Daniel Seburn
Advisor: Dr. Mark Hanson
ENVR 4500 – Project Proposal
Course Co-ordinator: Dr. Rick Baydack
2
1)
Develop laboratory Typha spp. germination and
root/shoot elongation assay to generate useful
experimental data regarding salinity impairment of
ecologically important emergent macrophytes
2) Perform experimental outdoor in-situ mesocosm assays
as a supplement to laboratory data
3) Review local environmental monitoring data to make
recommendations for further investigations
Typha spp.
(cattail)
Characterize
salinity toxicity
3
• Aquatic Macrophytes important for:
• Nutrient removal (cleaning up Lake Winnipeg algae bowl)
• Provide food/habitat for variety of fauna
• Used for phytoremediation of organic and inorganic
pollutants in treated wetlands
• SETAC 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
4
• Excess salinization to wetlands typically occurs as a result of:
• Saline sewage effluents
• Road salt de-icing compounds in runoff water
• Poor 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 2004
• Goal to improve the reporting and management of road salt usage among
municipalities, provincial governments, and other frequent applicators
(Environment Canada, 2012)
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• Typha assay – Seed Preparation Method
• Based on methods from McNaughton (1968) and recent studies
• Female flowering spikes collected from Oak Hammock Marsh,
Manitoba in Fall 2013
• 1 litre Technicon Commercial Blender
• 700ml RO water
• 40g Fisher Brand Sparkleen powder soap
• 5 ml Sodium Hypochlorite (bleach)
• Added in order to separate seeds from debris and remove any
residual chemicals left on seeds
• Blended for 30 seconds, then mixture added to dilution vessel
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• 15 individual seeds added to split petri dishes, 4 reps/treatment
• Used 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 photoperiods
• Germination success counted and root/shoot growth measured
with Infinity1 microscope and image analysis software
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• Infinity Analyze Software – Calibration and Measuring
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• Similar germination success
between seeds we collected (MB)
and ordered seeds (UK)
100
MB Sourced Seeds
UK Sourced Seeds
Germination Success (%)
80
• Significantly less germination
success without light, lower temp
60
• Germination success (52 to 71%)
comparable to other studies
assessing Typha germination:
40
20
0
A
B
C
D
E
• 60 to 90% (Burgeois et al., 2012)
• 80 to 90% (Moore and Lock, 2012)
• 79 to 93% (Muller et al., 2000)
Exposure Condition *
A = No light exposure @ 25°C for 7 days
B = 12 hours light/day @ 20°C for 7 days
C = 12 hours light/day @ 25°C for 7 days
D = 18 hours light/day at 25°C for 7 days
E = 18 hours light/day at 25°C for 14 days
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• UK sourced Typha latifolia seeds exposed to:
• Three different laboratory grade salts
• NaCl, CaCl2 * 2H2O, and KCl
• Three different commonly used road salt compounds
• No Name Road Salt (RS#1), Sifto Safe Step (RS#2), and Meltz All (RS#3)
• Greatest concentration 32g/L, subsequent solutions prepared by serial dilution
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Table 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.
Target Concentration NaCl
CaCl2
KCl
RS#1
RS#2
RS#3
1 g/L
1.105
1.145
1.134
1.145
0.584
1.653
2 g/L
2.085
2.205
2.101
2.012
1.304
2.831
4 g/L
3.985
4.036
3.996
3.781
2.174
4.753
8 g/L
7.925
7.545
7.689
7.154
4.002
8.801
16 g/L
15.834
13.641 14.609
13.084
7.685
15.371
32 g/L
26.625
25.134 26.013
25.863
14.865
24.194
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• Seeds left in Conviron growth chamber for seven days at 25°C and 18 hour light to
six hour dark photoperiod
• Photosynthetically 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 µmol m-2 s-1
• Study Design:
• Six different compounds
• Six concentrations of each
compound (plus controls)
• Five repetitions per
concentration
• Twenty seeds per repetition
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Germination Success (%)
80
NaCl
CaCl
KCl
Road Salt #1
Road Salt #2
Road Salt #3
60
40
20
0
Control
1 g/L
2 g/L
4 g/L
8 g/L
16 g/L
32 g/L
Treatment
Figure 2: Average germination success for Typha seeds exposed to increasing
concentrations of laboratory grade salts and common road salt compounds.
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Average Shoot Length (mm)
25
NaCl
CaCl
KCl
Road Salt #1
Road Salt #2
Road Salt #3
20
15
10
5
0
Control
1 g/L
2 g/L
4 g/L
8 g/L
16 g/L
32 g/L
Treatment
Figure 3: Average shoot length for Typha seeds exposed to increasing concentrations
of laboratory grade salts and common road salt compounds.
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18
NaCl
CaCl
KCl
Road Salt #1
Road Salt #2
Road Salt #3
Average Root Length (mm)
16
14
12
10
8
6
4
2
0
Control
1 g/L
2 g/L
4 g/L
8 g/L
16 g/L
32 g/L
Treatment
Figure 4: Average root length for Typha seeds exposed to increasing concentrations of
laboratory grade salts and common road salt compounds.
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Control
CaCl – 1 g/L
NaCl – 4 g/L
RS#1 – 8 g/L
RS#2 – 8 g/L
RS#3 – 1 g/L
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• 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 Inhibition
Number of Emergent Typha Plants
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Control
25 g/L
50 g/L
100 g/L
250 g/L
500 g/L
10
8
6
4
2
0
0
10
20
30
40
Days After Treatment
50
60
Figure 5: Typha emergence rates for in-situ
mesocosm assays performed with 18 identical
simulated wetland systems.
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• Many herbicides and other potentially phytotoxic
compounds are frequently applied/released and detected
in local surface waters
• Due to widespread eutrophication
issues, characterizing phytotoxicity
and changes to nutrient uptake
dynamics are very important
• Much work remains to be done
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)
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• Comparison of our compounds and endpoints (with calculated EC50’s)
• Comparing our results to other experiments
• Studies assessing salinity impairment in other macrophytes
• Compare 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-1
• Recommendations for further investigations of emergent macrophyte
toxicity based on monitoring data and governmental reports
• Widely used herbicides in Manitoba include 2-4D, atrazine, bromacil,
dicamba, and glyphosate (Currie and Williamson, 1995)
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Arts G, Davies J, et al. 2010. AMEG: the new SETAC advisory group on aquatic macrophyte ecotoxicology. Environ Sci Pollut Res 17:820–823.
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 Stewardship
123 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. 2014. Unexpected declines in stream phosphorus concentrations across southern Ontario. Rapid Communication 71: 337–342.