Abstract
Many contaminants are introduced into freshwater ecosystems worldwide. Preliminary investigations that focus on apex predator/sentinel species like otter and mink can inform more targeted follow-up studies. The feces of these elusive animals can be collected non-invasively for analysis of contaminants and complimentary genetics. Conservation detection dogs were used to locate otter and mink feces along five rivers in Montana for analysis of heavy metals, anthropogenic organic contaminants (AOCs) including pharmaceuticals and personal care products (PPCPs), polybrominated (PBDE) flame retardants, and genetics. With highest find rates of 6 and 20 fecal matter finds per km for otter and mink, respectively, and detection of all three focal contaminants in some fecal samples, this proved an excellent application of dogs. Recommendations for follow-up investigations are also provided.
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Notes
- 1.
Carbon-based compounds.
- 2.
Typically, used to treat seizures and nerve pain.
- 3.
Antibacterial/antifungal agents that are added to consumer products (e.g., toothpaste, lotions, soaps) to limit bacterial contamination.
- 4.
Partition describes the distribution of a solute between two immiscible solvents, for example organic and aqueous phases.
- 5.
Treated sewage sludge.
- 6.
These are not to be confused with fire retardants, typically sprays and foams, which are aerially applied in Montana and other states to combat forest fires (e.g., USDA et al. 2015).
- 7.
Signed in 2001 and made effective in 2004, the Stockholm Convention on Persistent Organic Pollutants is an international treaty implemented with the objective of restricting or eliminating the production and use of persistent organic pollutants (POPs) such as PBDE flame retardants. See: http://chm.pops.int/
- 8.
Repelled, rather than attracted, to water.
- 9.
Wherein low/trace concentrations of contaminants acquired or ingested by prey organisms are successively absorbed and magnified by their predators, according to their dietary predilections (i.e., of contaminated prey) and their ascending order in the food chain.
- 10.
An attempt to gather information from the same individual repeatedly.
- 11.
The Superfund is a program of the US government that was implemented to support site cleanup of areas that have been contaminated with contaminants and other hazardous substances.
- 12.
By Frankline Nwanguma, under the supervision of Dr. Chad Kinney.
- 13.
- 14.
- 15.
Also called ‘confirmation bias’, wherein a survey may be influenced by pre-conceived or pre-existing perceptions of the surveyor. In this context, it might mean favoring areas because the surveyor expects to find samples there, based on prior experience. However, other samples present in other parts of the surveyed area may be overlooked as a result of this focus.
- 16.
Samples were not homogenized, so subsamples cannot be considered an ‘aliquot’. In future studies, the collected fecal samples should be homogenized prior to analysis.
- 17.
The results of the river water sample analyses, and a discussion regarding the viability of water and fecal matter relative to one another is considered in the final report summarizing this work (Richards/WD4C, unpublished data).
- 18.
Of which one otter fecal samples was submitted in duplicate due to moisture exposure concerns.
- 19.
Sourced from 35 latrines, see Table 6.2.
- 20.
A term coined by M. Ben-David, see Ben-David et al. (2005).
- 21.
Interestingly, Murphy et al. (2003) also found that sex identification in the feces of the target organism could be affected by the sex of the prey consumed.
- 22.
- 23.
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Acknowledgements
Special thanks to Megan Parker, Director of Research for Working Dogs for Conservation, and to Pepin, for their contribution to the field survey component of this study. Much appreciation is extended to Kristy Pilgrim at the National Genomics Center for Wildlife and Fish Conservation in Missoula, Montana, for diligently performing the genetics analyses, enthusiastically sharing her knowledge and providing invaluable feedback to earlier versions of this chapter. Likewise, Heiko Langner, formerly in the Department of Geosciences at the University of Montana, is thanked for regularly lending his advice and expertise to the heavy metals component of this work. The creativity, hard work, and good humor of Matt Young, also at the Department of Geosciences (water sample processing), and of Thor Halldorson in the Department of Chemistry at the University of Manitoba (for his part in the PBDE sample analyses), is gratefully acknowledged. Thanks to Alan Ramsay, Marirose Kuhlman, and Ray Vinkey for giving their time to participate in the surveyor performance comparison trials.
