BUREAU OF CLEAN WATER. Appendix C Biological Field Methods C2. Benthic Macroinvertebrates DECEMBER 2013

Transcription

1 BUREAU OF CLEAN WATER Appendix C Biological Field Methods C2. Benthic Macroinvertebrates DECEMBER 2013

2 BENTHIC MACROINVERTEBRATES This chapter describes field and laboratory methodology for sampling and processing benthic samples collected using the Department s Instream Comprehensive Evaluation (ICE, 2011) and habitat specific stream protocols for riffle/run freestone (2012), limestone (2009), and multi-habitat pool/glide (2007) streams. The following definitions are applied accordingly: Freestone streams: Streams that normally have higher gradient so as to be dominated by riffle/run habitat consisting of various particle-sized substrates of unconsolidated ( free stone) sand, gravel, and cobble. Further, freestone commonly refers to streams with lower alkalinity and habitat conditions that do not meet the limestone stream definition. Note: Some freestone streams may be limestone influenced where they exhibit high alkalinities but still display unconsolidated freestone substrates not characteristic of true limestone streams. Limestone streams: Cold water streams that originate from limestone springs (or are very strongly influenced by limestone springs) and are usually characterized with alkalinities > 140 mg/l; a water temperature range of F ( C); and drainage areas < 20 sq. miles. Limestone streams will often display low-gradient flow conditions and are almost completely void of clearly defined freestone habitat beds. Low-Gradient/Multihabitat streams: Lower gradient streams where cobble/gravel substrate (riffle/run) habitat does not dominate (may sometimes be absent) but is also characterized by snags (submerged woody debris), pools, and depositional areas of coarse-particulate organic matter (CPOM), sand, and other fine sediments. Riffle/Run habitat: Riffle conditions are demonstrated when the water flowing over the substrate is shallow enough to create a broken, rough, and turbulent white water surface. Runs are stream segments where deeper flowing water keeps the gravel/cobble substrate completely submerged, which creates a smoother, less turbulent, non-white water surface. Riffle/Run habitats are generally considered to represent a stream s most productive macrobenthic areas to sample. A. Net Mesh Considerations One area of concern relating to the Quality Control of statewide biological sampling is standardization of the mesh size on various types of benthic macroinvertebrate sampling gear. Without standardization of mesh size, standardization of overall methods is of limited value. Benthic macroinvertebrates have historically been defined as animals large enough to be retained by a U.S. Standard No. 30 sieve (595 micron openings). A review of sampling equipment that was in use and commercially available during the early development of DEP s water quality program indicated that the 595 micron criterion was very seldom met. DEP hand-screens have mesh with about micron (µ) openings. Standard Surber nets have mesh openings of 1024µ (silk) or 1050µ (nylon). Surber nets of 728µ and 850/900µ are also available. Standard D-frame nets had 800 x 900µ openings. It was apparent from the above discussion that the most common mesh size in use for many years was in the µ range. Consequently, this size range was adopted and has been DEP s standard for many years. Multifilament nylon screen cloth with µ mesh was used for kick screens to ensure consistency. This 850/900µ mesh size was also the standard for replacement Surber nets. 1

3 In recent years, many state water quality programs, federal agencies (e.g. EPA, USGS), and other water quality monitoring organizations began using net sampling devices with 500µ µ adopted for D-frame nets used in the Department s PaDEP-RBP sampling protocols. The one exception is the PaDEP-RBP protocol for limestone streams where an µ mesh D-frame net is used because of the nature of sampling true limestone streams (described below). Except as noted above for limestone streams, future references to the D-frame sampler in the document assume 500µ mesh netting. The net mesh size of other screen samplers and limestone stream employed D-frames have not changed and still are to be µ. Because of differing net mesh size requirements of various PaDEP protocols, the mesh size of the sampler and sampling protocol used must be noted on field and bench identification sheets for the collected benthic sample. B. Qualitative Methods The type of sampling gear used is dependent on survey type and site-specific conditions. The recommended gear for sampling wadeable streams is 3 x 3 ft. flexible kickscreens and 12 inch diameter round D-frame nets. In larger streams or rivers, grab-type samplers may be used to obtain qualitative samples. While generally thought of as quantitative devices, Eckman, Peterson, or Petite Ponar grab samplers can also be used to obtain qualitative data. The type of gear, dimensions, and mesh size must be reported for all collections. When more than one gear type is used, the results must be recorded separately. Physical variables should be matched as closely as possible between background and impact stations when selecting locations for placement of the sampling gear within each station. Matching these variables helps minimize or eliminate the effects of compounding variability. Macrobenthos often exhibit clustered distributions, and if the sampling points are selected in close proximity to each other, a single clustered population may be obtained rather than a generalized measure of the overall population within the selected subhabitat. Spacing the sampling points as far apart as possible within the sub-habitat can minimize the problem of clustered distributions. B.1. Kick-screen. A common qualitative sampling method uses a simple hand-held kick-screen. This device is designed to be used by two persons. However, with experience, it may be used by one person and still provide adequate results. The kickscreen is constructed with a 1x1m piece of net material ( µ mesh size) fastened to two dowel handles (approximately 1 d. X 4 long). B.1.a. Traditional Method. Facing up stream, one person places the net in the stream with the bottom edge of the net held firmly against the streambed. An assistant then vigorously kicks the substrate within a 3x3 ft area immediately upstream of the net to a depth of 3-4 (approximately 10cm). The functional depth sampled may vary due to ease of disturbance as influenced by substrate embeddedness. The amount of effort expended in collecting each sample should be approximately equivalent in order to make valid comparisons. The effort, expressed as area, must be reported for all collections. 2

