Stable Isotope Analysis of Aquatic Macroinvertebrates, Stream Water, and Algae in Nine Mile Run, Pittsburgh, PA Kassia Groszewski with Arielle Woods Troy Ferland Zachary Tieman March 25, 2015
Brief Introduction to Nitrogen & Stable Isotopes Isotopes are forms of an element with the same number of protons but different number of neutrons Stable isotopes are isotopes that do not undergo radioactive decay Isotopes of an element behave very similarly chemically, but have a small mass difference This small mass difference causes isotopes to fractionate processes that affect the relative abundance of isotopes in a liquid or gas
Brief Introduction to Nitrogen & Stable Isotopes Our lab studies nitrogen in the urban environment Nitrogen is naturally occurring, but human activities contribute large amounts of N to the environment through fossil fuel use, fertilizer application, and in sewage and waste Excess nitrogen can cause a cascade of negative effects Image: Dr. Emily Elliott http://www.pitt.edu/~eelliott/research.html
Brief Introduction to Nitrogen & Stable Isotopes We use stable isotopes to differentiate nitrate (NO 3- ) sources. NO 3- is just one form of nitrogen in the environment Nitrogen: 14 N and 15 N Oxygen: 16 O, 17 O, and 18 O Different NO 3- sources fractionate isotopes differently. Can result in unique 15 N/ 14 N and 18 O/ 16 O ratios among NO 3- sources, allowing for source apportionment The isotopic ratio is represented as a delta value : δ (in ) = x 1000 (R) sample (R) standard (R) standard
Introduction to Nine Mile Run Urban stream located in Frick Park, Pittsburgh, PA Watershed drains ~19.4 km 2 area with 38% impervious cover Underlain by two different sewer systems 36% Combined Sewer 52% Municipal Separate Sanitary Sewer 12% Frick Park, sewer mains only 9.8 km 2 of the upper watershed buried underground in the early 1900 s
Introduction to Nine Mile Run Daylighted into a concrete culvert 3.5 km upstream of its confluence with the Monongahela River Severe historical degradation: Urban Stream Syndrome Flashy water level rises and falls quickly during storms Increased runoff due to impervious surfaces concrete, roofs, roads, parking lots, etc prevent water from moving through soil Reduced water quality Loss of biota few macroinvertebrates, no fish observed from 1941-1999 Major restoration project conducted 2002-2006 by the US Army Corps of Engineers Chris Condello, 2013 University of Pittsburgh News Services
Research Expectations and Hypotheses Previous research using stream nitrogen concentration and isotope data has shown that sewage-influenced water is ubiquitous in Nine Mile Run during both normal stream flow and stormflow (Divers et al. 2013, 2014) Sewage contains high concentrations of N and has a distinct isotopic signal 0 to 20 δ 15 N -15 to +15 δ 13 C We wanted to find another line of evidence to support our findings Macroinvertebrates are a tool often used in bioassessment studies Aquatic macroinvertebrates are animals that live in the stream: insects, crustaceans, mollusks, arachnids, and annelids Known relationship between stable isotopes in a consumer s tissues and their diet If diet contains an unique isotope signal, then it should be traceable Multidisciplinary approach using tools from the fields of geochemistry, environmental science, and ecology
Research Expectations & Hypotheses We hypothesized that the stable isotope (δ 15 N, δ 13 C) ratios of aquatic macroinvertebrates: collected closest to sewage inputs would display values more similar to a sewage source may be a useful tool for tracing sewage inputs and effects in Nine Mile Run NMR-3 Pipe
Macroinvertebrate Sampling Based on EPA and PA DEP Rapid Bioassessment protocols D-Nets used in streams to collect macroinvertebrates; We scuffled and kicked up sediment upstream so they would detach and go into the net This was supposed to be done in a 1 m 2 area for quantitative analysis Ended up qualitatively sampling various areas of the stream and using the d-net where we could
