Chromatographic (TLC) Differentiation of Grizzly Bear and Black Bear Scats

1. What they did

Where grizzly bears and black bears share the same country, telling their scats apart is a major problem for researchers. Scats are often collected days or weeks after they were left, when no other field sign remains, and scat diameter confuses black bears with small grizzly bears. Earlier attempts to separate the two species with thin-layer chromatography (TLC), a simple lab method that separates the chemicals in a sample into a pattern of spots on a plate, had failed.

The authors tried again, this time using all the spots on the plate rather than only those thought to be bile acids:

• Preparing the scats. Ground scat samples were soaked in hexane and methanol to remove plant pigments, then the extract was spotted on TLC plates at two concentrations, beside lanes of known chemical standards. The plates were photographed under visible and ultraviolet (UV) light, and the position of each spot was turned into computer data.
• Building a key. They used 356 profiles from 178 scats of known species, provided by the Interagency Grizzly Bear Study Team and the Montana Fish, Wildlife and Parks Department. They built a first key from 67% of the known data, tested it on known scats not in the key, and revised it more than 20 times until all the known data were included.
• Testing what affects the profiles. They ran 13 chemical standards (bile acids, sex hormones, cholesterol and others), 44 samples of 11 common bear food plants, and scats of different ages, drying methods and storage times.
• Blind test. The final key (Scat key 11.0) was tested on 77 known grizzly bear and black bear scats from the North Continental Divide and Greater Yellowstone Ecosystems.

2. What they found

Chemistry and diet

• Bile acids were only weakly related to the profiles and of little value for telling the species apart.
• Scat profiles had fewer spots than the plant foods, so they were not just a copy of what the bears ate. Scats containing moose had fewer spots than scats containing vegetation.

What changes the profiles

• Drying method (air dried, oven dried, or frozen then oven dried) made no major difference.
• Grass left out in good mountain weather for up to 7 days showed no major changes, but 24 hours of snow melt reduced the number of spots.
• Scats 1, 2 and 3 weeks old still correlated with 1-day-old scats (r = 0.7 or greater), suggesting some information was lost to weathering, probably through rain or snow melt.
• Scats stored for up to 5 years showed no major changes, though older stored collections seemed to give more unidentifiable scats.
• Profiles changed with the seasons, probably following changes in food as plants matured and dried.

The key

• 80% of the known scats were from grizzly bears and 20% from black bears.
• When the key was tested on known and unknown scats not used to build it, 15% were given an ambiguous identification (a tie between the four readings: two concentrations under visible and UV light). In the blind tests of known scats, the ambiguous rate was much higher, 24 to 54%.
• For scats collected 1 June to 7 July, the key was correct for 66.7% of the scats it identified, which was not significantly better than chance.
• For scats collected 8 July to 31 August, it was correct for 94% of the scats it identified (17 of 18), which was significant. However, 21 of the 39 scats (54%) in this period were ambiguous.
• Profiles that had never given a wrong match could identify only 30 to 40% of scats, so there is a trade-off between accuracy and how many scats get an identity.
• The two species’ scats differed most often at a few particular spot positions on the plates.

3. What they concluded

• No single TLC profile reliably shows the species, but grizzly bear and black bear scats differ enough to allow more reliable separation than previous methods.
• The method is robust to the minor variations of lab work, but species differences are partly masked by diet and other factors. Using the strength of each spot, not just whether it is there, would probably improve it.
• The method cannot tell a bear’s sex.
• More known black bear scats are needed: the key found a new black bear profile for every 3 new scats, against 1 for every 8 grizzly bear scats.

4. How tracking was or was not used

No tracking was done in this study. The paper is about identifying one kind of sign, scat, to species in the lab when field identification is not possible. The authors point out that field sign of the species is often absent when scats are collected days or weeks after deposition, and that scat diameter, scat volume and soil content are not reliable for telling the species apart.

5. Tracker or observer certification

Not reported. The method’s accuracy rests on the scats “of known identity” supplied by the Interagency Grizzly Bear Study Team and the Montana Fish, Wildlife and Parks Department, but the paper does not say how their species was known. Six people prepared scats and helped read the plates.

6. Other technologies, skills or innovations

• Thin-layer chromatography with p-anisaldehyde staining, read under visible and UV light from photographic slides.
• Two separate keys, one from visible light and one from UV light, combined as four votes per scat.
• A computer matching program that compares unknown scats against a library of known profiles, which “learns” as new profiles are found.
• The authors mention that High Performance Liquid Chromatography or immuno-detection might detect excreted hormones, and that DNA had been extracted from bear scats but might be hard to separate from bacterial DNA.