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The Trophic Diatom Index: A User's Manual. Revised Edition

1. 11 Action plan reference number Refers to relevant Region Area documentation The following Task Risk Assessments are included 1 Collection of diatom samples from freshwaters including deployment of polypropylene rope for diatom colonisation 2 Addition of preservative to diatom sample 3 Digestion of diatom samples using sulphuric and oxalic acids and potassium permanganate 4 Digestion of diatom samples using hot hydrogen peroxide 5 Preparation of permanent microscope slides 6 Identification and enumeration of diatoms from permanent microscope slides Task Risk Assessments are followed by example COSHH Assessment Forms for specific activities 1 Addition of iodine to diatom samples and subsequent laboratory procedures involving diatom samples preserved in iodine solutions 2 Laboratory and field procedures involving use of 4 formaldehyde solution diluted from 40 stock solution 3 Decanting stock solution of methylated spirit industrial 4 Decanting 37 hydrochloric acid from stock to glass dropping bottle and subsequent addition to samples during chemical digestion process 5 Preparation of saturated oxalic acid Decanting saturated oxalic acid into glass dropping bottle and subsequent use in chemical digestion of diatom samples 6 Addition of potassium permanganate crystals to beaker containing rope sulphuric acid and chemical digestion of samples 7 Decanting sulphuric acid into glass drop
2. Kelly M G 1998 Use of the trophic diatom index to monitor eutrophication in rivers Water Research 32 236 242 Kelly M G 1999 Progress towards Quality Assurance of benthic diatom and phytoplankton analyses in the UK In Use of Algae for Monitoring Rivers III ed J Prygiel B A Whitton amp J Bukowska pp 208 215 Agence de l Eau Artois Picardie Douai R amp D TECHNICAL REPORT E2 TR2 76 Kelly M G 2000 Identification of Common Benthic Diatoms in Rivers Field Studies 9 583 700 Kelly M G 2001 Use of similarity measures for quality control of benthic diatom samples Water Research 35 2784 2788 Kelly M G amp Whitton B A 1995a Plants for Monitoring Rivers A Report of a Workshop held at University of Durham 26 27 September 1994 34pp R amp D Note 366 National Rivers Authority Bristol Kelly M G amp Whitton B A 1995b The Trophic Diatom Index a new index for monitoring eutrophication in rivers Journal of Applied Phycology 7 433 444 Kelly M G Cazaubon A Coring E Dell Uomo A Ector L Goldsmith B Guasch H H rlimann J Jarlman A Kawecka B Kwandrans J Laugaste Lindstr m E A Leitao M Marvan P Padisak J Pipp E Prygiel J Rott E Sabater S van Dam H amp Vizinet J 1998 Recommendations for the routine sampling of diatoms for water quality assessments in Europe Journal of Applied Phycology 10 215 224 Kelly M G Penny C J amp Whitton B A
3. Activity undertaken by authorised personnel only Equipment maintenance record Electrical testing Restricted access to working area Laboratory CoP Laboratory induction procedure Activity undertaken by authorised personnel only 100 9 OPTIONS FOR IMPROVED RISK CONTROL 10 PRIORITY OF ACTIONS REQUIRED H M L C6 Identification and enumeration of diatoms from permanent microscope slides PART 1 WORK ACTIVITY TASK IDENTIFICATION 1 REGION AREA Insert as appropriate 2 TEAMS JOB TITLES Biology Team Leader Biology Biologists Student Biologists Temporary Biologists 3 TASK SUMMARY Identification and enumeration of diatoms from previously prepared microscope slides container Return of slide to storage container following analysis 6 TOOLS amp EQUIPMENT Microscope slides and coverslips Microscope immersion oil 8 ASSESSORS Insert as appropriate 11 AUTHORISATION CHECKED BY SIGNED R amp D TECHNICAL REPORT E2 TR2 101 2 DESCRIPTION OF TASK ELEMENTS 3 IDENTIFICATION OF HAZARD Removal return of microscope slide from to storage container Addition removal of microscope immersion oil Identification enumeration of diatoms using compound microscope 6 EXISTING PLANNED RISK CONTROL MEASURES Restricted access to working area Laboratory CoP Laboratory induction procedure Activity undertaken by authorised personnel only See control measures in 1 above Chemi
4. 3 Betts Avenue Martlesham Heath Ipswich IP5 7RH tel 01473 623995 fax 01473 612148 At the time of writing it costs 5 00 VAT and P amp P for 15 ml e Microscope slides Note that most microscope slides are washed in detergent prior to sale Slides for the preparation of permanent diatom mounts should not be washed Either purchase orders should specify unwashed slides or slides should be washed in ethanol prior to use to remove any residues of detergent e Coverslips Shape and size are not critical but for long term storage it is necessary for the mountant to cover the entire coverslip This is easiest to achieve using small e g 19 mm or less round coverslips but square coverslips can also be used so long as this criterion is fulfilled thinnest practical coverslips thickness 0 are recommended as these minimise the distance between objective and diatom but thickness 1 and 1 can also be used e Disposable Pasteur pipettes e Ethanol e Forceps spade ended are best for holding coverslips e A source of heat such as a Bunsen burner spirit lamp or hotplate Different stages of the process require different intensities of heat so more than one heat source is recommended R amp D TECHNICAL REPORT E2 TR2 22 3 4 2 Method As for other stages in the preparation of diatom slides there is no right way to prepare permanent slides so long as the finished slides fulfil certain criteria There is a definit
5. 9 GLOSSARY OF TERMS Definitions are provided for words in bold type Analytical Quality Control AQC Audit Benthos n benthic adj BMWP score Boulder Centric diatom Cobble Diatom Quality Index DQI Epilithon Epipelon Ephiphyton Episammon Eutrophic Eutrophication Formaldehyde Procedures to control errors in laboratory analyses within specified limits An independent measurement of the quality of the laboratory analysis of samples or the quality of the AQC inspection Those organisms which live on the bottom of a lake river or other water body Cf plankton Biological Monitoring Working Party score a biotic index of organic pollution based on the presence or absence of benthic invertebrate taxa in a sample Stones with a diameter gt 256 mm A diatom showing radial pill box or petri dish symmetry In the latest classification represented by the Class Coscinodiscophyceae Stones with a diameter gt 64 lt 256 mm A biotic index of environmental quality calculated as 100 TDI The community living on the surface of a rock Literally rock The community living on soft surfaces mud and silt The community living on but not usually fed by aquatic plants Literally on plant The community living attached to sand grains Literally on sand An ecosystem containing high concentrations of those inorganic nutrients which usually limit plant growth e
6. Cymbella delicatula 113 Cymbella microcephala Cymbella minuta silesiaca Cymbella sinuata mE See Reimeria sinuata Transferred to Encyonema Cymbella large forms 4 2 caespitosa lanceolata mE Cymbella other Includes Encyonema spp Other Denticula spp are halophilous New taxon Split from Navicula small same s and v values but Diadesmis is tolerant to desiccation Diatoma tenue 22 Diatoma vulgare Diatoma other 2 1 Didymosphenia geminata 23 Diploneis R amp D TECHNICAL REPORT E2 TR2 Nominally gt 70 Includes aj N U N gt Oo Oo Tam Ellerbeckia arenaria 4 2 Encyonema minutum 3 2 moved from Cymbella silesiacum Encyonema other see Cymbella other Epithemia 2 Eucocconeis o3 Eunotia Fragilaria brevistriata see Pseudostaurosira Fragilaria capucina Fragilaria crotonensis New taxon Plankton Fragilaria pinnata see Staurosirella Fragilaria vaucheriae Fragilaria other 2 l Staurosira and Fragilariforma now placed in separate categories 1 New taxon Formerly included in Fragilaria other Fragilariforma KO eos nM Nm Frustulia bo Gomphocymbella Gomphoneis see Gomphonema other Gomphonema angustatum Gomphonema minutum New taxon Gomphonema olivaceoides Gomphonema olivaceum Gomphomema parvulum Gomphonema other Gyrosigma Hannaea arcus Hantzschia Karayevia ew taxon Split from Achnant
7. Shading record estimated percentage Left bank None Broken Dense Right bank None Broken Dense Habitat Pool Run Riffle Slack Water clarity Clear Cloudy Turbid Bed stability Firm Stable Unstable Soft Time since last spate lt 3 days 3 Tdays 7 14days gt 14 days not known Photograph Facing upstream Facing downstream ____ Use the reverse of this sheet for sketch map and other comments R amp D TECHNICAL REPORT E2 TR2 83 This page has been left blank intentionally R amp D TECHNICAL REPORT E2 TR2 84 APPENDIX RISK ASSESSMENTS FOR ACTIVITIES ASSOCIATED WITH THE TROPHIC DIATOM INDEX This section of the manual includes a series of Task Risk Assessments and associated COSHH Risk Assessments covering all aspects of the procedures outlined in chapters 2 4 These have been prepared in line with the method described in the Environment Agency s Health and Safety Risk Management Manual which provides a straightforward way of assessing and recording a wide range of risks using the five main steps as recommended by the Health and Safety Executive e Identify hazards e Identify all those who may be affected by the hazards estimate the level of risk taking into account the adequacy and reliability of existing or planned risk control measures e Decide if new risk control measures are needed and investigate what new measures can be introduced having regard
8. A replicate count audit count is made under identical conditions to the primary count If a stratified count procedure was adopted for the primary count then this must also be used for the audit count Results from the primary and audit counts are entered into a spreadsheet and used to calculate the following statistics Bray Curtis similarity Hill s diversity gains losses difference in TDI ATDI If the TDI calculation was modified by removing one or more dominant taxa then the statistics should be calculated both with and without this modification A sample is considered to have acceptable quality if the Bray Curtis similarity gt 60 and gains and losses are each 2 If such conditions are fulfilled then ATDI is usually 5 If Hill s Nz diversity is 3 then the quality is acceptable if Bray Curtis similarity is gt 70 These criteria should also apply to modified TDI calculations if appropriate Audit results of failed batches that are reanalysed should not be used when detecting lab quality Reanalysed samples must be subject to audit in normal way A summary of the audit results must be returned to the primary analyst along with comments from the auditor on possible reasons for any deviations from acceptable quality A copy of the audit results should also be sent to the line manager All samples belonging to the batch from which this sample was drawn are considered to b
9. SAFE USE OF PPE EYE PROTECTION SKIN PROTECTION R amp D TECHNICAL REPORT E2 TR2 121 Operation should be undertaken within fume cupboard Chemical resistant gloves safety spectacles amp laboratory coat Only required for spillage clean up facemask appropriate filter Restricted access to working area Laboratory CoP Laboratory induction procedure Activity undertaken by authorised personnel only Chemical inventory storage arrangements Chemical spillage procedures Eye wash facilities safety shower Use of glass dropping bottle reduces risk of spillage during chemical digestion process YES YES NO Spillage procedures are outlined in the laboratory s Chemical Spillage Procedures Correct storage arrangements are identified in the laboratory Chemical Inventory amp Storage Arrangements R amp D TECHNICAL REPORT E2 TR2 122 IMPLEMENTATION OF FURTHER ACTIONS ACTION BY WHOM TARGET DATE No further actions required all identified risks are covered by existing procedures etc R amp D TECHNICAL REPORT E2 TR2 123 TEAMS BIOLOGY REVIEW PERIOD Addition of potassium permanganate crystals to beaker containing rope sulphuric acid and chemical digestion of Permanganic acid potassium salt 100 ESCRIBE ANY HAZARDOU Contact with skin and eyes causes severe irritation and PROPERTIES OFT THE S UBSTA NCES possible burns CAUSING RISKS TO HEALTH May causes gastroint