4 Collect a minimum of four screens at each site or until no new taxa are collected. Initial sampling should be conducted in riffle areas. Collection in additional habitats to generate a more complete taxa list can be conducted at the discretion of the investigator. Initial analysis of the data must be limited to the riffle data for standardization. A second analysis including other habitats may be conducted as needed. Data observations shall be recorded on a Flowing Waterbody Field Form (Appendix A) created for each station sampled. Record the relative abundance of each recognizable Family in each individual collection in the field. Relative abundance categories, with the observed total ranges indicated in parenthesis, include: rare (0-3), present (3-10), common (11-24), abundant (25-99), and (occasionally) very abundant (100+). The investigator, at his/her discretion, may elect to enumerate certain target taxa. The results of each collection are recorded to document conditions of the site for future decision making. a. A stressed or enriched community often exhibits little variability in community structure over an area while a healthy community should have a more complex structure. If varied taxa are found on each screen, the community is probably complex, while the presence of only a few dominant taxa on every screen indicates the community is a simple one. b. Collecting intolerant taxa in a majority of screens is a good indication of an unstressed community. However, collecting intolerant taxa in only one out of four screens may be an indication that the intolerant taxa have only a marginal existence at that location. A comparison of the composited taxa lists for each location may not indicate the rarity of the intolerant taxa, but this rarity would be readily apparent if the taxa lists for individual screens were compared. c. Separate screen taxa lists provide information concerning the distribution of taxa. For example, mayflies are taken in one of four screens at the background station and in none of the four screens at the impact station. All the other taxa collected at both the stations are tolerant forms. Based on a composited taxa list for each station, one might conclude that the impact station is depressed due to the absence of mayflies. However, the individual screen taxa lists would indicate that the mayflies may have a clumped distribution and there is a possibility that the collector simply missed the clumps at the impact station. This will be apparent to the biologist while in the field and he/she can continue collecting until comfortable that mayflies are indeed absent or less abundant at the impact station. Later, it can be reported, for example, that 4 of 10 screens contained mayflies at the background station while only 1 of 10 screens contained mayflies at the impact station. This is an instance when the collector, while still in the field, may choose to count the mayflies in each screen (especially if the background screens had many mayflies while the impact screens only had one or two). d. Separate screen data can lend weight to an analysis when classification techniques (ordination or clustering) are used. Results that cluster or score the individual background screens differently than the individual impact screens indicates a difference between the locations. When the classification technique scores 3

5 background and impact screens in an apparent random manner, then it is likely that there is no impact or that the natural variability is large and masks any impacts. Individuals of representative taxa for a station may be composited in a single vial and preserved for later laboratory verification or identification. Generally, the level of taxonomic identification would follow that as listed in Section E.1. Answers to several questions that require collector judgment and other taxa notes, can be useful in subsequent analysis and can be stored with the taxa lists as remark fields. Such questions include but are not limited to: What are the dominant and rare taxa? Are there any taxa that are found to be unusually abundant? B.1.b (SSWAP) Assessment Method. This method was used for assessments conducted as part of the Statewide Surface Waters Assessment Program (SSWAP). It employed the same kick screen gear, physical disturbance techniques, and relative abundance determinations as the traditional method (B.1.a). The main difference is that only two kicks were usually required and macroinvertebrate identifications were done streamside to family level taxonomy with hand-held lens (10X) if necessary. Data was recorded on field forms (Appendix A). This method is no longer used for official use Assessment purposes but is kept here for historical reference and as a quick field assessment tool during survey planning reconnaissance. B.2. D-Frame. The handheld D-frame sampler consists of a bag net attached to a halfcircle ( D shaped) frame that is 1 ft. wide. The net s design is that of an extended, tapering bag (routinely 500µ mesh size except for true limestone streams µ mesh size). B.2.a Sampling Riffle/Run Habitat. This D-frame methodology is basically the same as with the kick-screen - except for the following points: The net is employed by one person facing downstream and holding the net firmly on the stream bottom. One Dframe effort is defined as such: the investigator vigorously kicks an approximate area of 1m 2 (1X1 m) immediately upstream of the net to a depth of 10cm (or approximately 4, as the embeddedness of the substrate will allow) for approximately one minute. All benthic dislodgement and substrate scrubbing should be done by kicks only. Substrate handling should be limited to only moving large rocks or debris (as needed) with no hand washing. Since the width of the kick area is wider than the net opening, net placement is critical in order to assure all kicked material flows toward the net. Avoiding areas with crosscurrents, the substrate material from within the 1 m 2 area should be kicked toward the center of the square meter area above the net opening. B.2.b Sampling Low-gradient Habitat. Geomorphological processes in low-gradient streams create a variety of habitats (Multihabitat conditions) that cannot be sampled like riffle/run habitats. Therefore, with the exception of still collecting stationary D-frame kicks (described in B.2.a above) from any riffle/run habitat that may be present, for the other habitat types encountered, the D-frame net is used to sweep or jab through a given area of substrate. A Multihabitat sample consists of a compilation of 10 D-frame collections - jabs and riffle/run kicks - as needed: distributed proportionally from the available habitat types within each 100 meter sample reach. Each jab consists of a 1- meter-long sweep of a 0.3-meter wide area, using a D-frame dip net (500 micron mesh). 4

