4-Attachment B Scope of Work Phycology2026.pdf

UTAH DIVISION OF WATER QUALITY 1

Scope of Work Phycology Taxonomy RFP

Introduction

The Division of Water Quality (DWQ) seeks a contractor with phycology expertise to identify and enumerate organisms from periphyton, phytoplankton, and potentially zooplankton samples. Samples will be submitted to the contractor in the fall, with the expectation that taxonomic results will be returned to DWQ prior to April 30th the following year so the data can be processed prior to the next collection season.

The contractor will assist DWQ over a 5-year period by conducting the following activities:

• Taxonomic identification and enumeration of ~500 phytoplankton samples (as many as 100 samples/year) collected by DWQ staff from lakes throughout Utah.

• Taxonomic identification and enumeration of periphyton (“soft” algae and diatoms) from ~300 samples (as many as 60/year) collected from streams throughout Utah.

• Conduct taxonomic identification and enumeration of a variable number of zooplankton samples depending on the year-to-year programmatic needs of DWQ.

• Ongoing taxonomic harmonization to ensure taxonomic output aligns with modern nomenclature and facilitates the integration of taxonomic results with state and national assessment tools.

• Conduct a one-time harmonization effort to ensure that taxonomic results provided by the contractor are consistent with historic taxonomic results from samples previously collected by DWQ.

• Provide ongoing consultation services to DWQ on the use and interpretation of these data on an as-needed basis.

Background

DWQ engages in a wide range of activities to protect and improve water quality, including several monitoring and reporting programs to determine the condition of, and stressors to, the waters of the state. A core element of our assessment program is the evaluation of aquatic life use attainment in riverine and lake environments. DWQ uses a combination of water chemistry, physical habitat, and the composition of biological assemblages—to assess support of aquatic life uses. With respect to the latter, periphyton and phytoplankton are among the assemblages used to conduct these bioassessment activities and the focus of this solicitation.

DWQ uses bioassessment information to make regulatory decisions and meet delegated federal Clean Water Act requirements. As a result, it is critical that samples are processed in a scientifically rigorous manner that includes robust QA/QC procedures. The solicitor should be prepared to summarize their sample processing procedures and demonstrate that they are consistent with current best practices.

Raw taxonomic results are not amenable to resource management decision making, so sample results need to be provided to DWQ in a way that facilitates their integration into state and federal assessment tools by including standardized taxon identification codes, community-level summary statistics, and bioassessment metrics.

Similarly, sometimes utilization of these tools requires sample results to be reported in a specific format. If

Attachment B Scope of Work

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customization is needed, it is anticipated that data delivery will remain consistent over the 5-year period of the contract.

DWQ has been collecting periphyton and phytoplankton samples for over 20 years and the contractor will be required to ensure that the ongoing data provided is comparable to these existing datasets. This process will require a taxonomic harmonization effort to ensure that the taxonomic names from their analyses are consistent with historic data provided by other labs. In some cases, this process may also involve the conversion of historic collection results to make the sample results comparable to those generated by the contractor.

Most of DWQ’s monitoring and assessment programs are routine, which means that sample data can be delivered in a consistent format. However, unforeseen programmatic needs sometimes require adjustments to anticipated analytical work and a more thorough evaluation of taxonomic results. When such circumstances arise, DWQ may require expert phycology consultation.

Work Elements and Anticipated Outputs

This section summarizes the types of samples that the contractor will potentially be processing, considerations for sample quality assurance and quality control (QA/QC) and reporting expectations. Work Element 1: Taxonomic identification and enumeration of benthic periphyton species from samples collected at Utah streams (~100 samples/year)

Work Element Background: DWQ collects periphyton samples from streams throughout Utah as part of its biological assessment program. Biological assessment sites are selected to meet several programmatic objectives. Reference sites are sampled periodically to quantify undisturbed conditions and update biological assessment tools. Other sites are established where restoration activities have occurred to quantify water quality improvements over time. Finally, randomly selected locations are identified to quantify the condition of waters statewide and identify impaired stream segments that are no longer meeting biological designated uses.

Objective: The objective of this work element is to obtain taxonomic results from laboratory-derived subsamples of diatom and “soft algae” taxa from the composite field samples. The taxonomic results require additional analysis from DWQ scientists, so some of the activities are intended to ensure that the laboratory analytical results are more easily incorporated into agency biological assessment tools. Finally, elements are included to ensure taxonomic data integrity and year-to-year comparability of taxonomic results.

Work Activities:

1. Diatom Taxonomy. A minimum of 600 valves/sample will be identified, counted, and measured according to standardized and documented processing procedures. Taxa will be identified to the lowest practical taxonomic level (typically species or genus, if the species cannot be determined).