10. it is also useful to be able to relocate actual specimens if a second opinion is not available immediately If taxonomic assistance is available in house noting co ordinates on the microscope s Vernier scale may be sufficient If another microscope is likely to be used then a facility to record the absolute position of the R amp D TECHNICAL REPORT E2 TR2 25 specimen may be necessary One such device is an England Finder which ensures that the same point on a slide can be located on a different microscope which means that a slide could be posted to another diatomist who also needs to have an England Finder for a second opinion England Finders are available from Graticules Limited Soverign Way Tonbridge Kent Tel 01732 359061 4 3 Preliminary steps 4 3 1 Taxonomic criteria The minimum acceptable level of taxonomy is the checklist of TDI taxa provided in Chapter 5 This is the level of taxonomy that is covered by Quality Assurance protocols outlined in Chapter 7 Identification to species is not necessary for calculation of the TDI but may be appropriate for some other purposes It may also aid subsequent data interpretation 4 3 2 Sample size The sample size quoted in the first edition of the TDI manual was 200 valves This guideline has been changed and the recommendation now is that the sample size should be 300 valves of which at least 200 must contribute information on the trophic status of areach Taxa that would no
11. 1 some of these will vary at a site on time scales ranging from days to months 6 2 2 Assessing the reliability of the TDI The first edition of the manual included the TDI along with a second value Percent Pollution Tolerant Valves PTV The original rationale was that PTV helped to identify sites where the primary factor controlling community composition was organic pollution The taxa which contributed to the PTV were Gomphonema parvulum Navicula gregaria N lanceolata small Navicula species and Nitzschia other This proved to be useful up to a point however it soon became clear that the terminology was flawed and that PTV was being used by some as an index of organic pollution rather than as an indication of the reliability of the TDI as an estimate of eutrophication This was compounded by the fact that some of the taxa included in the PTV sum were also abundant at sites where organic pollution was not a serious problem Such instances included samples where Navicula lanceolata was abundant see 6 5 1 below and some upland sites where Gomphonema parvulum thrived In addition Nitzschia other was a very broad dumping ground largely due to the difficulties associated with identifying Nitzschia spp This category included taxa such as N palea that favoured organic pollution along with taxa such as N fonticola which preferred cleaner conditions Despite these limitations the principle of using a second characteristic of the di
12. 2 The lowest N P typically occurs in the summer when algal growth is most prolific More importantly Fig 6 2 gives an indication for this particular river system of target available P concentrations assuming remains unchanged if a floristic change 1 to be expected If diatom assemblages are to change in response to P reduction then post stripping concentrations should be low enough to ensure that N P 15 for most of the year including the summer biomass Increase in P loading Fig 6 1 Schematic representation of stages in response of photosysnthetic community to increase in nutrient loading Modified from Sas 1989 The response to P loading depends upon the zone in which the initial P concentration is found I Increase in biomass and floristic change in response to increased P loading II Increase in biomass but no floristic change III No biomass response to increase in P loading R amp D TECHNICAL REPORT E2 TR2 46 100 10 N P 0 1 1 10 ortho P Fig 6 2 Relationship between ortho P concentration and N P as TON and ortho P respectively in the River Stour above and below Ashford STW Kent Vertical lines indicate P concentrations representing N P ratios of 10 and 15 See text for more details e Factors other than inorganic nutrients that might influence competitive interactions between species The first edition of this manual stressed the importance of distinguishing the direct
13. 5 10 ml of the suspension to a beaker Add approximately 20 ml of hydrogen peroxide and heat on a hotplate set at 90 5 C until all the organic material has been oxidized typically 1 3 hours Care is needed while pouring cold concentrated hydrogen peroxide onto organic rich material and aquatic plants and also during the heating process Note If rope or macrophytes samples are placed directly into beakers for digestion then the volume of oxidising agent may need to be increased to about 50 ml per sample The oxidation process involves a reaction between hydrogen peroxide and carbon atoms bound into organic molecules to produce water plus carbon dioxide The reaction is complete when the evolution of bubbles of carbon dioxide ceases If there is still organic matter present then more hydrogen peroxide should be added Remove the beaker from the heat and allow to cool in the fume cupboard Run a few drops of hydrochloric acid down the side of the beaker to remove any remaining hydrogen peroxide and any carbonates This stage can be omitted if the sample comes from a region where carbonates are unlikely to be present in the water Wash down sides of beaker with distilled water Transfer contents of the beaker to a centrifuge tube top up with distilled water to 1 cm below the rim of the centrifuge tube and centrifuge The purpose of centrifugation is to separate solid and liquid phases The speed and time are not critical A speed of
14. THE SUBSTANCE IS SAFE USE OF PPE EYE PROTECTION SKIN PROTECTION R amp D TECHNICAL REPORT E2 TR2 133 Operation should be undertaken within fume cupboard Chemical resistant gloves safety spectacles amp laboratory coat Only required for spillage clean up facemask appropriate filter Restricted access to working area Laboratory CoP Laboratory induction procedure Activity undertaken by authorised personnel only Chemical inventory storage arrangements Chemical spillage procedures Eye wash facilities safety shower Use of glass dropping bottle reduces risk of spillage during chemical digestion process YES YES NO Spillage procedures are outlined in the laboratory s Chemical Spillage Procedures Correct storage arrangements are identified in the laboratory Chemical Inventory amp Storage Arrangements R amp D TECHNICAL REPORT E2 TR2 134 IMPLEMENTATION OF FURTHER ACTIONS ACTION BY WHOM TARGET DATE R amp D TECHNICAL REPORT E2 TR2 135
15. and if necessary clean the eyepiece lenses using a piece of lens tissue 2 Place a slide on the stage and copy relevant information from the slide label to the record sheet or computer program The minimum information is sample number river name site name sample date Other essential data are the date of the analysis and analyst s name 3 Select an appropriate starting position on the slide The edge of the dried sample suspension is recommended if horizontal or vertical traverses are used but if this approach is adopted ensure that there are no significant edge effects An alternative to the use of traverses 15 to use random fields If this approach is adopted then random fields should be located using the Vernier scales on the microscope in conjunction with either tables of random numbers or random number functions within computer programs or electronic calculators 4 Using a 100x objective identify all valves present in the first field of view Use the fine focus mechanism to differentiate between a single valve and an intact frustule Record an intact frustule as two units An intact frustule will have two distinct planes of focus when the striae raphe and other structures will be clearly visible Careful use of the fine focus mechanism should enable these to be differentiated An intact frustule also often has different Optical properties to a single valve R amp D TECHNICAL REPORT E2 TR2 29 The list of taxa used in
16. biomass may change at site C but the TDI will not detect this e Sites immediately upstream of STWs site B in Fig 6 4 are typically downstream of the urban area served by the STW and are often influenced by urban runoff storm sewers and trade effluents in the town e Sites immediately downstream of STWs site C in Fig 6 4 often have high levels of other constituents of the sewage effluent see Table 6 2 that will exert their own influence on the benthic diatom flora The recommendation for monitoring programs is to include an additional site upstream of the urban area site A in Fig 6 4 in order to indicate the contribution of the town itself to water quality at the upstream site plus an additional site further downstream from site C site D in Fig 6 4 Site D should be replaced in the recovery zone and it should be located on the basis of local knowledge Such a design may not always be practicable e g in highly urbanised areas and may be further complicated by confluences and other factors which affect water quality It may not even be necessary at all sites in some cases sites immediately upstream and downstream of STWSs sites and show little influence of urban runoff or serious organic pollution perhaps reflecting efficient water collection and or treatment and or good dilution ratios However this situation must not be assumed R amp D TECHNICAL REPORT E2 TR2 48 A look up table Table 6 3 is
17. drop will spread out more Room temperature or room temperature supplemented by gentle warming from a desk lamp s bulb is recommended Evaporation should take place in a warm dust free environment and takes upwards of one hour depending upon ambient conditions Once all liquid has been evaporated the result should be a thin whitish grey film over about half of the cover slip Common reasons for uneven strews of diatoms following evaporation include too rapid evaporation caused by high temperatures or because there is still some residual acid left in the suspension Some clumping of diatoms is acceptable so long as all valves can be identified reliably and density criteria are not exceeded 3 The density of valves should now be checked by placing the coverslip face down on a slide and examining this under medium power objective 1 e 400 x Assuming a maximum final density of about 15 valves per field of view at 1000 x magnification this dry mount should have a maximum of roughly 30 valves per field of view If the density is too high repeat step 2 but with a more dilute suspension Note that very gritty samples may necessitate dilute suspensions to ensure that the valves are not obscured by mineral particles 4 Once a satisfactory density of valves has been achieved put a small drop of naphrax onto a warm slide e g using a warm hotplate and place the coverslip face down on top of it Heat the slide until the naphrax spreads
18. each taxon are then totalled and expressed as a percentage of the total number of valves counted In practice there are of course many subtleties of which a beginner needs to take account Some of these are discussed below 4 2 Apparatus e Compound light microscope equipped with a mechanical stage and 1000x oil immersion lens Use of a phase contrast or differential interference Nomarski condenser is recommended The microscope must incorporate facilities for measurements e g an eyepiece graticule with a resolution of at least 1 um Apparatus for photomicroscopy or video capture are useful for taking pictures of difficult specimens e Floras identification guides and iconographs appropriate to the habitats under consideration The Field Studies Council s AIDGAP key Kelly 2000 was written specifically for users of the TDI Other useful identification guides are listed in Table 4 1 e Immersion oil and dispenser e Lens tissue e Facilities for recording data as it is collected This can be a pro forma count sheet with a list of taxon names and space beside each on which the counts can be made a laboratory notebook organised in such a way that taxon identities and numbers can be clearly recorded or a computer program with facilities for direct entry of data e Facilities for verifying the identity of difficult specimens This can take several forms high quality photomicrographs or captured video images may suffice However
19. for good practice e Prioritise the measures to be introduced and plan their implementation The Task Risk Assessments included in this manual have been developed as best practice and are necessarily generic in nature It is the responsibility of managers to produce Task Risk Assessments that are relevant to local circumstances and to implement the appropriate arrangements for safe working in their work locations Task Risk Assessments are sub divided into a number of columns 1 Item 2 Description of task elements Common to all laboratories performing diatom analyses 3 Identification of hazard harmful Common to all laboratories performing diatom effect analyses 4 Identification of person s Common to all laboratories performing diatom affected analyses 5 Initial risk assessment Common to all laboratories performing diatom analyses Suggested risk control measures Others may also apply 7 Level of risk This is based on a preliminary evaluation bearing in mind responses in columns 3 6 8 Risk control measures adequate If the risk assessment in column 7 is greater than R amp D TECHNICAL REPORT E2 TR2 85 222 2 2 0 that in column 5 then the response must 9 Options for improved risk control If the response in column 8 is then remedial action should be described here 10 Priority of action s required Prioritise actions based on evaluation of all responses in column 9
