daniel seburn advisor: dr. mark hanson envr 4500 – project proposal

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Characterizing the toxicity of surface water salinity contamination to emergent macrophytes using a laboratory cattail ( Typha spp. ) seedling germination and growth assay. Daniel Seburn Advisor: Dr. Mark Hanson ENVR 4500 – Project Proposal Course Co-ordinator: Dr. Rick Baydack. - PowerPoint PPT Presentation


Characterizing the toxicity of common surface contaminants to wetland plants

Daniel SeburnAdvisor: Dr. Mark HansonENVR 4500 Project ProposalCourse Co-ordinator: Dr. Rick Baydack

Characterizing the toxicity of surface water salinity contamination to emergent macrophytes using a laboratory cattail (Typha spp.) seedling germination and growth assayDevelop laboratory Typha spp. germination and root/shoot elongation assay to generate useful experimental data regarding salinity impairment of ecologically important emergent macrophytesPerform experimental outdoor in-situ mesocosm assays as a supplement to laboratory dataReview local environmental monitoring data to make recommendations for further investigationsPurpose of Research

Typha spp. (cattail) Characterize salinity toxicity

22Aquatic Macrophytes important for:

Background SummarySETAC Global Plants Advisory Group recently identified the need to develop standardized methods for aquatic macrophyte assays, other than Lemna spp. assays (Arts et al., 2010) Use Typha seedling bioassays to characterize salinity impairment to rooted, emergent macrophytes

Nutrient removal (cleaning up Lake Winnipeg algae bowl)Provide food/habitat for variety of faunaUsed for phytoremediation of organic and inorganic pollutants in treated wetlands3Salinity Hazards to WetlandsExcess salinization to wetlands typically occurs as a result of:Saline sewage effluents Road salt de-icing compounds in runoff waterPoor hydraulic engineering in coastal lagoon areas

Raney and Eimers (2014) recently analyzed trends in water quality data in southern Ontario over the past 30+ years They found that 58 of the 64 sites had significantly increasing chloride concentrations, with the highest concentrations always occurring during spring melt near urban centers (coinciding with road salt applications)

Similar trends found in surface waters in northeast USA (Kaushal et al. 2005)

In response to the environmental hazards posed by road salts, a code of practice for the proper management of road salts was developed under CEPA in April 2004Goal to improve the reporting and management of road salt usage among municipalities, provincial governments, and other frequent applicators (Environment Canada, 2012)4Materials and Methods Typha assay Seed Preparation MethodBased on methods from McNaughton (1968) and recent studiesFemale flowering spikes collected from Oak Hammock Marsh, Manitoba in Fall 20131 litre Technicon Commercial Blender700ml RO water 40g Fisher Brand Sparkleen powder soap5 ml Sodium Hypochlorite (bleach) Added in order to separate seeds from debris and remove any residual chemicals left on seedsBlended for 30 seconds, then mixture added to dilution vessel

5Seed Preparation Flow Diagram

6Background Germination and Growth15 individual seeds added to split petri dishes, 4 reps/treatmentUsed seeds from Oak Hammock Marsh as well as imported seeds from plant-world-seeds.com (UK)Seeds left in Conviron growth chamber for seven or fourteen days under varying temperatures and photoperiodsGermination success counted and root/shoot growth measured with Infinity1 microscope and image analysis software

7Measuring Germination and Growth

Infinity Analyze Software Calibration and Measuring8Background Germination ResultsSimilar germination success between seeds we collected (MB) and ordered seeds (UK)

Significantly less germination success without light, lower temp

Germination success (52 to 71%) comparable to other studies assessing Typha germination:

60 to 90% (Burgeois et al., 2012)80 to 90% (Moore and Lock, 2012) 79 to 93% (Muller et al., 2000)A = No light exposure @ 25C for 7 days B = 12 hours light/day @ 20C for 7 days C = 12 hours light/day @ 25C for 7 days

D = 18 hours light/day at 25C for 7 daysE = 18 hours light/day at 25C for 14 days9Preparing Exposure Solutions

UK sourced Typha latifolia seeds exposed to:Three different laboratory grade saltsNaCl, CaCl2 * 2H2O, and KCl Three different commonly used road salt compoundsNo Name Road Salt (RS#1), Sifto Safe Step (RS#2), and Meltz All (RS#3)Greatest concentration 32g/L, subsequent solutions prepared by serial dilution10Conductivity of Exposure SolutionsTarget ConcentrationNaClCaCl2KClRS#1RS#2RS#31 g/L1.1051.1451.1341.1450.5841.6532 g/L2.0852.2052.1012.0121.3042.8314 g/L3.9854.0363.9963.7812.1744.7538 g/L7.9257.5457.6897.1544.0028.80116 g/L15.83413.64114.60913.0847.68515.37132 g/L26.62525.13426.01325.86314.86524.194Table 1: Measured conductivity (mS/cm) of each stock solution used in experiment. Control stock solution had a measured conductivity value of 0.014 mS/cm. 11Environmental Chamber Conditions

Seeds left in Conviron growth chamber for seven days at 25C and 18 hour light to six hour dark photoperiodPhotosynthetically active radiation (PAR) levels were measured at five random locations in the growth chamber with an Apogee Quantum Flux Photometer (Model MQ-200)243 to 285 mol m-2 s-1Study Design:Six different compounds Six concentrations of each compound (plus controls)Five repetitions per concentrationTwenty seeds per repetition12Salinity Impairment Germination

Figure 2: Average germination success for Typha seeds exposed to increasing concentrations of laboratory grade salts and common road salt compounds. 13Salinity Impairment Shoot GrowthFigure 3: Average shoot length for Typha seeds exposed to increasing concentrations of laboratory grade salts and common road salt compounds.