To ensure taxonomic and nomenclatural consistency, the website the "Diatoms of the United States" (westerndiatoms.colorado.edu) will be used when possible as a continually updated, comprehensive reference to establish the exchange of accurate diatom information between UDWQ and the contractor. Coordination with this website not only establishes consistency, but is an opportunity for both parties to utilize and contribute to the effort to create "a publicly accessible resource to provide accurate information on diatoms" so that "analysts will have the ability to create consistent and accurate data in support of Federal and state compliance with the Clean Water Act” (http://westerndiatoms.colorado.edu/about). This resource supports a pictorial taxonomic diatom key that will serve as a library to document diatom taxa reported by the contractor, including specimens collected and analyzed under this contract.

Taxonomic results should also include sample identification information provided by DWQ and sample processing metadata such as magnification, total valves counted, taxon name with authority, North American Diatom Ecological Database codes (NADED; DS329), and any QC flags. Laboratory sample processing methods should be provided to DWQ yearly, with a document that highlights any changes from previous years, as needed.

about:blank https://pubs.usgs.gov/ds/ds329/

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2. Soft Algae Taxonomy. The laboratory will take additional subsamples from each periphyton sample to identify and quantify the relative abundance of non-diatom algae taxa. A search of multiple subsamples will be conducted until the examination of a new subsample does not reveal new taxa. For each subsample, all individuals encountered will be identified to lowest possible level of taxonomic resolution (typically genus or species) and classified into a relative abundance class (i.e., 1=Abundant, 2=Common, 3=Rare) (see “Qualitative” section in Attachment 1 for details).

3. Laboratory QA/QC. Laboratories are required to have documented QA/QC procedures to ensure taxonomic consistency among analysts and standardized processing procedures. These procedures should include internal QC procedures, recount rules and other corrective actions. Such procedures differ among laboratories, but DWQ recommends procedures that involve the preparation of ~5% duplicate slides that are blindly shared among taxonomic analysts. Laboratory QA/QC procedures should be provided to DWQ yearly, along with a memorandum that briefly summarizes the results of the QA/QC work.

4. Archival Materials. For some samples, DWQ may request that the prepared slides be archived.

Laboratories should specify how prepared slides will be archived. It is anticipated that these will be stored by the contractor, at an existing herbarium, or with DWQ. If DWQ is to be the repository the contractor will be expected to provide associated labeling and tracking information so that samples can be retrieved as needed.

5. Taxa Translator File. DWQ has developed tools that translate the diatom taxonomic results into metrics that can be more easily interpreted in the context of making water quality programmatic decisions. This requires linking the taxonomic results with pre-existing tables from several sources. To assist with these efforts, the laboratory will provide an electronic translator file that creates a crosswalk between its unique taxon-specific name/ID (clean names used for analysis and reporting) and the following: original names with authority and the corresponding name/ID from Diatoms of North America (DONA), Rimet et al.

(2019), and Carlisle et al. (2022). DWQ can assist with access to the lookup tables from other sources, but the contractor will be expected to identify any updates and provide an updated table with yearly sample results and provide a written summary of any changes to the lookup table.

6. Voucher Flora. To assist with regional and national taxonomic standardization efforts, a voucher flora file will be established for DWQ samples and made publicly available through a regional public archive (e.g., https://diatoms.org/practitioners/what-is-a-voucher-flora). A collection of high-resolution images with scale bars, taxonomic names, and codes will be established for each unique taxon identified from Utah samples. These images will be compiled into a common document that can be made publicly available in a regional or national archive. After this is established during the first year of sample processing, it will be updated during subsequent years. A brief memorandum summarizing updates from previous years will be provided to DWQ along with the taxonomic results.

7. Timing. Submitted samples are anticipated to be processed and reported, along with associated files, within a reasonable time, generally within six months upon receipt.

Anticipated Work Activity Outputs:

a. Taxonomic Results. Work Activities 1 & 2. A report will be prepared to summarize the results of the above tasks for each sample. Associated sortable spreadsheets (MS Excel or a tab-delimited text file) will include identification and enumeration of all diatom and soft algae taxa observed in each sample and associated sample identification (unique sample ID, DWQ site name, DWQ monitoring location ID, sample date) and processing metadata. Sample results will also include a summary table with commonly https://diatoms/

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assemblage-level summary statistics such as species richness, species evenness, and Shannon-Wiener diversity. Additional interpretive information and lab notes are also encouraged to be attached to the electronic file at the discretion of the contractor.

b. QA/QC Results. Work Activities 1 & 6. A document that describes the QA/QC procedures that were implemented during sample processing and the results of these procedures should be submitted along with the results. In addition, voucher flora files should be initiated or updated (as appropriate). In addition, any updates from previous voucher flora submissions should be summarized so that previous results can be updated, if appropriate.

c. Taxa Translator File. Work Activity 5. An Excel table that matches each unique diatom taxon identified from DWQ samples with the names/IDs from tables provided by the other sources. The initial translator table will be created to accompany year one taxonomy results. In subsequent years the table will be modified to accommodate any new taxa or updated taxonomic nomenclature. The updated table will be submitted to DWQ, along with a brief document (memorandum) summarizing the changes, why they were made, and in circumstances where taxonomic names are updated, a recommendation about how to update historical taxonomic records.

d. Electronic Data Submission. Reports and data files for diatom results, as described above, will be submitted in electronic form by email to DEQ. The contractor will create a file naming convention that clearly indicates that all associated files are part of that year’s data submission.

