STABLE ISOTOPIC AND FATTY ACID EVIDENCE FOR UPTAKE OF FISH FARMING INDUCED ORGANIC POLLUTANTS BY FILTER-FEEDING MUSSELS (PERNA VIRIDIS) IN A POLYCULTURE SYSTEM Kevin Q F Gao1, S G Cheung1, G H Lin2,3; S P Chen2; Paul K S Shin1 1Department of Biology and Chemistry, City University of Hong Kong. 2Laboratory of Quantitative Vegetation Ecology, Institute of Botany, the Chinese Academy of Sciences. 3Department of Global Ecology, Carnegie Institution of Washington, USA. 1. Good afternoon. My talk is about the study on the consumption of fish farming wastes by bivalve mussels using stable isotopes and fatty acids as trophic tracers.

Introduction: Fish farming is an important economic activity world-wide. 2. Fish farming is an important economic activity world-wide, especially facing to the problem of overfishing by capture fisheries.

Introduction: Fish farming leads to pollution due to uneaten feed, faeces and excretion. 3. (click) From environmental viewpoint, fish farming is a polluter because fish farming leads to nutrient enrichment even eutrophication due to the release of wastes in forms of uneaten fish feed, fish faeces and excreta.

Introduction: Bivalve mussels can filter particulate matter in high efficiency. 4. Bivalves, such as mussels, can filter particulate matter from surrounding waters in high efficiency due to their high population density and (Click) filtration rate.

Introduction: Polyculture, combining fish and mussels, can achieve an environmental and economic Win-Win solution. 5. Mussels have high nutritional value, so polyculture combining fish and mussel can get environmental and economic WinWin solution.

Introduction: Trophic markers Stable Isotopes: Changes in stable isotope ratios (e.g., 13C/12C and 15N/14N) are predictable when matter is transported along trophic levels; they can thus be used as markers to trace matter flow: food or pollutant sources and fates. 6. Changes in stable isotope ratios (e.g., 13C/12C and 15N/14N) are predictable when matter is transported along trophic level, they can thus be used as markers to trace matter flow: food or pollutant sources and fates.

Introduction: Trophic markers Fatty Acid Profile Sink-specific and conservative in transportation and transformation Use of Multiple Markers Improve the accuracy of determining source and sink 7. Fatty acid profiles from different biogeochemical sinks are different and they are transported and transformed in conservative patterns during biogeochemical processes, so they can be used as trophic markers. Use of multiple marker can improve the tracing accuracy.

Objectives 1 To quantify the contribution of potential food sources to the mussel ration. 2 To evaluate the feasibility and capability of filter-feeding bivalves as biofilters for organic wastes from fish farming activities in a polyculture system. 8. Objectives of our study are: To quantity the contribution of potential food sources to the mussel ration; (Click) To evaluate the feasibility and capability of filter-feeding bivalves as biofilters for organic waste from fish farming activities in a polyculture system.

Experimental site: Kau Sai marine fish culture zone - a semi-enclosed bay 9. The experimental site Kau Sai fish culture zone is a semi-closed bay in eastern Hong Kong.

Methods 10. Methods: First, mussels from same population were transplanted to fish raft and reference site. The reference site is 1km away from fish farms, avoiding effects from fish farms; 1 Mussels transplanted to fish rafts and a reference site (without effects from fish farms).

Methods 2. Sampling from fish raft and reference site: mussels, particulate matter, fish feed and fish faeces after 3 months in the field. 3. Sample analysis: 2 aliquots for all samples Aliquot 1 for stable isotope ratios (13C/12C and 15N/14N) with EA-IRMS; Aliquot 2 for fatty acid profiles with GC-FID 11. After 3-month adaptation, mussels, particulate matter, fish feed and fish faeces were sampled from fish raft and reference site; (Click) Each sample was divided into two aliquots, one for stable isotope measurement with a Finnigan Isotopic Ratio Mass Spectrometer interfaced with Elemental Analyzer, another for fatty acid determination of fatty acids with a Gas `Chro`matograph . (Click) Isotope values of mussel tissue between fish raft and reference site were compared with Student’s t-test; The contributions of food sources to mussel ration were evaluated with isotope mixing model; PCA ordination method was used to compare the fatty acid profiles. 4. Data analysis: Comparison: Mussel tissue between fish raft and reference site with t-test. Isotope mixing model: Contribution of food sources to mussel tissue. Principle Component Analysis (PCA) ordination: For fatty acid profiles.