14Salinity Impairment Root GrowthFigure 4: Average root length for Typha seeds exposed to increasing concentrations of laboratory grade salts and common road salt compounds.

15Comparing Compounds and Endpoints


CaCl 1 g/L

NaCl 4 g/L

RS#1 8 g/L

RS#2 8 g/LRS#3 1 g/L

16Calculating Dose-Response Curves Four parameter log-logistic concentration-response curves were generated for each compound and each endpoint using the Rstudio statistical software program (R Core Team, 2011) and our experimental data Ten and fifty percent inhibition concentrations (EC10 and EC50, respectively) with standard error and 95% confidence intervals were subsequently calculated from the concentration-response curves

Dose-Response Curve generated for RS#2 Shoot Length Inhibition17In-Situ Floating Typha spp. Assays

Figure 5: Typha emergence rates for in-situ mesocosm assays performed with 18 identical simulated wetland systems. 18Hazards to Local WetlandsMany herbicides and other potentially phytotoxic compounds are frequently applied/released and detected in local surface waters

Figure 2: Calculated hazard quotients for pesticides detected in Lake Winnipeg between the years of 1999 to 2012. Numbers indicate total number of detections and dashed line indicates HQ > 1. (Manitoba Conservation, unpublished data) Due to widespread eutrophication issues, characterizing phytotoxicity and changes to nutrient uptake dynamics are very importantMuch work remains to be done

19DiscussionComparison of our compounds and endpoints (with calculated EC50s)Comparing our results to other experiments Studies assessing salinity impairment in other macrophytesCompare to the Scientific Criteria Document for the Development of the Canadian Water Quality Guidelines for the Protection of Aquatic Life Chloride Ion (CCME, 2011)Guideline currently recommended for short term exposures at 640 mg Cl- * L-1 and long term exposures at 120 mg Cl- * L-1Recommendations for further investigations of emergent macrophyte toxicity based on monitoring data and governmental reportsWidely used herbicides in Manitoba include 2-4D, atrazine, bromacil, dicamba, and glyphosate (Currie and Williamson, 1995)


21ReferencesArts G, Davies J, et al. 2010. AMEG: the new SETAC advisory group on aquatic macrophyte ecotoxicology. Environ Sci Pollut Res 17:820823.

Bourgeois, B. r., S. Hugron, et al. 2012. Establishing a moss cover inhibits the germination of Typha latifolia, an invasive species, in restored peatlands. Aquatic Botany 100(0): 76-79.

Carlson JC. Anderson JC, et al. 2013. Presence and hazards of nutrients and emerging organic micropollutants from sewage lagoon discharges into Dead Horse Creek, Manitoba, Canada. Science of the Total Environment 445-446: 64-78.

CCME. 2011. Canadian water quality guideline for chloride: Scientific criteria document (draft). Canadian Council of Ministers of the Environment, Winnipeg, MB, Canada.

Currie RS, Williamson DA. 1995. An assessment of pesticide residues in surface waters of Manitoba, Canada. Manitoba Environment, Water Quality Management Section, Winnipeg, MB, Canada.

Environment Canada. 2012. Five-year Review of Progress: Code of Practice for the Environmental Management of Road Salts. Environment Canada, Ottawa, ON, Canada.

Kaushal S, Groffman P, et al. 2005. Increased salinization of fresh water in the northeastern United States. PNAS 102(38): 13517-13520.

Manitoba Conservation. Unpublished Data. Water Quality Management Section. Manitoba Conservation and Water Stewardship123 Main Street, Suite 160 Winnipeg MB R3C 1A5.

McNaughton SJ. 1968. Autotoxic Feedback in Relatin to Germination and Seedling Growth in Typha Latifolia. Ecology 49(2): 367-369.

Moore, M. T. and M. A. Locke . 2012. Phytotoxicity of Atrazine, S-Metolachlor, and Permethrin to Typha latifolia (Linneaus) Germination and Seedling Growth. Bulletin of Environmental Contamination and Toxicology 89(2): 292-295.

Muller SL, Huggett DB, et al. 2001. Effects of Copper Sulfate on Typha latifolia Seed Germination and Early Seedling Growth in Aqueous and Sediment Exposures. Archives of Environmental Contamination and Toxicology 40(2): 192-197.

Raney S, Eimers C. 20


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