Work Element 2: Taxonomic identification and enumeration of phytoplankton taxa in samples collected from lentic waters throughout Utah. (as many as 300 samples over 5 years)

Work Element Background: DWQ collects phytoplankton samples from priority lakes and reservoirs throughout Utah. At each reservoir, samples are collected at the beginning and end of the growing season, resulting in two samples per location. Additional phytoplankton samples are collected from Great Salt Lake wetlands, with varying frequency, for special investigations associated with efforts to ensure the long-term conservation of these important ecosystems.

Objective: To identify and enumerate phytoplankton samples collected by DWQ in Utah waterbodies for monitoring water quality and trophic conditions.

1. Phytoplankton Taxonomy. Phytoplankton will be identified, counted, and measured according to standardized processing procedures. Taxa are identified to the lowest taxonomic level possible, typically genus or species. Contractors should specify any groups of organisms where higher levels of taxonomic resolution are used as operational taxonomic units. Laboratory sample processing methods should be provided to DWQ yearly, with a document that highlights any changes from previous years, as needed.

Taxonomic results should be organized using taxonomic hierarchy and should include a field that provides a unique name or identification number for each taxon found in DWQ phytoplankton samples.

Taxonomic results should also include sample identification information provided by DWQ and sample processing metadata such as taxonomist and any QC flags. Finally, the laboratory should include assemblage-level metrics to assist with data interpretation, such as the relative abundance of cyanobacteria in comparison with other phytoplankton taxonomic groups. Other examples include commonly used community richness, evenness and diversity metrics.

2. Laboratory QA/QC. The laboratory should have established procedures for ensuring that samples are processed accurately and taxa are identified consistently.

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3. Timing. Submitted samples are anticipated to be processed and reported, along with associated files, within a reasonable time, generally no longer than six months upon receipt.

Anticipated Work Element Outputs:

a. Taxonomic Results. Work Activity 1. A report will be prepared to summarize the results of the above tasks for each sample. Associated sortable spreadsheets (MS Excel or a tab-delimited text file) including sample identification information provided by DWQ (location ID, location name, sample date), identification and enumeration of phytoplankton taxa and laboratory processing metadata (i.e., unique sample ID, taxonomist. Sample results will also include a summary table with commonly assemblage-level summary statistics such as species richness, species evenness, and Shannon-Wiener diversity.

Additional interpretive information and lab notes are also encouraged to be attached to the electronic file at the discretion of the contractor.

b. QA/QC Results. Work Activity 2. An annual report summarizing QA/QC activities conducted for phytoplankton analyses and the associated results for Utah samples will be provided to DWQ. The report will also document current sample processing procedures and include a brief memorandum describing any changes from previous processing methods.

c. Electronic Data Submission. Reports and data files for phytoplankton results, as described above, will be submitted in electronic form by email to DEQ. The contractor will create a file naming convention that clearly indicates that all associated files are part of that year’s data submission.

Work Element 3: Ongoing Consultation

Work Element Background: DWQ sometimes collects phytoplankton or periphyton samples for special studies or in response to specific water quality concerns such as spill. In such circumstances the Division sometimes requires additional expertise to provide a more detailed analysis and interpretation of the taxonomic results to meet programmatic objectives.

Objective: DWQ and the contractor will work collaboratively to further our understanding of the ecological connections between water quality, aquatic ecology, and phycology. In addition, during the transition to the new laboratory, consultation services may be required for harmonization of results with historic datasets.

Work Activities: DWQ staff may consult with the contractor on aquatic ecology and phycology research questions such as:

1. Ensuring comparability with Historic Taxonomic Records. DWQ has been collecting diatom information for decades and it is important that previously collected data are comparable to ongoing taxonomic processing results. The first year of the study the contractor will also need to harmonize unique taxa results with those previously used by DWQ.

The harmonization effort will likely involve a database exercise where the contractor takes DWQ historical data records and translates the files into a format consistent with the records they will be submitting to DWQ on an ongoing basis. DWQ can provide prospective contractors with up-to-date files of historic sample results if this information is needed to accurately scope this work element.

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This harmonization effort will need to be updated in subsequent years, but these ongoing updates should be incorporated into routine updates of lookup tables as described in Work Element 1, Activity 5.

2. In Depth-Characterizations of Taxonomic Results. On occasion, DWQ may require more detailed analysis and interpretation of taxonomic results to meet specific programmatic needs. When such circumstances arise, DWQ will meet with the contractor to discuss specific requirements and to develop an appropriate scope of work for each request.

