This Standard Methodologies Bank is a collection of standard operating procedures and methods that are used by PDE and its partners for freshwater mussel restoration, propagation, monitoring, and research. Many of these methods have been peer reviewed and are generally accepted. This resource is geared toward researchers, municipalities, environmental groups, and teachers to restore and research freshwater mussels throughout the Delaware Estuary. Monitoring and research techniques are split into two categories: “professional” and “public.” Professional methodologies generally require technical expertise, specialized equipment, and/or can be expensive. Public methodologies, however, are often less expensive and are suited for all user groups such as citizen scientists and students.

The table below organizes methodologies by category, metric, user group, and searchable keyword. Use the search option in the top right corner of the table to search the most appropriate method for your project, user group, and funding availability. Available resources and a brief description for each method are described below the table. The professional and public columns on the right contain reference numbers that correspond to reference numbers in the Standard Methods Resource Table. These links direct you to large user manuals, websites geared toward helping you find more resources, and specific methods (written by PDE staff and other organizations).

Use the search bar on the right to quickly locate your desired metrics.
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CategoryMetricMethod OptionProfessionalPublicKeywords
BiologicalAnimal Collection & TaggingCollection, Tagging, and Release7, 14
BiologicalAnimal Collection & TaggingIdentification11
BiologicalAnimal CountShoreline & Stream Bank Survey111
BiologicalAnimal CountWading Survey111
BiologicalAnimal CountSnorkel Survey111
BiologicalAnimal CountKayak Surkey111
BiologicalAnimal CountRough Estimate Survey12
BiologicalAnimal CountPopulation Density13
BiologicalReproductionGravidity Check15
BiologicalReproductionGlochidia Syringe Extraction Method16
BiologicalReproductionGlochidia Warming Extraction Method16
BiologicalReproductionViability Test
BiologicalMorphometricsVernier Caliper Measurements
BiologicalMorphometricsMicroscopy Measurement
BiologicalMorphometricsMetric Ruler
BiologicalMorphometricsWeight Measurements
BiologicalMorphometricsScope of Growth
BiologicalFeedingFiltration Rate25
BiologicalFeedingAbsorption Efficiency25
BiologicalExcretion MatterDefecation Efficiency25
BiologicalExcretion MatterAmmonia Excretion25
BiologicalRespirationRespiration Rates25
BiologicalProductivityOxygen Consumption:Ammonia Excretion Rates25
PhysicalHabitatObservation2, 32, 3
PhysicalHabitatBenthic Habitat Survey81, 3
PhysicalSoil TypeSediment Analysis63
PhysicalStream FlowCurrent-Meter43
ChemicalSalinityProfessional InstrumentSee Below, 10
ChemicalDissolved OxygenProfessional InstrumentSee Below, 10
ChemicalPhProfessional InstrumentSee Below, 10
ChemicalPhDigital Titration2, 32,
ChemicalTemperatureProfessional InstrumentSee Below, 10
ChemicalTemperatureThermometer 33
ChemicalConductivityProfessional InstrumentSee Below, 10
ChemicalOrganic ContentWeight-On-Ignition19, 23
ChemicalProtein ContentPierce BCA Test Kit20
ChemicalLipid CompositionLipid Extraction and Class Analysis21
ChemicalCarbohydrate ContentPhenol-Sulfuric Acid Method22
ChemicalCondition IndexCalculation of Condition Index23
ChemicalAlgal BloonChlorophyll a Field Procedure28
ChemicalTotal Suspended SolidsHemacytometer18, 24
ChemicalTotal Suspended SolidsCoulter Counter18, 24
ChemicalTotal Suspended SolidsTransparency Tube2, 32, 3
ChemicalTotal Suspended SolidsSecchi Disc33
ChemicalLaboratory SamplesSample Preparation17
ChemicalLaboratory SamplesWater Collection26
ChemicalWater Column Nutrients & ContaminationGrab Sample Analysis (Water Column Nutrients & Contamination)52
ChemicalWater Column Nutrients & ContaminationE. Coli. Laboratory Analysis (CFUs)27


Metric Descriptions and Standard Method Procedures:

Benthic Habitat Survey: Benthic habitat properties such as type of sediment (e.g. mud, sand, or silt) present and the presence or absence of vegetation can determine if freshwater mussels can survive. Scientists study benthic habitats to learn more about habitat requirements for mussels and improve restoration efforts. Habitats can be characterized by professionals and trained citizen scientists.
Professional: Freshwater Mussel Benthic Habitat Characterization
Public: Volunteer Stream Monitoring: A Methods Manual or Mussel Survey Program

Biochemical Analyses: A variety of biochemical properties of biological samples can be analyzed to determine the amount of protein, carbohydrate, and lipid in a sample. Data can be used to determine how much food is available in nature as well as how healthy animals were at the time they were sacrificed. Below are specific methods for scientists. These methods requires specific reagents, specialized laboratory equipment, and trained professionals.
Professional: Biochemical Analyses I. Protein, Biochemical Analyses II. Lipid, and Biochemical Analyses III. Carbohydrate