20. g nitrogen or phosphorus Cf oligotrophic The biological effects of an increase in concentration of plant nutrients usually nitrogen and phosphorus but sometimes others such as silicon potassium calcium iron or manganese on aquatic ecosystems Harper 1992 Popular name for methanal HCHO A gas that is highly soluble in water and widely used as a preservative It is R amp D TECHNICAL REPORT E2 TR2 12 Formalin Frustule Girdle band Girdle view Glide Heterotrophic Macrophyte Mantle Mean Trophic Rank MTR Oligotrophic Pebble Pennate diatom Percent Pollution Tolerant Valves PTV Phytoplankton Plankton n planktonic adj Quality Assurance Quality Control highly toxic A 40 solution of formaldehyde The collective term for the silicious components of a diatom cell Includes valves and girdle bands A complete frustule is composed of two valves plus a number of girdle bands Silicious bands wrapped around the outside of a frustule The number depends upon the species and can vary from 2 to 50 or more They enclose and protect the cell whilst permitting an increase in volume during the cell cycle The side view of a diatom frustule in which the mantle and girdle bands may be visible but not the valve face A term describing a river reach where water moves effortlessly in a smooth fashion Raven et al 1998 Applied to organisms which have a mode of met
21. in revised version a new method for auditing permanent slides has replaced the method in the 1 edition which proved to be difficult to use in practice Health and safety now collected together in Appendix C details of risk associated with procedures outlined in this manual have been included in this edition along with examples of best practice for managing these risks None of these changes affect the integrity of data collected following procedures outlined in the first version of the manual although in a few instances samples may need to be reanalysed in order to use the modifications to the index proposed here The revised TDI can be used alongside chemical and macrophyte data to prepare robust cases for the designation of sensitive areas by providing information about R amp D TECHNICAL REPORT E2 TR2 5 e the state of the river upstream of the sensitive area with respect to inorganic nutrients and organic pollution e whether or not there is a floristic change downstream of a qualifying discharge e whether or not this change can be attributed to nutrients alone or to other components of the discharge and e whether or not nutrient removal is likely to lead to a floristic change R amp D TECHNICAL REPORT E2 TR2 6 2 SAMPLE COLLECTION 2 1 Overview of sampling strategy Diatom communities in rivers are neither simple nor homogenous Although some workers have attempted to define discrete epilithon ep
22. is required If the sample is overwhelmingly dominated by a cosmopolitan taxon then a stratified procedure should be adopted Once the initial target has been reached the proportion of the dominant taxon is noted and the analyst continues counting including all taxa except this taxon until the revised target is reached For the TDI this revised target is 300 valves excluding the dominant taxon from the sum The numbers of the dominant taxon are then scaled up in proportion to its representation after the first target had been achieved 7 For some purposes it is useful to continue to scan the slide after the required number of diatom units has been counted and any taxa encountered that were not included in the count should be identified and recorded as present A further scan using a medium power magnification e g 400 x may also be appropriate in order to note any larger taxa e g Gyrosigma Didymosphenia which can escape detection at higher magnifications 8 At the end of the analysis the slide should be removed from the mechanical stage and excess immersion oil wiped from the objective and slide R amp D TECHNICAL REPORT E2 TR2 30 5 CALCULATING THE TROPHIC DIATOM INDEX 5 1 Introduction This chapter describes how the TDI is calculated from data analysed as described in the preceding chapters Full details of how the original version of the TDI was derived are given elsewhere Kelly and Whitton 1995b Kelly 1998 Sinc
23. it causes problems when counting or identifying diatoms Even distribution of valves with minimal edge effects and no valves Obscured by detritus or other foreign matter At present the definition of intermediate classes is subjective and scores of 3 or more are considered acceptable for routine purposes Some of the criteria such as the presence of large quantities of foreign matter may be beyond the control of the analyst and are not necessarily grounds for rejecting a slide The auditor must make a note of the reasons for rejection Density of valves 1 Too sparse Less than one valve per field of view Counting is time consuming and or it is not possible to count 300 valves 2 Sparse 1 5 valves per field of view Presents no problems for counting 3 Good 5 15 valves per field of view 4 Dense 16 25 valves per field of view Some difficulties in enumeration and identification due to overlapping valves etc 5 Very dense gt 25 valves per field of view R amp D TECHNICAL REPORT E2 TR2 65 The density of valves should be assessed by scanning the slide using a high power objective The appropriate score should be based on a count of valves visible in at least five separate fields of view When making judgements auditors must place themselves in the position of the primary analyst and avoid any areas of the slide that are unusually dense Classes 2 4 are acceptable for routine purposes with 3 preferred Class 1 may
24. of keeping the end of the rope submerged however this practice may not be suitable in all rivers particularly if there is a risk of injury to the public The function of the nail 1 to keep the rope floating just under the surface the weight should not be sufficient to bring the rope into contact with the river bed Minimising losses where possible inform river users of the use of the ropes one Region bailiffs were consulted for advice on the best places for deployment and also asked to keep an eye on the ropes during the exercise Local angling clubs were informed of the Agency s activities with emphasis given to the long term goal of restoring fisheries in the catchment A little extra effort to place the ropes out of sight of footpaths and away from places with easy access to the river is well worthwhile 2 8 Sampling emergent macrophytes In general samples should be collected from emergent macrophytes such as Sparganium only if there are portions that remain permanently submerged but which are not contaminated by the bottom sediments 1 As far as possible comparative studies in rivers should be based on samples collected from the same macrophyte species or group of closely related species 2 Details of the species of macrophytes used should be included with field sampling notes 3 At least five stems should be cut at water level and a plastic sampling bottle or glass jar put upside down on the underwater stem Th
25. only partially visible in a field of view If sample analysis is unlikely to be completed in a single session then it is useful to record the position of each traverse using the Vernier scale This ensures that subsequent traverses do not overlap with those already completed Additional precautions are required if more than one microscope is likely to be used for analysis as positions on Vernier scales may differ between microscopes 4 3 4 Treatment of broken and other unidentifiable diatoms Include a valve in a count only if both the central area and one pole are present see Fig 4 1 Otherwise omit For Asterionella Nitzschia Diatoma and other taxa with no clear central area count the poles and divide by two This avoids the possibility of double counting d D C d Fig 4 1 Two examples of how to treat broken valves in TDI analyses Fragilaria vaucheriae and b and Nitzschia dissipata c and d a shows a broken valve of F vaucheriae with one pole and a central area which should be included in a count whilst b shows a broken valve with one pole but no central area This should not be included N dissipata does not have an obvious central area Comparison with intact specimens will indicate that the specimen in diagram should be included whilst that in d should be excluded R amp D TECHNICAL REPORT E2 TR2 28 Because physical damage during sampling and preparation is likely to be negligible the pres
26. permanganate crystals to beaker containing sample sulphuric acid hydrochloric acid Addition of oxalic acid to beaker containing sample sulphuric acid and potassium permanganate Transfer of contents of beaker to centrifuge tube and subsequent centrifugation R amp D TECHNICAL REPORT E2 TR2 3 IDENTIFICATION OF HAZARD HARMFUL EFFECTS 4 IDENTIFICATION 9 OF PERSONS AFFECTED INITIAL RISK LEVEL H M L Biology Team H 6 Biology Team H 6 Biology Team L 2 Biology Team H 6 Biology Team H 6 Biology Team M 4 Biology Team H 6 Biology Team M 4 Biology Team M 4 Contact with swallowing oxalic acid crystals and splashing spillage of solution containing oxalic acid refer to COSHH assessment Splashing spillage of oxalic acid sulphuric acid and hydrochloric acid solutions refer to COSHH assessments Splashing spillage of solution containing iodine refer to COSHH assessment Splashing spillage of hydrochloric acid and solution containing iodine hydrochloric acid refer to COSHH assessments Splashing spillage of solution containing iodine hydrochloric acid refer to COSHH assessments Splashing spillage of sulphuric acid refer to COSHH assessment Contact with swallowing potassium permanganate crystals inhalation of vapours and splashing spillage of solution containing refer to COSHH assessment Splashing spillage of oxalic acid beaker containing mixt
27. problems of overwhelming dominance of some taxa To bring manual in line with recommendations draft European standard Advice of Project Board DQI to be recommended only for reporting for UWWTD purposes Information on calculation of DQI is included in 5 3 Many new insights gained through widespread use of the TDI A greater reliance on external contractors than envisaged when the manual was first written Audit procedure extended to cover quality of slide preparation New audit procedure for comparison of slides Audit procedure for checking data added QA issues when using contractors added R amp D TECHNICAL REPORT E2 TR2 To ensure that the slide archive is of high quality and to minimise enumeration problems caused by poor quality slides Previous system proved to be unworkable in practice This new system has been tested extensively As used in other procedural manuals More widespread use of contractors than envisaged when 1 edition written This page has been left blank intentionally R amp D TECHNICAL REPORT E2 TR2 82 APPENDIX B FIELD RECORD FORM River Site Date NGR Sample collected by Physical records measure as for RIVPACS Width Depth Substrate record estimated percentage bedrock boulders cobbles a pebbles gravel sand silt clay peat Estimate percentage of boulders and cobbles covered Cladophora and other filamentous algae other macrophytes
28. provided to help with data analysis Adjacent sites are entered into the appropriate boxes on the table and joined by arrows A vertical movement then indicates a change in the TDI whilst a horizontal movement indicates changes due to other factors Table 6 4 shows an example of the look up table as applied to the situation described in Fig 6 3 TDI values that fall in the left hand column should be considered as reliable estimates of the level of eutrophication at a particular site although such conclusions need corroboration from replicate surveys see section 6 5 Look up charts have been proved to be useful in many situations and Harding and Kelly 1999 contain examples of look up charts that have been used to prepare UWWTD designations However they should be used as tools for exploring spatial patterns in the data rather than as standalone evidence of the effect of nutrients on a river Upstream of town TOWN Downstream of town including storm sewers but upstream of STW STW Immediately downstream of STW Further downstream in recovery zone of STW Fig 6 3 A catchment based approach to detecting major sources of nutrients A D sampling stations STW sewage treatment works See text and Kelly 1998 for more details R amp D TECHNICAL REPORT E2 TR2 49 Hints for data interpretation 4 spatial variation in TDI Prior to a comparison test the assumption that there is no reason to expect the flora
