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8.d) Zavorals Creek September 2013 Technical Summary Macroinvertebrae-WCDAnalysis of a biological sample: Technical summary of methods and quality assurance procedures Prepared for Washington County Conservation District Erik Anderson, Project Manager December 13, 2013 by W. Bollman, Chief Biologist Rhithron Associates, Inc. Missoula, Montana METHODS Sample processing One macroinvertebrate sample collected from Zavoral's Creek was delivered to Rhithron's laboratory facility in Missoula, Montana on September 20, 2013. The sample arrived in good condition. The sample was decanted prior to shipment; preservative was replenished immediately upon arrival at the laboratory. A chain of custody document containing sample identification information was provided by the Washington County Conservation District (WCD) Project Manager. Upon arrival, the sample was unpacked and examined, and checked against the WCD chain of custody. An inventory spreadsheet including project code and internal laboratory identification numbers was uploaded into the Rhithron database prior to sample processing. A subsample of a minimum of 300 organisms was achieved using methods consistent with Minnesota Pollution Control Agency protocols (MPCA 2004). A Caton sub -sampling device (Caton 1991), divided into 30 grids, each approximately 6 cm by 6 cm was used. The sample was thoroughly mixed in its jar, poured out and evenly spread into the Caton tray, and individual grids were randomly selected. The contents of each grid were examined under stereoscopic microscopes using 10x -30x magnification. Grid selection, examination, and sorting continued until at least 300 organisms were sorted. All aquatic invertebrates from each selected grid were sorted from the substrate, and placed in 80% ethanol for subsequent identification. After the target number of organisms was obtained in the subsample, a large/rare search was performed: the Caton tray was scanned for additional organisms that were not collected in the subsample. These organisms were placed in a separate vial and labeled as "Large/Rare Organisms". Unsorted sample fractions were retained and stored at the Rhithron laboratory. Organisms were individually examined by certified taxonomists, using 10x — 80x stereoscopic dissecting scopes (Leica S8E) and identified to target taxonomic levels consistent with MPCA stream protocols (MPCA 2004), using appropriate published taxonomic references and keys. Identification, counts, life stages, and information about the condition of specimens were recorded on electronic bench sheets. Organisms that could not be identified to the taxonomic targets because of immaturity, poor condition, or lack of complete current regionally -applicable published keys were left at appropriate taxonomic levels that were coarser than those specified. To obtain accuracy in richness measures, these organisms were designated as 'snot unique" if other specimens from the same group could be taken to target levels. Organisms designated as "unique" were those that could be definitively distinguished from other organisms in the sample. Taxonomists examined organisms in the Large/Rare vial and determined there were not any unique taxa collected. Identified organisms were preserved in 80% ethanol in labeled vials, and archived at the Rhithron laboratory. Chironomids were carefully morphotyped using IN — 80x