6 One jab equals disturbing and capturing organisms from an area of ~0.23 m 2 (12 x 30 ). C. Semi-Quantitative Methods (PaDEP-RBP Sampling Protocols): In Plafkin (1989), EPA presented field-sampling methods designed to assess impacts normally associated with pollution impacts, cause/effect issues, and other water quality degradation problems in a relatively rapid manner. These are referred to as Rapid Bioassessment Protocols (RBPs). Barbour et. al. (1999) made revisions to these RBPs and expanded them to include Multihabitat methodology. The PaDEP-RBP methods are bioassessment techniques involving systematic field collection and subsequent lab analysis to allow detection of benthic community differences between reference (or control) waters and waters under evaluation. The PaDEP-RBPs are modifications of the EPA RBP III (Plafkin, 1989; Barbour, et al 1999 revisions) and Multihabitat methodology (Barbour, et al 1999) designed to be compatible with Pennsylvania's historical database. Modifications include: 1) the use of a D-frame net as the collection gear for all PaDEP- RBP sampled stream and habitat types, 2) different laboratory sorting procedures, 3) elimination of the CPOM (coarse particulate organic matter) sampling (except for the Multihabitat protocol), 4) number of multihabitat jabs; and 5) metrics substitutions. Unlike the EPA s RBP III methodology, no field sub-sample sorting is done. Only larger rocks, detritus, and other debris are rinsed and removed while in the field before the sample is preserved. Further, because the debris and detritus fraction of the samples may at times be extremely voluminous, rinsing and discarding as much of the materials as practical in the field prior to jar preservation and transport is strongly encouraged as it will facilitate easier and more efficient lab processing. While EPA s RBP methods were designed to compare impacted waters to reference conditions (cause/effect approach), the PaDEP-RBP modifications were designed for un-impacted waters, as well as impacted waters. C.1. Sample Collection. The purpose of the standardized PaDEP-RBP collection procedure is to obtain representative macroinvertebrate fauna samples from comparable stations. For most of the PaDEP-RBP sampling protocols, the riffle/run habitat is targeted for sampling as it is routinely the most productive habitat. Exceptions to this rule are discussed in the Limestone Stream and Multihabitat protocols. Within each station reach, the habitat is sampled in a downstream-to-upstream direction using the D-frame net method described above. For all sampling methods, when compositing materials collected from multiple D-frame kicks, care must be taken to minimize wear and tear on the collected organisms when compositing the materials. It is recommended that the benthic material be placed in a bucket filled with water to facilitate gentle stirring and mixing. The number of D-frame efforts is dependent on the type of survey conducted as described below: C.1.a. Wadeable Freestone Stream Surveys. Wadeable freestone surveys include but not are limited to; Antidegradation, Aquatic Life Use, Existing Use, Instream Comprehensive Evaluation (ICE) and Cause and Effect surveys. For these surveys, it is necessary to characterize macroinvertebrate fauna communities from an area larger than a single riffle. Therefore, an Antidegradation, Aquatic Life Use, Existing Use, ICE, or Cause and Effect survey station is defined as a stream reach of approximately 100 meters in length. At each station, six D-frame efforts are 5

