1. Whole Effluent Toxicity Basics Betty Jane Boros-Russo NJDEP, Office of Quality Assurance Christopher J. Nally American Aquatic Testing

2. 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

3. 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

4. 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

5. Test Species

6. 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

7. 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

8. Ceriodaphnia dubia • Female • approximately 2 mm Photo compliments of Marinco Bioassay Laboratory

9. Pimephales promelas Photo by Karen McCabe from Animal Soup

10. 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

11. Mysidopsis bahia Female approximately 6 mm in length Photo compliments of Marinco Bioassay Laboratory

12. New Jersey Freshwater Chronic Test Species Invertebrates: • Ceriodaphnia dubia Fish • Pimephales promelas Fathead Minnow Algae • Selenastrum capricornutum

13. 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

14. Test Methods

15. 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

16. 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

17. USEPA Methods Documents • Health and safety • Quality assurance • Facilities, equipment and supplies • Test organisms and culture methods • Dilution water

18. USEPA Methods Documents(cont.) • Effluent sampling and handling • Endpoints and data analysis • Individual test methods • Report preparation and test review

19. Test Types • Acute and Short-term Chronic Tests • Static non-renewal • Static renewal • Flow through • Test Species dependent • Use dependent

20. 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)

21. Perspective is Everything

22. Test Conditions and Acceptability Criteria

23. 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

24. 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

25. 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

26. 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

27. Method Specific Test Conditions • Test type and duration • Temperature, light, DO, salinity • Chamber size and volume • Species selection, age and feeding

28. Method Specific Test Conditions (cont.) • Dilution water • Dilution series • Sampling • Test acceptability criteria • Test measurements

29. 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

30. 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

31. Data and Endpoints

32. 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

33. 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. 6.25 % Effluent 12.5 % Effluent 25.0% Effluent 50.0% Effluent 100.0% Effluent Control 0% Mortality 0% mortality 20 % Mortality 40% Mortality 80% Mortality 100% Mortality

34. 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).

35. Chronic Test Data % % Average EffluentMortalityDry 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%

36. 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

37. 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

38. Standard Reference ToxicantProgram

39. Standard Reference Toxicants (SRT’s) • Purpose • Frequency • Acceptability Criteria • Control Charts

40. Control Charts

41. Sample Collection

42. 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

43. 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

44. 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

45. 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.

46. 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

47. Preservation • For toxicity testing only temperature preservation permitted • Refrigeration during sampling optional • Refrigeration or icing immediately upon collection required

48. 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

49. New Jersey Toxicity Testing Program

50. 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