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Published on December 30, 2007

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Whole Effluent Toxicity Basics:  Whole Effluent Toxicity Basics Betty Jane Boros-Russo NJDEP, Office of Quality Assurance Christopher J. Nally American Aquatic Testing History:  History 16th century - scientists began testing the lethality of chemical compounds on animals prior to their use on humans for therapeutic purposes 1930’s - some of the first uses of aquatic organisms for testing to determine the causes of observed fish kills 1945 - some of the first methods for conducting toxicity tests were published Use of Toxicity Testing in Water Quality Based Toxics Control:  Use of Toxicity Testing in Water Quality Based Toxics Control To characterize and measure the aggregate toxicity of an effluent or ambient waters To measure compliance with whole effluent toxicity limits As an investigative tool and to measure progress in a toxicity reduction program As an ambient instream measure of toxicity to identify pollution sources NJ WET Program History:  NJ WET Program History Early 1980’s - Acute monitoring and limits used on a routine basis 1989 - Began use of chronic monitoring and chronic limits 1993 - Group permit challenge on chronic WET 1996 - Settlement and initial chronic WET program revisions 1997 - Final program revisions adopted Test Species:  Test Species Species Selection:  Species Selection Sensitive species which are easily cultured and readily available year round Must provide consistent and reproducible response Also encourage ecologically, commercially and or recreationally important No one species is always the most sensitive Species used is dependent upon salinity of receiving water and the state standards New Jersey Freshwater Acute Test Species:  New Jersey Freshwater Acute Test Species Invertebrates: (Daphnids) Ceriodaphnia dubia Daphnia magna Daphnia pulex Fish Pimephales promelas Fathead Minnow Oncorhynchus mykiss Rainbow Trout Salvelinus fontinalis Brook Trout Ceriodaphnia dubia:  Ceriodaphnia dubia Female approximately 2 mm Photo compliments of Marinco Bioassay Laboratory Pimephales promelas:  Pimephales promelas Photo by Karen McCabe from Animal Soup New Jersey Saline Acute Test Species:  New Jersey Saline Acute Test Species Invertebrates: Mysidopsis bahia Opossum Shrimp (Americamysis bahia) Fish Cyprinodon variegatus Sheepshead Minnow Menidia beryllina Inland Silversides Menidia peninsulae Tidewater Silversides Menidia menidia Atlantic Silversides Mysidopsis bahia:  Mysidopsis bahia Photo compliments of Marinco Bioassay Laboratory Female approximately 6 mm in length New Jersey Freshwater Chronic Test Species:  New Jersey Freshwater Chronic Test Species Invertebrates: Ceriodaphnia dubia Fish Pimephales promelas Fathead Minnow Algae Selenastrum capricornutum New Jersey Saline Chronic Test Species:  New Jersey Saline Chronic Test Species Invertebrates: Mysidopsis bahia Opossum Shrimp Fish Cyprinodon variegatus Sheepshead Minnow Menidia beryllina Inland Silversides Menidia peninsulae Tidewater Silversides Menidia menidia Atlantic Silversides Other Arbacia punctulata Sea Urchin Champia parvula Red Macroalgae Test Methods:  Test Methods Rules for Conducting Toxicity Tests:  Rules for Conducting Toxicity Tests 40 CFR 136.3 - Table 1A Effective November 15, 1995 Amended November 19, 2002 and effective December 19, 2002 Methods must be followed as they are written Incorporate by Reference:  Incorporate 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-R-02-012 Short-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-R-02-013 Short-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-02-014 USEPA Methods Documents:  USEPA Methods Documents Health and safety Quality assurance Facilities, equipment and supplies Test organisms and culture methods Dilution water USEPA Methods Documents (cont.):  USEPA Methods Documents (cont.) Effluent sampling and handling Endpoints and data analysis Individual test methods Report preparation and test review Test Types:  Test Types Acute and Short-term Chronic Tests Static non-renewal Static renewal Flow through Test Species dependent Use dependent Test Design:  Test Design 5 Concentrations + Control Serial dilution’s of effluent and “control water” (also termed “dilution water”) Dilution series of 0.5 or greater Single concentration test Replicates Randomization (organisms/chambers) Perspective is Everything:  Perspective is Everything Test Conditions and Acceptability Criteria:  Test Conditions and Acceptability Criteria Acute Toxicity Tests:  Acute