7 collected. Make an effort to spread the samples out over the entire reach. Choose the best riffle habitat areas and be certain to include areas of different depths (fast and slow) and substrate types that are typical of the riffle. C.1.b. Limestone Stream Surveys. For limestone stream surveys, two paired D- frame efforts are collected from each station - one from an area of fast current velocity and one from an area of slower current velocity within the same riffle. Limestone streams have low gradient often making it difficult to locate well developed riffles. If there are no riffles in the sample area, a run or the best rock substrate available is sampled. The resulting D-frame efforts (two) are composited into one sample jar (or more as necessary). Because limestone samples are very abundant in organic material, the sample is preserved in 95 % ethanol and returned to the lab for processing. C.1.c. Low-gradient Multihabitat Surveys. Aquatic macroinvertebrate samples are collected using a multihabitat sample collection method modified from that described in Barbour et al (1999). Organisms are collected from a maximum of five different habitat types within the sample reach. Table 1 describes the five habitat types and explains the different sampling techniques. Sample collection consists of 10 D-frame jabs: 2 from each of five habitat types or distributed proportionally from the available habitat types within each 100 meter sample reach. Each jab consists of a 30-inch-long sweep of a 0.3-meter wide area, using a D-frame dip net (500 micron mesh). Table 1. Stream Habitat Types and Sampling Techniques for Multihabitat Surveys Habitat Type Description Sample Technique Cobble/Gravel Substrate Snag (includes beaver dams) Stream bottom areas consisting of mixed gravel and larger substrate particles; Cobble/gravel substrates are typically located in relatively fast-flowing, erosional areas of the stream channel Snag habitat consists of submerged sticks, branches, and other woody debris that appears to have been submerged long enough to be adequately colonized by aquatic macroinvertebrates; Preferred snags for sampling include small to medium-sized sticks and branches (preferably < ~4 inches in diameter) that have accumulated a substantial amount of organic matter (twigs, leaves, uprooted aquatic macrophytes, etc.) that is colonized by aquatic macroinvertebrates. Macroinvertebrates are collected by placing the net on the substrate near the downstream end of an area of gravel or larger substrate particles and simultaneously pushing down on the net while pulling it in an upstream direction with adequate force to dislodge substrate materials and the aquatic macroinvertebrate fauna associated with these materials; Large stones and organic matter contained in the net are discarded after they are carefully inspected for the presence of attached organisms which are removed and retained with the remainder of the sample; One jab consists of passing the net over approximately 30 inches of substrate. When possible, the net is to be placed immediately downstream of the snag, in either the water column or on the stream bottom, in an area where water is flowing through the snag at a moderate velocity; The snag is then kicked in a manner such that aquatic macroinvertebrates and organic matter are dislodged from the snag and carried by the current into the net; If the snag cannot be kicked, then it is sampled by jabbing the net into a downstream area of the snag and moving it in an upstream direction with enough force to dislodge and capture aquatic macro-invertebrates that have colonized the snag; One jab equals disturbing and capturing organisms from an area of ~0.23 m 2 (12 x 30 ). 6

8 Habitat Type Description Sample Technique Coarse Particulate Organic Matter (CPOM) Submerged Aquatic Vegetation (SAV) Sand/Fine Sediment Coarse particulate organic matter (CPOM) consists of a mix of plant parts (leaves, bark, twigs, seeds, etc.) that have accumulated on the stream bottom in depositional areas of the stream channel; In situations where there is substantial variability in the composition of CPOM deposits within a given sample reach (e.g., deposits consisting primarily of white pine needles and other deposits consisting primarily of hardwood tree leaves), a variety of CPOM deposits are sampled; However, leaf packs in highervelocity ( erosional ) areas of the channel are not included in CPOM samples. Submerged aquatic vegetation (SAV) habitat consists of rooted aquatic macrophytes. Sand/fine sediment habitat includes stream bottom areas that are composed primarily of sand, silt, and/or clay. CPOM deposits are sampled by lightly passing the net along a 30-inch long path through the accumulated organic material so as to collect the material and its associated aquatic macroinvertebrate fauna; When CPOM deposits are extensive, only the upper portion of the accumulated organic matter is collected to ensure that the collected material is from the aerobic zone. SAV is sampled by drawing the net in an upstream direction along a 30-inch long path through the vegetation; Efforts should be made to avoid collecting stream bottom sediments and organisms when sampling SAV areas. Sand/fine sediment areas are sampled by bumping or tapping the net along the surface of the substrate while slowly drawing the net in an upstream direction along a 30-inch long path of stream bottom; Efforts should be made to minimize the amount of debris collected in the net by penetrating only the upper-most layer of sand/silt deposits; Excess sand and silt are removed from the sample by repeatedly dipping the net into the water column and lifting it out of the stream to remove fine sediment from the sample. The resulting number of D-frame efforts as required per survey type are composited into one sample jar or as few as necessary. Care must be taken to minimize wear and tear on the collected organisms when compositing the materials. It is recommended that the benthic material be placed in a bucket and filled with water to facilitate gentle stirring and mixing and reiterated that as much of the rocks, debris, and detritus be rinsed and discarded as practical in the field prior to jar preservation and transport. The sample is preserved in ethanol and returned to the lab for processing. C.2. Sample Processing. Samples collected with a D-frame net are generally considered to be qualitative. However, the preserved samples can be processed in a manner that yields data that is semi-quantitative - data that was collected by qualitative methods but gives information that is almost statistically as strong as that collected by quantitative methods. The following procedures are adapted from EPA 1999 RBP methodology and used to process qualitative D-frame samples so that the resulting data can be analyzed using benthic macroinvertebrate biometric indices (or metrics ). Equipment needed for the benthic sample processing are: 2 large laboratory pans gridded into 28 squares* (more gridded pans may be necessary depending on the size of the sample) DEP Central Office staff use Rubbermaid 2 Gallon White Dur-x Container 18" x 12" x 3 1/2" from US. Plastic Corp. (catalog #6575) 7

