Whole Effluent Toxicity Presentation to: KWWOA April 09, 2014 Department for Environmental Protection Environmental & Public Protection Cabinet To Protect and Enhance Kentucky’s Environment

Whole Effluent Toxicity The aggregate toxic effect of an effluent measured directly by a toxicity test.

Toxicity Test A procedure using live organisms to determine whether a chemical or an effluent is toxic. A toxicity test measures the degree of the effect of a specific chemical or effluent on exposed test organisms.

Toxic Substance A substance that is bioaccumulative, synergistic, antagonistic, teratogenic, mutagenic, or carcinogenic; and causes death, disease, a behavioral abnormality, a physiological malfunction, or a physical deformity in an organism or its offspring or interferes with normal propagation.

Representative Indicator Organism An aquatic organism designated for use in toxicity testing because of its relative sensitivity to toxicants and its widespread distribution in the aquatic environment.

Pimephales promelas (fathead minnow) Adults are small in size and can range from 43 to 102 millimeters in length. Fathead minnow is a freshwater fish widely distributed in the United States. They are tolerant of high turbidity, high temperatures and low oxygen concentrations.

Pimephales promelas (fathead minnow) Adult Larvae

Ceriodaphnia dubia (water flea) Water flea occur in littoral area lakes, ponds and marshes throughout most of the world. They are a vital link in aquatic food chains. A significant source of food for small fish. They have a short life cycle and are easy to culture in a laboratory. Sensitive to a broad range of contaminants. Require small amounts of test water.

Ceriodaphnia dubia (Water flea) Ceriodaphnia dubia image

Daphnia Pulex Most common species of water flea. Occurs in a wide range of aquatic habitats, but is most closely associated with small shaded pools.

D. magna (water flea) Daphnia magna is principally a lake dweller and is restricted to waters in northern and western North America. Usually five to six millimeters in length.

WET Certification Components Personnel Qualifications Laboratory facilities Equipment & Instrumentation Sample collection, handling & preservation Test methodology General lab practices Quality Assurance/Quality control Reference toxicant data Records & data reporting Test acceptability criteria

General Wet Lab Facility Requirements Should have at least 150-200 square feet of lab space and 15-20 linear feet of lab bench space. Should have adequate lighting, cooling and heating to maintain appropriate environmental conditions for culturing organisms/toxicity tests. Hot & cold running water. Separate areas for culturing, toxicity testing and other chemical analyses. Well ventilated and free of toxic fumes. Secure and maintained in a clean, organized manner.

Sample Handling, Preservation & Shipping Sample holding time begins when a grab sample is collected or when a composite sampling period is completed. Maximum holding time prior to initial use of an effluent sample for toxicity testing shall be 36 hours after the completion of sample collection. Time of initial use is defined as the point in time when organisms have been introduced into test chambers for all tests.

Sample Shipping & Receiving Effluent samples shall be shipped with ice or under other refrigerated conditions (blue ice shall not be used). Sample temp shall not exceed 6°C upon arrival at the performing laboratory unless sample is received within 2 hours of the end of the collection period and sample used for testing upon arrival or stored immediately. Upon receiving the temp & pH of the sample, date & time of receipt, and initials of laboratory personnel receiving the sample shall be recorded. Samples shall be stored at < 6°C (without freezing) in the dark until used and when not in use.

WET Methods & Manuals Methods for Measuring Acute Toxicity to Freshwater and Marine Organisms 5th Edition: 2002.0 Ceriodaphnia dubia acute 2021.0 Daphnia Pulex & D. magna acute 2000.0 Pimephales promelas acute Short-term Methods for Estimating Chronic Toxicity to Freshwater Organisms 4th Edition: 1000.0 Pimephales promelas (larval survival & growth) 1002.0 Ceriodaphnia dubia (survival & reproduction)

USEPA Methods Document Health & safety Quality assurance/Quality Control Facilities, equipment and supplies Test organisms and culture methods Dilution water Food quality Effluent sampling & handling Endpoints and data analysis Individual test methods Report preparation & test review

Toxicity testing labs must: Maintain a qualified staff. Develop, implement & maintain a QA/QC program. Develop data quality objectives. Develop & maintain detailed written standard operating procedures for all areas of toxicity testing. Use approved testing methods. Provide adequate lab facilities & equipment. Notify regulatory authority of any changes in equipment or personnel that would affect the labs ability to meet toxicity test criteria.