The following are several examples where additional consultation may be needed:

i.The characterization of individual water bodies in cases where focused analysis is required beyond scope of a single data point.

ii.Review and make recommendations of diatom multimetric indices and other assessment tools developed by DWQ and collaborators.

iii.Additional review and interpretation of information needed to ensure data comparability between taxonomic labs beyond the initial effort to harmonize with historic DWQ data.

a. Historic Taxonomic Record Comparability. Work Activity 1. Proposals should include a separate cost estimate for harmonizing ongoing taxonomic results with historic DWQ sample results. Cost associated with ongoing updates to this initial harmonization effort should be incorporated into per sample laboratory processing bids.

b. Additional Consultation. Work Activity 2. Specific outputs for this work element will need to be determined on an ongoing basis. For proposal purposes, contractors should consider providing examples of similar work conducted for other clients and a proposed hourly rate for additional consultation activities.

Work Element 4 (optional): Taxonomic identification and enumeration of zooplankton

Work Element Background: Zooplankton samples are not routinely collected by DWQ. However, the need to process zooplankton samples is occasionally needed for special projects.

Objective: To identify and enumerate zooplankton samples collected by DWQ from Utah wetlands and lakes for monitoring water quality and trophic conditions.

1. Zooplankton Taxonomy. Zooplankton will be identified, counted, and measured according to standardized processing procedures. Standardized procedures used by laboratories should be provided to DWQ and updated, if appropriate, on an annual basis. Zooplankton taxa will be identified to the lowest taxonomic level possible, or for some taxonomic groups to an established and agreed upon higher level of taxonomic resolution. Taxonomic results will include site/sample metadata provided by DWQ, sample processing metadata, hierarchical taxonomic information (i.e., order, family, genus, species) and a unique name/ID for each zooplankton taxon and counts/relative abundance.

2. Laboratory QA/QC. Work Activity 2. An annual report summarizing QA/QC activities conducted for zooplankton analyses and the associated results for Utah samples will be provided to DWQ. The report

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will also document current sample processing procedures and include a brief memorandum describing any changes from previous processing methods.

3. Timing. Zooplankton sampling will be part of special projects and expectations for delivery of taxonomic results may vary, but DWQ anticipates that sample results will be returned to DWQ within six months of sample submission to the contractor.

a. Taxonomic Results. Work Activity 1. A report will be prepared to summarize the results of the zooplankton taxonomic analysis. Associated sortable spreadsheets (MS Excel or a tab-delimited text file) will include identification and enumeration of all zooplankton taxa observed in each sample and associated sample identification (unique sample ID, DWQ site name, DWQ monitoring location ID, sample date) and processing metadata. Sample results will also include a summary table with commonly assemblage-level summary statistics such as species richness, species evenness, and Shannon-Wiener diversity. Additional interpretive information and lab notes are also encouraged to be attached to the electronic file at the discretion of the contractor.

b. QA/QC Results. Work Activity 2. An annual report summarizing QA/QC activities conducted for zooplankton analyses and the associated results for Utah samples will be provided to DWQ. The report will also document current sample processing procedures and include a brief memorandum describing any changes from previous processing methods.

c. Electronic Data Submission. Reports and data files for zooplankton results, as described above, will be submitted in electronic form by email to DEQ. The contractor will create a file naming convention that clearly indicates that all associated files are part of that year’s data submission.

Project Requirements

The contractor will provide any equipment needed to properly identify, quantify and create archival materials for stream biota, including any computer software and hardware needed to conduct the work. The contractor will also provide DWQ with a Chain of Custody and similar materials needed to track samples.

DWQ will provide agreed upon field collection materials, including sample bottles and sample preservation materials. DWQ will also ensure that any necessary metadata and chain of custody forms are submitted with the samples each fall.

Prospective contractors are encouraged to propose a collaborative workflow for conducting the work, but DWQ envisions that the workflow will proceed in three phases:

Phase 1: Project Kickoff To be completed within the first month of the start of the contract.

DWQ has attempted to provide sufficient detail in this scope of work to provide prospective contractors with sufficient detail to accurately scope the project when preparing bids. However, the agency acknowledges that additional details will need to be worked out collaboratively to ensure the work element outputs are delivered as accurately and efficiently as possible.

• Review sample preservation and shipping procedures

• Review data submission forms and requisite metadata

• Establish communication procedures and appropriate contacts

• Establish and agree upon data submission formats

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Phase 2: Taxonomic Harmonization To be completed within the first year of the start of the contract.

DWQ will provide the contractor with historic data so that the contractor can develop a workplan to complete the harmonization of historic data with the ongoing taxonomic analyses conducted over the five years of the contract. Given the complexities of this element of the contract, DWQ envisions that the contractor will work with the agency to develop a refined work plan that includes a detailed workflow to ensure that this work is completed within the first year of the contract. This work plan will include interim completion dates for work elements and will also specify what data and information will be provided by the agency to ensure that the interim and final deadlines are met.

Phase 3: Yearly Sample Processing Ongoing By the end of March, each year of the contract, monitoring staff will develop a work plan for the upcoming field season, including an estimate of the number and type of samples to be collected during the upcoming field season to the solicitor. While unforeseen circumstances are always possible, the plan will also include foreseeable anticipated consultation and non-routine sample processing requests.

At the end of each field season (approximately October 31), DWQ will deliver samples, preserved via solicitor protocols and requisite Chain of Custody forms to the solicitor.

