Presentation on theme: "Whole Effluent Toxicity Basics"— Presentation transcript:
1Whole Effluent Toxicity Basics Purpose of the training course is to provide an overview of whole effluent toxicity test methods and implementationProvide a better understanding of scientific underpinnings of WETDescribe uses and limitations,implementations requirementsdescribe methods including a demonstration of a tox testpurpose of a Toxicity Reduction EvaluationBetty Jane Boros-RussoNJDEP, Office of Quality AssuranceChristopher J. NallyAmerican Aquatic Testing
2History16th century - scientists began testing the lethality of chemical compounds on animals prior to their use on humans for therapeutic purposes1930’s - some of the first uses of aquatic organisms for testing to determine the causes of observed fish killssome of the first methods for conducting toxicity tests were publishedMost earlier work was with single chemicals and mostly freshwaterJune, 1945, Hart, Doudoroff and Greenbanck published “The evaluation of the toxicity of industrial wastes, chemicals and other substances to fresh-water fish. Included a detailed method.By the late 1960’s, people began noting differences between groups of organisms living in streams based on the absence or presence of wastewater inputs. CA and MI were beginning using it in reg program.FWPCA of it is the national policy that the discharge of toxic pollutants in toxic amounts be prohibited. Maintained in 1977 and 1987.1975 EPA published its “Methods for Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians.”Federal Water Pollution control Act Amendments (Clean Water Act) section 101(a)3 in 1977 stated that it is the national goal that the discharge of toxic pollutants in toxic amounts be prohibited.This began the use of WET tests for the control of the discharge of toxics into the environment in the 70’s and 80’s First EPA acute manual1984 and 85 EPA strengthened and outlined its policy for the technical approach for assessing and controlling the discharge of toxic substances TSD published
3Use of Toxicity Testing in Water Quality Based Toxics Control To characterize and measure the aggregate toxicity of an effluent or ambient watersTo measure compliance with whole effluent toxicity limitsAs an investigative tool and to measure progress in a toxicity reduction programAs an ambient instream measure of toxicity to identify pollution sourcesWET is used as part of the water quality based approach to toxics controlIt measures the response of exposed aquatic organismsUsed in all these ways / purposes in NJUsed in ambient investigations in Delaware for ultimately development of WQBELs.Statements of policy are found in the Surface Water Quality Standards at N.J.A.C 7:9B-1.5(a)3 and the table at 1.14(c) 12
4NJ WET Program HistoryEarly 1980’s - Acute monitoring and limits used on a routine basisBegan use of chronic monitoring and chronic limitsGroup permit challenge on chronic WETSettlement and initial chronic WET program revisionsFinal program revisions adoptedAcute only to start since chronic short term tests weren’t available. Many states started with acute and chronic together later.In late 1992, adjudicatory hearing requests by a group of municipal dischargers on chronic limits and related TRE requirements1996 Agreed to disagree on the science. Agreed that changes in program implementation were necessary.Prior to 1997 several of limit calculation procedures and program implementation requirements were out of date and inconsistent with USEPA guidance and policy.That adjudication forced NJDEP to reevaluate and update its rules governing WET.Limit expression and calculation, TRE requirements. Program requirements became more specific, increased oversight of lab activity.Group permit modification of 100 permits, 11 removed limits.
6Species SelectionSensitive species which are easily cultured and readily available year roundMust provide consistent and reproducible responseAlso encourage ecologically, commercially and or recreationally importantNo one species is always the most sensitiveSpecies used is dependent upon salinity of receiving water and the state standardsEcologically cerio mysid and kelpCommercially abaloneRecreationally troutFor NJ - whether or not to use a freshwater or saltwater test species is dependent upon the salinity of the receiving water that the discharge flows to.If the discharge is to a storm sewer, you determine the salinity of the receiving water that the storm sewer goes to.N.J.A.C. 7:9B-1.5(f) specifically statest that “the objective of the Department is to use test species for toxicity testing bioassays that are representative of the more sensitive aqutic biota from the different trophic levels in question. Test species need not be indegenous to , nor occur in the wtaters inqsuetion.Freshwater means all nontidal and tidal waters generally having a salinity, due to natural sources, of less than or equal to 3.5 ppt at mean high tide. Saline is grater than 3.5 ppt at mean high tide.
7New Jersey Freshwater Acute Test Species Invertebrates: (Daphnids)Ceriodaphnia dubiaDaphnia magnaDaphnia pulexFishPimephales promelas Fathead MinnowOncorhynchus mykiss Rainbow TroutSalvelinus fontinalis Brook TroutDaphnids have only been used for acute testing in recent years.Trout - though they have been in the regulations governing acute testing since the program began, have never been used in NJ.
8Ceriodaphnia dubia Female approximately 2 mm The freshwater test organism used both in NJ and most widely across the nation, mostly for chronic testing.Closely related to Daphnia, but a smaller and have a shorter generation time.Inhabits lakes ponds and marshes throughout most of the worldPopulation consist almost entirely of females, the males appear in fall. Production of males appears to be induced primarily by low water temp, high population densities and/or a decrease in the available food.Cerio’s reproduce only by cyclic parthenogenesis in which the males contribute to the genetic makeup of the young during the sexual state of reproduction.Filter feeders.Clutch of 4-10 eggs is released into the brood chamber where they hatch and are released, when the female molts, grows and a new clutch of eggs is released into the brood chamber.Photo compliments of Marinco Bioassay Laboratory
9Pimephales promelas Photo by Karen McCabe from Animal Soup Adults are small fish typically 43 mm to 102 mm, and aceraging about 50 mm, in total length.Widely distributed in North America. In muddy brooks, streams creeks, ponds and small lakes, is uncommon or absent in streams of moderate and high gradients and in most of the larger and deeper impoundments, and is tolerant of high temperature and turbidity, and low oxygen concentrations.bait fishbreeding males develop a conspicuous, narrow, elongated, gray, fleshy pad of spongy tubercles on the back, anterior to the dorsal fin, and two or three rows of strong nuptial tubercles across the snout.Photo by Karen McCabe from Animal Soup
10New Jersey Saline Acute Test Species Invertebrates:Mysidopsis bahia Opossum Shrimp(Americamysis bahia)FishCyprinodon variegatus Sheepshead MinnowMenidia beryllina Inland SilversidesMenidia peninsulae Tidewater SilversidesMenidia menidia Atlantic SilversidesOf the fish, the sheepshead minnow is the most widely used but in general there are very few saltwater fish used for routine monitoring.