Results: Stable Isotopes δ13C=[(13C/12C)smpl/ (13C/12C)std-1]X1000‰ δ15N=[(15N/14N)smpl/ (15N/14N)std-1]X1000‰ 12. This slide shows the comparisons in the Delta C13 and Delta N15 values of mussel tissue between fish raft and reference site. For both C13 and N15, the Delta values in fish raft site (blue bars) are lager than reference site (purple bars), indicating the effects of isotopically heavy fish feed and fish faeces on mussel tissue in fish raft site. This will also be shown in next slide. Comparison of stable isotope values in mussel tissue between fish raft and reference site

Results: Stable Isotopes Contributions of food sources to mussel ration: Based on isotope mixing model, POM: 68.3%; Fish feed: 27.5%; Fish faeces: 4.2%, 13. This dual isotope plot showed the isotope values of the mussel tissue and potential food sources, including particulate matter, fish feed and fish faeces. Along X-axis, Delta C13 of mussel tissue is between those of POM, fish feed and fish faeces; along Y-axis, Delta N15 of mussel tissue is also between those of POM, fish feed and fish faeces. This indicates that all these three sources can be consumed by mussels. Based on isotope mixing model, the contributions of POM, fish feed and fish faeces to mussel food are 68.3, 27.5 and 4.2%, respectively. Dual isotope plot showing the food source of mussels at fish raft site

Results: Overall Fatty Acid Profiles Fatty Acids MFR MRS PFR PRS FFD FFC Saturated Fatty Acids 11:0 0.00 0.01±0.00 12:0 0.01 0.17±0.02 14:0 7.55±0.51 5.96±0.45 3.41±0.35 3.06±0.22 5.27±0.88 1.17±0.68 15:0 0.90±0.22 0.71±0.03 0.24±0.02 0.42±0.09 0.72±0.06 0.23±0.01 16:0 30.31±3.82 35.04±3.22 29.41±2.33 24.34±2.15 32.70±1.66 8.80±1.11 18:0 10.59±0.78 7.53±0.46 2.48±0.30 4.26±0.48 9.16±1.10 7.72±0.35 17:0 1.73±0.55 2.36±0.55 0.46±0.03 0.90±0.08 1.01±0.05 0.41±0.02 20:0 0.97±0.08 0.55±0.06 0.49±0.07 0.83±0.07 0.75±0.06 21:0 0.13±0.01 0.76±0.05 0.02 1.24±0.16 22:0 0.24±0.01 subtotal 52.18±1.85 52.92±1.66 36.72±1.58 33.00±1.55 49.87±1.34 20.32±1.22 14~15. This table shows the overall fatty acid profile of mussel tissue and potential food sources. MFR-mussel of Fish Raft, MFS-Mussel of Ref. Site, PFR-POM of Fish Raft PFS-POM of Ref. Site, FFD- Fish Feed, FFC-Fish faeces (meanSD, N=3-9)

Results: Overall Fatty Acid Profiles Monounsaturated Fatty Acids 15:1n5 0.00 0.84±0.03 1.79±0.01 0.04 17:1n7 0.21±0.01 0.68±0.02 3.16±0.16 7.25±0.22 0.60±0.03 0.64±0.03 16:1n7 10.65±0.68 8.47±0.56 17.76±1.23 17.73±1.33 8.28±1.02 8.67±0.89 18:1n9 4.66±0.16 2.27±0.09 2.38±0.09 2.11±0.11 12.75±1.35 24.86±1.88 18:1n7 3.85±0.11 3.52±0.15 7.67±0.22 9.78±0.36 3.89±0.11 3.55±0.15 20:1n9 2.35±0.08 3.21±0.18 0.16±0.02 22:1n9 0.45±0.05 0.61±0.03 0.39±0.05 subtotal 22.16±1.55 18.75±1.63 31.80±2.15 38.66±2.30 25.71±2.10 38.11±1.59 Polyunsaturated Fatty Acids 18:2n6 1.46±0.12 1.50±1.08 1.58±0.15 0.35±0.01 0.91±0.09 1.47±0.12 18:3n6 0.20±0.01 0.05 0.28±0.01 20:2n6 0.72±0.03 0.48±0.05 0.21±0.02 20:3n3 0.14±0.01 20:4n6 3.30±0.22 2.97±0.36 0.77±0.05 3.42±0.25 5.26±0.26 20:5n3 8.24±0.22 12.21±1.23 16.06±1.25 14.37±1.36 7.00±0.79 7.74±0.88 22:2n6 0.92±0.05 0.25±0.02 22:6n3 11.59±0.98 10.04±0.88 12.41±1.23 13.62±1.33 13.07±1.56 26.84±2.32 25.66±1.52 28.32±2.21 31.48±3.32 28.34±2.35 24.45±2.01 41.59±3.32