Macroinvertebrate Sampling Also did qualitative picks of leaf packs and stream bed substrate All grabs placed into a plastic tray and hand sorted using forceps Picks likely heavily biased towards larger macroinvertebrates due to inexperience Estimated that ~10 to 15% of macroinvertebrates in the stream were actually sampled Likely missed large numbers of macroinvertebrates as well as unique taxa
Results Macroinvertebrates Chironomidae NMR-1 Order Family Count Tolerance (phylum-class) Mollusca-Gastropoda Physidae 1 8 (phylum) Plathyhelminthes (Genus) Planaria 33 9 Diptera Chironomidae (red) 15 6 Diptera Chironomidae (non-red) 18 8 Diptera Empididae 1 6 Plecoptera Unknown 2 n/a NMR-2 Order Family Count Tolerance (phylum) Plathyhelminthes (Genus) Planaria 1 9 Diptera Simuliidae 6 6 Diptera Empididae 14 6 Diptera Chironomidae (non-red) 34 6 Diptera Chironomidae (red) 24 8 Ephemeroptera Baetidae 18 6 Trichoptera Hydropsychidae 3 5 Trichoptera Unknown 10 n/a NMR-3 Order Family Count Tolerance (phylum) Plathyhelminthes (Genus) Planaria 24 9 Diptera Chironomidae (non-red) 14 8 Diptera Chironomidae (red) 35 6 Diptera Simuliidae 5 6 Ephemeroptera Baetidae 13 6 Trichoptera Hydropsychidae 1 5 Baetidae http://aquaticinsectsofcentralvirginia.blogspot.com/ Hydropsychidae
Results - Macroinvertebrate Analysis - Metrics Metrics NMR-1 NMR-2 NMR-3 Total Collected Taxa 5 7 5 Total Organisms Collected 79 110 92 No. EPT Taxa 1 3 2 No. EPT Individuals 2 31 14 % EPT 2.53 28.18 15.22 Hilsenhoff Biotic Index 7.09 Poor 5.89 Fairly Poor 6.36 Fairly Poor % Tolerant Individuals (PVT 8-10) 65.82 22.73 41.30 One EPT taxon at NMR-1 The two individuals were very crushed/smeared; definitely of the same family but actual family ID impossible Plecopterans generally found in less-polluted environments, but some taxon are highly tolerant of many different conditions Hisenhoff Biotic Index and % Tolerant Individuals matches what we saw with the habitat quality well Expected that NMR-3 would be better than NMR-1 because it s more downstream but saw that the habitat at NMR-3 consisted of just algae covered cobbles. NMR-2 habitat quality was better; more substrate diversity; influx from Fern Hollow
Sources of Error During Macroinvertebrate Analysis Some organisms partially destroyed were able to be identified to the order level, but not family (e.g., Trichoptera but not family) An unknown number of Tubifex sludge worms (Order Oligochaeta, subfamily Tubificinae) misidentified as red chironomids Tubifex: pollution tolerance value 10 Likely misidentified at every site HBI scores likely higher in reality than what we report, but other metrics would have remained the same
Results Stream Water Nitrogen 3.00 2.50 2.00 Average Concentration (mg/l NH4 as N) mg/l as N 1.50 Average Concentration (mg/l NO2 as N) IC 1.00 Average Concentration (mg/l NO3 as N) IC 0.50 Total N (mg/l as N) 0.00 NMR 1 NMR 2 NMR 3 NMR 3 Tunnel NMR 3 drain
Results Stream Water Nitrogen
Tracing Sewage through the Food Chain Family level IDs too broad to be used for general trophic level or functional feeding groups However, we could break down our samples into predators, omnivores, and non-predators δ 15 N values observed in reference streams (Walker Branch, South Fork Eel River) much lower: 1 st order producers: -2 to +3 Top-level predators (trout): +8
Results Macroinvertebrate δ 13 C, δ 15 N -19-21 -23-25 Algae -27 δ 13 C ( ) -29-31 -33-35 -37-39 Plants NMR-1 Macroinvertebrates NMR-1 Algae NMR-1 Plants NMR-1 Bladder Snail NMR-2 Macroinvertebrates NMR-2 Algae NMR-2 Plants NMR-3 Macroinvertebrates NMR-3 Algae NMR-3 Plants Sewage Effluent (Northington & Hershey 2006) NMR-1 Terrestrial Caterpillar 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 δ 15 N ( )