11Mysidopsis bahia Female approximately 6 mm in length Most frequently used acute and chronic saltwater test species.Shrimplike crustaceans (Americamysis bahia)Occurs in salinities primarily above 15 ppt found in greatest abundance in salinities 30ppt. Eggs develop for about 20 days in the marsupium before the yound are released as jubeniles; broods are released at night during molting.Very cannibalisticadults 4.4mm to 9.4 mm. Females usually larger than the male. Somewhat transparent but tinted, yellow, brown or black.Female approximately 6 mm in lengthPhoto compliments of Marinco Bioassay Laboratory
12New Jersey Freshwater Chronic Test Species Invertebrates:Ceriodaphnia dubiaFishPimephales promelas Fathead MinnowAlgaeSelenastrum capricornutumThough algae are contained in the program, they are not used.
13New Jersey Saline Chronic Test Species Invertebrates:Mysidopsis bahia Opossum ShrimpFishCyprinodon variegatus Sheepshead MinnowMenidia beryllina Inland SilversidesMenidia peninsulae Tidewater SilversidesMenidia menidia Atlantic SilversidesOtherArbacia punctulata Sea UrchinChampia parvula Red MacroalgaeArbacia and Champia are not used in the program.
15Rules for Conducting Toxicity Tests 40 CFR Table 1AEffective November 15, 1995Amended November 19, 2002 and effective December 19, 2002Methods must be followed as they are writtenFirst acute manual was published in 1975.USEPA added new biolgical testing methods to the list of approved and standardized analytical method for testing wastewater pollutants. This information was published in the Federal Register as a final amendment to 40 CFR 136 and became effective November 15, 1995.Prior to this none were in EPA regulation though the documents were published.Standardized 17 procedures, 7 acute tests 4 freshwater and 6 marine chronic.These methods do not apply to marine waters of the Pacific Ocean.
16Incorporate by Reference Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms. 5th Edition, USEPA, Office of Water, October 2002, EPA 821-RShort-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. 4th Edition, USEPA, Office of Water, October 2002, October 2002, EPA 821-RShort-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms. 3rd Edition. USEPA, Office of Water, October 2002, EPA 821-R
17USEPA Methods Documents Health and safetyQuality assuranceFacilities, equipment and suppliesTest organisms and culture methodsDilution waterEach method document covers at a minimum a section on each topic,Two chronic methods documents are set up the same way though the acute is somewhat differentMethods include both requirements and recommendations. The intent of the manuals are to use for a wide range of uses. Requirements where necessary for standardization but much of some sections is left to the implementing authority.
18USEPA Methods Documents (cont.) Effluent sampling and handlingEndpoints and data analysisIndividual test methodsReport preparation and test review
19Test Types Acute and Short-term Chronic Tests Test Species dependent Static non-renewalStatic renewalFlow throughTest Species dependentUse dependentStatic non-renewal tests: The test organisms are exposed to the same test solution for the duration of the test. Pros: simple, minimum physical disturbance to organisms. Cons: toxicants may volatilize or degrade during testing.Static renewal tests: The test organisms are exposed to a fresh test solution of the same concentration of sample every 24-hrs or other prescribed interval by transferring the test organism from one test chamber to another or by replacing all or a portion of solution in the test chambers. Adv.: addresses toxicant degradation/volatilization issue. Disadv: more labor intensive, more physical disturbance to organisms.Flow through tests: sample is pumped continuously from the sampling point directly to the dilutor system or a grab or composite samples are collected periodically and then placed in a tank to the dilutor system. Adv.: produces exposure more like that of stream organisms. Disadv Significantly more labor intensive and expensive.
20Test Design 5 Concentrations + Control Replicates Serial dilution’s of effluent and “control water” (also termed “dilution water”)Dilution series of 0.5 or greaterSingle concentration testReplicatesRandomization (organisms/chambers)EPA recommends multi-concentration test with a minimum of 5 test concentrations plus a control.Dilution factor a minimum of Attempt to bracket any critical concentration.Single concentration test = pass/fail test not generally recommended.Replicates 2- 10Randomization of test chambers, organisms, throughout the test
23Acute Toxicity Tests Test Procedures Advantages Disadvantages 96 hours or less (species specific)Mortality is the measured endpointFor daphnia mortality determined by immobilizationAdvantagesless expensive and time consuming than chronicendpoint is easy to quantifyDisadvantagesindicates only lethal concentrationsonly the effects of fast acting chemicals are exhibitedShort Term relative to life cycle.Acute means a stimulus severe enough to rapidly induce an effect: in toxicity tests, an effect observed in 96 hours or less is typically considered acute. An acute effect is not always measured in terms of lethality.Acute refers to the exposure not the effect.Acute tests are conducted on effluents and receiving waters.
24Acute Test Acceptability Criteria Minimum control survival at least 90%Temperature 20 +/- 1o CMaximum test organism age at start:14 days for fish5 days for Mysid shrimp24 hours for daphnidsTest acceptability criteria determine the validity and acceptability of the test based on the control typically survival,
25Short-term Chronic Toxicity Tests Test Procedurestypically 4-10 daysMortality, growth, fecundity, reproductionAdvantagesmore sensitive than acute, assess parameters other than lethalitymay better reflect real worldLimitationsmore costly and time intensive than acutemore sensitive to low level contaminationChronic means a stimulus that lingers or continues for a relatively long period of time, often one-tenth of the life span or more. Chronic should be considered a relative term depending on the life span of an organism. The measurement of a chronic effect can be reduced growth, reduced reproduction, etc.., in addition to lethality.Chronic tests are conducted on effluents and receiving waters.