9 egory%5fname=20369&product%5fid=16540 slips of paper or other uniform objects (numbered from 1 to 28) for drawing random numbers, and forceps (or any tools that can be used to pick floating benthic organisms), Grid cutters made from tubular material that approximates an inside area of 4 in 2 *. * EPA s (1989) gridding techniques suggested using 5 cm x 5 cm (2 x2 ) grids. Existing equipment consisted of 14 x8 x2 pans which were conducive to dividing into 2 x2 grids and thus, contained 28 squares. The 4 in 2 grid cutters conform to these pan dimensions. While pan size is not critical, the number of grids (28) must be maintained if any basic density comparisons wish to be made between samples. Grid cutters (or similar subsampling devices) used with different sized pans should conform to the pans grid dimensions. The procedures described below begin with the premise that the collected samples have been properly composited according to the type of survey. For Antidegradation, Aquatic Life Use, Existing Use, ICE and Cause-Effect surveys, a station sample represents a composition of six D-frame efforts (collected from fast and slow riffle areas in a 100 meter reach). For Limestone surveys, a station sample is a composition of two D-frame efforts. For Multihabitat surveys, a station sample is a composite of 10 D-frame jabs. Following the steps listed below; process each composited D-frame sample to render a sub-sample size targeted for the specific survey type. The targeted sub-sample size for the various PaDEP-RBP sampling protocols are presented below: Table 2. Targeted Sub-Sample Sizes for PaDEP-RBP Sampling Protocols Sub-Sample Size Requirements of Identifiable Organisms^ 20% Sampling Protocol Range Target Required 20% Preferred # Standard (Andtidegradation, Aquatic Life Use, Existing Use, Cause/Effect, Multihabitat) Limestone Stream ^ Identifiable Organisms organisms identified to the finest required taxon resolution per Section E.1. This also excludes pupae, larval bodies missing too many critical structures to render confident IDs, extremely small instar larvae, empty shells or cases, and non-benthic taxa. # PaDEP-RBP IBI and metric scoring thresholds are based on the respective subsample target + 20% size range of identifiable organisms. However, at times, it has been found that the lower end of the targeted range may not render enough identifiable organisms. The sample processor may have sorted just over the lower 8

10 -20% of the target range only to find out later during the ID step that some individuals could not be identified to genus and had to revert to their family taxonomic level. If a taxonomic Family count is represented by several genera counts and a count of organisms identified to the Family level, either the Family total count or the genus counts can be used in the IBI and metrics analysis. For example, all the genera counts must be bumped up to be included in the Family count or only the genera counts can be used and the Family counts left out. Either of these scenarios results in weaker scores or, in the case where excluding Family counts drops the sub-sample total below the acceptable lower range limits, invalidate the IBI or metrics analysis. In order to assure IBIs or metrics analyses are usable, sub-sampling to the narrower range is preferred and recommended to minimize the probability of these invalidating sub-sampling scenarios occurring. Therefore, it is prudent to exceed the 200 organism target to ensure adequate individuals that can be identified. C.2.a Processing Riffle/Run Habitat Samples collected by PaDEP-RBP Antidegradation, Aquatic Life Use, Existing Use, ICE and Cause/Effect Protocols. 1) The composited sample is placed in a 28-square gridded pan (Pan1). It is recommended to remove fine materials and residual preservative prior to subsampling by rinsing the sample through a sieve (Standard USGS or sieve bucket) that matches the mesh size (or smaller) of the net gear used to collect the sample. 2) The sample is gently stirred to disperse the contents evenly throughout Pan1 as thoroughly as possible. (In order to ease mixing and to minimize wear-and-tear on delicate organisms, water may be added to the pan to the depth of the sample material before stirring) 3) Randomly select a grid using the 28 random number set and, using the grid cutters, remove the debris and organisms entirely from within the grid cutter (centered over the selected grid and cut into the debris) and place removed materials in a second gridded pan (Pan2). 9

11 Figure 1. Pan1 with 4 grid cutters in randomly selected grids. i. Float and pick, count, and sub-total all identifiable organisms (as noted above in Table 2) from each cut grid placed in Pan2. Repeat until at least 4 grids have been sub-sampled from Pan1. If, after 4 Pan1 grids have been sorted, the subtotal is less than the respective preferred sub-sample size (Table 2), then continue to remove and sort grids one at a time until the preferred sub-sample size is obtained from Pan2. If the benthic organism yield from the 4 Pan1 grids exceeds the targeted sub-sample size, then proceed to Step ii. ii. With all of the 240+ (or 360+) identifiable organisms remaining in Pan2, randomly select one grid and back count (removing) all the organisms from that grid. Repeat one grid at a time until the bug count remaining in Pan2 satisfies the targeted 200 or % rule ( or preferred as noted in Table 2). iii. Because limestone samples are often characterized by dense invertebrate concentrations, it is important not to exceed the upper boundary range (360+) of the limestone targeted 300 subsample size. If it appears that the number of benthic organisms from the last grid will cause the sub-sample to exceed its target size by more than 20% (>360 organisms), count this last grid and place in a separate clean gridded pan (Pan3) with enough water to facilitate gentle stirring and even distribution. Randomly select grids from Pan3 and remove individuals until the count of organisms remaining in Pan3 allows the subsample count to fall below the 360 upper limit. 4) If not identified immediately, the sub-sample should be preserved and properly labeled for future identification. 5) The benthic material remaining (Pan1) after the target sub-sample has been picked can be returned to its original sample jar and preserved. They shall be retained in accordance with QA retention times as specified for this respective survey type. 10