The solicitor will send sample results and associated metadata, in agreed upon formats, to DWQ prior to the field season the following year (early May).

Ongoing Project Deliverables

Taxonomic Results (yearly): Taxonomic summary tables providing relative abundance of taxa, identified to the lowest practical level of taxonomic resolution will be submitted electronically to the agency for samples collected the previous year prior to beginning of the next field season.

Taxonomic Harmonization Tables (by the end of first year): Tables will be provided to DWQ that translate historic taxonomy results into files that are comparable with the taxonomic results provided by the contractor.

Project Budget

The number of samples submitted for processing will vary year-to-year based on changing programmatic needs.

As a result, DWQ requests that contractors provide per sample bids for routine sample processing.

Pricing is guaranteed for one year. All future requests for increase must be submitted with market research and receive approval from the agency. Each increase will be guaranteed for one year.

Literature Cited

Carlisle, D.M., Spaulding, S.A., Tyree, M.A., Schulte, N.O., Lee, S.S., Mitchell, R.M. and Pollard, A.A., 2022. A web-based tool for assessing the condition of benthic diatom assemblages in streams and rivers of the conterminous United States. Ecological indicators, 135, p.108513.

Rimet, F., Gusev, E., Kahlert, M., Kelly, M.G., Kulikovskiy, M., Maltsev, Y., Mann, D.G., Pfannkuchen, M., Trobajo, R., Vasselon, V. and Zimmermann, J., 2019. Diat. barcode, an open-access curated barcode library for diatoms. Scientific Reports, 9(1), p.15116.

Standard Operating Procedures:

Protocols for the Preparation and Analysis of

Periphyton, Diatom and Phytoplankton Samples

Rushforth Phycology, LLC

Attachment 1

Total Phytoplankton Filtration Protocol

CONTENTS

Sample Types and Procedures

Analysis of Periphyton Samples – Qualitative

Preparation of Diatom Sample Material

Preparation of Diatom Slides

Analysis of Diatom Samples – Quantitative

Preparation of Phytoplankton Sample Material

Analysis of Phytoplankton Samples – Quantitative

References

Appendix I - Stages of Laboratory Processing and Analysis of Algal Sample

Appendix 2 – EPL Counting Software for Quantitative Analysis

Appendix 3 – Example Report, Periphyton Composition Analysis

Appendix 4 – Example Report, Phytoplankton/Diatom Quantitative Digital Data Matrix

Appendix 5 – Example Phytoplankton/Diatom Quantitative Text Report

Appendix 6 – Phytoplankton Filtration Protocol

Equipment

Process

Logging Samples

Labeling sample tubes

Filtering Samples

Other sample types

Archiving Samples

Preservation

SAMPLE TYPES AND PROCEDURES

1. Purpose

Our research involves analysis of three distinct types of algal samples with specific modifications for subsample type. Periphyton samples, phytoplankton samples, and diatom samples are each treated with separate protocols regarding processing, examination, and data analysis. Protocols for all stages of each type of algal subsample analysis are presented herein.

For the purposes of studies including diatom and benthic soft algae, we subsample each original periphyton sample into a single soft algae periphyton subsample and a single diatom subsample. The qualitative perphytion sample will be prepared for an analysis of attached algae, including diatoms to the classification of pennate or centric. The diatom sample will undergo further preparation (see “Diatom Cleaning” section) for analysis of diatoms at the species level. For studies involving phytoplankton populations, samples are subsampled following protocol for net plankton or total plankton samples (see “Analysis of Phytoplankton Samples” section).

2. Definitions

Qualitative periphyton subsamples: Qualitative periphyton samples are collected from attached algal communities in streams and rivers. Samples are obtained by scraping stones and other attached substrata directly from the river bed. A representative piece of any visible algae at the collection site is included in the sample. In analysis of qualitative periphyton samples, algal forms are identified to the species level, and scored according to relative abundance. Due to the variety of growth patterns of attached algae in periphyton samples, subsamples must be made carefully to ensure that all algae present in the original sample are represented in the subsample.

Diatom Subsamples: Diatom subsamples are divided from the original sample and prepared to facilitate accurate identification of diatoms to the lowest taxonomic category possible. Identifications are made based on the structure of the cell wall, symmetry, valve structure, and other physical characteristics (Patrick and Reimer 1966). A careful analysis of diatom cell structures relies on frustules being cleared of organic matter. We conduct a nitric acid digestion procedure for each sample to clear internal and external organic matter in order to clearly view the silica cell walls of all diatoms present. We prepare, analyze, and archive permanent slides for each diatom sample. Analysis of diatom samples is quantitative based on careful measurements during slide preparation and precise counts from microscope enumeration.

Phytoplankton Samples: Phytoplankton samples are collected either as net plankton, total plankton, or surface samples. Studies of phytoplankton involve direct observation and enumeration of the dominant algae present in the water column sampled. For total plankton samples, phytoplankton floras are examined from both the upper and lower halves of the euphotic zone of the sample location. We determine the number of each alga present in each sample and the number of each alga per milliliter of water. We also determine the biovolume of the total number of each individual organism in cubic micrometers, the relative density of each taxon according to its biovolume, and the in-sample rank of each taxon according to biovolume (biomass) in each sample. We also perform several descriptive statistical assessments of each sample.