26Chronic Test Acceptability Criteria Minimum control survival 80%Minimum control dry weight (average):0.25 mg for fish0.20 mg for Mysid shrimpMinimum of 15 young (average) for control C. dubiaTemperature 25 +/- 1o CMaximum test organism age at start:48 hours for fish7 days for Mysid shrimp24 hours for daphnidsTest acceptability criteria determine the validity and acceptability of the test based on control survival and weight gain.Example:The term short-term is used in the WET program> or = 80& survivalminimum 3 of young / surivivng control female> or = 0.25 mg wt.
27Method Specific Test Conditions Test type and durationTemperature, light, DO, salinityChamber size and volumeSpecies selection, age and feedingThe test method specifies the minimum number of test organism and the number of replicates required for each test concentration, the range of salinity tolerance of the orgnaism and the test temperature.The test temperature is specified for each methods and is typically within a narrow ranges. 25+/-1 degree C. Criticalfor chronic can significantly affect growth and reproduction with organisms. Shock due to large increase in temperature.Light quality intensity and photoperiod are specified for each method.Do. requirements are specified for each method.DO concentration in the effluent samples should be near saturation prior to used. Aeration may be necessary to bring the DO into equilibrium with air, minimizeDO demand and stabilize the pH. Aeration can reduce the apparent toxicity of the test solutions by stripping them of highly volatile toxic substances, or increase the toxicity by altering the pH. However, the heDO in the test solutions must not be allowed to fall below 4.0 mg/L. If aeration is necessary the rate is specified in the test and must be conducted equally across all test chambers.
28Method Specific Test Conditions (cont.) Dilution waterDilution seriesSamplingTest acceptability criteriaTest measurements
29Test MeasurementsDissolved oxygen cannot fall below 4 mg/l (initial and final)pH (initial and final)conductivitytotal residual chlorinetotal hardness and alkalinitysalinitytemperatureMeasurements are performed daily to ensure that the test is meeting the requirements of the methods. Some of these can influence test results or mask toxicants.Low DO caused by high levels of BOD can cause adverse effects to test organisms and mask the presence of chemical toxicants.pH can modify the toxicity of some compounds. For instance, ammonia is more toxic at a higher pH.Conductivity can be a general indicator of toxicity associated with total dissolved solids.TRC is very toxic and can mask the presence of other toxicants.Hardness of the water can influence the expression of certain compounds. For instance, the bioavailability and thus the toxicity of copper decreases as hardness increases.For SW tests, the appropriate salinity level must be maintained for the specific marine organisms used in the test.T is critical in several ways. Test organisms can suffer shock by sudden changes in T. During chronic tests, T can significantly influence growth and reproduction of the test organisms.
30Selection of Dilution Water May be either a standard laboratory water or the receiving waterChoice of water is dependent on the objectives of the testAbsolute toxicity use standard waterEstimate of toxicity in uncontaminated receiving water, use receiving waterContaminated receiving water, use laboratory waterAge - EPA policy is to protect the most sensitive life stages of fish and aquatic life. EPA recognizes that the sensitivity of organisms to toxicants generally decreases significantly with age beyond the early life stage and therefore reduced ages of test organisms in the latest revision from 90 days to 14 days. Fatheads and inland silversides.Use of receiving water may require a second set of controls with culture water.Acclimation of organisms to salinityforty fathomsBrine from saltwaterSpring waterDI waterUse of receiving water in a test for controls generally requires the use of a second set of controls using standard laboratory water.Effluents are typically freshwater and are not appropriate for the use of saline test organisms. This necessitates the adjustment of the effluent with dry artificial sea salts. Can result in artifactual toxicity. Increase pH least to increase in ammonia.
32Acute Test EndpointsLC50 - Concentration of effluent that is lethal to 50 percent of the exposed organisms at a specific time of observation (e.g. 96 hr LC50), (expressed as % effluent)NOAEC - No Observed Adverse Effect ConcentrationLowest concentration at which survival is not significantly different from the controlalways set equal to 100% effluentEC - Effect ConcentrationLC = Lethal Concentration - point estimate of the toxicant concentration that is lethal to 50% of the organisms during a specified test period. when mortality is measuredEC = Effect Concentration - point estimate of toxicant concentration that would cause an observable adverse effect such as death orimmobilization or serious incapacitation in a given percentage of test organisms. usually for quantal data such as or fertilization
33Test DataTypical dose response where mortality increases as the concentration of effluent in the mixture increases.LC50 would be somewhere between 25% effluent and 50% effluent.calculated point estimate or aPass fail test where a concentration, usually that which is considered a critical concentration of effluent in the recieving water orambient toxicity test measured against a controlExamples of Pass/Fail Acute testInstream waste concentration equals 75%statistical evaluation using a student-t test compares mortality rates of ambient or IWC sample against a controlIs there a “significant statistical difference between the two results”6.25 % Effluent12.5 % Effluent25.0% Effluent50.0% Effluent100.0% EffluentControl0% Mortality0% mortality20 % Mortality40% Mortality80% Mortality100% Mortality
34Chronic Test Endpoints IC25 - Inhibition Concentration - Concentration of effluent which has an inhibitory effect on 25% of the test organisms for the monitored effect, as compared to the control (expressed as % effluent).NOEC - No Observable Effect Concentration - Highest concentration of effluent tested which shows no statistically significant effect on the organisms as compared to the control (expressed as % effluent).IC = Inhibition Concentration - % reduction is nonquantal continuous measurements such a length, weight gain, reproduction.Advantages include CV an SDResults are often higher for the IC25 than they are for an NOECLOEC lowest Observed Effect Concentration the lowest concentration of an effluent or a toxicant that results in observable adverse effects in teh aquatic test organisms.NOEC and LOEC are hypotheseis testing techniques while the ICp values are point estimates.NOEC may represent a different amount of effect from test to tests.NOEC is a lower concentration level than the LOECNOEC do not allow calculateion of coefficients of variation or confidence intervals.ICp you can calculate CVs and CIs.Always estimating the same effect.Need to specify the biological effect (the value of p)
35Chronic Test Data % % Average Effluent Mortality Dry weight % w/Eggs NOEC % % %IC % % %Exclusion of data from animals that do not survive to the end of the test would bias the results in favor of animals that do.