12 6) Any grid chosen must be picked in its entirety (If picking the last grid causes the subsample to exceed its target size by more than 20% (>240 or >360 organisms), follow back counting step C.2.a.3.iii above). 7) Record the final grid counts selected for each gridding phase (Pan1, Pan2, and Pan2 back counting as necessary) on the lab bench ID sheet for the sample. C.2.b Processing Samples collected by the PaDEP-RBP Multihabitat Protocol. Multihabitat samples will usually require multiple sample jars because of the complexity and degree of detritus and inorganic materials that characterize these samples. Because of this, multihabitat samples need to be processed in a different manner than those samples collected by other protocols. Initial Processing of Multihabitat Macroinvertebrate Samples 1) Similarly as in C.2.a Step 1 above, multihabitat samples should be rinsed in a standard USGS No. 35 sieve (or sieve bucket) to remove fine materials and residual preservative prior to sub-sampling. Because of the nature of multi-habitat samples (i.e. greater volumes of finer silts, organic, and particulate material), rinsing while retaining the macroinvertebrates may pose extra challenges. Using larger containers (such as five-gallon buckets) and rinsing smaller aliquots of material may be necessary. All multihabitat sample cleansing steps should use gentle and careful rinsing and agitation to minimize organism damage. 2) Once the total sample has been properly rinsed, transfer the contents of the sieve into a clean, white, 3.5 deep rectangular gridded pan (Pan1) (measuring 14 long x 8 wide on the bottom of the pan) marked off into 28 four-square inch (2 x 2 ) grids and follow Steps 2-7 as listed above in C.2.a to target a 200 sub-sample ( preferred) as indicated in Table 2. C.2.c Processing larger, excessive amounts of D-frame sample debris and multihabitat samples Hopefully, the collector will rarely have very large amounts of D-frame materials to process. The reduction of large materials by careful removal, inspection, and rinsing in a bucket or using a sieve prior to field preservation or at the lab is encouraged. However, if the amount of material composited in the field jars exceeds the functional sorting capacity of Pan1, then follow this guidance: a) Evenly distribute the material between as many pans as necessary. b) From each pan (Pan1a, Pan1b, etc.), remove debris and organisms from 4 random grids and place in Pan2 as described in Step C.2.a.3.i above. c) Once the required 4 grids from each Pan1(a, b... etc.) have been placed in Pan2, evenly and gently redistribute the materials as in Step C.2.a.3.ii. d) Then, resume processing, again as described in Step C.2.a.3.iii, selecting a grid from Pan2 and placing the materials into a gridded Pan3. e) Process this material and repeat as described in Step C.2.a.i until the preferred subsample size is obtained from Pan3. 11

13 f) If, after processing 4 grids, the +20% upper limit (240+ or 360+) is exceeded, follow back counting method in Step C.2.c.ii or iii (for limestone samples) g) Once the targeted sub-sample is reached, continue with Step C.2.a.4-7. D. Quantitative Methods The type of sampling gear used is dependent on survey type and site-specific conditions. The recommended gear includes Surber-type samplers (with micron mesh), artificial substrate (multi-plate) samplers (meeting specifications in Section D.2), and grab-sample devices. The type of gear, dimensions, and mesh size must be reported for all collections. When more than one gear type is used, the results must be recorded separately. In order to limit the complications arising from compounding variability, follow the guidelines discussed in Section II when selecting background stations or reference water bodies and impact stations. Physical variables should be matched as closely as possible between the background and impact stations when selecting locations for placement of the sampling gear within each station. This helps minimize or eliminate the effects of compounding variability. An additional consideration is locating the samplers in areas where current, depth, and substrate are optimal for gear efficiency. It should be clearly noted on field forms when sampling gear is used under sub-optimal conditions (i.e. slow current, bedrock etc.). Macrobenthos often exhibit clustered distributions and if the sample points are selected within close proximity, only a single clustered population sample may be obtained rather than a generalized measure of the overall population within the selected sub-habitat. Spacing the sampling gear as far apart as possible within the sub-habitat can minimize the problem of clustered distributions. D.1. Surber-type Samplers. Surber-type samplers are defined as samplers that delineate or confine an area of the stream bottom (usually 1 ft 2 ), which is to be sampled by disturbing the enclosed substrate. The dislodged materials (organisms, detritus, and other debris) are swept into an attached, tapering net. These samplers include the Surber Sampler, Portable Benthic Invertebrate Sampler (PBIS), and Hess Sampler. The sampling procedure for all these samplers and other similar devices are as follows: a. The substrate will be completely disturbed within the confines of the sampler frame to a depth of 3-4 (approximately 10cm). Larger rocks should be gently, but thoroughly scrubbed while being held in the net mouth. b. Collect a minimum of three quantitative samples at each site. Do not composite samples, but place each collection in a separate container. c. These quantitative samples may be processed in the manner described in Section C.2), except that once all organisms have been floated and picked from the debris, it is not necessary to pick a sub-sample from a gridded pan. Evaluations using organisms collected by these quantitative methods rely on the total number of individuals from the entire sample. d. The organisms may be identified to the taxonomic level deemed necessary by the collector and problem being investigated. Samples may be split to expedite processing. Procedures for sub-sampling are defined in Elliott (1977) and in EPA 12

