1. TVA-KINGSTON FLY ASH RELEASE ENVIRONMENTAL RESEARCH SYMPOSIUM MARCH 11 – 12, 2010 RICK M. SHERRARD, Ph.D. TENNESSEE VALLEY AUTHORITY Ecotoxicology Overview

2. TOPICS Ecotoxicology Studies conducted by TVA independent laboratories Brief summary results of studies to date Studies conducted by other laboratories Ongoing and future studies planned Laboratory challenges in working with ash Gaps in knowledge

3. What is Ecotoxicology? “In the broadest sense of the word, an ecotoxicologist is one who carries out toxicity testing on any component of any ecosystem.” Cairns, 1989 Ecotoxicity studies are designed to employ ecological attributes to assess toxicity Goal of the assessment – to protect entire ecosystems, not isolated components

4. TVA-Sponsored Studies Conducted by Independent Laboratories Phase I: April through June 2009  Vibracore®  Dredge Plume  Plant Stilling Pond Effluent (Outfall 001) Phase II: August 2009 to present  Dredge Plume  Plant Stilling Pond Effluent (Outfall 001) Focus:  Assess the potential for short-term effects associated with removing ash from the Emory River

5. Phase I Sampling and Analyses IV. Toxicological Monitoring A. Whole Sediment Toxicity Evaluation B. Elutriate Toxicity Evaluation C. Plume Toxicity Evaluation D. Polymer Toxicity Evaluation (Stilling Pond Effluent)

6. Samples  Vibracore ® upstream and downstream  Vb.1, Vb.2 – 3/17/09  Vb.3, Vb.4 – 6/11-12/09  Cores composited, homogenized  Stored in 19-L plastic buckets at 4ºC  Holding time = 8 weeks  Re-homogenized prior to use  Lab control CRM 189.0  Unaffected Emory River water controls, overlay, diluent

7. Hyalella azteca Survival and Growth Test for Sediments  Duration: 10 d  Temperature: 23 ± 1ºC  Photoperiod: 16 / 8  Sed. Volume: 100 mL  Water Volume: 175 mL  Renewal: 2 vol. add./d  Age: 7 – 14-d  No./Chamber: 10  No. Replicates: 8  Feeding: YCT 1 mL/d  Endpoints: Survival, Growth  Acceptance: ≥ 80%

8. Freshwater Juvenile Mussel Survival Test for Sediments (Lampsilis siliquoidea, L. cardium)  Duration: 5 d & 10 d  Temperature: 24 ± 1ºC  Photoperiod: 24 dark  Sed. Volume: 20 mL  Water Volume: 200 mL  Renewal: 5d-0 10d-1  Age: < 8 d  No./Chamber: 10  No. Replicates: 5  Feeding: every 3 d  Endpoint: Survival  Acceptance: ≥ 90%

9. Lumbriculus variegatus Survival Test for Sediments (qualifying test for 28-d bioaccumulation test)  Duration: 4 d  Temperature: 23 ± 1ºC  Photoperiod: 16 / 8  Sed. Volume: 100 mL  Water Volume: 175 mL  Renewal: 2 vol. add./d  Age: Adults  No./Chamber: 10  No. Replicates: 4  Feeding: None  Endpoint: Burrowing, Survival  Acceptance: ≥ 90%

10. Corbicula fluminea Bioaccumulation Test for Sediments  Duration: 28 d  Temperature: 20 ± 1ºC  Photoperiod: 16 / 8  Sed. Volume: > 5 L  Water Volume: 15 L  Renewal: 50% 3/wk  Size: 0.5-1.5 g WTW  No./Chamber: ~ 30  No. Replicates: 5  Feeding: None  Endpoint: Bioaccum.  Acceptance: Mass

11. Ceriodaphnia dubia Survival Test for Elutriates  Duration: 96 h  Temperature: 25 ± 1ºC  Photoperiod: 16 / 8  Water Vol.: 15 mL min  Renewal: None  Age: < 24 h  No./Chamber: 5  No. Replicates: 5  Feeding: Prior / 48 h  Concentrations: Non- Centrifuged 5 (0-100%); Centrifuged 1 (100%)  Controls: ERW, MHSW  Endpoint: Survival  Acceptance: ≥ 90%

12. Pimephales promelas Survival Test for Elutriates  Duration: 96 h  Temperature: 25 ± 1ºC  Photoperiod: 16 / 8  Water Vol.: 200 mL min  Renewal: None  Age: < 24 h  No./Chamber: 10  No. Replicates: 5  Feeding: Prior / 48 h  Concentrations: Non- Centrifuged 5 (0-100%); Centrifuged 1 (100%)  Controls: ERW, MHSW  Endpoint: Survival  Acceptance: ≥ 90%

13. Freshwater Juvenile Mussel Survival Test for Elutriates (Lampsilis siliquoidea, L. cardium)  Duration: 10 d  Temperature: 24 ± 1ºC  Photoperiod: 24 dark  Sed. Volume: 20 mL clean  Water Volume: 200 mL  Renewal: 1 (day 6)  Age: < 8 d  No./Chamber: 10  No. Replicates: 5  Feeding: every 3 d  Concentrations: Non- Centrifuged 5 (0-100%); Centrifuged 1 (100%)  Controls: ERW, MHSW  Endpoint: Survival  Acceptance: ≥ 90%