36Toxicity ValuesLC50, IC25, NOAEC: As a limit these values will INCREASE as the limit becomes more stringentThese are minimum limitsLC50, IC25: When evaluating data, exhibit more toxicity as the values decreaseToxic Units: Maximum limitsAs values increase as limits, they become less stringent
37Toxic Units (TU’s) Reciprocal of the fractional LC50, NOEC, IC25 value Calculated by dividing the value into 100TUa = 100/LC50TUc = 100/IC25Examples of Toxic UnitsAcute (TUa)Assuming LC50=28%TUa = 100/LC50100/28 = 3.6Assuming LC50=10%100/10 = 10Chronic (TUc)Assuming NOEC=50%TUc = 100/NOEC100/50 = 2Assuming IC25 = 30%TUc = 100/IC25100/30 = 3.3
39Standard Reference Toxicants (SRT’s) PurposeFrequencyAcceptability CriteriaControl ChartsThe most important part of the QC programMandated for chronic onlySRT program serves to track laboratory’s ability to consistently conduct the test over time, as well as to assess the health of the test organisms.Potassium chloride is used by most laboratories.Results of ongoing testing must fall within the control limits established by the data. The control limits are +/- two standard deviations around the mean.Results outside two standard deviations imply that something has changed.What is a SRT testsWhy do we run themWhat are the requirementsHow Frequently are they runWhat is a control chartWhat should you look for Assure that the labs can consistently perform the tests.
42Subchapter 9 N.J.A.C. 7:18 Subchapter 9 Sample Requirements Addresses collection, handling and preservation of environmental samplesSection 9.5 – Requirements for acute toxicity testing samples
43Grab vs. Composite Grab samples offer “snap shot” of effluent Composite samples offer “average view” of effluentNJDEP requires sampling based on discharge typeContinuous discharge – 24 hour composite sampleIntermittent discharge – grab or composite each day that is representative of dischargeType of sample and frequency of collection is dependent upon the use of the data and the discharge the test is intended to represent.Samples are not to be used after they have been held for 72 hours.Samples are to be chilled during or immediately upon collection to 4oC.
44Frequency and HoldingDaily for acute toxicity testing (single comp. for daphnids)Every 48 hours for chronic testingMinimum # of samples for 24 hour composite - 48 (every 30 minutes)Holding times24 hours to first use for acute and chronic testing72 hours to use three times, chronic testing only
45Effluent Sampling Containers Constructed of non-toxic materials:Glass – borosilicate, tempered or soda lime304 or 316 stainless steelMedical or food grade siliconePerfluorocarbons – Teflon, etc.Plastics – polyethylene, polypropylene, polycarbonate,polystyreneContainers rinsed with sample, used once and disposed of, or cleaned.Avoid splitting samples across weeks or months.Sampling days are an issue.
46Sampling LocationNJPDES sample location must be used for toxicity test samplingThis is generally the same sampling location required for all other parametersPrechlorination sampling may be requiredPost dechlorination sampling may be requiredLocation should always be specified in the permitHolding times for acute testing 24 hours to first use;Holding times for chronic testing 24 to first use, 3 uses in 72 hoursVery important to use NJPDES sample site to make data comparable to chemistries done
47PreservationFor toxicity testing only temperature preservation permittedRefrigeration during sampling optionalRefrigeration or icing immediately upon collection requiredSampling can be refrigerated, but not mandatory for NJDEP, sample must be iced immediately upon collection.
48SAMPLING DOCUMENTATION Chain of CustodyFacility informationDate, time, sample ID, sampler ID, sample location informationSignatures for custody transferSignatures are important!!Avoids confusionPrevents sampling from occurring when plant not operating normally
50Whole Effluent Toxicity Approach to Water Quality Based Toxics Control WET is used as an effluent parameter to measure the aggregate toxic effect of the discharge of toxic pollutants to surface watersGoal is to protect aquatic biota and achieve surface water quality standardsLimits are set to be met at the “End of the pipe” to satisfy the “No toxics in toxic amounts” narrative water quality standardMuch of the 70’s in environmental protection was spent evaluating the presence of individual chemicals in wastewater discharges.By the 1980’s it was widely recognized that a shift in environmental protection was necessary. Protection should be achieved through not only chemical specific limits but also whole effluent toxicity tests. Permittees could be in complianace with chemical specific limits but the effluetn would still be toxic.Cost effective approach to controlling toxicis.The test predicts the toxicity of the entire solution the environment taking into account matrix effects and the exposure instream.1980’s also brought about some of the TRE/TIE testing procedure developmentWET is used in discharge monitoring and permitting in combination with the chemical specific program where numeric criteria for various chemicals are used to limit the discharge.
51The Whole Effluent Approach Capabilities Toxicity of all effluent constituents are measured and the toxic effect can be regulated with one parameterImplements the national policy of no toxics in toxic amountsChemical interactions are assessedUnknown toxicants are addressedBioavailability of toxic constituents is assessed and the interactions of constituents accounted forCant limit everythingDon’t know what everything is.Where toxicants are additive can provide a better measureThe studies conducted establish results which are predictive of impacts if the appropriate assumptions are met.Biological criteriaReflect overall ecological integrity but the problem is that impacts have already occurred therefore they are not predictive, and difficult to interpret impacts.
52The Whole Effluent Approach Limitations No direct human health protectionCarcinogenicity, mutagenicity and bioaccumulations are not assessedNo direct treatmentPredictivity of results should be carefully assessedNo persistency on sediment coverageIncomplete knowledge of a causative toxicantNot designed to be quantitative predictors of ecosystem response though many studies have demonstrated significant associates between toxicity test results and ecosystem impacts.
53Program Structure Compliance Testing Program (Enforcement) Laboratory Certification Program for Acute and Chronic ToxicityPermit ProgramWhole Effluent Toxicity LimitsToxicity Testing Monitoring RequirementsToxicity Reduction EvaluationsCompliance Testing Program (Enforcement)All aspects covered that are recommended by EPALab cert program for acute since early 80s chronic was not until 2001Prior to that chronic was enforced through permits.Compliance testing program for toxicity has been in existence since the very early stages of the program, with one of the first industrial discharges to receive a limit, eventually paying penalties for violations of the toxicity limit.Who each program is run by
54Laboratory Certification Program Regulations Governing the Certification of Laboratories and Environmental Measurements (N.J.A.C. 7:18)Subchapter 7 contains test methodsSubchapter 9 contains the procedures governing sample collection and handlingFormal certification programNational and state programsGoverns many other programsBoth a local and national program NELAPLab must apply to the DepartmentPass an onsite inspectionReview of SOPs and Quality system
55WET Certification Components Personnel qualificationsLaboratory facilities and safetyEquipment and instrumentationSample collection, handling and preservationTest MethodologyGeneral lab practicesQuality controlReference toxicant dataRecords and data reportingTest acceptability criteriaComprehensive set of requirementsPersonnel minimum educational and experience requirements.Number of replicates, concentrations and controls.Types of water used for culturing and testing.Types of glassware which can be used as well as the cleaning procedures for that glassware.Sample types (composite or grab), holding times and temperatures.Minimum number of replicates, types of test organisms, age of organisms.Maintenance of records, QA manual, SOPs.
56Acute Toxicity Methods Methods contained in rule at N.J.A.C. 7:185 concentrations + controlreplicates96 hours or less (species specific)Mortality or immobilizationReceiving water for dilution preferredTest speciesAcute is more detailed in equipment, dilution water and sample collection. However, the test methods themselves are not significantly different.3.5 pptMost common freshwater test species are Ceriodaphnia dubia and Pimephales promelas (fathead minnow).Most common saline test species are Mysidopsis bahia and Cyprinodon variegatus (sheepshead minnow).Other test species are used in the program and are based on sensitivity and site characteristics, such as salinity.Static renewal and flowthroughDifferences from EPAProblems with dilution water collection
57Chronic Toxicity Methods Incorporated by reference in N.J.A.C. 7:18-7.1(a)Part V includes additional requirementsUSEPA Methods (40 CFR 136)Certified laboratoriesSame test species as acute testing“Part V.”This document specifies the methods applicable to the NJ WET program, outlines the Standard Reference Toxicant (SRT) Program and defines areas left to the NJDEP by EPA such as effluent sampling, use of dilution water, test acceptability criteria and reporting.Fecundity endpoint is not required for the mysid testSampling again is the main concern - issue3 samples collected during the test - once every other dayproblems
59Limit CalculationN.J.A.C. 7:14A-13 - Effluent Limitations for DSW PermitsUSEPA’s Technical Support DocumentAcute and Chronic WQBELsValues of 0.3 and 1.0 used to interpret narrative “no toxics” criteriaReasonable Potential determinations based on site specific dataEffluent toxicity standard at N.J.A.C. 7:9-5.7(a) is an LC50>50% effluentTechnical Support Document for Water Quality-based Toxics Control (EPA/505/ ) march 19___.In 7:14A specific section on determining RP, specifically states if max exceeds or max projected exceeds you must impose limitGet more data if insufficient data exists.Steady state mass balance - no accounting for background toxicity and generally complete mixing is assumed.Regulatory Requirements for Development of WQBELS for WET are found in 40 CFR (d)1.Collect data and determine reasonable potential to exceed Water quality standards using numeric or narrative criteria. Account for existing controls on point and nonpoint sources of pollution, the variability of the pollutant and the sensitivity of the test species.Effluent and receiving water flow, available mixingacute and chronic mixing zones, which is more protectivelimit adjusted based on data variabilitylimit is evaluated for RP
60Who Gets What??What gets imposed - acute / chronic, limits or monitoring only, is highly dependent on what type of data is availableGenerally, if no data exists a limit will not be imposed right away, unless the discharge is the result of a cleanupFinal limits are affected significantly by available dilution and the acute:chronic ratioEvery discharge gets evaluated to some extent for WET- data submitted with application.Only a single limit is imposedSame procedure used to calculate other WQBELsWhere only acute data is available, generally the permit will contain additional data collection requirements.What gets imposed is entirely dependent on what data is availableLimits not imposed without a Reasonable Potential AnalysisAffected significantly by available dilution and acute:chronic ratio (ACR)Where chronic data is available, adequate data is usually available to calculate and determine reasonable potential. It is in this case that a limit may be imposed.Short duration batch discharges generally do not receive chronic testing requirements.
61Limits - The Bottom Line Variability of data affects the final limit outcomeThe more data the betterEnsure input values are appropriateVariability of toxicity test results will influence permit developmentMinimal variability and high quality effluent will lower reasonable potentialReasonable potential is influenced by dilution, need to accurately assessed and modeledCollect data under all conditions, wet or dry weather, high or low flowDon’t stop at the minimumExperience, culture history and health (ref tox results), visit the lab, references, experience with TIEs,Be proactive
62Permit Requirements Limit and testing frequency Test species and methodReporting requirements (endpoints)Repeat testing requirementsCharacterization requirementsSplit samplesToxicity Reduction RequirementsCompliance schedule vs. triggerInterim vs. final limits3 or 5 yearsLimit, monitoring frequency and effective date in Part III, effluent limitations table.3.5 pptFor any permit containing a toxicity limit, there is an associated section in Part IV of the permit.Monitoring frequency is generally based on tables contained in rule at 7:14A-14. Quarterly testing for major facilities an semi-annual for minors. Can be reduced after several years of data has been collected if no reasonable potential can be supported.Where violations occur of an effective limit, the penalty rules at 7:14-8 require monthly monitoring. For limits not effective TRE requirement would apply.