14 (1990). In addition, a plankton splitter may be employed to sub-sample large numbers of chironomid larvae or other abundant taxa. e. Complete a coded field form to ensure that the associated physical data is recorded. D.2. Multi-plate Samplers. Multi-plate artificial substrate samplers (Figure 1, page 17) may be used where water depth and/or substrate prevents the use of other sampling techniques. A description of the sampler used including the type of artificial substrate, the individual component dimensions, and total surface area must be reported with each collection. In continuing investigations, the samplers must be uniform from year to year. Physical variables should be matched as closely as possible when using the samplers on an annual basis at the same location and between stations for cause/effect surveys. Matching these variables helps minimize or eliminate the compounding of variables. An additional consideration is locating the samplers in areas where the current, depth, and substrate are optimal for gear efficiency while minimizing problems of theft and disturbance. Multi-plate samples for WQN stations must conform to the following procedures: a. Place a minimum of two samplers at each site to help assure the retrieval of at least one. b. Leave the samplers in place for a minimum of 6 weeks. The amount of time the samplers are in place must be reported with each collection. c. Samplers should be enclosed in a net (of equal or smaller mesh size) or plastic bag before retrieval to prevent loss of organisms. When the use of nets or bags is not possible, retrieve the sampler with a smooth but rapid motion to minimize loss of organisms. The retrieved sampler should be immediately placed in a tray, scraped, and all the scrapings preserved. Do not composite the samples. Preserve each separately. d. The taxa should be identified to genus whenever possible. Samples may be split to expedite processing. Procedures for sub-sampling are defined in Elliott (1977) or EPA (1973). e. Complete a coded field form to insure that the associated physical data is recorded. D.3. Grab Samplers. Where standard shallow-water sampling methodology is not feasible, grab-sampling devices may be necessary. These include Ekman, Peterson, or Ponar-style grab samplers. They are designed for use in deeper waters or in areas that have soft, unconsolidated substrate. These samplers are somewhat cumbersome and labor intensive to use. They are heavy, by design, so that they can be dropped from a boat and penetrate the substrate. They often need a boom and pulley retrieval system. For specific discussions on the advantages, disadvantages, and sampling methodology, refer to EPA

15 E. Identification E.1. Taxonomic Level. The level of identification under magnification for most aquatic macroinvertebrates will be to genus based on the recommended references listed in the Taxonomy Reference List on the 2013 Assessment Methods webpage. Some individuals collected will be immature and not exhibit the characteristics necessary for confident identification. Therefore, with the exception of the Ephemeroptera, Plecoptera and Trichoptera (EPT) taxa, the lowest level of taxonomy attainable will be sufficient. EPT taxa must be identified to the Genus level. The following taxonomic groups, however, may be identified to a higher taxonomic level as follows: Snails (Gastropoda) - Family Clams, mussels (Bivalvia) - Family Flatworms (Turbellaria) identifiable planarids - genus or Family Planaridae others - Phylum Turbellaria Segmented worms (Annelida) aquatic earthworms & tubificids - Class Oligochaeta leeches - Class Hirudinea Moss animacules - Phylum Bryozoa Proboscis worms Phylum Nemertea Roundworms - Phylum Nematoda Water mites- Hydracarina (an artificial taxonomic grouping of several mite superfamilies) Midges (Chironomidae) Family Taxonomic references can be found in the Taxonomic Reference List included with the 2013 Assessment Methods. E.2. Verifications. For Quality Assurance purposes, certain laboratory invertebrate processing procedures should be checked routinely. Normally, a colleague may perform these spot checks. These include the floating/picking steps, taxonomic identifications, and total taxa list scans: a. Sorting. After the floating and picking has been completed for samples that require this treatment (Pa-RBP, Surber-type, multi-plate, and grab samples), the residue should be briefly scanned before discarding to assure that the sample has been sufficiently picked. This should be done for 10% of the samples (or at least one sample) per survey. b. Identification. For samples not involving litigation or enforcement issues, laboratory bench ID sheets for all samples should be reviewed. Any unusual taxa or taxa that are not typical to the type of stream or water quality condition that was surveyed, should be checked. For samples involving legal issues, representative specimens of each taxon may need to be verified by independent expert taxonomists. For each staff performing identifications, a minimum of 10% of the samples identified should be quality assured by another taxonomist. E.3. Sample Retention. For Quality Assurance purposes, identified benthic macroinvertebrate samples should be preserved and retained for later verifications. Based on the nature and purpose of the survey, retention times would vary: 14

