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

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

3. 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.

4. 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.

5. 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.

6. 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.

7. Pimephales promelas (fathead minnow)
Adult
Larvae

8. 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.

9. Ceriodaphnia dubia (Water flea)
Ceriodaphnia dubia image

10. 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.

11. 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.

12. 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

13. General Wet Lab Facility Requirements
Should have at least square feet of lab space and 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.

14. 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.

15. 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.

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

17. 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

18. 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.

19. 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.

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

21. 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.

22. 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.

23. Control Chart Example

24. 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)

25. 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 days old.

26. 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

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

28. 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.

29. 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.

30. 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.

31. References
Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms; 5th Edition October 2002 “EPA-821-R ”
Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms; 4th Edition October 2002 “EPA-821-R ”
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

32. Contact Information
Erich Cleaver
Laboratory Auditor
Division of Water 200 Fair Oaks Lane; 4th Floor
Frankfort, KY 40601
Phone: (502)
Fax: (502)