1. Survey of the Mercury Content of Earthworms on the South River Floodplain John Cianchetti and Dean Cocking, James Madison University, Harrisonburg, VA Bill Berti, DuPont Co., Central Research and Development, Newark, DE South River Science Team April 8, 2008

2. 2 Objectives of Study Conduct a survey of Hg concentrations in earthworms to understand the extent to which mercury bioaccumulates in earthworms. Evaluate the relationship between total mercury (THg) and methyl mercury (MeHg) concentrations in earthworm and paired soil samples.

3. 3 Floodplain Earthworm Sample Locations Grand Caverns Abbee Road Bridge Confluence Ridgeview Pk North Pk Crimora Hopeman Pkwy Robert Turk Rd Belvidere Rd Forestry Ctr Dooms Basic Pk CNF GGC BVD FOR CRM RTR DMS HPK BPK NPK ARB RVP Point source Waynesboro Staunton Control site

4. 4 Sample collection procedure A 10x10 square meter grid was established in each location and was subdivided into 1x1m quadrats 5 of the quadrats were randomly selected for sampling.

5. 5 Sample collection procedure  Excavated a surface layer of 50 cm at each quadrat to collect minimum of 30 individual earthworms.  Soil samples were collected from this surface layer at each quadrat.  The aliquots from each quadrat were homogenized within a plastic bag and placed in vials for total and methyl mercury analysis; five composite samples per location corresponding to each earthworm sample.  Residual soil from each quadrat was combined to create a composite sample at each location sample used for soil property characterization.

6. 6 Sample Processing: Earthworms The earthworms were transported to the laboratory in a cooler. Earthworm samples were sequentially rinsed with DI water in a series of six pyrex glass dishes. A sub sample of at least 12 earthworms from three quadrats from each location were processed as undepurated samples which were frozen immediately. A similar sized sample from each quadrat was depurated for 24 hours in a container with moist filter paper, and then frozen. All samples were shipped to Studio Geochemica for analysis

7. 7 Summary Earthworm and Soil Sampling Design at Each Location All Samples were shipped to Studio Geochemica for analysis

8. 8 Detection Limits With the exception of methyl mercury in earthworms at the control site, analyzed samples all had values well above minimum detection limit (MDL) and the practical quantitative limit (PQL) for the fresh weight samples. (Provided by Studio Geochemica)

9. 9 RVP ARB NPK BPK HPK DMS RTR CRM FOR BVD GGC CNF Downstream River Kilometer (0 = Waynesboro Point Source) Mean Total Mercury (THg) and Methyl Mercury (MeHg) in Soil of the South River, VA, Floodplain ± 1 SEM

10. 10 RVP ARB NPK BPK HPK DMS RTR CRM FOR BVD GGC CNF Downstream River Kilometer (0 = Waynesboro Point Source) Mean Mercury (THg & MeHg) in Depurated Earthworms Living on the South River, VA, Floodplain ± 1 SEM

11. 11 Downstream River Kilometer (0 = Waynesboro Point Source) RVP ARB NPK BPK HPK DMS RTR CRM FOR BVD GGC CNF A Comparison of THg and MeHg Concentrations in Earthworms Living on the South River, VA, Floodplain Wet Weight Basis (ww) ± 1 SEM

12. 12 Wet Weight Basis (ww) THg in Soil of the South River, VA, Floodplain RVP ARB NPK BPK HPK DMS RTR CRM FOR BVD GGC CNF Downstream River Kilometer (0 = Waynesboro Point Source)

13. 13 Wet Weight Basis (ww) ± 1 SEM Mean THg in Soil of the South River, VA, Floodplain Downstream River Kilometer (0 = Waynesboro Point Source) RVP ARB NPK BPK HPK DMS RTR CRM FOR BVD GGC CNF

14. 14 Mean THg in Depurated Earthworms Living on the South River, VA, Floodplain Downstream River Kilometer (0 = Waynesboro Point Source) RVP ARB NPK BPK HPK DMS RTR CRM FOR BVD GGC CNF Wet Weight Basis (ww) ± 1 SEM