Toxicity Tests Test Procedures 96 hours or less (species specific) Mortality is the measured endpoint For daphnia mortality determined by immobilization Advantages less expensive and time consuming than chronic endpoint is easy to quantify Disadvantages indicates only lethal concentrations only the effects of fast acting chemicals are exhibited Acute Test Acceptability Criteria:  Acute Test Acceptability Criteria Minimum control survival at least 90% Temperature maintained @ 20 +/- 1o C Maximum test organism age at start: 14 days for fish 5 days for Mysid shrimp 24 hours for daphnids Short-term Chronic Toxicity Tests:  Short-term Chronic Toxicity Tests Test Procedures typically 4-10 days Mortality, growth, fecundity, reproduction Advantages more sensitive than acute, assess parameters other than lethality may better reflect real world Limitations more costly and time intensive than acute more sensitive to low level contamination Chronic Test Acceptability Criteria:  Chronic Test Acceptability Criteria Minimum control survival 80% Minimum control dry weight (average): 0.25 mg for fish 0.20 mg for Mysid shrimp Minimum of 15 young (average) for control C. dubia Temperature maintained @ 25 +/- 1o C Maximum test organism age at start: 48 hours for fish 7 days for Mysid shrimp 24 hours for daphnids Method Specific Test Conditions:  Method Specific Test Conditions Test type and duration Temperature, light, DO, salinity Chamber size and volume Species selection, age and feeding Method Specific Test Conditions (cont.):  Method Specific Test Conditions (cont.) Dilution water Dilution series Sampling Test acceptability criteria Test measurements Test Measurements:  Test Measurements Dissolved oxygen cannot fall below 4 mg/l (initial and final) pH (initial and final) conductivity total residual chlorine total hardness and alkalinity salinity temperature Selection of Dilution Water:  Selection of Dilution Water May be either a standard laboratory water or the receiving water Choice of water is dependent on the objectives of the test Absolute toxicity use standard water Estimate of toxicity in uncontaminated receiving water, use receiving water Contaminated receiving water, use laboratory water Data and Endpoints:  Data and Endpoints Acute Test Endpoints:  Acute Test Endpoints LC50 - 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 Concentration Lowest concentration at which survival is not significantly different from the control always set equal to 100% effluent EC - Effect Concentration Test Data:  Test Data Typical dose response where mortality increases as the concentration of effluent in the mixture increases. LC50 would be somewhere between 25% effluent and 50% effluent. 0% Mortality 0% mortality 20 % Mortality 40% Mortality 80% Mortality 100% Mortality 6.25 % Effluent Control 12.5 % Effluent 25.0% Effluent 50.0% Effluent 100.0% Effluent Chronic Test Endpoints:  Chronic 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). Chronic Test Data:  Chronic Test Data % % Average Effluent Mortality Dry weight % w/Eggs 0 2.5 0.418 69.6 6.25 7.5 0.371 68.8 12.5 10.0 0.348 50.0 25.0 10.0 0.308 28.6 50.0 17.5 0.248 0.0 100.0 100.0 0.0 0.0 NOEC 50.0% 12.5% 12.5% IC25 55.7% 23.2% 10.7% Toxicity Values:  Toxicity Values LC50, IC25, NOAEC: As a limit these values will INCREASE as the limit becomes more stringent These are minimum limits LC50, IC25: When evaluating data, exhibit more toxicity as the values decrease Toxic Units: Maximum limits As values increase as limits, they become less stringent Toxic Units (TU’s):  Toxic Units (TU’s) Reciprocal of the fractional LC50, NOEC, IC25 value Calculated by dividing the value into 100 TUa = 100/LC50 TUc = 100/IC25 Standard Reference Toxicant Program:  Standard Reference Toxicant Program Standard Reference Toxicants (SRT’s):  Standard Reference Toxicants (SRT’s) Purpose Frequency Acceptability Criteria Control Charts Control Charts:  Control Charts Sample Collection:  Sample Collection Subchapter 9:  Subchapter 9 N.J.A.C. 7:18 Subchapter 9 Sample Requirements Addresses collection, handling and preservation of environmental samples Section 9.5 – Requirements for acute toxicity testing samples Grab vs. Composite:  Grab vs. Composite Grab samples offer “snap shot” of effluent Composite samples offer “average view” of effluent NJDEP requires sampling based on discharge type Continuous discharge – 24 hour composite sample Intermittent discharge – grab or composite each day that is representative of discharge Frequency and Holding:  Frequency and Holding Daily for acute toxicity testing (single comp. for daphnids) Every 