Quality Assurance (QA) Quality assurance addresses all activities affecting the quality of the final effluent toxicity data: evaluation of effluent sampling and handling, source and condition of test organisms, equipment condition, test conditions, instrument calibration, replication, use of reference toxicant, record keeping, data and other aspects of the test and testing procedures.

Quality Control (QC) Quality control is the set of focused, routine, day-to-day activities carried out as part of an overall QA program.

Reference Toxicant Testing Used for initial and ongoing demonstration of performance of laboratory personnel. A check of the sensitivity of test organisms and the suitability of the test methodology in a toxicity test. Monthly or side by side testing. Acceptance criteria. Not a “de facto criterion” for test rejection. Labs should evaluate Coefficient of Variation (CVs) based on national values.

Control Charts A statistical tool used in quality control to analyze and understand process variables, determine process capabilities and monitor effects of variables on the difference between target and actual performance. Control charts shall be prepared for each combination of reference toxicant, test species, test conditions and endpoints. Toxicity endpoints from five or six tests are adequate for establishing control charts. Control chart shall be maintained using 20 most recent data points.

Control Chart Example

Chain of Custody The chain of custody shall at a minimum contain the following information: Identification, location date & time of collection; Sample collector’s name; Preservative added & shipping requirements; Container type & volume; Sample type & analysis requested; Any special remarks concerning the sample; Custody transfer (released by/received by)

Acute Test Acceptance Criteria Control survival of at least 90% Test temperature must not deviate by more than 3°C during the test. (20 +/- 1°C or 25° +/- 1°C) Daphnids <24 hours old minnows 1-14 days old.

Chronic Test Acceptance Criteria Minimum control survival of 80% Test temperature must not deviate by more than 3°C during the test (25° +/- 1°C). Average15 young for control C. dubia & 60% of control organisms must produce 3 broods Minimum control dry weight (average) of 0.25 mg for minnow larvae. Maximum test organism age of 48 hours for minnows & 24 for daphnids

Test Measurements Dissolved Oxygen Temperature Conductivity Salinity or pH Alkalinity/Total Hardness Total Residual Chlorine Must use 40 CFR 136.3 approved method

Phase I of WET lab audit Preliminary contact between lab & regulating authority to determine agreeable dates. Submission of all requested pre-audit documents within the determined time frame. (Pre-survey information forms, QAP, SOPs DMR-QA studies, etc.) to allow adequate review time by auditor. Appropriate lab staff are made aware of the audit and made available to auditor.

Phase II (On-site visit) Opening conference with all relevant lab staff. Tour of the facility. Review & verify items completed on pre-survey forms. Have one-on-one discussions with technical staff. Complete all relevant WET review checklists. Close out meeting with all relevant staff to discuss deviations/recommendations.

Phase III (Final Report) Submission of a final report to the laboratory listing all deviations, recommendations and required corrective actions. Corrective action plan (CAP) is submitted by the lab and reviewed by the auditor. A final determination is provided to the lab along with request for any required documentations.

References Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms; 5th Edition 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 October 2002 “EPA-821-R-02-013” Manual for the Evaluation of Laboratories Performing Aquatic Toxicity Tests; January 1991 “EPA/600/4-90/031 401 KAR 5:002. Definitions for 401 KAR Chapter 5 Whole Effluent Toxicity Basics by Betty Jane Boros-Russo & Christopher J. Nally

Contact Information Erich Cleaver Laboratory Auditor Division of Water 200 Fair Oaks Lane; 4th Floor Frankfort, KY 40601 Phone: (502) 564-3410 Fax: (502) 564-2741 Email: erich.cleaver@ky.gov