Jump to main content or area navigation.

Contact Us

Water: Monitoring & Assessment

Sampling Methods and Analysis: Macroinvertebrates and Amphibians

Below is a detailed description of the sampling methodology and analysis process used by Ohio EPA for macroinvertebrates and amphibians. Also included are the lessons learned for sampling these assemblages.

Amphibian and macroinvertebrate taxa were selected as potential indicators of wetland condition. On the recommendation of many field professionals, experienced in amphibian monitoring, funnel traps were used. The funnel traps also proved to be extremely effective at sampling wetland macroinvertebrate communities. Therefore, the same sampling protocols are used for both amphibians and macroinvertebrates in Ohio EPA's study.

oh1frog oh1trap

Funnel Trap

The funnel traps used in this project have cylinders constructed of aluminum window screen and funnel ends made from fiberglass window screen. The funnel traps are similar in design to commercially available minnow traps. The cylinders are 18 inches long and 8 inches in diameter. The two funnel ends are attached to the cylinders and begin 8 inches in diameter and taper inward 5 inches to a 1 3/4 inch opening. A string handle that runs from end to end is attached to the two seams where the cylinder and funnels ends join.

To date, Ohio sampling data has come from depressional wetland systems in the Eastern Corn Belt Plains (ECBP) ecoregion. The Eastern Corn Belt Plains ecoregion spans most of the western half of Ohio and accounts for about 40 percent of Ohio's land mass. Within the ECBP, depressional study wetlands that demonstrate a gradient of disturbance levels, from least impacted to greatly disturbed, were selected.

Selected wetlands are sampled for amphibians and macroinvertebrates three times during the year (early, middle, and late spring). An early spring sample is collected in the period from late February to late March. This early spring sample run allows sampling of adult Ambystomatid salamanders and early spring macroinvertebrates (e.g., fairy shrimp), which are only present in wetlands for a limited time. Adult salamanders enter wetlands to breed in early spring following the first few warm, rainy nights of late winter and early spring. The actual timing of their arrival in Ohio is highly weather dependent and varies greatly by year and location.

oh1salm oh1shrmp

A second sample is collected during the month of April in order to collect adult frogs entering wetlands to breed, as well as amphibian larvae already present, and to sample for macroinvertebrates. Mosquito larvae, an important prey item for many predators, are abundant in April in Ohio wetlands.


The final sampling is conducted between May 15 and June 15. Salamander larvae and frog tadpoles, in addition to the resident macroinvertebrates, are collected. This last sampling run occurs late enough in the breeding cycle to allow collection of larvae from all breeding amphibians. However, it is still early enough that even in drought years temporary wetlands will not have dried up.

oh1cray oh1tadp

Generally, ten funnel traps are installed at each wetland. Prior to installing the first funnel trap, the perimeter of the area where standing water is present is measured using a hip chain or by pacing. The total perimeter length is then divided by ten and ten funnel traps are installed uniformly around the perimeter of the wetland at intervals of ten percent of the total perimeter distance. Each funnel trap location is permanently marked with flagging for use throughout the sampling season.

The funnel traps are installed on the bottom at a location deep enough to submerge the trap. The traps are left in the wetland for twenty-four hours in order to ensure unbiased sampling of animals with diurnal and nocturnal activity patterns. The design of the traps results in the collection of any amphibians and macroinvertebrates that swim or crawl into the funnel openings.

oh1pic2 Upon retrieval, the traps are emptied by inverting one of the funnels and dumping and shaking the contents into a white sorting pan. Organisms that can be readily identified in the field are counted and recorded in the field notebook and released. The remaining organisms are transferred to a one-liter plastic bottle and preserved with 70 percent ethanol. The contents of each trap are kept in separate, clearly marked bottles for individual analysis in the laboratory. If large numbers of amphibians are kept for identification in the lab, the samples are transferred to formalin for long-term storage. All organisms collected are identified in the lab using appropriate keys and the results are recorded.

Dip Net

Qualitative collections are made concurrently with funnel trapping at each wetland once during each of the three sampling periods. Qualitative sampling involves the collection of amphibians and macroinvertebrates from all available natural wetland habitat features. This is achieved by using triangular ring frame 30-mesh dip nets and manual picking of substrates with field forceps. The goal is to compile a comprehensive species/taxa list of macroinvertebrates and amphibians at the site. A minimum of thirty minutes is spent collecting the qualitative sample. Sampling continues until the field crew determines that further sampling effort will not produce new taxa. At least one specimen of all taxa collected during the qualitative sampling is preserved in a jar of ethanol for positive identification in the laboratory.

oh1dip1 oh1dip2

Hester Dendy Artificial Substrate Sampler

Five Hester Dendy (HD) artificial substrate samplers were tied to the top of a concrete block and placed in wetlands where they remained submerged for six weeks. The samplers were collected and preserved in formalin. All the macroinvertebrates colonizing the samplers were counted and identified.

Analytical Methods: Macroinvertebrates and Amphibians

Macroinvertebrates were identified to genus or species. Amphibians were identified to species except for some small salamander larvae which were identified to genus. Each funnel trap collection was analyzed individually so that location-specific information was not lost by pooling all samples from a site.

The number of individuals collected in the traps was divided by the number of hours trapped to give a relative abundance consisting of number of individuals per trap hour. Results from the different wetland study sites were examined for faunal differences in distribution and abundance. Analysis of the data for potential biological indicators of human disturbance are underway.

Lessons Learned for Macroinvertebrates and Amphibians

  1. Funnel traps consistently collected an average of ten more macroinvertebrate taxa than qualitative sampling using dip-nets. Funnel traps were much more effective in sampling amphibians and fish than sampling with dip nets.
  2. Qualitative sampling collected somewhat more Mollusca and Chironomidae taxa than funnel traps.
  3. Funnel traps collected more leech taxa, Hemiptera taxa, Coleoptera taxa, Odonata taxa, and Crustacea taxa than qualitative sampling.
  4. Hester-Dendy artificial substrate samplers were ineffective for sampling most wetland macroinvertebrates except oligochaetes, Chironomidae, and Mollusca.
  5. A 24-hour sampling period for funnel traps is preferred as it allows for the collection of nocturnal species that are infrequently collected by daytime sampling methods.

Jump to main content.