63Toxicity Reduction Evaluations (TRE) Specific TRE language is included at N.J.A.C. 7:14A-13.17(a)Language to exclude test results not considered representative included at N.J.A.C. 7:14A-13.14(a)2Permittees responsibilityNJDEP oversight roleSeries of stepped requirementsApply whether limit in effect or notCompliance schedules of three years are routinely incorporated into permits.The TRE requirements consist of a series of stepped requirements based on an initial trigger of 2 violations in 6 consecutive tests. The first step (Toxicity Characterization Phase) is a period of more frequent monitoring. The second step (Preliminary Toxicity Investigation) is an evaluation of in plant toxicity controls and assessments. The third step (Comprehensive Toxicity Investigation) is a detailed formalized Toxicity Identification Evaluation.Intent of the requirement is that the limit has been imposed based on site specific data which has exhibited “reasonable potential to violate water quality standards.” So if the permittee begins monitoring under a new permit, the three year compliance schedule provides adequate time to determine if an intensive TIE is necessary. The permit allows for extension of compliance schedules to five years where permittee is in a “good faith” TRE.
64Regulatory Issues Recent Developments Toxicity test precision can be compared to analytical chemical precision.Analytical chemical precision similar to toxicity test inter-laboratory.Most commonly used test spec. Ceriodaphnia dubia. Many issues raised regarding culturing, methods and data analysis. Presence of males microscopic examination Test termination, 60% of 3 brood.
65USEPA Support for WETEPA National Policy for WQBEL development for Toxic PollutantsCFR Revised for WQBELsTechnical Support Document for Water Quality-based Toxics ControlWET Control Policy UpdatedIncorporation of WET methods in 40 CFR 1361984 Policy for the Development of Water Quality based Permit Limits for Toxic pollutant addressed the use of biological and chemical methods to assure that toxic whole effluent discharges were regulated consistent with federal and state requirements. Based on sections 101(a) and 308 of the CWA, Who should be reviewed for WET what data to consider how to do reasonable potential monitoring and compliance schedules.1989 revised federal regs which contained non-specific requirement that NPDES permits contain any more stringent requirements necessary to achieve state WQS. Codified in 40 CFR. The preamble detailed explanation of the legal and policy support for WET testing and limits.1991 TSD revised with detailed guidanceJuly 1994 updated policy in (d) governing the development of limits for WET reaffirming their strong commitment to control WET under existing regs.October 16, CFR updated effective November ‘95.
66October 26, 199540 CFR revised to establish standard protocols for conducting WET testsIncorporates acute and chronic test method manuals by referenceSupplemental Information Document provides responses to comments raisedRevisions to Part V to reference 40 CFR 136In November 1995 USEPA issued a final rule standardizing WET testing procedures for use in NPDES monitoring. The rule set uniform WET test procedures for determining the Toxicity of the overall effluent. 17 different procedures, 4 freshwater chronic, 6 marine chronic and 7 acute.In comments received on the draft rule as well as the response to the final rule, there were complaints of inconsistency of the methods and that the methods were to variable for use.6 different litigants sued EPA in 5 different courts of appeal. (Western Coalition of Arid States (WESTCAS) and the Edison Electric)
67NJ WET Program HistoryEarly 1980’s - Acute monitoring and limits used on a routine basisBegan use of chronic monitoring and chronic limitsGroup permit challenge on chronic WETSettlement and initial chronic WET program revisionsFinal program revisions adoptedIn late 1992, adjudicatory hearing requests by a group of municipal dischargers on chronic limits and related TRE requirements1996 Agreed to disagree on the science. Agreed that changes in program implementation were necessary.Prior to 1997 several of limit calculation procedures and program implementation requirements were out of date and inconsistent with USEPA guidance and policy.That adjudication forced NJDEP to reevaluate and update its rules governing WET.Limit expression and calculation, TRE requirements. Program requirements became more specific, increased oversight of lab activity.Group permit modification of 100 permits, 11 removed limits.
68Settlement Agreement Requirements July 24, 1998Variability Guidance DocumentMethod Guidance DocumentInterlaboratory Variability StudyRulemaking actionsLaw suit settled by 3 different settlement agreements, each requiring specific EPA actions to complete several products.It is not a consent decree, which would be independently enforceableexisting test manuals to clarify mandatory items to assure consistency in measurements and clarify discretionary options to optimize successful test completion.Rulemaking to convert discretionary provisions:Ceriodaphnia test randomization and blocking by parentageDevelopment of valid dose concentration response as a re-requisite for a valid testpH control / shiftPrepare guidance and recommendations regarding adjustments to statistical error rate assumptions, confidence intervals, available dilution waters and permutation of the valid dose-response curve. The agreement require that these actions be announced in January 2000In JANUARY Revise manuals to incorporate new requirements and recommendationsCurrent methods provide sufficient direction and flexibility until that time.Recognize variability arises from effluents, analytical procedure and the analystDevelop guidance on how to take analytic variability into account for determining the need for and derivation of the WET Limit by April 2000
69Results 8 of 10 methods had test completion rates >90% Test completion rate of 82% for CeriodaphniaSuccessful test completion rate of approximately 64% for Selenastrum7 of 10 test with no false positives9 of 10 methods had false positives<5%82% = 8 labs failed 50% or more of tests initiated, 24 labs had no failuresfor selenastrum - possible explanations for test failuresStock culture health - tests initiated on the same day in a laboratory either both passed or both failedstock culturing technique - labs that culture organisms with EDTA often failed a high percentage of tests without EDTA, labs that cultured organisms without EDTA often failed a high percentage of tests with EDTACVs were below 50% for all tests except selenastrumLower than average values cited in the method manuals for the same methods at the time of method approval.
70ConclusionsWET Variability Study results confirmed EPAs conclusions that WET methods provide sufficient precision and can be reliably used in permitsIn September 2001, EPA proposed to ratify its previous approval of the methods evaluated in the studyHope that this study will advance the WET debate from questions of method variability to approaches for improving the implementation of the WET program.