29. response of the flora to nutrients from responses to factors that were correlated with nutrients As the main source of P in lowland rivers is from sewage discharges it is particularly important to recognise the influence of other components of sewage effluents on benthic diatom assemblages Table 1 1 Kelly et al 1996 In broad terms the TDI is more likely to be sensitive to change e following nutrient removal in rivers where the levels of other physical and chemical stresses are minimal This corresponds to General Quality Assessment biological and chemical classes A and B 6 4 Variation in space Changes in the longitudinal distribution of diatom taxa along stream gradients occur even in the absence of human impacts Allen 1995 Molloy 1992 It is important when using indices such as the TDI that such changes are not confused with changes due to human impacts Most comparisons involving the TDI take place over relatively short spatial scale i e comparisons above and below a STW might use two sites km apart in which case it should be possible to assume that changes due to non human factors are negligible However this assumption must be evaluated separately for each comparison Factors such as a major change in physical attributes slope current speed temperature between sites might invalidate such an assumption R amp D TECHNICAL REPORT E2 TR2 47 Hints for data interpretation 3 threshold of sensitivity Analyse r
30. shaken vigorously in either stream water or distilled water to dislodge attached diatoms or they should be scraped using a toothbrush The rope R amp D TECHNICAL REPORT E2 TR2 13 should then be removed and the suspension decanted into a sample bottle This procedure can take place either in the laboratory or the field Best practice for the deployment of ropes The aim of rope substrates is to simulate fine leaved aquatic macrophytes such as Ranunculus or Potamogeton The length of rope required will depend upon water depth i e sufficient to allow sufficient rope to be sampled see 2 7 point 4 to be suspended just under the water surface Rope with a diameter of about 7 mm is recommended Polypropylene rope available from DIY stores has been used extensively but other types of rope may also be suitable No tests have been performed on the relative performance of different types of rope Attachment different Regions have experimented with the use of bricks to anchor one end of the rope and stakes to attach the rope to the river bed Scan the river bed first to ensure that there are no buried electric cables Further options include attaching ropes to bridge supports or to buoys in the case of navigable rivers The ropes should be placed far enough away from the bank that they do not become entangled with bankside vegetation and are not heavily shaded A nail pushed through the rope about 30 cm from the end has also been used as a means
31. slide R amp D TECHNICAL REPORT E2 TR2 64 Quality management is the responsibility of a designated Quality Manager who may be the relevant Team Leader This person does not have to be trained in diatom analyses but does need an awareness of general QA and AQC issues 7 3 2 Ensuring the quality of permanent slides The facility to produce permanent slides in TDI analyses is an important benefit These slides if correctly prepared and stored represent an archive of conditions that can be consulted or even re analysed in the future For this reason it is important that the slides themselves are subjected to quality assurance procedures At least 10 of slides must be subjected to this audit Two semi quantitative scales are used for this to evaluate the quality of the preparation itself and the density of valves These scales are as follows Quality of preparations v poor 3 acceptable 5 v good Organic matter not completely removed from valves and or mountant not properly cured and or many air bubbles in mountant and or mountant not spreading right to edge of coverslip and or distribution of valves on coverslip clumped and or large quantities of foreign matter that cause problems in identification or enumeration of the specimens Mountant is properly cured and spread to edges of coverslip air bubbles absent distribution of valves shows some variability including edge effects but any clumping is not so serious that
32. that ultrapure water typically has a pH of about 5 6 so it is not possible to obtain a completely neutral solution 6 When the acid has been removed transfer the cleaned material to a small glass vial using a clean Pasteur pipette Add a small amount of distilled water to wash material from the wall of the tube This sample must be labelled with waterbody name reach name and sample date as a minimum and should be stored prior to slide preparation A preservative a few drops of formaldehyde or alcohol should be added to prevent microbial growth 7 See 3 5 for details of slide preparation 3 3 Hot peroxide method This method is suitable for all types of sample described in Chapter 2 including circumstances where entire rope or macrophytes samples need to be digested 3 3 1 Reagents and apparatus required Figures in square brackets give some indication of the quantities required for a typical sample All of these reagents are stable under normal conditions and if you are planning to look at lots of diatom samples in the next few months then it is worth preparing large batches of the reagents The limiting step in terms of efficient use of time is the number of digestion vessels that can fit on the hotplate Analytical grade reagents are not necessary for this procedure e 30 100 volume hydrogen peroxide H202 solution 20 ml per sample more if digesting rope or macrophytes samples see note after 3 3 2 point 1 e Dilut
33. to the taxonomic burden this genus has been retained within Achnanthes other Similarly not all of the new genera that have been split from Navicula in recent years e g Hippodonta Geissleria Mayamaea Eolimna Fistulifera are distinguished in the TDI and users should continue to use either Navicula other or Navicula small for these as appropriate Table 5 1 Nutrient sensitivities s and indicator values v for taxa included in the TDI No entry in comment column indicates no change since previous version of index Taxon sjy Comment LL NN demens 0 0 Moved om io anton Wowemsdes See under kargea Moved Seer Parmar aera Wowewkspbeess WeRdei inanes bromides ee nier Parmar stones Achnanthidium Previously Achnanthes minutissima type Includes A minutissimum A microcephalum A biasolettiana Achnanthes other Excluding taxa now moved to Achnanthidium Planothidium Psammothidium Karayevia Kolbesia but including Rossithidium R amp D TECHNICAL REPORT E2 TR2 32 Tm Com Bacar pater i Wwuwm Malle Bache ES Me dons Ceratoneis arcus see Hannaea Chaetoceras 00 Cocconeis pediculus 4 2 Cocconeis placentula 32 Cocconeis other 22 Craticula New taxon Motile Split from Navicula 2 ho wm to other Ctenophora pulchella Formerly Synedra pulchella Cyclostephanos 00 Cyclotella 00 Cymatopleura O3 Cymbella affinis 113
34. 1995 Comparative performance of benthic diatom indices used to assess river water quality Hydrobiologia 302 179 188 Kelly M G Whitton B A amp Lewis A 1996 Use of diatoms to monitor eutrophication in U K rivers In Use of Algae to Monitor Rivers ed B A Whitton E Pipp and E Rott pp 79 86 Universitat Innsbruck Innsbruck Krammer K amp Lange Bertalot H 1986 1991 Die S sswasserflora von Mitteleuropa 2 Bacillariophyceae 4 Teil Stuttgart Gustav Fischer Verlag Lancaster J Real M Juggins S Monteith D T Flower R J amp Beaumont W R C 1996 Monitoring temporal changes in the biology of acid waters Freshwater Biology 36 179 201 Magurran A E 1988 Ecological Diversity and its Measurement Croom Helm London Marker A F H 1976 The benthic algae of some streams in southern England Biomass of the epilithon in some small streams Journal of Ecology 64 343 358 May R M 1975 Patterns of species abundance and diversity pp 81 120 Ecology and Evolution of Communities ed M L Cody and J M Diamond Belknap Press Cambridge Mass Molloy J M 1992 Diatom communities along stream longitudinal gradients Freshwater Biology 28 56 69 Peterman amp MoOonigle M 1992 Statistical power analysis and the precautionary principle Marine Pollution Bulletin 24 231 234 Prygiel J amp Coste M 2000 Guide M thodologique pour la mise en oeuvre de l Indice Biologique Dia
35. 2 111 AREA TEAMS BIOLOGY DESCRIBE WHY IT IS NOT REASONABLY PRACTICABLE TO USE A SAFER SUBSTITUTE LIST OF ASSOCIATED ACTIVITIES AND POSSIBLE CAUSES OF EXPOSURE DESCRIBE ANY HAZARDOUS PROPERTIES OTHER THAN THOSE CAUSING RISKS TO HEALTH AND CONFIRM THAT CONTROLS ARE SPECIFIED IN THE TASK RISK ASSESSMENT DESCRIBE ANY Hi R amp D TECHNICAL REPORT E2 TR2 No lower risk alternative identified DATE Decanting stock solution of methylated spirit industrial IMS is a lower risk substitute for formaldehyde which could be used for the Highly flammable Vapour air mixture explosive Incompatible with various chemicals storage arrangements specified in task risk assessment Biology Team Temporary staff students have less experience of working with chemicals Others in lab e g building contractors cleaners visitors Emergency Services and other EA staff in rest of building in event of major spill or fire 112 aL BY 1 Jel 57 SMALL __ LARGE REFER TO THE TABLE ON P 94 TO FIND THE LEVEL OF CONTROL YOU NEED TICK THE APPROPRIATE BOX TO IDENTIFY THE CONTROL LEVEL NEEDED SAFE USE OF PPE EYE PROTECTION SKIN PROTECTION R amp D TECHNICAL REPORT E2 TR2 113 RISK CONTROL RISK CONTROL APPROACH GENERAL VENTILATION Natural or forced ventilation within the range of 5 and 15 air changes per hour to dilute the contaminant to a safe level Working pro
36. 2 TR2 Preparation of saturated oxalic acid Decanting saturated oxalic acid into glass dropping bottle and subsequent use in Contact with skin and eyes causes severe irritation and possible eye burns Causes gastrointestinal tract burns Causes severe pain nausea vomiting diarrhoea and shock May cause haemorrhaging of digestive tract Overexposure can cause hypocalcemia and kidney injury May cause severe irritation of respiratory tract with pain burns and inflammation Incompatible with combustible materials and certain other chemicals including oxidising agents storage conditions specified in task risk assessment Contamination of waste water Chemical digestion is an essential pre cursor to microscopic examination of diatom samples Chemical digestion with oxalic acid and sulphuric acid has been identified as the safest option and replaces the previous use of hydrogen peroxide Breakage leakage of stock solution 120 Biology Team Temporary staff students have less experience of working with chemicals only staff deemed competent will be permitted to undertake tasks using this substance Others in lab e g building contrators cleaners visitors access to lab restricted to authorised personnel only al Bufe 1 si raas or millilitres SMALL LARGE row MEDIUM HIGH IDENTIFY HOW DUSTY OR VOLATILE THE SUBSTANCE IS
37. 3000 5000 rpm for 3 5 minutes has been used successfully in past but the efficiency depends upon the gravitational forces exerted which is function of the rotor properties as well as the speed and users must satisfy themselves that these conditions are suitable for their own equipment before proceeding Decant the supernatant treating the waste liquid appropriately If the reaction is complete then the hydrogen peroxide should all be oxidised so supernatant can be washed down the sink with plenty of water Resuspend pellet with distilled water to cm below the rim of the centrifuge tube and repeat centrifugation stage twice more R amp D TECHNICAL REPORT E2 TR2 21 5 When all traces of hydrogen peroxide and acid have been removed transfer the cleaned material to a small glass vial using a clean Pasteur pipette Add a small amount of distilled water to wash material from the wall of the tube This sample must be labelled with waterbody name reach name and sample date as a minimum and should be stored prior to slide preparation A preservative a few drops of formaldehyde alcohol or hydrogen peroxide should be added to prevent microbial growth 3 4 Preparation of permanent slides 3 4 Apparatus and reagents Brand new glassware slides coverslips Pasteur pipettes must be used to minimise risks of contamination e Naphrax This is a diatom mountant supplied in toluene It can only be bought from Northern Biological Supplies
38. 4 ml per 100 ml 9 If samples are to be stored for some time the suspension can be concentrated by allowing it to settle overnight decanting the supernatant and transferring the sediment to a smaller e g 60 100 ml bottle R amp D TECHNICAL REPORT E2 TR2 12 2 6 Method for sampling vertical man made structures in situ 1 The apparatus see 2 4 should be scraped along the surface to be sampled at a depth of about 30 cm to allow for fluctuating water levels and wave action The diatom film dislodged as a result of this scraping falls into the net and bottle 2 This scraping should take place at five different places to simulate the five replicates in 2 5 The total area covered must be at least 10 If the diatom film is sparse then this area should be increased 3 See 2 5 points 6 to 9 for instructions on labelling preservation transport and storage of samples 4 The net and collecting bottle used must be well rinsed between samples 27 Method for installing and using frayed polypropylene rope as an artificial substrate Precise details of the deployment of rope substrates cannot be given This is an area where the ingenuity of the sampler needs to be exploited to the full The following points represent principles that must be followed on all occasions These are followed by some examples of best practice developed within the Agency over the past five years and from the literature Cattaneo amp Ami
39. ATION OF HAZARD HARMFUL EFFECTS 4 IDENTIFICATION S OF PERSONS INITIAL AFFECTED RISK LEVEL H M L Biology Team M 3 Biology Team L 3 Biology Team L 2 Biology Team L 3 Biology Team L 2 Splashing spillage of any preservatives added to the sample vial refer to COSHH assessment Contact of skin eyes with hydrogen peroxide solution refer to COSHH assessment Electric shock scalding from hotplate Cuts from breaking cover slip Splashing spillage of Naphrax solution refer to toluene COSHH assessment Cuts from breaking microscope slide and or coverslip Electric shock Cuts from breaking microscope slide and or coverslip Cuts from breaking microscope Slide and or coverslip R amp D TECHNICAL REPORT E2 TR2 6 SUGGESTED RISK CONTROL MEASURES 7 LEVEL OF RISK H M L 8 RISK CONTROL MEASURES ADEQUATE YES NO Restricted access to working area Laboratory CoP Laboratory induction procedure Activity undertaken by authorised personnel only Chemical spillage procedures Fume cupboard Equipment maintenance record Hydrogen peroxide COSHH Risk Assessment Eye wash facilities safety shower Latex gloves safety spectacles laboratory coat As 1 above Electrical safety testing Hot plate operating instructions As 1 above Naphrax toluene COSHH Risk Assessment Restricted access to working area Laboratory CoP Laboratory induction procedure
40. D TECHNICAL REPORT E2 TR2 42 Table 6 1 Some non nutrient factors which can influence the composition of the diatom assemblage Factor 1 nature of substrate 2 presence of filamentous algae or macrophytes 3 current speed 4 grazing pressure 5 organic pollution 6 heavy metals 7 eutrophication 8 salinity 9 pH 10 temperature other Response hard surfaces favoured by attached forms silty surfaces favour motile forms certain taxa are characteristically epiphytic sluggish currents favour loosely attached filaments fast currents favour closely adpressed forms some species are more resistant to grazing than others some species are capable of heterotrophic growth some species have morphological aberrations in the presence of heavy metals some species can develop tolerance some taxa have a competitive advantage at low nutrient concentrations others grow well at higher concentrations some taxa are characteristic of brackish conditions some taxa favoured by low pH some taxa have a competitive advantage at low temperatures R amp D TECHNICAL REPORT E2 TR2 43 Examples of taxa favoured by these conditions Gomphonema Navicula Nitzschia Rhoicosphenia Melosira Cocconeis Cocconeis placentula Nitzschia palea Fragilaria capucina Achnanthidium minutissimum many Eunotia spp Diatoma vulgare Pleurosigma Melosira nummuloides Eunotia exi
41. Data used in calculation of TDI and for site on River Browney on 5 July 1994 Gomphonema olivaceum R amp D TECHNICAL REPORT E2 TR2 38 76 e2 ee O2 un N e QJ lt AB j lt O3 N QN 2 12 4 23 38 54 34 4 3 45 1 1 1 E gt E comment 25 5 Un 12 4 5 22 2 14 Motile EE ae 6 2 84 21 J 4 UA 2 un E EN j lt ho e QC QC WMS Xasv 1528 388 3 97 TDI WMS 25 25 3 97 x 25 25 99 2 25 74 2 74 100 TDI 100 74 26 R amp D TECHNICAL REPORT E2 TR2 39 Ymotile taxa 38 203 x 100 18 7 Interpretation The data indicates a fairly eutrophic site TDI 2 74 See Chapter 6 for more details on interpretation R amp D TECHNICAL REPORT E2 TR2 40 6 INTERPRETING THE RESULTS 6 1 Introduction In this chapter a brief overview is given of how diatoms through the TDI can be used to help make decisions for the management of nutrients in rivers Our understanding of eutrophication in running waters as opposed to lakes is relatively crude and we have little experience of using phosphorus stripping on large sewage works in this country Whilst a great deal of spatial monitoring has taken place since the first edition of this manual was published in 1996 there has been little oppo
42. It is considered in more detail in 6 5 1 The latter option is considered here R amp D TECHNICAL REPORT E2 TR2 44 As a rule of thumb any situation where extended counting protocols are invoked see 4 4 point 6 is a candidate for modification of the TDI The assumption underlying the modification is that sub dominant and other taxa present in such a sample are more likely to give an indication of the trophic status than the dominant organism The TDI is therefore recalculated with the dominant taxon removed This strategy is worth following so long as the following conditions are met e The count size must be increased or a stratified counting strategy used see section 4 4 point 6 so that the modified TDI calculations are based on a statistically valid sample size e TDI results must be presented with and without modification and the methods section of any report must state clearly what has been done e Any report should also include an ecological justification for the modification written in terms that non biologists can understand e It is also useful to present a scatter chart showing the relationship between the original and modified TDI However there are also circumstances where diversity is low but the trophic status indicated by the diatom assemblage is clear Assemblages with low diversity but dominated by Eunotia or Achnanthidium typically represent relatively low nutrient concentrations within the context of UWWTD rela
43. Morphology of the Diatom Frustule Scientific Publication of the Freshwater Biological Association U K No 44 Battarbee R W 1986 Diatom analysis In Handbook of Holocene Palaeoecology and Palaeohydrology ed B E Berglund pp 527 570 Wiley Chichester Biggs B J F amp Lowe R L 1994 Responses of two trophic levels to patch enrichment along a New Zealand stream continuum New Zealand Journal of Marine and Freshwater Research 28 119 134 Cattaneo amp Amireault M C 1992 How artificial are artificial substrata for periphyton Journal of the North American Benthological Society 11 244 256 Chesters R K 1980 Biological Monitoring Working Party The 1978 Testing Exercise Department of the Environment Water Data Unit Technical Memorandum 19 1 37 Coring E 1993 Zum Indikationswert benthischer Diatomeengesellschaften basenarmen Fliesgew ssern Reihe Biologie Verlag Shaker Aachen Coring E Schneider S Hamm A amp Hofmann G 1999 Durchgehendes Trophiesystem auf der Grundlage der Trophieindikaation mit Kieselalgen 219pp Deutscher Verband f r Wasserwirtschaft und Kulturbau e V Koblenz Coste M Bosca C Dauta A 1991 Use of algae for monitoring rivers in France In Use of Algae for Monitoring Rivers ed B A Whitton E Rott amp G Friedrich pp 75 88 E Rott Innsbruck Cox E J 1996 Identification of Freshwater Diatoms from Live Material Chapman amp Hall London Dell
44. R2 V O N nN Aner m ie QUALITY ASSURANCE 7 1 Introduction 7 2 Training requirements 7 3 Quality control and audit procedures 7 4 Use of contractors LIST OF ABBREVIATIONS AND ACRONYMS LIST OF FIGURES LIST OF TABLES GLOSSARY REFERENCES APPENDIX A CHANGES TO THIS EDITION OF TDI MANUAL APPENDIX B FIELD RECORD FORM APPENDIX C RISK ASSESSMENTS FOR ACTIVITIES ASSOCIATED WITH THE TROPHIC DIATOM INDEX R amp D TECHNICAL REPORT E2 TR2 vi 71 72 75 79 1 85 1 INTRODUCTION 1 1 Purpose and scope This manual sets out the procedures for collecting examining analysing and interpreting benthic diatom samples for use in the assessment of trophic conditions in streams and rivers with respect to the Urban Wastewater Treatment Directive UWWTD European Community 1991 staff undertaking such work must follow these procedures This includes staff within the Environment Agency and contractors working for it Methods described in this manual may also be appropriate for other monitoring purposes It is recommended that these methods are used whenever benthic diatom samples are collected in order to ensure consistency within and between laboratories and comparability of survey results Good management practices must be followed at all stages to ensure that the results meet this criterion If procedures in this manual are not followed comparisons of data may be misleading and could lead to erroneous conclusions Before emba
45. T E2 TR2 2 DESCRIPTION OF TASK ELEMENTS 3 IDENTIFICATION OF HAZARD HARMFUL EFFECTS Road traffic accidents breakdown Road traffic hazards Attacks trampling by livestock Hostile landowners pets Fractures etc from tripping falling including crossing fences gates Hypothermia frost bite Sunburn skin cancer over heating heat exhaustion Steep banks deep and fast flowing water Fractures drowning from tripping falling slipping Cuts from scissors Tripping falling on uneven river bed Cuts from glass etc on river bed Submersion Poisoning infection through contact with contaminated polluted water Weil s Disease Cuts from sharp edges of natural substrates stones etc Poisoning infection through contact with contaminated polluted water Weil s Disease 88 4 IDENTIFICATION 5 OF PERSONS INITIAL AFFECTED RISK LEVEL H M L 1 Loading unloading sampling safety Road traffic hazards Biology Team M 3 equipment from vehicle Lifting injuries Biology Team H 6 Biology Team M 3 Biology Team M 4 Biology Team M 3 Biology Team M 3 R amp D TECHNICAL REPORT E2 TR2 6 SUGGESTED RISK CONTROL MEASURES 7 LEVEL OF RISK High visibility jackets Training in manual handling Regularly serviced maintained vehicles Authorised drivers only annual licence checks Field worker monitoring procedure High visibility jackets Hostile situations training Do
46. The Trophic Diatom Index A User s Manual Revised Edition Research and Development Technical Report E2 TR2 ENVIRONMENT AGENCY The Trophic Diatom Index A User s Manual Revised edition R amp D Technical Report E2 TR2 M G Kelly C Adams A C Graves J Jamieson J Krokowski E B Lycett J Murray Bligh S Pritchard amp C Wilkins Research Contractor Bowburn Consultancy Publishing Organisation Environment Agency Rio House Waterside Drive Aztec West Almondsbury BRISTOL BS32 4UD Tel 01454 624400 Fax 01454 624409 Website www environment agency gov uk Environment Agency 2001 May 2001 ISBN 1 857 05597 7 All rights reserved No part of this document may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording or otherwise without the prior permission of the Environment Agency The views expressed in this document are not necessarily those of the Environment Agency Its officers servants or agents accept no liability whatsoever for any loss or damage arising from the interpretation or use of the information or reliance upon views contained herein Dissemination Status Internal Released to Regions External Released to Public Domain Statement of Use This report guides users in appropriate methods of collection preparation identification and enumeration of benthic diatoms from running waters and of calculation an
47. Uomo A 1996 Assessment of water quality of an Apennine river as a pilot study for diatom based monitoring of Italian watercourses In Use of Algae for Monitoring Rivers ed B A Whitton amp E Rott pp 65 72 Universitat Innsbruck Innsbruck Department of the Environment Transport and Regions and Welsh Office 2000 Urban Waste Water amp Nitrate Directives Sensitive Areas Eutrophic and Polluted Waters Eutrophic Guidance Note on Information Gathering for Future Designation Reviews Departmetn of the Environment Transport and Regions London R amp D TECHNICAL REPORT E2 TR2 75 Eloranta P amp Andersson K 1997 Diatom indices in water quality monitoring of some South Finnish rivers Verhandlungen Internationale Vereinigung f r Theoretische und Angewandte Limnologie 27 1213 1215 Engelberg K 1957 Die Diatomeen Z nose in eimem Mittelgebirgsbach und die Abgrenzung jahreszeitlicher Aspekte mit Hilfe der Dominanz Identitdt Archiv f r Hydrobiologie 110 217 236 European Community 1991 Council directive of 21 May 1991 concerning urban waste water treatment 91 271 EEC Official Journal of the European Community Series L 135 40 52 Extence C A Balbi D M amp Chadd R P 1999 River flow indexing using British benthic macroinvertebrates a framework for setting hydroecological objectives Regulated Rivers Research amp Management 15 543 574 Fairweather P G 1991 Statistical power and design requ