Chlorophyll a Field Procedure: Water samples are taken at various depths within a body of water and scientists analyze the amount of chlorophyll a in each sample. Chlorophyll a is a pigment found in phytoplankton which means it is related to the overall abundance of phytoplankton. This analysis can be used to estimate the productivity of water bodies which can ultimately be linked to nutrient concentrations.
Professional: Standard Operating Procedure for Chlorophyll a Sampling Method Field Procedure

Collection, Tag, and Release: Freshwater mussels can be collected, tagged, and released in a variety of ways associated with different restoration techniques. Collections are performed in such a way to protect wild populations. Tags are attached to mussels to aid in finding mussels again. Mussels are released in a number of ways that provide scientists data on how best to restore mussels. These techniques require scientific permits as well as specialized tagging and tracking equipment.
Professional: Fresh Water Mussel Collection or Freshwater Mussel Tagging, Release, and Monitoring

Colorimeter: An inexpensive method for estimating water pH levels via a paper color intensity scale. The pH level of water reveals acidity; this is critical for sensitive species.
Professional/Public: Volunteer Stream Monitoring: A Method Manual

Condition Index: This is a measure of the overall health of a freshwater mussel. The measurements for dry tissue weight, total wet weight, and dry shell weight are used to calculate the condition index. To perform this analysis, trained professionals use technical laboratory equipment. Condition index is best understood when comparing among seasons.
Professional: Determination of Condition Index and Tissue Preparation for Biochemical Analysis of Bivalve Molluscs

Current-Meter: A current meter can measure the speed of flowing water which can be used to estimate important physical conditions such as the volume of water flowing in a stream or river. Shorelines, stream banks, and benthic habitats may all be impacted by the volume and speed of flowing water. These measurements help managers make decisions about best management practices. Flow measurements can be taken in a variety of locations in a number of ways. There are methods to suit all user groups.
Professional: Discharge Measurements at Gaging Stations
Professional/Public: Volunteer Stream Monitoring: A Methods Manual

E. Coli. Lab Analysis: Escherichia Coli bacteria is a bacteria found in the intestinal tract of warm-blooded animals. When this bacteria is found in the water column, it indicates fecal pollution and suggests the presence of other pathogens. This lab analysis uses petri dish counts to determine pathogen presence in units of Colony Forming Units (CFU).
Professional: Method 1103.1: Escherichia coli (E. coli) in Water by Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar (mTEC)

Enumeration: Once glochidia (freshwater mussel larvae) are extracted from females, obtaining a total count is valuable to reproduction data. Trained professionals can determine total counts in the laboratory using specialized equipment.
Professional: Glochidia Extraction and Assessment

Glochidia Extraction: Glochidia is the scientific term for freshwater mussel larvae. There are multiple methods for professionals to assess and extract glochidia. Two methods are the syringe method (using reverse pliers and a syringe) and the warming method (using temperature-controlled water). Methods vary in effectiveness depending on the species of mussel.
Professional: Glochidia Extraction and Assessment

Gravidity Check: A gravid mussel is assumed to be a female with fertilized eggs that have developed into viable larvae (glochidia) in their gills. Freshwater mussels can be checked for gravidity. Trained professionals can use reverse pliers to observe a suspected gravid mussel’s gills without harming the animal.
Professional: Freshwater Mussel Gravidity Check

Identification: Scientists who study freshwater mussels must be able to accurately identify mussel species. Shell color, shape, and general location found can be helpful for mussel identification. Both professionals and citizen scientists can participate in identification. It is important to remember that mussels are sensitive animals and live mussels should be left alone by citizen scientists. Many states require special scientific permits in order to handle live mussels.
Professional/Public: Mussel Survey Program

Measurement of Freshwater Mussel: Collecting consistent and accurate data on mussel size is important to growth and heath studies. Measuring mussels in a standardize manner with calibrated measuring tools is imperative to mussel research. Typical mussel measurements include shell length, shell height, total animal wet weight, and dry shell weight. Several different measuring devices can be used including vernier calipers, microscopy measurements, and a ruler. Freshwater mussels are sensitive animals and may not be handled without proper permitting. Please do not pick up live mussels.
Professional: Measuring Freshwater Mussels

Mussel Population Size and Density Survey: There are multiple different available methodologies for studying mussel populations. Surveys vary in effort and intensity from walking shorelines to snorkeling. These surveys vary in intensity and area that they cover. These surveys can be done by both professionals or citizen scientists.
Professional: Qualitative Freshwater Mussel Survey or Semi-Quantitative Freshwater Mussel Survey or Quantitative Freshwater Mussel Survey
Public: Mussel Survey Program

Observation: Recording general field observations provides information that may otherwise not be captured such as the overall condition of vegetation, stream bed, water surface appearance, and the weather conditions. This information may prove to be valuable when interpreting data results. Citizen scientists are encouraged to share any observations they have on their local streams.
Professional/Public: Volunteer Stream Monitoring: A Method Manual or Volunteer Estuary Monitoring: A Method Manual