14. Ceriodaphnia dubia Survival and Reproduction Test for Emory River Plume and Outfall 001  Duration: 3 broods  Temperature: 25 ± 1ºC  Photoperiod: 16 / 8  Water Vol.: 15 mL min  Renewal: Daily  Age: < 24 h  No./Chamber: 1  No. Replicates: 10  Feeding: Once daily  Concentrations: 5 (0-100%)  Controls: ERW, MHSW  Endpoints: Survival, Reproduction  Acceptance: ≥ 80% survival, ≥15 neos/survivor

15. Pimephales promelas Survival and Growth Test for Emory River Plume and Outfall 001  Duration: 7 d  Temperature: 25 ± 1ºC  Photoperiod: 16 / 8  Water Vol.: 250 mL min  Renewal: Daily  Age: < 24 h  No./Chamber: 10  No. Replicates: 4  Feeding: Twice daily  Concentrations: 5 (0-100%)  Controls: ERW, MHSW  Endpoints: Survival, Growth  Acceptance: ≥ 80% survival, 0.25 mg growth

16. Phase II Sampling and Analyses  Weekly (Aug – Sept)  Biweekly (Oct – present)  Plume: grab samples  Outfall 001: 24-h composites  Unaffected Emory River water controls, diluent

17. Ceriodaphnia dubia Survival Test for Emory River Plume and Outfall 001  Duration: 96 h  Temperature: 25 ± 1ºC  Photoperiod: 16 / 8  Water Vol.: 15 mL min  Renewal: At 48 h  Age: < 24 h  No./Chamber: 5  No. Replicates: 5  Feeding: Prior / 48 h  Concentrations: 5 (0-100%Controls: ERW, MHSW  Endpoint: Survival  Acceptance: ≥ 90%

18. Pimephales promelas Survival Test for Emory River Plume and Outfall 001  Duration: 96 h  Temperature: 25 ± 1ºC  Photoperiod: 16 / 8  Water Vol.: 200 mL min  Renewal: At 48 h  Age: < 24 h  No./Chamber: 10  No. Replicates: 5  Feeding: Prior / 48 h  Concentrations: 5 (0-100%)  Controls: ERW, MHSW  Endpoint: Survival  Acceptance: ≥ 90%

19. Vb.1, Vb.2 Ash Composites Vb.3, Vb.4 Ash Composites Whole ash  H. azteca: adverse effects  L. variegatus: can’t burrow  L. siliquoidea: effects limited to 1 of 4 tests  C. fluminea: no bioaccumulation Elutriates: no effects Whole ash  H. azteca: adverse effects  L. variegatus: can’t burrow  L. cardium: effects in one 5-d test and one 10-d test  C. fluminea: no bioaccumulation Elutriates: mixed bag TVA Phase I Summary Results: Ash Samples

20. TVA Phase I & II Summary Results: Plume and Outfall 001 Samples No adverse effects in tests with exposures to plume samples Only one Outfall 001 sample to date resulted in decreased survival to C. dubia (but not P. promelas) Pathogen interference is being dealt with appropriately through parallel testing of UV-treated and untreated test solutions

21. Other Ecotoxicological Studies  USACE – ERDC  10-d larval fish elutriate bioassay  10-d juvenile fish elutriate bioassay  Bioaccumulation & health indicators  USGS, USFWS  Whole sediment and elutriate studies with benthic invertebrates  H. azteca, Villosa iris, Lampsilis fasciola, Chironomus dilutus  10-d, 28-d

22. Studies Planned by TVA  Independent laboratory bioavailability study with H. azteca and C. dubia  Whole ash, porewater only  With and without resin treatment  ORNL early life stages effects study with fish  Task 1: Fish embryo-larval toxicity tests of fly ash  Task 2: Longer-term exposures to fly ash in the laboratory  Task 3: Evaluating early life stage success in fly ash exposed fish populations

23. Challenges: Ecotoxicological Studies of Kingston Fly Ash Site  Site location  Water characteristics of converging rivers  Native sediment characteristics  Behavior of ash in storage and test chambers  Pathogen interference in Emory River water

24. Reference Control Sediment  Currently using clean sediment from Clinch River Mile 189.0 for comparisons  To date, TVA has:  Attempted to formulate sediment  Considered ash washing  Incorporated ion-exchange resin treatment

25. Homogenizing Whole Ash Samples  In storage, ash settles and compacts, porewater surfaces  Water poured off  Stainless steel beater with teeth on bottom chips away at ash  Water content decreases toward bottom of container  Entire contents blended into pourable slurry

26. Pathogen Interference in P. promelas Exposures with Emory River Water  Well-documented in Kingston Fossil Plant NPDES WET monitoring history  Interrupted concentration- response  High variability in mortality between replicates  Greater effect on Emory River controls and lower concentrations

27. Ultraviolet Treatment of Test Solutions Prepared with Emory River Water  All P. promelas tests with Emory River water  Parallel exposures (with and without UV treatment)  Dilutions prepared first, split, then treated individually  Treatment durations are 2, 3.5, or 5 min based on turbidity  Interrupted concentration- response and high variation between replicates - invalidated

28. Pathogen Interference in L. cardium Exposures with Vb.3 and Vb. 4 Laboratory Reported:  Organism stress as early as 48 h, including MHSW controls  At 96 h, large masses of “debris” (fungal filaments with colonies of protozoa)  Test organisms entangled in debris  Sporadic mortality among replicates in most test treatments  Source could be test organism supplier or Emory River water

29. Suggestions  Avoid “tunnel vision” – other constituents can cause effects  Better understanding of fate characteristics of ash constituents  Ash is not a natural sediment but models that we use to predict toxicity and assess risks are based on studies with natural sediments  Remember to use a weight of evidence approach  Conduct studies in a scientifically-defensible manner