48 hours for chronic testing Minimum # of samples for 24 hour composite - 48 (every 30 minutes) Holding times 24 hours to first use for acute and chronic testing 72 hours to use three times, chronic testing only Effluent Sampling Containers:  Effluent Sampling Containers Constructed of non-toxic materials: Glass – borosilicate, tempered or soda lime 304 or 316 stainless steel Medical or food grade silicone Perfluorocarbons – Teflon, etc. Plastics – polyethylene, polypropylene, polycarbonate,polystyrene Containers rinsed with sample, used once and disposed of, or cleaned. Sampling Location:  Sampling Location NJPDES sample location must be used for toxicity test sampling This is generally the same sampling location required for all other parameters Prechlorination sampling may be required Post dechlorination sampling may be required Location should always be specified in the permit Preservation:  Preservation For toxicity testing only temperature preservation permitted Refrigeration during sampling optional Refrigeration or icing immediately upon collection required SAMPLING DOCUMENTATION:  SAMPLING DOCUMENTATION Chain of Custody Facility information Date, time, sample ID, sampler ID, sample location information Signatures for custody transfer Signatures are important!! Avoids confusion Prevents sampling from occurring when plant not operating normally New Jersey Toxicity Testing Program:  New Jersey Toxicity Testing Program Whole Effluent Toxicity Approach to Water Quality Based Toxics Control:  Whole 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 waters Goal is to protect aquatic biota and achieve surface water quality standards Limits are set to be met at the “End of the pipe” to satisfy the “No toxics in toxic amounts” narrative water quality standard The Whole Effluent Approach Capabilities:  The Whole Effluent Approach Capabilities Toxicity of all effluent constituents are measured and the toxic effect can be regulated with one parameter Implements the national policy of no toxics in toxic amounts Chemical interactions are assessed Unknown toxicants are addressed Bioavailability of toxic constituents is assessed and the interactions of constituents accounted for The Whole Effluent Approach Limitations:  The Whole Effluent Approach Limitations No direct human health protection Carcinogenicity, mutagenicity and bioaccumulations are not assessed No direct treatment Predictivity of results should be carefully assessed No persistency on sediment coverage Incomplete knowledge of a causative toxicant Program Structure:  Program Structure Laboratory Certification Program for Acute and Chronic Toxicity Permit Program Whole Effluent Toxicity Limits Toxicity Testing Monitoring Requirements Toxicity Reduction Evaluations Compliance Testing Program (Enforcement) Laboratory Certification Program:  Laboratory Certification Program Regulations Governing the Certification of Laboratories and Environmental Measurements (N.J.A.C. 7:18) Subchapter 7 contains test methods Subchapter 9 contains the procedures governing sample collection and handling Formal certification program National and state programs WET Certification Components:  WET Certification Components Personnel qualifications Laboratory facilities and safety Equipment and instrumentation Sample collection, handling and preservation Test Methodology General lab practices Quality control Reference toxicant data Records and data reporting Test acceptability criteria Acute Toxicity Methods:  Acute Toxicity Methods Methods contained in rule at N.J.A.C. 7:18 5 concentrations + control replicates 96 hours or less (species specific) Mortality or immobilization Receiving water for dilution preferred Test species Chronic Toxicity Methods:  Chronic Toxicity Methods Incorporated by reference in N.J.A.C. 7:18-7.1(a) Part V includes additional requirements USEPA Methods (40 CFR 136) Certified laboratories Same test species as acute testing Permitting:  Permitting Limit Calculation:  Limit Calculation N.J.A.C. 7:14A-13 - Effluent Limitations for DSW Permits USEPA’s Technical Support Document Acute and Chronic WQBELs Values of 0.3 and 1.0 used to interpret narrative “no toxics” criteria Reasonable Potential determinations based on site specific data Effluent toxicity standard at N.J.A.C. 7:9-5.7(a) is an LC50>50% effluent Who Gets What??:  Who Gets What?? What gets imposed - acute / chronic, limits or monitoring only, is highly dependent on what type of data is available Generally, if no data exists a limit will not be imposed right away, unless the discharge is the result of a cleanup Final limits are