71Technical Corrections Notice February 2, 1999 (64 FR 4975)Incorporated into the WET final rule an errata documentcorrects minor errors and omissionsprovides clarificationestablished consistency among the methods manuals and the final rule
72Variability Guidance Document July 18, 2000 (65 FR 44528)Guidance to regulatory authorities, permittees, and testing labs on measurement variability in WET testingExplains the toxicity test protocol, organisms, chemical and physical conditions, renewals, dilution series, test design, measurements (mortality reproduction) data analysis and test endpointsUnderstanding and Accounting for Method Variability in WET Applications Under the NPDES Program.Quantifies the variability of WET methods and provides guidance for addressing and minimizing variability.Contains CVs for promulgated methodsEvaluate statistical methods described in the Technical Support Document for determining RP and deriving WET permit conditions.EPA prepared a document which was then peer reviewed the document 5 independent experts not identified to EPA staff.Peer reviewed conclusions: substantial agreement with approach detailed comments greatly improved the document.Rather extensive document.
73Method Guidance Document July 28, 2000 (65 FR 46457)% Minimum Significant DifferenceConfidence intervalsConcentration response relationshipDilution series selectionDilution water selectionProvides specific technical guidance on issues of WET test conduct and result interpretationCompleted July 28, 2000%MSD calculated for any test should be less than or equal to a a based on test species should be considered for all test resultsCR must be considered in all cases, Not all tests will produce ideal, classical CR curves. Examples of CR curves and guidance on whether to use the data.The evaluation of CR relationships is not always clear-cutLook at variability study data, 698 samples 30 were flagged and recommended resettingTypical 6.25, 12.5, 25, 50, 100 or 0.5 dilution factor not required don’t be afraid to change, consider the effluent limit, data variability, objectives of the test.Dilution water of consistent quality, meets objectives of the test, organisms perform adequately. Adequate flow year round, support adequate performance of test organisms, consistent quality without contaminants.Dual controls - how to evaluated the data.Incorporate guidance into final changes to methods.
74Laboratory ErrorsErrors in the analysis and reporting of WET test results were prevalent.Errors ranged from single data entry or rounding errors to errors in statistical method selection for use.Most errors had minor effects on test results.Lab data submitted for interlaboratory study were reviewed to evaluate the accuracy with whidch the labs routinely analyzed and reported WET test results.Most errors wer made in the calculation or reporting of results.Errors ranged from simple data entry or computational errors to misinterpreation of test endpoints or misuse of statstical methods.Many of these errors were minor and resulted in little or no difference in the reported result.These results indicate a need for increased lab quality control and personnel training, with an increased emphasis on state and regional lab cert programs, and increased client attention to lab selection and result reporting.
75Guidance to Testing Laboratories Maintain QC control charts for IC25 PMSD.Routinely plot average treatment responses and replicate data to identify anomalies and excessive variability.Ensure that the upper PMSD is not exceeded.Use at least four replicates for minnow tests.Additional topics: lab quality control, standardizing reference toxicants, acceptance limits for ref-tox test results.
76Guidance to NPDES Permittees Use one laboratoryReview your laboratory’s control chartsCheck test acceptability criteriaCheck sample holding times and Chain of custody’sObtain at leas 10 data points over >1 year to characterize effluent variability
77Guidance to Regulators Review the test reportsEvaluate PMSD as well as TACConduct routine lab auditsReview SRT control charts
78Proposed Rule Amendments September 28, 2001 (66 FR 49794)Specific revisions to the test methods and proposed to ratify its previous approval of the methodsComment period scheduled to end on November 27, 2001, extended to January 11, 2002
79Final Rule Issued November 19, 2002 Vol. 67. No. 223, 40 CFR 136 Effective December 19, 2002Ratified most of the previously adopted methodsAmended the table containing the toxicity methodsAll of the methods commonly used in New Jersey were adopted.
80Ratification of Ten Methods Methods are repeatable and reproducibleAvailable and applicableRepresentativeVariability study showed high rate of successful completionDo not often produce false positive resultsExhibit precision comparable to chemical methods approved at 40 CFR 136methods are repeatable and reproducible (i.e.. Exhibit adequate within-laboratory and between-laboratory precision)Available and applicable (I.e., adaptable to a wide variety of laboratories and use widely available organisms and suppliesrepresentative (predictive of receiving system impacts.Variability study showed high rate of successful completiondo not often produce false positive results,exhibit precision comparable to chemical methods approved at 40 CFR 136
81Withdrawal of Two Methods Holmesimysis costata Acute Testwest coast test organismChampia parvula Reproduction TestMethods can still be usedEPA was unable to obtain interlaboratory precision data for these methods in the WET Interlaboratory Variability study due to laboratory unavailability.EPA was unable to contract with a minimum of 6 labs qualified and willing to conduct these test methods within the time frame of the study.By withdrawing methods from 136, EPA does not reject their use on more limited bases.Agency has not validated the methods for national useEPA continues to support the use for applications other than for the determination of compliance with NPDES permit limits, as well for limited, localized, or regional use where the methods have been validated by other entities.Holmesimysis costata pacific ocean test organism, test organisms not listed in 40 cfr 136.