stereoscopic dissecting microscopes (Leica S8E) and representative specimens were slide mounted and examined at 200x — 1000x magnification using an Olympus BX 51 or Leica DM 1000 compound microscope. Slide mounted organisms were archived at the Rhithron laboratory. Quality control procedures Quality control procedures for initial sample processing and subsampling involved checking sorting efficiency. An independent observer microscopically re-examined 25% of sorted substrate from the sample. Quality control procedures proceeded as follows: The quality control technician poured the sorted substrate from the processed sample out into a Caton tray, redistributing the substrate so that 25% of it could be accurately lifted out by removing entire grids in a random fashion. Grids were selected, and re-examined until 25% of the substrate was re -sorted. All organisms that were missed were counted and this number was added to the total number obtained in the original sort. Sorting efficiency was evaluated by applying the following calculation: SE = n, x 100 n, +n2 where: SE is the sorting efficiency, expressed as a percentage, nl is the total number of specimens in the first sort, and nZ is the total number of specimens expected in the second sort, based on the results of the re -sorted 25%. Quality control procedures for taxonomic determinations of invertebrates involved checking accuracy, precision and enumeration. All organisms were re-identified and counted by an independent taxonomist. Taxa lists and enumerations were compared by calculating a Bray - Curtis similarity statistic (Bray and Curtis 1957), Percent Taxonomic Disagreement (PTD) and Percent Difference in Enumeration (PDE). Routinely, discrepancies between the original identifications and the QC identifications are discussed among the taxonomists, and necessary rectifications to the data are made. Discrepancies that cannot be rectified by discussions are routinely sent out to taxonomic specialists for identification. However, taxonomic certainty for identifications in this project was high and no external verifications were necessary. Data analysis Taxonomic data and counts were entered into the Rhithron laboratory database, and an uploadable spreadsheet file, consistent with MPCA stream data requirements, including taxonomy, counts, life stages, sample metadata, and other information was generated. Taxa lists and counts were constructed and standard metric calculations for aquatic invertebrate assemblages were made using Rhithron's customized database software. A sites -by - taxa matrix in spreadsheet format was also created. RESULTS Quality Control Procedures Sorting efficiency was 95.40%, taxonomic precision for identification and enumeration was 98.01% (Bray Curtis), 0.93% PTD and 1.07% PDE, and data entry efficiency was 100%. These similarity statistics fall within acceptable industry criteria (Stribling et al. 2003). Data analysis Taxa list and metric summary pages are given in the Appendix. Electronic spreadsheets were provided to the WCD Project Manager via email. 2 REFERENCES Bray, J. R. and J. T. Curtis. 1957. An ordination of upland forest communities of southern Wisconsin. Ecological Monographs 27: 325-349. Caton, L. W. 1991. Improving subsampling methods for the EPA's "Rapid Bioassessment" benthic protocols. Bulletin of the North American Benthological Society. 