3. Methods

A “Subsample/Sample Volume Form” is printed for the sample set. This form tracks assigned subsample ID’s for periphyton and diatom subsamples, the volume of the original sample, and subsample volumes.

The contents of the sample are poured into a graduated cylinder to measure the original volume prior to subsampling. This volume is recorded on the Subsample/Sample Volume Form assigned to the project.

A subsample ID number is assigned for both the qualitative periphyton subsample and the diatom subsample taken from the original sample. This ID consists of 9 letters from the site name of the originals sample followed by a –D for diatom samples and a –P for periphyton samples. In cases where a storette number is provided in the original sample, it will be included in the subsample ID.

The algae in the sample is suspended by shaking or swirling before the subsample volumes are carefully transferred to each corresponding container labeled with the subsample ID number. If visible algae occur in the sample, a portion is included in the periphyton subsample. Periphyton subsamples are stored under dark refrigeration until the time of micrscopic analysis. Protocol for the further preparation of diatom samples continues in the following two sections, “Diatom Cleaning by Nitric Acid Digestion,” and “Preparation of Diatom Slides.”

ANALYSIS OF PERIPHYTON SAMPLES – QUALITATIVE

Qualitative periphyton samples are collected from attached algal communities in streams and rivers. Samples are obtained by scraping stones and other attached substrata directly from the river bed. A representative piece of any visible algae at the collection site is included in the sample. Samples are typically gathered in 200ml bottles and if they are not returned to the laboratory on the day of collection, they may be preserved in formalin solution. This protocol describes the process of qualitative community analysis of such periphyton samples.

Analysis of these communities provides a broad view of the algae present in the habitat of the sampling site and can produce important information regarding extant water quality. Examining changes through time in periphyton communities provides data concerning changes in water quality and may result in conclusive findings regarding taste or odor problems at the collection site.

Periphyton samples are examined microscopically. Algae encountered are identified to the lowest taxonomic level possible, and scored according to relative abundance (see “methods” below for elaboration). Diatoms are categorized as pennate or centric. If the original sample has been subsampled for diatom analysis, diatoms will be identified to the species level in a separate procedure (see “Analysis of Diatoms” section).

The examination of periphyton samples is a qualitative process intended to generate a comprehensive list of algal taxa in a sample, providing an estimate of the species composition of the habitat from which the sample was collected. As specified in the “Philadelphia Academy of Natural Sciences’ “Protocols for the Analysis of Algal Samples Collected as Part of the U.S. Geological Survey National Water-Quality Assessment Program,” identification of every algal taxa in a sample will most likely not occur in a qualitative analysis of a periphyton sample. It is generally agreed, however, that a majority of the species in a sample will be encountered in a “reasonable search” (Academy of Natural Sciences 2002).

2. Methods

A “Periphyton Community Composition Analysis Form” is printed and filled in with the periphyton subsample ID# created in the subsampling process.

A wet mount is prepared. A segment of each piece by visible algae should be included.

Segments are placed on a 1 X 3 inch glass microscope slide using tweezers, and a cover slip is carefully placed over the material. Several drops of subsample water are placed on the same slide. Multiple wet mounts may be necessary to effectively analyze each type of visible algae in a subsample.

Filamentous, colonial, and single celled algal forms are identified to the lowest taxonomic level possible at 400 X magnification using a Nikon Eclipse E200 microscope equipped with Nikon's CFI60 infinity optical system. In cases where the small number of cells or the absence of reproductive cells prevent identification at the species level, taxa are identified to the generic level and listed with “species” following the generic name.

On the “Periphyton Community Composition Analysis Form,” the relative abundance of each algal form encountered in each wet mount is recorded. Relative abundance is recoded as trace, rare, common, or abundant. In general, if a taxon is observed as a single or very rare specimen, it is recorded as trace. If a taxon is present in less than approximately 10% of the microscopic examination fields, it is recorded as rare. If a taxon is present in approximately 10-20% of the microscopic examination fields, it is recorded as common, and if a taxon is present in more than approximately 20% of the microscopic examination fields, it is recorded as abundant.

A second and third wet mount are prepared and the process repeated. If after analysis of the third wet mount new forms are still encountered, a fourth wet mount is prepared and an additional analysis is conducted. If no new forms are encountered in the third wet mount, results are documented on the Composition Form and the microscopic analysis is complete.

Samples are assessed to estimate total biomass (low, moderate, or high), conspicuous odor, and the presence of vascular plants. This information is recorded in the box labeled “notes” at the bottom of page 2 of the “Periphyton Community Composition Analysis Form.”

Algal forms that are generally rarely encountered or that being encountered for the first time in a wet mount will be digitally photographed for reference.

Analysis of a single periphyton sample take an average of 2 hours to complete. Samples with high species diversity take longer.