15. 15 Mean MeHg in Soil of the South River, VA, Floodplain Downstream River Kilometer (0 = Waynesboro Point Source) RVP ARB NPK BPK HPK DMS RTR CRM FOR BVD GGC CNF Wet Weight Basis (ww) ± 1 SEM Outlier

16. 16 Mean MeHg in Depurated Earthworms Living on the South River, VA, Floodplain Downstream River Kilometer (0 = Waynesboro Point Source) RVP ARB NPK BPK HPK DMS RTR CRM FOR BVD GGC CNF Wet Weight Basis (ww) ± 1 SEM

17. 17 Mean THg in Soil vs Concentration in Depurated Earthworms Wet Weight Basis (ww)

18. 18 Wet Weight Basis (ww) Mean MeHg in Soil vs Concentration in Depurated Earthworms R 2 =.4201

19. 19 Downstream River Kilometer (0 = Waynesboro Point Source) RVP ARB NPK BPK HPK DMS RTR CRM FOR BVD GGC CNF Wet Weight Basis (ww) BAF = [earthworm] / [soil] Bioaccumulation Factor (BAF) for Total Mercury (THg) Using Depurated Earthworm Data

20. 20 Downstream River Kilometer (0 = Waynesboro Point Source) RVP ARB NPK BPK HPK DMS RTR CRM FOR BVD GGC CNF Wet Weight Basis (ww) Bioaccumulation Factor (BAF) for Methyl Mercury (MeHg) Using Depurated Earthworm Data BAF = [earthworm] / [soil]

21. 21 Downstream River Kilometer (0 = Waynesboro Point Source) RVP ARB NPK BPK HPK DMS RTR CRM FOR BVD GGC CNF A Comparison of THg and MeHg Concentrations in Earthworms Living on the South River, VA, Floodplain Wet Weight Basis (ww)

22. 22 Soil Characteristics

23. 23 How much Hg is tied up in earthworms on the floodplain? 5-yr floodplain - 2094 acres = 8.5 E+6 sq m Mean earthworm fresh wt = 20.6 g / sq m Estimated earthworm fresh wt 5-yr floodplain= 1.7 E+8 g Mean THg in undepurated earthworms = 2447 ng/g Mean MeHg in undepurated earthworms = 66 ng/g Total Hg in earthworms on 5-yr floodplain = 4.3 E+17 ng = 426 grams Total MeHg in earthworms on 5-yr floodplain = 1.2 E+16 ng = 11.6 grams For the large amount of area involved, this appears to be a very small proportion of the Hg present on the floodplain. SOURCES OF ERROR IN THIS ESTIMATE: Not all of the floodplain is suitable earthworm habitat (eg parking lots); un-weighted mean biomass and THg/MeHg may be over or under estimates of true values

24. 24 Summary Both forms of mercury are present in the soil and earthworms of the floodplain at an elevated level. The pattern of total mercury distribution is superficially similar to that previously observed in other organisms in the river. The methyl mercury concentrations do not follow a discernable pattern. Total mercury concentrations in earthworm tissues are proportional to the total mercury concentration of the soil, however this is not the case in the methyl mercury concentrations.

25. 25 Path Forward What is the food chain position of earthworms within these ecosystems? What is the relationship between these results and those from studying other organisms along the South River? How do these results compare with studies of the uptake of Hg by earthworms from other terrestrial sites including sewage sludge deposits and industrial wastes. It is of interest to pursue what causes the fluctuations in MeHg within the earthworm tissues in relation to soil MeHg content. While soil is likely a contributing factor, other variables may also play a role: microhabitat time of sampling, earthworm species composition (affects feeding habits) endogenous vs exogenous origin of MeHg etc. While it appears that the observed patterns of Hg content in earthworms are not unexpected, there are some questions of passing interest: