1. CERP: F2 Experiment Focused on exposing adult mummichogs to 4 levels of contaminated sediments Clean and contaminated food njwrri.rutgers.edu/graduate/

2. Mortality estimates Measure growth in length and weight Quantify egg production II. Egg performance experiment (EP) Measure egg size, hatching success, and size at hatch for larvae Objectives I. F2 experiment: ehpnet1.niehs.nih.gov/docs/ 1996/104-9/innov.html

3. Sediment treatment Elizabeth River mud diluted to 6.25 % (ERL), 12.5 % (ERM), and 25 % (ERH) Fishing Bay (FB) mud as control Same as F1 experiment Clean/contaminated food treatment Contaminated clams: Live clams were placed out in the ER river on 2 occasions for 6-7 weeks (T) Clean clams: live clams were bought from a local seafood market (Chester and Ware Rivers) (C) Clams were mixed with fish gel and water to make clam jello Example ERMT =Elizabeth River 12.5 % (medium) dilution and toxic food treatment F2 Experiment

4. Characteristics of clean & contaminated food PAH Accumulation in Mercenaria (note scale change) Control Elizabeth River

5. F2 Experiment 24 50-gallon flow-through tanks fitted with manifolds that released incoming flow just above the sediment surface to mix water column 3 replicates per treatment Extra aeration was added to each tank 56 fish were added to each tank ranging from 40-73 mm (~F1) Fish were fed 0.03 g/g/day of their body weight for the first 30 days then switched to 0.06 g/g/day after day 30 (same as low food from F1)

6. 4 sampling days: Days 0, 30, 59, and 74 Experiment ran from 6/11/03- 8/22/03 Sub-samples of fish removed for biomarker expression, chemical analysis, bioenergetic partitioning, and growth Eggs collected from each tank Egg production (daily), hatchability (weekly), and egg/larvae size estimates (weekly) F2 Experiment

7. Fish were fed twice a day (by hand) Salinity, temperature, DO measurements taken daily Egg collectors and flow rates checked daily Tanks were checked daily for dead/dying fish F2 Experiment

8. Environmental Parameters Salinity: 7.5 –11.1 ppt 2002: 11.3-20.9 ppt Temperature: 20 – 24 o C 2002 18.3-22.4 o C DO: - 5.1-8.4 mg L -1 - No problems with hypoxia or anoxic mud this time! Flow rate: 300 mL min –1 - same as higher flow in 2002 www.elizabethriver.org/monitoring/ mummichogfacts.htm F2 Experiment

9. B B B B A A A A P<0.0001 A B A B C C B B A A AB AB P<0.0001 Day 74 Day 59 Adult Fundulus mortality by treatment Sediment Food Sediment

10. Mortality comparisons from the last day of sampling for F1 and F2 experiments

11. Disease prevalence: fish with red fins and nape Problems with seawater system may have contributed– times when there was no water flow for half a day, several days in a row Algal bloom? Food differences? Pellet food vs clam jello No energy effect Brood effects? Other expts using fish from same collections experienced higher than expected mortalities Other expts using different fish experienced high morts as well Differences between F1 and F2 experiment:

12. Adult Fundulus mean length by treatment

13. Adult Fundulus mean absolute growth rate in length (+/- SD) for day 59 and 74 No sediment or food effect on growth

14. Mean growth on the last day of sampling for F1 and F2 experiment Slightly slower growth in 2003 than 2002

15. Mean egg production by week throughout the F2 experiment

16. Mean egg production for F1 and F2 experiments

17. Total egg production (+/- SD) from F2 experiment B A B A Food effect: p=0.0096 Sediment effect: p=0.0527

18. Total egg production for F1 and F2 experiments

19. Eggs were collected daily from collectors in each tank. Eggs were subsequently grouped by week # of the F2 experiment Eggs incubated for 14 days in standard culture media, exposed to air for 2 hours and then placed into highly aerated seawater for 30 minutes This hatching process occurred for 3 days, eggs unhatched on the 3 rd day of the hatching process were termed unhatched or dead www.miljolare.no/virtue/img/Species/ pages/Fish%20eggs.php Egg Performance (EP) experiment:

20. EP experiment: Mean area (+/- SD) of eggs by week collected from F2 experiment

21. EP experiment: Mean length (+/- SD) of larvae at hatch by week

22. EP experiment: Mean % hatching success (+/- SD) of eggs by week collected from F2 experiment

23. Egg and larval traits from EP experiments F2 showed similar results with F1 that there appears to be no difference among sediment or food treatments: Larval length Egg area Hatching success

24. Conclusions: Growth appeared to be affected by the lower food level at the start of the expt and the presence of disease Egg production Higher and longer time of egg production than F1 Egg production was significantly higher in the toxic tanks Dose response curve with higher total egg production in the FB tanks (marginally significant) There were no apparent differences among sediment or food treatments with larval and egg size or traits www.whoi.edu/fishtox/ bonyfish.html