Results Macroinvertebrate δ 13 C, δ 15 N -19-21 -23-25 -27 δ 13 C ( ) -29-31 -33-35 -37-39 NMR-1 Macroinvertebrates NMR-1 Algae NMR-1 Plants NMR-1 Bladder Snail NMR-2 Macroinvertebrates NMR-2 Algae NMR-2 Plants NMR-3 Macroinvertebrates NMR-3 Algae NMR-3 Plants Sewage Effluent (Northington & Hershey 2006) NMR-1 Terrestrial Caterpillar 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 δ 15 N ( )
Results Macroinvertebrate δ 13 C, δ 15 N -19-21 -23-25 δ 13 C ( ) -27-29 -31-33 -35-37 -39 Simuliidae, Predator NMR-1 Macroinvertebrates NMR-1 Algae NMR-1 Plants NMR-1 Bladder Snail NMR-2 Macroinvertebrates NMR-2 Algae NMR-2 Plants NMR-3 Macroinvertebrates NMR-3 Algae NMR-3 Plants Sewage Effluent (Northington & Hershey 2006) NMR-1 Terrestrial Caterpillar 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 δ 15 N ( )
Results Macroinvertebrate δ 13 C, δ 15 N -19-21 δ 13 C ( ) -23-25 -27-29 -31-33 -35-37 -39 Non-red Chironomids, likely predatory or omnivorous Simuliidae, Predator NMR-1 Macroinvertebrates NMR-1 Algae NMR-1 Plants NMR-1 Bladder Snail NMR-2 Macroinvertebrates NMR-2 Algae NMR-2 Plants NMR-3 Macroinvertebrates NMR-3 Algae NMR-3 Plants Sewage Effluent (Northington & Hershey 2006) NMR-1 Terrestrial Caterpillar 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 δ 15 N ( )
Results Macroinvertebrate δ 13 C, δ 15 N -19 δ 13 C ( ) -21-23 -25-27 -29-31 -33-35 -37-39 Non-red Chironomids, likely predatory or omnivorous Trophic enrichment δ 15 N of a consumer typically 2 to 3 higher than its source of N Simuliidae, Predator NMR-1 Macroinvertebrates NMR-1 Algae NMR-1 Plants NMR-1 Bladder Snail NMR-2 Macroinvertebrates NMR-2 Algae NMR-2 Plants NMR-3 Macroinvertebrates NMR-3 Algae NMR-3 Plants Sewage Effluent (Northington & Hershey 2006) NMR-1 Terrestrial Caterpillar 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 δ 15 N ( )
Results Macroinvertebrate δ 13 C, δ 15 N -19 δ 13 C ( ) -21-23 -25-27 -29-31 -33-35 -37-39 Non-Predators Non-red Chironomids, likely predatory or omnivorous Simuliidae, Predator NMR-1 Macroinvertebrates NMR-1 Algae NMR-1 Plants NMR-1 Bladder Snail NMR-2 Macroinvertebrates NMR-2 Algae NMR-2 Plants NMR-3 Macroinvertebrates NMR-3 Algae NMR-3 Plants Sewage Effluent (Northington & Hershey 2006) NMR-1 Terrestrial Caterpillar 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 δ 15 N ( )
δ 15 N ( )
δ 15 N ( )
δ 15 N ( )
δ 15 N ( )
δ 15 N ( )
δ 15 N ( ) Sewage Range
δ 15 N ( ) Top Predator: Rainbow trout 1 st Order (-2 to +3 )
δ 15 N ( ) Algae
25 23 d15n 21 19 17 15 13 11 9 Algae Water Deviation (Algae - Water) 7 5 NMR 1 NMR 2 NMR 3
Conclusions + Implications Stable isotope analysis of macroinvertebrates never before done in Nine Mile Run Results indicate that macroinvertebrates may be a useful tool in determining the extent of sewage-derived nitrogen Sewage derived nitrogen observed in all of the stream water samples, macroinvertebrates, and algae. Clear evidence that macroinvertebrates are incorporating sewagederived nitrogen into their diets Nine Mile Run stream water, algae, and macroinvertebrate stable isotope values resemble those of sewage and WWTP effluent Stream restoration efforts haven t focused on reducing the impact of sewage, and the macroinvertebrate communities and inorganic nitrogen data reflect this
Future Work Evaluate sewage incorporation throughout the ecological food web: More detailed sampling of food web base: Fine and coarse particulate organic matter Periphyton and bacteria Expert analysis of organisms needed to make conclusions on functional feeding groups, trophic levels, food webs Use macroinvertebrates as a tool to identify sewage seeps Only collect/analyze non-predators direct incorporation of sewage-derived nitrogen, rather than trophic enrichment
Acknowledgements Arielle Woods Troy Ferland Zach Tieman Dr. Emily Elliott Katie Redling Geol G2525 Stable Isotopes Class