16 a. Cause/effect surveys: until all legal issues have been resolved. b. Monitoring surveys: 1) WQN - 2 years 2) Reference WQN - 5 years c. Stream Redesignation and Use-attainability surveys: until any proposed stream classification changes become final (approximately 2 years). d. Enforcement/compliance surveys: until all legal issues, including appeals and related litigation have been resolved. 15

17 References Barbour, M.T., J. Gerritsen, B.D. Snyder, and J.B. Stribling Rapid Bioassessment Protocols for Use in Streams and Wadeable Rivers: Periphyton, Benthic Macroinvertebrates and Fish. Second Edition. EPA/841-B U.S. EPA, Office of Water, Washington, D.C. Elliott, J. M.; Statistical Analysis of Samples of Benthic Invertebrates. Freshwater Biological Association, Publication No. 25. Environmental Protection Agency Biological Field and Laboratory Methods. Office of Research and Development. EPA; Cincinnati, OH. EPA-670/ Macroinvertebrate Field and Laboratory Methods for Evaluating the Biological Integrity of Surface Waters, Office of Research and Development publication: EPA/600/4-90/030, Nov Plafkin, J.L, M.T. Barbour, K.D. Porter, S.K. Gross, and R.M. Hughes Rapid Bioassessment Protocols for Use in Streams and Rivers: Benthic macroinvertebrates and fish. EPA/440/ U.S. Environmental Protection Agency, Office of Water, Washington, D.C. 16

18 Figure 1. Multi-plate sampler diagram 17

19 Appendix A Flowing Waterbody Field Form 18

20 3800-FM-WSFR0086 Rev. 12/2008 COMMONWEALTH OF PENNSYLVANIA DEPARTMENT OF ENVIRONMENTAL PROTECTION BUREAU OF WATER STANDARDS AND FACILITY REGULATION FLOWING WATERBODY FIELD DATA FORM (Information and comments for fields boxed in double lines are required database entries. Other fields are optional for personal use.) Date-Time-Initials* Example XYZ Secondary Station ID - - Date Time Initials *Date as YYYYMMDD, time as military time, and your initials uniquely identify the stream reach. Survey Type Watershed Code (HUC) Surveyed by: Stream Code SWP Watershed (1) Basin Survey, (2) Cause / Effect, (3) Fish Tissue, (4) Instream Comprehensive Evaluation [ICE], (5) Point-of-First-Use, (6) SERA, (7) Antidegradation [Special Protection], (8) Toxics, (10) Use Attainability, (11) WQN, (12) Limestone, (13) Low-gradient [Multihabitat] Location County: Municipality: Topo Quad: Location Description: Ch. 93 Use Land Use Residential: % Commercial: % Industrial: % Cropland: % Pasture: % Abd. Mining: % Old Fields: % Forest: % Other: % Land Use Comments: Canopy cover: open Collectorsequence # partly shaded mostly shaded fully shaded Temp ( 0 C) Field Meter Readings: DO (mg/l) ph Water Quality Cond. (umhos) Alkalinity mg/l Water Appearance/Odor Comments: (^see bottom of back for common descriptors) Bottle Notes (N-normal, MNF-metals nonfiltered, MF-metals filtered, B-bac t, Others: indicate) Findings Not Impaired: Impaired biology? Impaired habitat? Is impact localized? Reevaluate designated use? Decision comments. Describe the rationale for your Not Impaired or Impaired decision; reach locations for use designation reevaluations; special condition comments; etc.: IBI Score: Total Habitat Score:

21 3800-FM-WSFR0086 Rev. 12/2008 Macroinvertebrate sampling Sampling method: Std. kick screen: D-frame: Surber: Other: method?: Comments/Abundance Notes: Habitat Impairment Thresholds Metric Score #3 Riff/Run: embeddedness or #3 Glide/Pool: substrate character + #6 Sediment Deposition = 24 or less (20 or less for warm water, low gradient streams) #9 Condition of Banks + #10 Bank Vegetation = 24 or less (20 or less for warm water, low gradient streams Total habitat score 140 or less for forested, cold water, high gradient streams (120 or less for warm water, low gradient streams) Habitat Comments: Special Condition Use this block to describe conditions that justify attainment/impairment of stations with IBI score <63 and >53. ^Common descriptors: Water Odors - none normal sewage petroleum chemical other; Water Surface Oils - none slick sheen globs flecks; Turbidity - clear slight turbid opaque; NPS Pollution - no evidence some potential obvious; Sediment Odors - none normal sewage petroleum chemical anaerobic; Sediment Oils - absent slight moderate profuse; Deposits none sludge sawdust paper fiber sand relict shells other. Are the undersides of stones deeply embedded black?