The work described in this chapter was made possible through the generous support of the Kenney Brothers Foundation (Wick Fund), the Cinnabar Foundation (Montana’s Conservation Fund), the Arthur L. ‘Bud’ and Elaine V. Johnson Foundation, and the Animal Welfare Institute, via a Christine Stevens Wildlife Award. N. Richards and D. (Smith) Woollett extend their sincere thanks to these funders for their generosity and backing.
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Appendices
Appendix 1
6.1.1 Summary of Methods Used for Polybrominated Diphenyl Ether (PBDE) Flame Retardant Analyses of Otter and Mink Fecal Samples
In Year 1/2013 (i.e., Method 1), weighed samples (between 1.0 and 6.0 grams) were spiked with 10 μL of a 1 ng/μL BDE-71, 126, and 13C-209 as a recovery internal standard (RIS) solution, and homogenized using a Polytron hand-blender.
Homogenized tissue was transferred to a test-tube and 30 mL of the extracting solvent dichloromethane—DCM: hexane:acetone (45:45:10) was added. Samples processed in Year I were sonicating for 20 minutes.
In Year II/2014 (i.e., Method 2), a Precellys tissue homogenizer (PrecellysS24) was used for extracting target analytes. In this case, 2 mL of the extracting solvent was added to a 5 mL Precellys tube and the contents homogenized by agitating at 5000 rpm for 30 secs. In both cases, extracts were removed, reduced in volume and filtered (1 μm PTFE syringe filters, Gelman Laboratory, MI). An aliquot of each extract was evaporated to dryness and lipid weights were determined gravimetrically. Lipid was removed from the remaining extract using automated gel permeation chromatography (J2 Scientific, Columbia, Missouri, USA) on a column (29.5 mm i.d. × 400 mm) packed with 60 g (dry weight) of 200–400 mesh S-X3 Envirobeads (ABC Instruments, MO) that had been soaked in DCM/hexane overnight. Further purification was achieved on a gravity flow column (300 mm × 10.5 mm i.d., not attached to an LC pump) of reagent-grade Florisil (1.2% deactivated (w/w), 8 g, 60–100 mesh size, Fisher Scientific). Native PBDEs and the suite of RIS added to the samples were eluted off the column using 40 mL of hexane followed by 35 mL of hexane:DCM (85:15). The collected fraction was reduced in volume to 100 μL by a gentle stream of ultra-high purity (UHP) N2 and spiked with 10 μL of 1 ng/μL of BDE-156 as an instrument performance internal standard prior to high resolution gas chromatography electron capture negative ion mass spectrometry analysis (GC/MS).
All analyses were performed on an Agilent 5973 GC-MS fitted with a 10 m × 0.25 mm id DB-5 capillary column (0.25 μm film thickness, J&W Scientific, CA). UHP helium was used as the carrier gas at a flow rate of 2 mL/min. Splitless injections of 2 μL were made by a 7683 Agilent autosampler with the injector set isothermally at 280 °C. The initial oven temperature was set at 90 °C with no hold time, ramped at 20 °C/min to 310 °C and held for 5 minutes. MS analysis was performed in the electron capture negative ionization mode using methane as the buffer gas. Source and quadruple temperatures were both set to 150 °C. Detection of PBDE congeners was done under selected ion monitoring conditions (i.e., SIM) using the [Br]¯ ions (m/z 79, 81). An external standard solution containing the PBDE congeners was used to quantify the samples.
Instrument blanks were injections of hexane run after every five samples and were used to monitor possible PBDE contamination between GC injections. Method (or procedural) blanks were derived by extraction of anhydrous sodium sulfate—Na2SO4. Method blanks were used to monitor the potential for contamination to occur during extraction and work-up of the sample.
Two criteria were used for confirmation of compounds on the GC. First, the elution time of a compound had to be within ± 2 sec in the sample and external standard. Second, the ratio of the m/z 79 and m/z 81 ions had to be within ± 15% of the theoretical value.