82Amendment to 40 CFR 136.3 Table 1A Clarified mysid test method does not apply to Holmesmysis costataAdded method numbers to acute testsModified footnotes and references to cite the updated version of the method manualsRevise the parameter measured in marine tests to refer to organisms “of the Atlantic Ocean and Gulf of Mexico”
83Impact of the Adoption Blocking by parentage Ceriodaphnia test endpointpH driftDilution seriesDilution waterPathogen interferenceVariability criteriaMinimum number of replicatesTest requirements / recommendationsReference toxicant testingSample collection and holding timesSampling holding temperatureBiomassTotal residual chlorineAdditional minor correctionsStates no one dilution series is required. Recommendations only.No single dilution water type is required. Synthetic or nature waters may be used as long as they meet the requirements for acceptable dilution water which is one which supports adequate performance of the test organisms with respect to survival growth, reproduction or other responses that may be measures in a test, is of consistent quality and doesn’t contain contaminants that could produce toxicity.Mandates the use of variability criteria for five chronic methods. Recommends use of point estimation.TRC only required where expected to be present
84Ceriodaphnia dubia Chronic Toxicity Test Mandated use of a very specific procedure of “Blocking by Known Parentage” with at least six neonatesNeonates from a single parent may be used to initiate more than one testElimination of use of fourth brood organismsBlocking requires the use of at least six neonates from each of at least ten separate parents.If more than 6 neonates from a given parent remain after allocating organisms to a test, those remaining neonates may be discarded, used in future culture organism, or used in another test initiated on the same day (must be 24 hr age requirements.
85pH Drift Changes between proposal and adoption Permitted in chronic methods onlySpecific procedures to demonstrate need for pH controlSpecific procedures for pH control during testingClarifies that when the test objective is to determine the absolute toxicity of the effluent, the target pH to maintain in a pH-controlled test is the pH of the sample upon completion of collection.Clarify that in pH-controlled tests, pH should be maintained within +/-.2 for fw and .3 for SW.Guidance on interpreting the results in parallel testing.Clarified that they did not believe that pH drift alone is not a test interference if pH is within the organism’s tolerance range. The Degree of pH drift typically observed in effluent samples should generally only interfere with test results if the sample contains a compound with toxicity that is pH dependent and at a concentration that is near the toxicity threshold. Because EPA did not receive data to suggest otherwise, EPA removed any reference to pH drift interference in the absence of pH dependent toxicants.No pH control guidance is included in the acute test methods because those methods pH drift may be remedied by more frequent test renewals or the use of follow-through testing.
86PMSD Percent Minimum Significant Difference Test Method: Endpoint 10th PMSD 90th PMSDFathead Minnow GrowthC. dubia ReproductionSheepshead minnow Growth (6.3) (23)Inland Silverside GrowthMysid GrowthPMSD values calculated with Dunnett’s test must be between within the range established by the 10th and 90th PMSD values.PMSD = (MSD x 100) / Control Mean (This value is calculated via the Dunnett’s program)
87Test Requirements / Recommendations Modification of tables summarizing test conditionsNew section on test reviewMandatory review of concentration response relationship by regulatory authorityMandatory use of variability criteria for data reported as an NOEC
88Reference Toxicant Testing Used for initial and ongoing demonstration of performance and to assess sensitivity and health of test organismsMonthly or side by side testingUse of suppliers five most recent testsNot a “de facto criterion” for test rejectionLabs should evaluate CVs based on national values
89Sample Collection & Holding Maintained default maximum of 36 hrs for first use of sampleClarified these samples may be used for later renewalsPermitting authority may allow continued use of most recent sampleCollection on days one, three and five recommended (not required)
91Serious Violation Limit (% Effluent) > or = 80 and < or = 100 Result Difference> or = 20> or = 15> or = 10> or = 9What is deemed a violation of a toxicity limit is defined in N.J.A.C 7: Civil Administrative Penalty for Violation of Whole Effluent Toxicity Limits.A Serious Violation has a mandatory minimum penalty of $1000 for the first violation and $5000 for each subsequent violation. Penalty maximum can be not be greater than $50,000 for a serious violation. Non serious penalty maximums can not be greater than $40,000.
92Affirmative DefenseN.J.A.C. 7: violator is entitled to an affirmative defense to liability for a violation occurring as a result of an upset, an anticipated or unanticipated bypass, or a testing or laboratory error.
93Testing or Laboratory Error A violator asserting a testing or laboratory error as an affirmative defense shall also have the burden to demonstrate that a violation involving the exceedance of an effluent limit was the result of unanticipated test interference, sample contamination, analytical defects, or procedural deficiencies in sampling or other similar circumstances beyond the violators control.
95Toxicity where you don’t expect it?? StormwaterCooling WaterFilter backwashHigh or low TDSChlorine
96Naturally low pH water N.J.A.C. 7:18-9.5(a)2vii. If the receiving water has a natural pH below 5.0 units, then the dilution water samples shall be adjusted to a pH of 5.0 prior to their use in test organism acclimation and/or toxicity testing.
97Where can I get toxicity data on various chemicals? 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.
98Other Issues NMAT to NOAEC How much data is enough? Dilution estimates When is data too old?What are other states doing?Intermittent discharges?Sampling issues for intermittent discharges.
99How does increasing the difference in test concentration dilutions affect the prediction of response?Better resolution around threshold effect concentrationReducing the distance between effluent dilutions should be encouragedminimum set of dilutions, i.e. no wider than 0.5 dilutions between concentrationsTest design should maximize test concentrations around the instream waste concentration, in order to minimize the need for interpretation of effects between tested concentrationsBetter resolution around threshold effect concentration provide better input to mathematical models to predict point estimations of effect and reduce uncertainty in hypothesis test of effect. Reducing the distance between effluent dilutions should be encouraged. There may be some confusion about EPA’s specification of dilution series in these cases. The methods specify a minimum set of dilutions, i.e. no wider than 0.5 dilutions between concentrations. No limitations on added concentrations within that range exist. Experimental design should account for concentrations of concern and should attempt to maximize resolution in that range. Test design should maximize test concentrations around the effect conc.. Of concerns, I.e the instream waste concentration or limited concentrations of a discharging facility, in order to minimize the need for interpretation of effects between tested concs.
100My effluent tests indicate there may be a problem but I can see fish in the area of my discharge, is there really a problem?Observations of organisms in the area of the outfall does not mean that more subtle impacts are not occurring or that the organisms that are present are sensitive enough to represent most organisms instream.Reduction of a food sourcePresence of pollution tolerant organismsmore sensitive organisms are already goneSometimes difficult to establish agreement between tox test results and instream conditions.Limits are based upon available dilution, effluent variability.