affected significantly by available dilution and the acute:chronic ratio Limits - The Bottom Line:  Limits - The Bottom Line Variability of data affects the final limit outcome The more data the better Ensure input values are appropriate Permit Requirements:  Permit Requirements Limit and testing frequency Test species and method Reporting requirements (endpoints) Repeat testing requirements Characterization requirements Split samples Toxicity Reduction Requirements Compliance schedule vs. trigger Interim vs. final limits 3 or 5 years Toxicity Reduction Evaluations (TRE):  Toxicity 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)2 Permittees responsibility NJDEP oversight role Series of stepped requirements Apply whether limit in effect or not Regulatory Issues:  Regulatory Issues Recent Developments USEPA Support for WET:  USEPA Support for WET 1984 - EPA National Policy for WQBEL development for Toxic Pollutants 1989 - 40 CFR 122.44 Revised for WQBELs 1991 - Technical Support Document for Water Quality-based Toxics Control 1994 - WET Control Policy Updated 1995 - Incorporation of WET methods in 40 CFR 136 October 26, 1995:  October 26, 1995 40 CFR 136.3 revised to establish standard protocols for conducting WET tests Incorporates acute and chronic test method manuals by reference Supplemental Information Document provides responses to comments raised Revisions to Part V to reference 40 CFR 136 NJ WET Program History:  NJ WET Program History Early 1980’s - Acute monitoring and limits used on a routine basis 1989 - Began use of chronic monitoring and chronic limits 1993 - Group permit challenge on chronic WET 1996 - Settlement and initial chronic WET program revisions 1997 - Final program revisions adopted Settlement Agreement Requirements:  Settlement Agreement Requirements July 24, 1998 Variability Guidance Document Method Guidance Document Interlaboratory Variability Study Rulemaking actions Results:  Results 8 of 10 methods had test completion rates >90% Test completion rate of 82% for Ceriodaphnia Successful test completion rate of approximately 64% for Selenastrum 7 of 10 test with no false positives 9 of 10 methods had false positives<5% Conclusions:  Conclusions WET Variability Study results confirmed EPAs conclusions that WET methods provide sufficient precision and can be reliably used in permits In September 2001, EPA proposed to ratify its previous approval of the methods evaluated in the study Technical Corrections Notice:  Technical Corrections Notice February 2, 1999 (64 FR 4975) Incorporated into the WET final rule an errata document corrects minor errors and omissions provides clarification established consistency among the methods manuals and the final rule Variability Guidance Document:  Variability Guidance Document July 18, 2000 (65 FR 44528) Guidance to regulatory authorities, permittees, and testing labs on measurement variability in WET testing Explains the toxicity test protocol, organisms, chemical and physical conditions, renewals, dilution series, test design, measurements (mortality reproduction) data analysis and test endpoints Method Guidance Document:  Method Guidance Document July 28, 2000 (65 FR 46457) % Minimum Significant Difference Confidence intervals Concentration response relationship Dilution series selection Dilution water selection Laboratory Errors :  Laboratory Errors Errors 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. Guidance to Testing Laboratories:  Guidance 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. Guidance to NPDES Permittees:  Guidance to NPDES Permittees Use one laboratory Review your laboratory’s control charts Check test acceptability criteria Check sample holding times and Chain of custody’s Obtain at leas 10 data points over >1 year to characterize effluent variability Guidance to Regulators:  Guidance to Regulators Review the test reports Evaluate PMSD as well as TAC Conduct routine lab audits Review SRT control charts Proposed Rule Amendments :  Proposed Rule Amendments September 28, 2001 (66 FR 49794) Specific revisions to the test methods and proposed to ratify its previous approval of the methods Comment period scheduled to end on November 27, 2001, extended to January 11, 2002 Final Rule:  Final Rule Issued November 19, 2002 Vol. 67. No. 223, 40 CFR 136 Effective December 19, 2002 Ratified most of the previously adopted methods Amended the table containing the toxicity methods Ratification of Ten Methods:  Ratification of Ten Methods Methods are repeatable and reproducible Available and applicable Representative Variability study showed high rate of