8(3): 317-319. MPCA, 2004. Laboratory Analysis — Invertebrate Sample Processing and Identification. State of Minnesota Standard Operating Procedure. Stribling, J.B., S.R Moulton II and G.T. Lester. 2003. Determining the quality of taxonomic data. J.N. Am. Benthol. Soc. 22(4): 621-631. 3 APPENDIX Taxa list and metric summary Washington County Conservation District, MN Zavoral's Creek September 2013 Taxa Listing Yes RAI No.: WCD13ZC2001 5 Client ID: 0.31% Date Coll.: 9/17/2013 No. Jars: 1 Taxonomic Name Count Other Non -Insect Yes Acari 11 Nemata 1 Turbellaria 2 Asellidae CG Caecidotea sp. 3 Gammaridae 4 Gammarus sp. 69 Physidae Unknown Physa sp. 22 Oligochaeta Yes Oligochaeta 1 Ephemeroptera 12.62% Baetidae Larva Baetis sp. 41 Leptophlebiidae Yes Paraleptophlebia sp. 2 Plecoptera 4.92% Capniidae Larva Early Instar Capniidae 16 Nemouridae Yes Amphinemura sp. 1 Trichoptera 0.31% Glossosomatidae Larva Glossosoma sp. 1 Hydropsychidae Yes Diplectrona modesta 2 Hydropsychidae 6 Lepidostomatidae Larva Early Instar Lepidostoma sp. 2 Limnephilidae Yes Hesperophylax designatus 2 Limnephilus sp. 3 Psychomyiidae Larva Lype diversa 4 Rhyacophilidae Yes Rhyacophila sp. 9 Friday, December 13, 2013 Project ID: WCD13ZC2 RAI No.: WCD13ZC2001 Sta. Name: Zavoral's Creek STORET ID: PRA Unique Stage Qualifier BI Function 3.38% Yes Unknown 5 PR 0.31% Yes Unknown 5 UN 0.62% Yes Unknown 4 PR 0.92% Yes Unknown 8 CG 21.23% Yes Unknown 4 SH 6.77% Yes Unknown 8 SC 0.31% Yes Unknown 10 CG 12.62% Yes Larva 5 CG 0.62% Yes Larva 1 CG 4.92% Yes Larva Early Instar 1 SH 0.31% Yes Larva 2 SH 0.31% Yes Larva 0 SC 0.62% Yes Larva 0 CF 1.85% No Larva Early Instar 4 CF 0.62% Yes Larva 1 SH 0.62% Yes Larva 5 SH 0.92% Yes Larva 3 SH 1.23% Yes Larva 2 SC 2.77% Yes Larva 1 PR Taxa Listing STORET ID: RAI No.: WCD13ZC2001 Client ID: PRA Date Coll.: 9/17/2013 No. Jars: 1 Taxonomic Name Count Diptera 0.31% Ceratopogonidae Larva Ceratopogoninae 1 Dixidae 2.46% Dixa sp. 8 Empididae 1 Empididae 1 Neoplasta sp. 1 Ptychopteridae 6 Ptychoptera sp. 1 Simuliidae Larva Simulium sp. 17 Chironomidae 0.31% Chironomidae Larva Corynoneura sp. 2 Eukiefferiella sp. 20 Heterotrissocladius sp. 1 Heterotrissocladius sp. 1 Micropsectra sp. 4 Odontomesa sp. 3 Orthocladiinae 2 Parametriocnemus sp. 1 Parametriocnemus sp. 16 Paratendipes sp. 1 Polypedilum sp. 2 Prodiamesa sp. 3 Synorthocladius sp. 2 Tanytarsus sp. 1 Tanytarsus sp. 1 Thienemanniella sp. 2 Thienemannimyia Gr. 1 Tvetenia sp. 34 Zavrelimyia sp. 1 Sample Count 325 Friday, December 13, 2013 Project ID: WCD13ZC2 RAI No.: WCD13ZC2001 Sta. Name: Zavoral's Creek STORET ID: PRA Unique Stage Qualifier 131 Function 0.31% Yes Larva Early Instar 6 PR 2.46% Yes Larva 1 CG 0,31% No Larva Early Instar 6 PR 0.31% Yes Larva 5 PR 0.31% Yes Larva 7 CG 5.23% Yes Larva 6 CF 0.62% Yes Larva 7 CG 6.15% Yes Larva 8 CG 0.31% No Pupa 0 CG 0.31% Yes Larva 0 CG 1.23% Yes Larva 4 CG 0.92% Yes Larva 4 CG 0.62% No Pupa Damaged 6 CG 0.31% No Pupa 5 CG 4.92% Yes Larva 5 CG 0.31% Yes Larva 10 CG 0.62% Yes Larva 6 SH 0.92% Yes Larva 3 CG 0.62% Yes Larva 2 CG 0.31% No Pupa 6 CF 0.31% Yes Larva 6 CF 0.62% Yes Larva 6 CG 0.31% Yes Larva 5 PR 10.46% Yes Larva 5 CG 0.31% Yes Larva 8 PR Project ID: WCD13ZC2 RAI No.: WCD13ZC2001 Ste. Name: Zavoral's Creek Client ID: STORET ID Coll. Date: 9/17/2013 Latitude: Abundance Measure: Sample Count: Sample Abundance: Coll. Procedure: Sample Notes: Longitude: 325 1,950.00 16.67% of sample used Taxonomic Composition Category R Terrestrial 7 Other Non -Insect 6 