Organic Content Analysis: This method provides information on the amount of organic material in any biological sample. Information about organic material helps scientists understand mussel health and nutrition available in nature. Trained personnel using special laboratory equipment perform these types of analyses.
Professional: Biomass Measurement II. Dry and Ash-Free Dry Weight or Determination of Condition Index and Tissue Preparation for Biochemical Analysis of Bivalve Molluscs

Professional Instrument: There are a number of professional instruments at varying price ranges which give instant measurements for dissolved oxygen, salinity, pH, conductivity, temperature, etc. These are handheld instruments that can yield immediate results in the field. Some companies which manufacture these instruments include href=”” target=”_blank”>YSI, Hydromet, Fischer Scientific, Eureka Waterprobes etc. The Partnership for the Delaware Estuary uses a water quality sonde connected to a data collector.
PDE’s Professional Instrument Methods: Water Quality Monitoring Using a Handheld Sonde for in situ Measurements

Physiological Experiments: This method outlines physiological experiments using bivalve mollusks. Data are gathered on bivalve physiological processes such as feeding, waste production, and respiration rates. Rates are scaled to the body size of bivalves. These data can be used to estimate various ecosystem services. Tests provide insight to nutrient availability and animal health. There are several specific methodologies within this document, all of which require professional expertise and specialized equipment.
Professional: Weight-specific Processing Rates and Fates of Suspended Microparticulate Matter Consumed by Bivalve Molluscs

Sample Preparation: This method organizes the various analyses of the chemistry and biochemistry of algae or seston samples (suspended sediments in water). Sample preparation requires laboratory equipment and trained professionals.
Professional: Preparation of Samples for Chemical and Biochemical Characterization of Algae and Seston

Secchi Disc: An easy to use, inexpensive, and accurate tool for measuring water clarity. Water clarity data provides proxies of suspended sediments in the water column. This method is suitable for all user groups.
Professional/Public: Volunteer Stream Monitoring: A Method Manual

Sediment Analysis: Sediment analysis may include a variety of components such as particle size and organic material content. Sediment composition and size are fundamental properties of soils; they can provide information about overall benthic conditions.
Professional: Techniques of Water-Resources Investigations of the United States Geologic Survey
Public: Volunteer Stream Monitoring: A Methods Manual

Scope of Growth: This is a measure of an animal’s physiological growth potential, which is used for describing the health and understanding the animals’ organismal condition in response to different treatments or sites. This method was developed as a health assessment metric for aquatic organisms, but it also represents a potentially useful way to examine how rates and fates of material processing can vary spatially and temporally. Several technical procedures must be performed to calculate this value.
Professional: Weight-specific Processing Rates and Fates of Suspended Microparticulate Matter Consumed by Bivalve Molluscs

Thermometer: Thermometers can vary in price and function. They can easily are used to measure the temperature of air and water.
Professional/Public: Volunteer Stream Monitoring: A Method Manual

Titration Kit: Inexpensive kits which can be used to measure dissolved oxygen, pH, NH3, NH4, and NHx by all user groups. Dissolved oxygen (Winkler Titration) and pH (Digital Titration) measurements help explain flora and fauna community distribution.
Professional/Public: Volunteer Stream Monitoring: A Method Manual or Volunteer Estuary Monitoring: A Method Manual

Total Suspended Solids: Procedures for counting particle concentrations utilize either a simple device such as a hemacytometer or a specialized device such as a particle counting machine. Scientists are interested in the amount of particles of all sizes because particle size relates to the quality of food available to filter feeders like freshwater mussels. The hemacytometer is relatively simple to use requiring a basic count using a microscope. The particle counting machine method is more accurate and precise but requires very expensive machinery and technical expertise. These highly technical methods require trained professionals and laboratory equipment.
Professional: Biomass Measurement I. Particle and Volumetric Concentration or Collection of Seston for Particle Counting, Weight-On-Ignition Analysis or Proximate Biochemical Analysis

Transparency Tube: Sometimes called a “turbidity tube,” tube readings are a proxy for total suspended solids and turbidity of a source of water, these reading compare relatively well to precise lab measurements.
Professional/Public: Volunteer Stream Monitoring: A Method Manual

Viability Test: his is a test used to determine the amount of healthy glochidia (freshwater mussel larvae). This test requires a dissecting microscope and professional training.
Professional: Glochidia Extraction and Assessment

Water Collection: Collecting water from field locations should follow strict protocols to avoid sample contamination. Water is consistently collected by hand or pump by trained personnel.
Professional: Water Collection for Seston Analysis and Bivalve Feeding

Water Sample Analysis: Contaminants, pathogens, nutrients, and other chemical measures are critical to monitoring and research. Since mussels rely on nutrients such as nitrogen and phosphorus to grow and reproduce, excess or insufficient availability can cause population to suffer. Drinking water contaminants can have dangerous health impacts on communities. There are a variety of water quality testing methods which are suited for a variety of user groups ranging from volunteers to professionals including: laboratory chemistry, titration, grab sampling, in situ tests, and much more.
Professional: Methods Approved to Analyze Drinking Water Samples to Ensure Compliance with Regulations
Public: Volunteer Stream Monitoring: A Method Manual