 90th PMSD values.

Test Requirements / Test Requirements / RecommendationsRecommendations

Modification of tables summarizing Modification of tables summarizing test conditionstest conditions

New section on test reviewNew section on test review– Mandatory review of concentration Mandatory review of concentration

response relationship by regulatory response relationship by regulatory authorityauthority

– Mandatory use of variability criteria Mandatory use of variability criteria for data reported as an NOECfor data reported as an NOEC

Reference Toxicant Reference Toxicant TestingTesting

Used for initial and ongoing demonstration Used for initial and ongoing demonstration of performance and to assess sensitivity of performance and to assess sensitivity and health of test organismsand health of test organisms

Monthly or side by side testingMonthly or side by side testing Use of suppliers five most recent testsUse of suppliers five most recent tests Not a “de facto criterion” for test rejectionNot a “de facto criterion” for test rejection Labs should evaluate CVs based on Labs should evaluate CVs based on

national valuesnational values

Sample Collection & Sample Collection & HoldingHolding

Maintained default maximum of 36 Maintained default maximum of 36 hrs for first use of samplehrs for first use of sample

Clarified these samples may be used Clarified these samples may be used for later renewalsfor later renewals

Permitting authority may allow Permitting authority may allow continued use of most recent samplecontinued use of most recent sample

Collection on days one, three and Collection on days one, three and five recommended (not required)five recommended (not required)

EnforcementEnforcement

Serious ViolationSerious Violation

Limit (% Effluent)Limit (% Effluent)

> or = 80 and < or > or = 80 and < or = 100= 100

> or = 50 and < 80> or = 50 and < 80

>10 and < 50>10 and < 50

< or = 10< or = 10

Result DifferenceResult Difference

> or = 20> or = 20

> or = 15> or = 15

> or = 10> or = 10

> or = 9> or = 9

Affirmative Affirmative DefenseDefense

N.J.A.C. 7:14-8.3 - violator is N.J.A.C. 7:14-8.3 - violator is entitled to an affirmative entitled to an affirmative defense to liability for a defense to liability for a violation occurring as a result violation occurring as a result of an upset, an anticipated or of an upset, an anticipated or unanticipated bypass, or a unanticipated bypass, or a testing or laboratory error.testing or laboratory error.

Testing or Laboratory Testing or Laboratory ErrorError

A violator asserting a testing or A violator asserting a testing or laboratory error as an affirmative defense laboratory error as an affirmative defense shall also have the burden to shall also have the burden to demonstrate that a violation involving demonstrate that a violation involving the exceedance of an effluent limit was the exceedance of an effluent limit was the result of unanticipated test the result of unanticipated test interference, sample contamination, interference, sample contamination, analytical defects, or procedural analytical defects, or procedural deficiencies in sampling or other similar deficiencies in sampling or other similar circumstances beyond the violators circumstances beyond the violators control.control.

FAQ’sFAQ’s

Toxicity where you don’t Toxicity where you don’t expect it??expect it??

StormwaterStormwater Cooling WaterCooling Water Filter backwashFilter backwash High or low TDS High or low TDS ChlorineChlorine

Naturally low pH waterNaturally low pH water

N.J.A.C. 7:18-9.5(a)2vii.N.J.A.C. 7:18-9.5(a)2vii. If the receiving water has a natural If the receiving water has a natural

pH below 5.0 units, then the pH below 5.0 units, then the dilution water samples shall be dilution water samples shall be adjusted to a pH of 5.0 prior to adjusted to a pH of 5.0 prior to their use in test organism their use in test organism acclimation and/or toxicity testing. acclimation and/or toxicity testing.

Where can I get toxicity Where can I get toxicity data on various data on various

chemicals?chemicals?

http://www.epa.gov/ecotox/http://www.epa.gov/ecotox/ The ECOTOX (ECOTOXicology) database provides single

chemical toxicity information for aquatic and terrestrial life. ECOTOX is a useful tool for examining impacts of chemicals on the environment. Peer-reviewed literature is the primary source of information encoded in the database. Pertinent information on the species, chemical, test methods, and results presented by the author(s) are abstracted and entered into the database. Another source of test results is independently compiled data files provided by various United States and International government agencies.

Other IssuesOther Issues

NMAT to NOAECNMAT to NOAEC How much data is enough?How much data is enough? Dilution estimatesDilution estimates When is data too old?When is data too old? What are other states doing?What are other states doing? Intermittent discharges?Intermittent discharges? Sampling issues for intermittent Sampling issues for intermittent

discharges.discharges.

How does increasing the difference How does increasing the difference in test concentration dilutions affect in test concentration dilutions affect

the prediction of response?the prediction of response?

Better resolution around threshold effect Better resolution around threshold effect concentration concentration

Reducing the distance between effluent Reducing the distance between effluent dilutions should be encourageddilutions should be encouraged

minimum set of dilutions, i.e. no wider than 0.5 minimum set of dilutions, i.e. no wider than 0.5 dilutions between concentrationsdilutions between concentrations

Test design should maximize test Test design should maximize test concentrations around the instream waste concentrations around the instream waste concentration, in order to minimize the need for concentration, in order to minimize the need for interpretation of effects between tested interpretation of effects between tested concentrationsconcentrations

My effluent tests indicate there may be a My effluent tests indicate there may be a problem but I can see fish in the area of problem but I can see fish in the area of my discharge, is there really a problem?my discharge, is there really a problem?

Observations of organisms in the Observations of organisms in the area of the outfall does not mean area of the outfall does not mean that more subtle impacts are not that more subtle impacts are not occurring or that the organisms occurring or that the organisms that are present are sensitive that are present are sensitive enough to represent most enough to represent most organisms instream. organisms instream.

WET ResourcesWET Resources

www.epa.gov/waterscience/WETwww.epa.gov/waterscience/WET www.epa.gov/ostwater/WET/www.epa.gov/ostwater/WET/

index.htmlindex.html www.setac.orgwww.setac.org www.toxicity.comwww.toxicity.com