Oligochaeta 1 Odonata 3 Ephemeroptera 2 Plecoptera 2 Heteroptera MTV Meqaloptera 3 Neuroptera 7 Trichoptera 7 Lepidoptera 1 Coleoptera Diptera 5 Chironomidae 15 Dominant Taxa 44.31 Category 78.77% Gammarus ■ Lepi dopter a Beets 692 Tvetenia 4.010 Phvsa ®Diptera Eukiefferiella 0.096 Simulium 29 Parametriocnemus Im Capniidae 30.15% Acari 8.31 Rhvacophila 3 Dixa 8.31% Hvdroosvchidae 53.85% Micropsectra 37.54% Lvpe diversa 1.000 Caecidotea 0.500 Functional Composition Category R Predator 7 Parasite E Ter r estr i al Collector Gatherer 17 Collector Filterer 3 Macrophvte Herbivore ■Dl i gochaeta Piercer Herbivore 27 Xvlophaqe MTV Scraper 3 Shredder 7 Omnivore 17 Unknown 1 A PRA E Richness Biolndex Description E Ter r estr i al 108 33.23% ■ Other No.-mee.t 1 0.31% ■Dl i gochaeta Montana DEQ Plains (Bukantis 1998) 27 ■ Dd..aie MTV Montana Revised Valleys/Foothills (Bollman 1998) 0 Eph-, pt.ra 43 13.23% DPle.optera 17 5.23% 0Heter.Pt Dominance 0 M egal ptar a 21.23% DominantTaxa (2) Percent Neu, opter e Dominant Taxa (3) Percent 44.31 T, i chapter a 78.77% Diversity ■ Lepi dopter a 29 692 Col eoptera 4.010 Marqalef D ®Diptera Simpson D 0.096 D Chi r on..i da. 29 8.92% Im 98 30.15% Predator Percent A PRA 69 21.23% 41 12.62% 34 10.46% 22 6.77% 20 6.15% 17 5.23% 17 5.23% 16 4.92% 11 3.38% 9 2.77% 8 2.46 6 1.85% 4 1.23% 4 1.23% 3 0.92% A PRA 27 8.31 0 Cal Iec rFi I terer 148 45.54% 0 Col l ector Gatherer M..r.phyte Herbiv re 27 8.31% 11110-w.re 1111P. -in. OPier- Herbivore E P r edamr 0s raper 27 8.31% ■ shredder 95 29.23% ■Unkn.wn 7) 0 Xyl .phage 1 0.31 Bioassessment Indices 38 E Richness Biolndex Description Score Pct Rating BIBI B -IBI (Karr at al.) 20 40.00% MTP Montana DEQ Plains (Bukantis 1998) 27 90.00% None MTV Montana Revised Valleys/Foothills (Bollman 1998) 10 55.56% Slight MTM Montana DEQ Mountains (Bukantis 1998) 11 52.38% Moderate Friday, December 13, 2013 Metric Values and Scores Metric Value Composition Taxa Richness 38 E Richness 2 P Richness 2 T Richness 7 EPT Richness 11 EPT Percent 27.38% All Non -Insect Abundance 109 All Non -Insect Richness 7 All Non -Insect Percent 33.54% Oligochaeta+Hirudinea Percent 0.31% Baetidae/Ephemeroptera 0.953 Hvdropsvchidae/Trichoptera 0.276 Dominance Dominant Taxon Percent 21.23% DominantTaxa (2) Percent 33.85% Dominant Taxa (3) Percent 44.31 Dominant Taxa It 0) Percent 78.77% Diversity Shannon H (loqe) 2.779 Shannon H (log2) 4.010 Marqalef D 6.439 Simpson D 0.096 Evenness 0.054 Function Predator Richness 7 Predator Percent 8.31 Filterer Richness 3 Filterer Percent 8.31% Collector Percent 53.85% Scraper+Shredder Percent 37.54% Scraper/Filterer 1.000 Scraper/Scraper+Filterer 0.500 Habit Burrower Richness 3 Burrower Percent 0.92 Swimmer Richness 3 Swimmer Percent 15.69% Clinqer Richness 6 Clinqer Percent 16.00 Characteristics Cold Stenotherm Richness 0 Cold Stenotherm Percent 0.00% Hemoqlobin Bearer Richness 2 Hemoqlobin Bearer Percent 0.92% Air Breather Richness 1 Air Breather Percent 0.31% Voltinism Univoltine Richness 16 Semivoltine Richness 0 Multivoltine Percent 47.08% Tolerance Sediment Tolerant Richness 1 Sediment Tolerant Percent 0.31 Sediment Sensitive Richness 1 Sediment Sensitive Percent 0.31 Metals Tolerance Index 3.316 Pollution Sensitive Richness 1 Pollution Tolerant Percent 30.46% Hilsenhoff Biotic Index 4.665 Intolerant Percent 15.08% Supertolerant Percent 14.77% CTQa 91.147 100% 80% 60% 40% 20% 0% BIBI MTM MTP MTV Bioassessment Indices