48. abolism that requires a supply of organic material from the environment Some diatoms are facultative heterotrophs with respect to carbon and or nitrogen Any aquatic plant recognisable with the naked eye Includes a few larger algae e g Cladophora Lemanea plus bryophytes and vascular plants Side part of the valve biotic index based on the relative abundance of macrophyte taxa in a survey length typically 100 An aquatic ecosystem containing very low concentrations of inorganic nutrients cf eutrophic Stones with a diameter gt 16 lt 64 mm A diatom showing longitudinal date box symmetry A measure of reliability of the Trophic Diatom Index used in the first version but not in this update Those photosynthetic taxa found primarily in the plankton Those organisms which live suspended in the water column Cf benthos Procedures to quantify and control or reduce errors See Analytical Quality Control R amp D TECHNICAL REPORT E2 TR2 73 Raphe Rapid Riffle Run Sensitive area eutrophic Trophic TDD Valve Diatom Index Longitudinal slit or furrow found in many pennate diatoms through which mucus for support or motility is extruded Divided into two parts arranged end to end with a gap at the centre An area of broken standing waves forming distinctive whitewater conditions normally over cobble or boulder substrate Associated with steep gradient rivers and streams Raven
49. actors Percentage of motile valves TDI lt 20 21 40 41 60 gt 60 0 9 10 19 20 29 30 39 40 49 50 59 60 69 79 80 89 gt 90 R amp D TECHNICAL REPORT E2 TR2 52 Table 6 4 Example of look up chart as applied to the situation described in Table 6 3 See text for further details Percentage of motile valves TDI lt 20 21 40 41 60 gt 60 40 49 50 59 60 69 70 79 80 89 R amp D TECHNICAL REPORT E2 TR2 53 a Cocconeis placentula gt 50 40 30 20 Percent 10 b Navicula lanceolata gt 25 Percent Fig 6 4 Seasonal occurrence of high proportions of Cocconeis placentula and Navicula lanceolata in benthic diatom samples from the UK Shaded area indicates the seasonal occurrence of all samples in the dataset 1861 samples and represents relative sampling effort Solid bars indicate percentage of occurrences exceeding threshold values set at roughly the upper 5 percentile In both cases there is a statistically significant relationship between sampling effort and the distribution pattern of the diatom in question R amp D TECHNICAL REPORT E2 TR2 54 6 5 2 Handling between survey variation Between surveys variation is apparent even if guidelines in sections 2 3 and 6 4 are followed closely This reflects the complexity of river and stream systems and the influence of non nutrient factors Although most spates treated individually have relatively l
50. aldehyde is used As formaldehyde is heavier than air a local exhaust ventilation system which draws air away at the bottom is required 10 AUTHORISATION CHECKED BY SIGNED R amp D TECHNICAL REPORT E2 TR2 90 PART 2 RISK ASSESSMENT SHEET 2 DESCRIPTION OF TASK ELEMENTS 3 IDENTIFICATION OF HAZARD 4 IDENTIFICATION 5 HARMFUL EFFECTS OF PERSONS INITIAL AFFECTED RISK LEVEL H M L Opening sample container Splashing spillage of Biology Team Addition of iodine solution to preservative refer to COSHH sample container Sealing of sample assessment container 6 SUGGESTED RISK CONTROL 7 LEVEL OF 8 RISK 9 OPTIONS 10 MEASURES RISK H M L CONTROL FOR PRIORITY MEASURES IMPROVED OF ADEQUATE RISK ACTIONS YES NO CONTROL REQUIRED H M L Restricted access to working area Laboratory CoP Laboratory induction procedure Activity undertaken by authorised personnel only Chemical inventory storage arrangements Chemical spillage procedures Fume cupboard or local exhaust ventilation facilities COSHH Risk Assessment for preservative Heavy duty rubber latex gloves safety spectacles laboratory coat Eye wash facilities safety shower R amp D TECHNICAL REPORT E2 TR2 91 C3 Digestion of diatom samples using sulphuric and oxalic acids and potassium permanganate PART 1 WORK ACTIVITY TASK IDENTIFICATION 1 REGION AREA Insert as appropriate 2 TEAMS JOB TITLES Biology Team Leader Biology Biologists Stu
51. all of the supernatant and treat the waste liquid appropriately 2 Add 5 ml concentrated sulphuric acid and agitate gently 3 Add a few drops of a saturated solution of potassium permanganate and agitate gently After this step the suspension should have a purple colour If this is not the case add a few more drops 4 Add 10 ml saturated oxalic acid slowly to the sample by trickling it down the wall of the centrifuge tube There will be much fizzing and effervescence which is why this must be done slowly The end product should be a clear solution with a bleached precipitate at the bottom of the tube If this is not the case and organic matter has not been fully oxidised add more oxalic acid until the colour change is complete Do not add more than 5 ml additional oxalic acid after the first 10 ml If there is still unoxidised organic matter you should centrifuge the sample then repeat steps to 5 Alternatively the procedure should be repeated with a smaller quantity of material 5 Add distilled water to 1 cm below the rim of the centrifuge mix the sample and centrifuge as before Decant the supernatant and treat the waste liquid appropriately Repeat this centrifugation stage twice more After the supernatant has been decanted for the third time test the pH of the remaining suspension with indicator paper If it is still strongly acid then the centrifugation step should be R amp D TECHNICAL REPORT E2 TR2 19 repeated Note
52. ammothidium subatomoides New taxon added Kolbesia Genus split from Achnanthes other Only one common species ploenensis Karayevia Genus split from Achnanthes Only one common species K clevei Bacillaria paxillifer New taxon added Craticula Genus split from Navicula other Diadesmis Genus split from Navicula other Diatoma mesodon New taxon added Diatoma moniliforme New taxon added Gomphonema minutum New taxon added Fragilariforma Genus split from Fragilaria other Luticola Genus split from Navicula other Navicula capitoradiata New taxon added Nitzschia fonticola New taxon added Nitzschia palea New taxon added R amp D TECHNICAL REPORT E2 TR2 37 5 3 Calculation of the TDI and 1 Calculate for each taxon abundance a x v and a x s x v 2 Add together all values for av to give Zav 3 Repeat for asv to give Zasv 4 Calculate Zasv to give WMS 5 Calculate TDI as WMS x 25 25 6 Add up the number of valves belonging to motile taxa and calculate this as a percentage of the total Calculation of the index can be easily automated via a spreadsheet or database package For some reporting purposes results should be presented as the Diatom Quality Index which is 100 TDI See Chapter 6 for details of how to interpret these results 5 4 Worked example A practical example River Browney upstream of Browney Sewage Treatment Works is presented below Table 5 3
53. and just begins to bubble Apply gentle pressure e g with the end of a pair of blunt forceps to remove air bubbles Repeat this 3 4 times then allow to cool Ensure that the mountant has been properly cured by checking to see if the coverslip moves when pushed with a R amp D TECHNICAL REPORT E2 TR2 23 fingertip If it cannot be moved then check the valve density again under a microscope 5 When a satisfactory mount has been prepared it must be labelled with the site location and date of collection and any information i e site codes to permit cross reference with computer records 3 5 Archiving diatom slides and samples Diatom slides provide a permanent historical record of conditions at a site and should be stored in order to ensure that they can be accessed for future analyses is recommended that at least two slides are prepared from each sample One of these should be lodged in the appropriate national herbarium for future reference These are England and Wales The Natural History Museum Cromwell Road London SW7 5BD Scotland The Royal Botanic Gardens Edinburgh EH3 5LR Northern Ireland Botanic Gardens Ulster Museum Belfast BT9 5AB samples to be lodged with herbaria should be labelled with the following information use a fine tipped pen to make the task of curation easier Name of collector if several are involved one name plus et al is appropriate Environment Agency is adequate Date of co
54. at the sites involved to be different if there were no human impacts on the river Variation in space needs to be considered in light of issues raised in 6 3 concerning the limits of sensitivity of the TDI Consider the possible impact of urban runoff storm sewers and trade effluents on sites upstream of STWs when designing a survey Be aware that the flora at sites immediately downstream of STWs may be impacted by factors other than nutrients Place additional sampling sites in the recovery zone if necessary Y Sample collection and storage is relatively cheap so it is better to collect too many samples from a river rather than too few Treat the first survey on a stretch of river as provisional and be prepared to change locations of sampling sites in light of experiences Consider the use of look up charts to aid data interpretation 6 5 Variation in time 6 5 1 Timing of surveys Although samples can be collected from any season although winter should be avoided see Chapter 2 the ideal time for a survey will depend upon local circumstances The main ecological factor influencing sampling is the seasonal dominance of weedy taxa which tend to obscure real patterns of variation within the reach Two taxa Cocconeis placentula and Navicula lanceolata cause particular problems under certain circumstances detailed below Other taxa Melosira varians Navicula gregaria Nitzschia other can c
55. ative pressure with safe discharge arrangements for extracted air Typically requiring permit to work maintenance procedures SPECIAL CONTROLS Controls designed by experts in occupational hygiene to provide site specific solutions for cases where an extreme risk to health exists PROTECTION OF SKIN amp EYES Special attention to control measures protecting the individual and ensuring the effective use of items of personal protective equipment Restricted access to working area Laboratory CoP Laboratory induction procedure Activity undertaken by authorised personnel only Chemical inventory storage arrangements Chemical spillage procedures GIVE DETAILS OF THE Fume cupboard CURRENT OR PROPOSED Eye wash facilities safety shower METHODS OF CONTROL PPE heavy duty rubber latex gloves laboratory coat safety spectacles R amp D TECHNICAL REPORT E2 TR2 106 IMPLEMENTATION OF FURTHER ACTIONS OF FURTHER ACTIONS ACTION BY WHOM TARGET DATE R amp D TECHNICAL REPORT E2 TR2 107 AREA TEAMS BIOLOGY REASONABLY PRACTICABLE TO LIST OF ASSOCIATED ACTIVITIES AND POSSIBLE CAUSES OF EXPOSURE DESCRIBE ANY HAZARDOUS PROPERTIES OTHER THAN THOSE DATE Laboratory and field procedures involving use of 4 formaldehyde solution diluted from 40 stock solution Need to fix preserve samples between time of collection and analysis seasonality of workload dictates that samples cannot be analys
56. atom assemblage to assess the confidence with which the TDI can be interpreted seemed fundamentally sound However this update of the TDI uses a slightly different criterion the percentage of valves belonging to motile taxa motile and the rationale for using this criterion is explained below There is some evidence that the growth forms of diatoms can provide insights into factors influencing community composition Sediments for example are typically dominated by motile diatoms which are able to avoid burial by fresh depositions Round et al 1990 The close proximity of oxidising and reducing conditions within sediments along with often relatively high concentrations of organic matter favours the evolution of traits such as tolerance to toxins and the capacity for facultative heterotrophy within sediment dwelling taxa contrast headwater streams are typically dominated by growth forms adapted to withstand or recover rapidly from disturbances Molloy 1992 Within a reach there will also be temporal and spatial heterogeneity in relative abundance of growth forms related to local hydrological conditions Marker 1976 Local spatial heterogeneity should not be a significant issue for users of the TDI if the guidelines in chapter 2 sample collection are followed However if there are differences in average hydrological conditions between two reaches then a shift in dominant growth forms might be encountered even if water R amp