PREPARATION OF DIATOM SAMPLE MATERIAL

In order to identify diatoms at the species level, organic matter must be removed from all diatoms in a sample, leaving clean frustules that allow for clear visibility of diatom structure. This protocol describes a procedure for clearing a sample of organic matter through nitric acid digestion and centrifuging.

If the organic matter contained in a sample is light, the acid/hotplate procedure described here may not be necessary. A hydrogen peroxide method will be used in this circumstance.

Diatom Cleaning by Nitric Acid Digestion

2. Methods

In the cleaning process of diatom slide preparation, depending upon the amount of organic matter and apparent diatom mass present approximately 25ml of each diatom subsamble is placed in a 100 ml beaker.

An amount of concentrated nitric acid (~15.8 M) equal to the amount of the sample is very carefully added to the beaker. A small amount of acid should be added to the sample to determine a potential hazardous reaction before the full amount is slowly added.

This solution is boiled for 30 minutes.

A very small amount of potassium dichromate is then slowly added to the solution until it turns slightly brown in color. The sample is removed from the hotplate and cooled.

When organic matter is low in a sample, adequate digestion of organic matter may be obtained by placing 25ml concentrated hydrogen peroxide in the beaker with the subsample and allowing 24-72 hours for digestion.

The subsample is transferred to a centrifuge tube and capped. Sample information is written on the outside of the centrifuge tube. The filled tube is placed in one of six slots in the Clinaspin centrifuge. The number of the centrifuge slot is recorded on the “Diatom Preparation Form” to ensure careful tracking of the sample through the transfer from the beaker to the centrifuge.

The solution is centrifuged for three to five minutes at 3150 RPM (maximum RCF = 1132 x

g) in the Clinaspin centrifuge.

The supernatant is decanted and discarded. Care is taken to ensure that all diatom-containing sediment remains in the centrifuge tube.

The sediment containing diatoms is re-suspended by adding distilled water to the centrifuge tube. This volume is again centrifuged for three to five minutes at 3150 RPM.

The diatom pellet is washed and centrifuged an additional two to three times in distilled water in order to remove all acid residues

After the final washing process, the material remaining in the centrifuge tube is carefully swirled. The contents are poured into a marked container labeled with sample information including the original sample ID, collection date, and the subsample ID created in the subsampling process and recorded on the “Subsample/Sample Volume Form.”

Using distilled water, the centrifuge tube is rinsed of any remaining material. The water from this rinse is used to bring the sample to 10-20 ml. At this point, samples are ready for slide preparation.

PREPARATION OF DIATOM SLIDES

The most common type of diatom slide mount is the strewn mount (Patrick and Reimer 1966). A strewn mount is a permanent mount made by allowing a small volume (several drops) of the prepared sample to dry on a coverslip. The coverslip is then reverserd and positioned on a warmed slide on which a drop of mountant has been placed.

When preparing diatom slides, it is important to use a mountant with a high refractive index to ensure visibility. The mounting agent used in the preparation of our diatom slides is Naphrax.

A “Diatom Slide Preparation Form” is printed. The diatom subsample ID created during the subsampling process is recorded and linked to a project slide number and a slide reference.

The “Diatom Slide Preparation Form” includes information about the digestion method, the slide preparation used, and the mounting medium used.

A label is printed with information from the “Diatom Slide Preparation Form” including a project slide number, the date of collection, the diatom subsample ID, and the slide reference. The label is placed on a clean 1X3 inch microscope slide.

A small portion, usually two to four drops of the stock solution depending upon the density of diatoms in the pellet, is diluted with distilled water in a separate culture tube.

Approximately l ml of the diluted solution is dispersed on a glass cover slip and air dried.

The cover slip is then mounted on a slide using Naphrax diatom mountant. The Naphrax is diluted with toluene and a drop is placed on a glass slide and brought to a low boil over an alcohol lamp. The cover slip with the diatom frustules is then placed over the hot Naphrax and pressed firmly against the slide until all air bubbles are forced out.

The slide is allowed to cool for five minutes, and excess mountant is scraped away with a razor blade. At this point, the slides are ready for examination.

ANALYSIS OF DIATOM SAMPLES – QUANTITATIVE

Diatoms species are often sensitive indicators of biotic and/or abiotic factors in aquatic environments. Quantitative information about diatom assemblages can be valuable in assessing current water quality and in establishing baselines for ecosystems that are in flux or in danger of perturbation.

A sizable literature has developed surrounding diatoms as water quality indicators, making analysis of quantitative samples valuable.

Prepared diatom slides are carefully examined and diatom species identified to the lowest taxonomic level possible.

Microscopy on diatom slides is performed using a Nikon Eclipse E200 microscope equipped with Nikon's CFI60 infinity optical system.

Identifications are performed using standard taxonomic works and personal reference slide collections.

Digital images are taken for any species that is generally rarely encountered or that is encountered for the first time. Images are then added to a database of diatom species from the region, which included information on average biovolume measurements, taxonomic characteristic ,nomenclature changes, authorities, notes on variance, etc.