Results: Overall Fatty Acid Profiles Mussels at fish raft Mussels at reference site Fish feed, fish raft only POM at reference site POM at fish raft 16. The overall fatty acid profiles may be illustrated with this PCA ordination plot. For POM, the fatty acid profiles of fish raft and reference site were overlapped, showing the identical composition POM in these 2 sites; (Click) The group of fish faeces is far away from other groups, showing the minor contribution of fish faeces to the mussel food; (Click) The fatty acid profile of mussel tissue from the 2 site are classified into two groups and the fatty acid profile of mussel from fish raft approaches more to fish feed (Click), indicating the effect of fish feed on the fatty acid composition of mussels in fish raft. Fish feces, fish raft only PCA ordination plot showing the Overall fatty acid profiles of mussels and their food sources

Results: Single Fatty Acids Sources identical No effect Negative Effect Positive Effect 17. This graph shows the comparison in energetically important fatty acids between mussel tissue and its food sources. Generally, fatty acids are assimilated in proportion to concentration in food sources. Please notice the effect of fish feed (red bar) on the mussels in fish raft (blue bar). For 14:0, 18:0 and 18:1n9, the concentration of fish feed is higher than POM (pink bars and green bars), as a result, the concentrations of these 3 acids of mussels in fish raft are higher than the mussels at reference site (purple bars). (Click) For 20:5n3 (EPA), the concentration of fish feed is lower than POM, so the concentrations of EPA of mussels in fish raft are lower than the mussels at reference site. (Click) For 16:0 and 18:1n7, fish feed does not showed obvious effects on mussel tissue. (Click) For 22:6n3 (DHA), the concentrations all sources are identical, so the concentrations of this acid in mussels from fish raft and reference site did not differ obviously. Mussel, fish raft Fish feed, raft only POM, fish raft POM, ref. site Mussel, ref site Effects of fish feed fatty acids on mussel tissue

Discussion 1. The distinct stable isotopic and fatty acid signatures of different sources can be used as trophic markers to trace the transfer of organic matter along the food chains. 2. Combined evidence of stable isotopes and fatty acid profiles showed that filter-feeding mussels could directly consume the organic wastes derived from fish farming activities. 3. Eutrophic nitrogen and phosphorus could be fixed by mussel assimilation before the waste mineralization. The distinct stable isotopic and fatty acid signatures of different sources can be used as trophic markers to trace the transfer of organic matter along the food chains. (Click) Combined evidence of stable isotopes and fatty acid profiles showed that filter-feeding mussels could directly consume the organic wastes derived from fish farming activities. (Click) Eutrophic nitrogen and phosphorus could be fixed by mussel assimilation before the waste mineralization. (Click) The mussels in a polyculture system can function as biofilters to reduce the nutrient released from uneaten fish feed and egested fish faeces in fish farms. 4. The mussels in a polyculture system can function as biofilters to reduce the nutrient released from uneaten fish feed and egested fish faeces in fish farms.

Acknowledgements Agriculture, Fisheries and Conservation Department (AFCD) of Hong Kong government for funding; Group colleagues for sampling assistance; Technicians in Institute of Botany, the Chinese Academy of Sciences; Department technicians We would like to thank AFCD of Hong Kong government, group colleagues and laboratory technicians for support and assistance.

Thank You!