57. ause similar problems Unfortunately in other situations they contribute valuable information to the TDI so these taxa cannot be excluded from the TDI altogether Two options for handling such circumstances are either to plan the survey to minimise the problem or to manipulate the TDI calculation subsequently see section 4 4 point 6 and section 6 2 2 former option is preferable but requires prior knowledge of the system under consideration Cocconeis placentula has a broad ecological range and is found in most running waters except those with low nutrients or acidic conditions It is tolerant of moderate organic pollution and also extends into brackish waters It is abundant on rocks but is also found on other surfaces such as filamentous algae and macrophytes There is also evidence Jacoby 1987 Rosemund et al 1993 Biggs amp Lowe 1994 that this species thrives under heavy grazing which may explain why it is dominant in some types of UK river during the late summer Fig 6 4a Under favourable conditions valves of C placentula can constitute gt 80 of the total For these rivers samples from spring may be more sensitive to nutrients than samples from summer or autumn R amp D TECHNICAL REPORT E2 TR2 50 Navicula lanceolata also has a broad ecological range but differs from C placentula in being motile and having a stronger tendency towards organically enriched conditions However it also appears to be able to grow at low t
58. be adopted over wide areas where suitable natural substrates are either lacking altogether or not available at all sites Whilst macrophytes can be sampled see 2 8 and 2 9 they are not recommended unless it is not possible to either sample hard surfaces or deploy artificial substrates The flow chart Fig 2 1 is designed to guide users to the most appropriate means of sampling under various circumstances and tries to balance the competing interests of standardisation and typicality of the substrate The method chosen should be applied to all sites included in a particular survey Although the chart should cover most situations there may be circumstances where ad hoc approaches not described in this chapter need to be adopted Such methods must be fully documented and should also be supported by preliminary experiments to provide a firm basis for subsequent data interpretation 2 3 Timing of surveys It is difficult to give precise guidelines on the time of sampling that apply to all circumstances however the following principles should be followed 1 Samples collected for specific comparisons i e upstream and downstream of major discharges should be collected on the same day 2 Repeat sampling should take place at approximately the same time each year Some discretion is required here major spates scour away much growth and reset communities to pioneer stages largely regardless of the time of year For this reason time of sampl
59. beaker and the diatoms allowed to settle before the supernatant is poured off Alternatively some lengths should be cut at random from submerged plants using a knife or scissors and the sections placed into a sampling bottle These can be fractionated further in the laboratory if required and the macrophyte sections plus attached diatoms placed directly in a flask for cleaning 6 4 2 See section 2 5 points 6 9 for guidance on labelling transfer to the laboratory and storage R amp D TECHNICAL REPORT E2 TR2 15 This page has been left blank intentionally R amp D TECHNICAL REPORT E2 TR2 16 3 PREPARATION OF DIATOMS FOR MICROSCOPY 3 1 Introduction This chapter describes how to prepare permanent slides from benthic diatom samples collected from rivers For ease of identification all internal contents of the frustule chloroplast cytoplasm etc need to be removed along with extraneous organic materials that were included in the sample The cleaned valves digestion usually separates the two valves of the frustule are then mounted in a special mountant with a high refractive index in order to make it easier to see surface ornamentation such as striae Permanent slides should be stored in herbaria for future reference They can also be easily posted from one laboratory to another so that identifications of difficult taxa can be checked and so that slides can be subjected to audits There is not a right way to c
60. both diatom and macrophyte indices of water quality used in UWWTD assessments This involved inverting the scale of the TDI so that low scores corresponded to high nutrient concentrations and high scores to low nutrient concentrations In order to avoid further confusion this new index was referred to as the Diatom Quality Index diatoms were used in conjunction with the macrophyte Mean Trophic Rank Holmes et al 1999 the was recommended for use with the TDI being retained for situations where comparison with other measures of eutrophication were necessary Table 1 1 Effects of organic and nutrient pollution on plants and the ecophysiological adaptations required for tolerance from Kelly et al 1996 Chemical effect of pollution Ecological response of tolerant plant elevated P concentration competitive advantage at high P concentrations and or low N P ratio Elevated ammonium tolerant to high ammonium concentration concentration reduced oxygen concentration capable of respiration at low oxygen concentrations elevated suspended solids shade tolerant or motile abundant organic matter capable of heterotrophic growth Routine use of diatoms for water quality monitoring in the UK at this point was further complicated by the lack of clear guides to taxonomy and identification in English Asa result the first version of the TDI manual also included keys to the genera along with illustrations and descriptions
61. cal inventory storage arrangements Chemical spillage procedures Microscope immersion oil COSHH Risk Assessment Latex gloves safety spectacles laboratory coat See control measures in 1 above 4 Equipment maintenance record Electrical testing R amp D TECHNICAL REPORT E2 TR2 7 LEVEL OF RISK HARMFUL EFFECTS Cuts from breaking microscope slide and or coverslip Cuts from breaking microscope slide and or coverslip Splashing spillage of microscope immersion oil refer to COSHH Risk Assessment Electric shock Eye strain Back strain 8 RISK CONTROL MEASURES ADEQUATE CONTROL 102 9 OPTIONS FOR IMPROVED RISK 4 IDENTIFICATION 5 OF PERSONS INITIAL AFFECTED RISK LEVEL H M L Biology Team Biology Team Biology Team 10 PRIORITY OF ACTIONS REQUIRED C7 Example COSHH assessment forms Note that step 3 makes reference to the following table relating information on quantity and dustiness volatility of the identified health hazard group see page 3 COSHH Risk Assessment Procedure Applying the five step process to find the level of control required HEALTH HAZARD LIKELIHOOD OF EXPOSURE DUSTINESS VOLATILITY DUSTINESS VOLATILITY DUSTINESS VOLATILITY DUSTINESS VOLATILITY DUSTINESS VOLATILITY DUSTINESS VOLATILITY R amp D TECHNICAL REPORT E2 TR2 103 Addition of iodine solution 1 w v in potassium iodide to diatom samples and subsequent labo
62. cedures defined and training and information provided to employees Restricted access to working ENGINEERING CONTROL Local exhaust ventilation applied at source to capture contaminants CONTAINMENT Closed systems possibly under negative pressure with safe discharge arrangements for extracted air Typically requiring permit to work maintenance procedures SPECIAL CONTROLS Controls designed by experts in occupational hygiene to provide site specific solutions for cases where an extreme risk to health exists PROTECTION OF SKIN amp EYES Special attention to control measures protecting the individual and ensuring the effective use of items of personal protective equipment Restricted access to working area Laboratory Code of Practice Laboratory induction procedure Activity undertaken by authorised personnel only Chemical inventory storage arrangements METHODS OF CONTROL Chemical spillage procedures Fume cupboard Equipment maintenance record PPE heavy duty rubber gloves safety spectacles laboratory coat DOES THE CURRENT METHOD OF CONTROL COMPLY WITH THE APPROACH IDENTIFIED IN R amp D TECHNICAL REPORT E2 TR2 114 IMPLEMENTATION OF FURTHER ACTIONS OF FURTHER ACTIONS ACTION BY WHOM TARGET DATE R amp D TECHNICAL REPORT E2 TR2 115 TEAMS BIOLOGY REVIEW PERIOD Decanting 37 hydrochloric acid from stock to glass dropping bottle and subsequent addition to samples during Chemical digest
63. correct problems as they arise rather than at the end of the project Audits for diatom analyses undertaken by contractors should be managed via a separate contract between the Agency and auditor and not by a sub contract from the contractor This avoids conflicts of interest that may arise if audits reveal poor quality Copies of audit results sheets should be sent to the Agency project manager and a copy forwarded to the contractor Descriptions of what is considered to be an acceptable standard of work must be included in tender specifications R amp D TECHNICAL REPORT E2 TR2 68 LIST OF ABBREVIATIONS AND ACRONYMS ATDI AIDGAP AQC BMWP CCA CEN DETR DQI FRP GDI IMS IPS LIFE MTR NRA Zomotile PTV QA QD SEPA STW TDI TWINSPAN UWWTD V WFD WMS Difference in TDI parameter used in AQC protocol Aids to Identification in Difficult Groups of Animals and Plants Analytical Quality Control Biological Monitoring Working Party Canonical Correspondence Analysis Comit Europ en de Normalisation European Committee for Standardization Department of Environment Transport and the Regions now Department of the Environment Food and Rural Affairs Diatom Quality Index Filtrable Reactive Phosphorus Indice Diatomique Generique Industrial methylated spirits Indice de polluosensibilit Lotic Invertebrates for Flow Evaluation Mean Trophic Rank macrophytes based index of water quality National Rivers Au
64. cs modified Background revised and updated Main changes to this version described Chapter 2 Three principles increased to four suffix of confidence dropped Flow chart for selecting sampling strategy updated Preferred artificial substrata changed from tiles to ropes Guidelines on sampling vertical man made structures included 2 6 Guidelines on use of emergent 2 8 and submerged 2 9 macrophytes included More detailed guidelines on time of sampling General tightening of guidelines to bring TDI manual into line with CEN standard R amp D TECHNICAL REPORT E2 TR2 Rationale Following practice of other Agency manuals Mainly minor changes in light of experience Original version details development of TDI until 1996 New version includes developments subsequent to this date For information Update in line with CEN standard the new principle was implied but not stated in the previous edition Not necessary The PTV provides a measure of the reliability of the TDI as a measure of trophic status To incorporate other changes see below Based on experience within the Agency over the past five years Ad hoc methods adopted in some regions need rationalisation method used here 1s that used in Thames Region Based on request from Anglian Region Adopted methods are taken from CEN Standard Based on experience within the Agency Not included in the CEN standard as best time for sampli
65. ct PROPERTIES OTHER THAN THOSE with water can cause violent liberation of heat and CAUSING RISKS TO HEALTH AND splattering of the material Containers may explode when Jn LS ARE heated or if contaminated with water Contact with metals may evolve flammable hydrogen gas Storage conditions are specified in task risk assessment Runoff from fire control or dilution water may cause pollution Chemical digestion is an essential pre cursor to microscopic examination of diatom samples Chemical digestion with oxalic acid and sulphuric acid has been identified as the safest option and replaces the previous use of hydrogen peroxide Breakage leakage of stock solution R amp D TECHNICAL REPORT E2 TR2 128 Biology Team Temporary staff students have less experience of working with chemicals only staff deemed competent will be permitted to undertake tasks using this substance Others in lab e g building contractors cleaners visitors access to lab restricted to authorised personnel only a B SMALL LARGE ga DUSTINESS VOLATILITY OF SOLID OF LIQUID R amp D TECHNICAL REPORT E2 TR2 129 Operation should be undertaken within fume cupboard Chemical resistant gloves safety spectacles amp laboratory coat Only required for spillage clean up facemask appropriate filter Restricted access to working area Laboratory CoP Laboratory induction procedure Activity unde
66. cted without bias to one side of the river or the other from areas which fulfil the microhabitat conditions described above and which have an obvious diatom film detected by either its brown colour or slimy texture Where suitable substrates are very abundant random or stratified sampling strategies may be appropriate within the defined reach Stones should be selected as far as possible from unshaded areas within the main flow and free from obvious filamentous algae or siltation see above R amp D TECHNICAL REPORT E2 TR2 11 2 Any loosely attached surface contamination e g organic debris should be removed by washing the stone briefly in the stream water The stones should be placed in a tray along with approximately 50 ml of river water 3 Wash a stiff toothbrush in clean river water and rub it on waders or a similar surface in order to remove any diatom contamination from previous samples Brush the upper surface of the stone vigorously to remove the diatom film rinsing the toothbrush periodically in the water in order to transfer the diatoms If there are filamentous algae or silt deposits on the stone try to remove diatoms from those parts of the stone which are free of such contaminants Sites should be sampled in an upstream downstream sequence This means that the small number of diatoms transferred between sites on toothbrushes will be those that might be washed down the river naturally 4 If 75 of stones smother