PREPARATION OF PHYTOPLANKTON SAMPLE MATERIAL

Cells from open water phytoplankton samples must be concentrated for purposes of observation. Cells per mL values are estimated based on concentration values derived from the original volume of a sample and the volume of a processed subsample.

Samples from each collecting locality are placed in 2-liter containers, iced and returned to the laboratory for quantitative analysis. Samples are stored under refrigeration in the dark until processing is performed.

At the time of processing, a 0.5 liter subsample is removed after mixing. This subsample is suction filtered through a 1.2 micrometer pore size Millipore filter. The algal cells retained on the filter are re-suspended in 5 ml of distilled water in a 50 ml beaker (see filtration protocols, Appendix 6).

ANALYSIS OF PHYTOPLANKTON SAMPLES – QUANTITATIVE

Biological water quality is reflected by phytoplankton floras in an aquatic ecosystem.

Studies of phytoplankton communities are conducted in order to determine extant biological water quality as well as changes in water quality through time. It is important to perform biological studies to establish a baseline of data for comparison with changes in lake and reservoir systems due to system perturbations that may occur over time.

Our phytoplankton analyses include both net plankton and total plankton samples.

Protocol for collection, processing, microscopy, and numerical analysis for each type of phytoplankton sample follows in the “Methods” section.

Both types of phytoplankton samples are examined microscopically. Algal forms are identified to the lowest possible taxonomic level (see “methods” below for elaboration). In phytoplankton analysis, the diatoms Asterionella formosa, Fragilaria crotonensis, Fragilaria virescens, Melosira granulata var. angustissima, Melosira granulata, Stephanodiscus niagarae, and Tabellaria fenestrata are identified to the specific level. In cases where identification to the species level is not possible, these diatoms are identified to the generic level and listed with “species” following the generic name. Other diatoms are categorized and counted as pennate or centric. If the original sample has been subsampled for diatom analysis, diatoms will be identified to the species level in a separate procedure (see “Analysis of Diatoms” section).

2.1 Total plankton samples

Total plankton samples are collected by withdrawing water from the euphotic zone. One sample is taken from the upper half of the euphotic zone as determined by measuring Secchi extinction depth and collecting a continuous column of water from the surface to extinction depth. A separate sample is collected from the lower euphotic zone from Secchi extinction depth to a depth 3 times the Secchi extinction depth.

2.1.1 Microscopy on total plankton samples is performed using a Nikon Eclipse E200 microscope equipped with Nikon's CFI60 infinity optical system.

2.1.2 Identifications are performed using standard taxonomic works and personal reference slide collections. Colonial and single celled algal forms are identified to the lowest taxonomic level possible at 400 magnifications. In cases where the small number of cells or the absence of reproductive cells prevents identification at the species level, taxa are identified to the generic level and listed with “species” following the generic name.

2.1.3 Aliquots are removed from subsamples and placed into a Palmer Counting Chamber for enumeration (Palmer and Maloney 1954).

The Palmer Cell is advantageous for counting total plankton samples since the algae can be studied at 400 magnifications rather than 160X. Counting at a greater magnification facilitates species identification, especially of smaller taxa. This increased resolution can be an important factor, especially for nannoplankton work.

Furthermore, studies of total plankton in standing water at high magnification are often important since they generally contain a more comprehensive suite of organisms than net plankton samples which tend to lose small organisms through the net mesh.

2.1.4 One or more transect from each Palmer cell subsample is studied to determine the mean number of cells per transect. The number of algal cells present per milliliter of reservoir water is then calculated by multiplying the mean number of cells per transect by appropriate multiplication factors.

2.1.5 Important species indices (ISIs) are calculated for each taxon by multiplying the percent frequency of the taxon by its average relative density (Kaczmarska and Rushforth 1983).

2.2 Net plankton samples

Net plankton samples are collected using a13 cm diameter plankton net with 50 micrometer mesh pores. Net plankton samples are collected by lowering the weighted plankton net to the bottom of the euphotic zone, then towing the net upward through the water column to the surface of the reservoir. The depth (length) of the tow is recorded and used in calculating biomass for net samples.

Euphotic zone depth is estimated by determining Secchi Disk extinction depth and multiplying this depth by three.

2.2.1 Net samples collected in this manner can be considered composite euphotic zone net plankton samples. After towing the net through the water column, the collecting vial attached to the net is flushed into a laboratory bottle for subsequent analysis.

2.2.2 Net plankton samples are returned to the laboratory and are stored under refrigeration in the dark until processing is performed.

2.2.3 The volume of each net plankton sample is measured in the laboratory using a graduated cylinder. After mixing, a subsample of each net plankton sample is placed in a Sedgewick-Rafter microscope chamber for counting (American Public Health Association 1995; Whipple et al. 1948). Algae are counted at 160 magnifications.

2.2.4 Microscopy on net plankton samples is performed using a Nikon Eclipse E200 microscope equipped with Nikon's CFI60 infinity optical system.

2.2.5 Identifications are performed using standard taxonomic works and personal reference slide…