successful completion Do not often produce false positive results Exhibit precision comparable to chemical methods approved at 40 CFR 136 Withdrawal of Two Methods:  Withdrawal of Two Methods Holmesimysis costata Acute Test west coast test organism Champia parvula Reproduction Test Methods can still be used Amendment to 40 CFR 136.3 Table 1A:  Amendment to 40 CFR 136.3 Table 1A Clarified mysid test method does not apply to Holmesmysis costata Added method numbers to acute tests Modified footnotes and references to cite the updated version of the method manuals Revise the parameter measured in marine tests to refer to organisms “of the Atlantic Ocean and Gulf of Mexico” Impact of the Adoption:  Impact of the Adoption Blocking by parentage Ceriodaphnia test endpoint pH drift Dilution series Dilution water Pathogen interference Variability criteria Minimum number of replicates Test requirements / recommendations Reference toxicant testing Sample collection and holding times Sampling holding temperature Biomass Total residual chlorine Additional minor corrections Ceriodaphnia dubia Chronic Toxicity Test:  Ceriodaphnia dubia Chronic Toxicity Test Mandated use of a very specific procedure of “Blocking by Known Parentage” with at least six neonates Neonates from a single parent may be used to initiate more than one test Elimination of use of fourth brood organisms pH Drift:  pH Drift Changes between proposal and adoption Permitted in chronic methods only Specific procedures to demonstrate need for pH control Specific procedures for pH control during testing PMSD:  PMSD Percent Minimum Significant Difference Test Method: Endpoint 10th PMSD 90th PMSD Fathead Minnow Growth 12 30 C. dubia Reproduction 13 47 Sheepshead minnow Growth (6.3) (23) Inland Silverside Growth 11 28 Mysid 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 / Recommendations:  Test Requirements / Recommendations Modification of tables summarizing test conditions New section on test review Mandatory review of concentration response relationship by regulatory authority Mandatory use of variability criteria for data reported as an NOEC Reference Toxicant Testing:  Reference Toxicant Testing Used for initial and ongoing demonstration of performance and to assess sensitivity and health of test organisms Monthly or side by side testing Use of suppliers five most recent tests Not a “de facto criterion” for test rejection Labs should evaluate CVs based on national values Sample Collection & Holding:  Sample Collection & Holding Maintained default maximum of 36 hrs for first use of sample Clarified these samples may be used for later renewals Permitting authority may allow continued use of most recent sample Collection on days one, three and five recommended (not required) Enforcement:  Enforcement Serious Violation:  Serious Violation Limit (% Effluent) > or = 80 and < or = 100 > or = 50 and < 80 >10 and < 50 < or = 10 Result Difference > or = 20 > or = 15 > or = 10 > or = 9 Affirmative Defense:  Affirmative Defense N.J.A.C. 7:14-8.3 - 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. Testing or Laboratory Error:  Testing 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. FAQ’s:  FAQ’s Toxicity where you don’t expect it??:  Toxicity where you don’t expect it?? Stormwater Cooling Water Filter backwash High or low TDS Chlorine Naturally low pH water:  Naturally 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. Where can I get toxicity data on various chemicals?:  Where can I get toxicity data on various chemicals? 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 Issues:  Other 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. How does increasing the difference in test concentration dilutions affect the prediction of response?:  How does increasing the difference in test concentration dilutions affect the prediction of response? Better resolution around threshold effect concentration Reducing the distance between effluent dilutions should be encouraged minimum set of dilutions, i.e. no wider than 0.5 dilutions between concentrations Test design should maximize test concentrations around the instream waste concentration, in order to minimize the need for interpretation of effects between tested concentrations My effluent tests indicate there may be a problem but I can see fish in the area of my discharge, is there really a problem?:  My 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. WET Resources:  WET Resources www.epa.gov/waterscience/WET www.epa.gov/ostwater/WET/index.html www.setac.org www.toxicity.com

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