Sample weight and percent lipid content | ||
|---|---|---|
Sample ID | Sample weight (g) | % lipid |
DENML3 | 1.83 | 1.2 |
DENOL1 | 4.71 | 0.9 |
PJMS4 | 2.12 | 49.6 |
PJMS6 | 1.18 | 3.4 |
RoundML2 | 3.04 | 5.9 |
RoundML4 | 5.61 | 1.2 |
WStnOL1 | 4.93 | 0.3 |
WStnOL5 | 4.74 | 0.5 |
YML 26S13 | 1.56 | 1.6 |
YOL28S13 | 5.25 | 0.7 |
4MOL19S13 | 4.98 | 0.4 |
6MLOL19S13 | 5.27 | 0.2 |
Appendix 2
6.1.1 Summary of Methods Used for Anthropogenic Organic Contaminant (AOC) Analysis of Otter and Mink Fecal Samples
In total, eight mink and five river otter fecal samples were submitted for analysis of AOCs. Given that this represents the first time that wildlife fecal matter has thus been analyzed, a screening method initially had to be developed and validated. Adopted and modified from the work of Burkhardt et al. (2005), this method relied on a series of samples consisting of a mixture of peat moss and topsoil as surrogate matrix spikes, with known quantities of the target AOCs. The method was validated using spiked fecal samples collected from captive otter and mink as ‘control’ matrices. The developed method utilized pressurized liquid extraction via a mixture of isopropanol and water as the extraction solvent followed by extract clean-up and pre-concentration using Oasis HLB (Waters Corp.) SPE cartridges and evaporation under nitrogen at 70 °C. The final extracts were subjected to analyte derivatization with bistrimethylsilyltrifluoroacetamide (BSTFA) and quantified by GC/MS operated in the selected ion monitoring (SIM) mode.
Appendix 3
6.1.1 Summary of Methods Used for Genetics Analyses of Otter and Mink Fecal Samples
Genomic DNA was extracted from the fecal samples using the QIAmp DNA Stool Mini Kit (Qiagen Inc.; Valencia CA). Species identification was performed using the control region of mitochondrial DNA (mtDNA). Three hundred and forty-four (344) bp of the left domain were amplified using primers L15926 and H16498 (Kocher et al. 1989; Shields and Kocher 1991). The quality and quantity of template DNA were determined by 1.6% agarose gel electrophoresis. PCR products were purified using ExoSap-IT (Affymetrix-USB Corporation, OH, USA) according to manufacturer’s instructions. DNA sequence data was obtained using the Big Dye kit and the 3700 DNA Analyzer (ABI; High Throughput Genomics Unit, Seattle, WA). DNA sequence data were viewed and aligned with Sequencher (Gene Codes Corp. MI) and compared with reference sequences available publicly (Genbank, NCBI).
Individual identification was performed on both the river otter and mink DNA extracts using microsatellite DNA markers. For river otter, primers Lut435, Lut457, Lut604, Lut701, Lut733, Lut782, Gg7, Gg14, Tt1, Mer022, and Mvi075 were used (see Dallas and Piertney 1998; Davis and Strobeck 1998; Fleming et al. 1999). For American mink, Mvi2243, Mvi1354, Mvi1302, Mvi1381, Mvi099, Mvi075, Mvi1341, and Mvi3402 were used (Fleming et al. 1999; Vincent et al. 2003). The samples were also tested using an SRX/SRY analysis to determine sex (Hedmark et al. 2004). The resultant products were visualized on a LI-COR DNA analyzer (LI-COR Biotechnology). All samples were amplified using the multi-tube approach (Eggert et al. 2003) and data was error checked using program Dropout (McKelvey and Schwartz 2005).
Appendix 4
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Richards, N.L., Tomy, G., Kinney, C.A., Nwanguma, F.C., Godwin, B., Woollett, D.A.(. (2018). Using Scat Detection Dogs to Monitor Environmental Contaminants in Sentinel Species and Freshwater Ecosystems. In: Richards, N. (eds) Using Detection Dogs to Monitor Aquatic Ecosystem Health and Protect Aquatic Resources. Palgrave Macmillan, Cham. https://doi.org/10.1007/978-3-319-77356-8_6
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