67. ctions R amp D TECHNICAL REPORT E2 TR2 94 C4 Digestion of diatom samples using hot hydrogen peroxide PART 1 WORK ACTIVITY TASK IDENTIFICATION 1 REGION AREA Insert as appropriate 2 TEAMS JOB TITLES Biology Team Leader Biology Biologists Student Biologists Temporary Biolo 3 TASK SUMMARY Chemical digestion of diatom algae samples in preparation for mounting on microscope slides Process involves addition of chemical 5 TASK FINISHES Final re suspension of pellet following centrifugation prior to preparation of 6 TOOLS amp EQUIPMENT Sample diatoms polypropylene rope with preservative Centrifuge tube or 100 ml glass beaker Hydrogen peroxide Hotplate Distilled water Hydrochloric acid specific gravity 1 18 Glass dropping bottles Centrifuge 7 LOCATIONS Entire task should be undertaken within defined laboratory area 8 ASSESSORS Insert as appropriate 11 AUTHORISATION R amp D TECHNICAL REPORT E2 TR2 95 4 IDENTIFICATION 5 HARMFUL EFFECTS OF PERSONS INITIAL AFFECTED RISK LEVEL H M L hydrochloric acid 1M to sample risk assessment hydrogen peroxide to sample risk assessment handling 4 Accidental spillage of digestion Risk of fume inhalation and Biology Team H chemicals burns Hot surface of hot plate Burns from touching hot Biology Team surfaces Danger from spillage of hydrogen peroxide onto hot plate 6 Leaving hot plate unattended Use of centrifuge of supernatant to
68. d interpretation of the trophic diatom index Keywords Algae Diatoms Streams In Natural Channels Water Quality Natural Waters Monitoring Eutrophication Trophic Diatom Index Research Contractor This document was produced under R amp D Project E2 2 by Bowburn Consultancy 11 Monteigne Drive Bowburn Durham DH6 S5QB Tel 0191 377 2077 Fax 0191 377 2077 Environment Agency s Project Manager The Environment Agency s Project Manager for Project E2 2 was Dr Jan Krokowski Head Office Waterside Drive Aztec West Almondsbury Bristol BS32 4UD Further copies of this report are available from Environment Agency R amp D Dissemination Centre c o We Frankland Road Swindon Wilts SNS SYF tel 01793 865000 fax 01793 514562 e mail publications wreple co uk R amp D TECHNICAL REPORT E2 TR2 FOREWORD Initial stages of development of the Trophic Diatom Index were funded by the National Rivers Authority NRA Following the formation of the Environment Agency 1 April 1996 the project was absorbed into the Agency s R amp D programme The first version of the TDI introduced benthic diatom analyses to many biologists within the Agency without first hand experience of diatoms There was a steep learning curve for all concerned adapting the principles outlined in the manual to a wide range of circumstances For the most part ideas contained in the first edition of the TDI manual were robust and indeed many of the
69. dent Biologists Temporary Biologists 3 TASK SUMMARY Chemical digestion of diatom samples in preparation for mounting on microscope slides Process involves addition of chemical oxidising agent and acid to samples 4 TASK COMMENCES Removal of sample from preservative 5 TASK FINISHES Final re suspension of pellet following centrifugation prior to preparation of microscope slide 6 TOOLS amp EQUIPMENT Sample diatoms polypropylene rope in iodine solution Centrifuge tube or 100 ml glass beaker Concentrated Sulphuric acid Oxalic acid crystals Potassium permanganate crystals Hotplate Distilled water Hydrochloric acid specific gravity 1 18 Glass dropping bottles Centrifuge 7 LOCATIONS Entire task should be undertaken in fume cupboard within defined laboratory area 8 ASSESSORS Insert name of Team Leader Biology 11 AUTHORISATION R amp D TECHNICAL REPORT E2 TR2 92 PART 2 RISK ASSESSMENT SHEET 2 DESCRIPTION OF TASK ELEMENTS Dissolve oxalic acid crystals in distilled water whilst stirring and heating Decanting oxalic acid sulphuric acid and hydrochloric acid into glass dropping bottles Scan sample and remove large pieces of silt and sand Sieve if necessary Add hydrochloric acid if any calcareous material present Pour off supernatant and decant sample into glass beaker Addition of sulphuric acid to glass beaker centrifuge tube containing sample Addition of potassium
70. e knack to preparing slides especially in judging the density of suspensions from which the slides are prepared This comes with experience and beginners may have to experiment to develop their technique The principle is that a drop of the cleaned suspension prepared according to section 3 2 3 3 is evaporated gently onto a coverslip which is then mounted onto a slide This slide will then be used for the analysis of the diatom assemblage and calculation of the TDI and for this reason it is important that the final suspension is of a low enough density to permit easy identification and counting and that factors which contribute to non random distributions such as edge effects are minimised 1 Shake a vial of material cleaned according to section 3 2 or 3 3 and hold it up to the light Fine particles should be just visible in the suspension If the suspension appears milky or turbid distilled water should be added to reduce their concentration If no particles can be seen then allow the suspension to settle and then carefully decant off some of the excess liquid Note that ethanol can be used to dilute the suspension but not more than 5 10 v v final concentration this reduces the density of the solution and allows the drop to spread out more on the coverslip 2 Use a Pasteur pipette to remove some of the shaken suspension from the vial and put a drop onto a clean cover slip Note if you on the coverslip first the
71. e e g 1 M hydrochloric acid about 5 ml per sample e Distilled water Ultra high purity water can also be used but is not necessary e Basic laboratory safety equipment fume cupboard gloves lab coats etc e Beakers or conical flasks one per sample 100 ml capacity recommended e Hot plate with a thermostat The hot plate must be capable of heating to 90 5 Note that it is also possible to perform the digestions using a water bath in place of a hot plate and boiling or centrifuge tube replacing the beakers e Means of measuring 20 ml volumes of oxidising agents e g small measuring cylinder e Clean Pasteur pipettes e Glass stirring rods one per sample you can also use disposable Pasteur pipettes R amp D TECHNICAL REPORT E2 TR2 20 Centrifuge plus appropriate tubes these need to be large enough to contain all the reagents listed above plus space for dilution with distilled water 30 50 ml capacity is recommended Check if using plastic tubes that they are resistant to the reagents involved before starting Sample vials for long term storage 3 3 2 Method Note that one advantage of this method is that rope and macrophyte samples can be placed directly into beakers for digestion removing the need for pre treatment described in section 2 7 and 2 8 If this approach is adopted then coarse material should be removed after 30 minutes oxidation l Homogenize the sample by shaking then transfer
72. e changes in trophic status These were as follows R amp D TECHNICAL REPORT E2 TR2 56 Date Jun 93 Jul 94 Jun 95 Sep 95 Jul 96 Bradley 36 48 49 66 48 U s Gaunless 54 60 60 67 76 Difference 14 12 11 1 28 A paired sample t test was performed with the null hypothesis that there was no significant difference between the two sites The resulting t value was 5 153 which was highly significant p lt 0 01 The TDI does in this example support the case for designation which was eventually made in 1998 Note that there are valid grounds for eliminating the September 1995 samples as they were taken during a long period of low flow and were not typical however it has been included for the sake of this exercise Example 2 A large non qualifying discharge enters the River Wear above the first QD and diatom samples were collected from above and below this works in order to examine its contribution to eutrophication upstream of the sensitive area Four TDI values were common to both sites Date Jun 93 Jul 94 Jun 95 Jul 96 Wolsingham 36 34 38 49 D s Bradley 36 48 48 48 Difference 0 14 10 1 Once again a paired sample t test was performed with the same null hypothesis as above However on this occasion there was no significant difference between the two sites t 2 960 P gt 0 05 However this immediately raises the question of whether or not the sample size was adequate to detect a difference if one was there and this is w
73. e of the TDI is reduced when other pollutants are present and when inorganic nutrient levels are sufficiently high that phosphorus is no longer the limiting nutrient Other environmental stresses e g periodic inundations with brackish water have not been tested but are also likely to influence results Efficiency of method Sampling natural substrates is quick and easy and can be combined with visits to sites for other purposes i e macrophyte survey invertebrate kick samples Sampling artificial substrates requires a preliminary visit in addition to the above to place the substrates Data interpretation should be based on a series of samples from the same sites Logistics of method Sampling by one operator two recommended at main river sites for safety reasons Length of time at site approximately 10 minutes Preparation of permanent slides allow 3 4 hours for batch of 6 8 samples Microscopic analysis 30 mins to 1 h per slide depending upon complexity of material and operator skill As a rule of thumb allow 2 3 h per sample for preparation and counting 1 in 10 samples 1 in 5 for newcomers to technique must be submitted for analytical quality control audit 1 3 Background The trophic diatom index TDI was developed as part of a National Rivers Authority Research Fellowship at the University of Durham between 1992 and 1995 Kelly amp Whitton 1995 Part of the objective of this Fellowship was to review existing meth
74. e stem should be cut below the mouth of the bottle then the bottle plus stem turned upside down and closed Diatoms should be removed from the stem in the laboratory by stirring scraping or gentle brushing If the diatom film is not delicate it may be possible to cut 5 6 stems and transfer R amp D TECHNICAL REPORT E2 TR2 14 these directly to a sampling container without the need to invert bottles over the stem See section 2 5 points 6 9 for guidance on labelling transfer to the laboratory and storage 2 9 Sampling submerged macrophytes l As far as possible comparative studies in rivers should be based on samples collected as far as possible from a single macrophyte species or group of closely related species that 1s sufficiently abundant at the reach to facilitate sampling of the same species in the future Genera such as Myriophyllum Ranunculus and Potamogeton are all suitable Five replicates should be sampled and placed into plastic bags for transfer to the laboratory Each replicate should consist of a single stem plus associated branches of the plant from the lowest healthy leaves to the tip Submerged leafless stems should not be included Diatom epiphytes should be present as a brown floc or film associated with the macrophytes In the laboratory the plants should be stirred or shaken vigorously in some distilled water in a large beaker to dislodge attached diatoms The macrophytes should then be removed from the
75. e then there have been a number of nomenclatural changes which affect freshwater diatoms summarised in Whitton et al 1998 Experience gained over the past five years has also indicated that some taxa previously lumped into broad generic categories are sensitive indicators under certain environmental conditions These two factors have resulted in a number of new categories in the TDI The TDI is based on the weighted average equation of Zelinka and Marvan 1961 n 2 459 1 index n j l where aj abundance or proportion of valves of species j in sample sj pollution sensitivity 1 5 of species j and indicator value 1 3 Values of sensitivity are as follows 1 favoured by very low nutrient concentrations 2 favoured by low nutrient concentrations 3 favoured by intermediate concentrations of nutrients 4 favoured by high concentrations of nutrients 5 favoured by very high concentrations of nutrients In addition a few taxa have TDI sensitivity values of zero These include a few taxa that are relatively rare in freshwaters and whose ecological preferences are not well defined along with planktonic taxa which are routinely excluded from calculations Chapter 6 describes situations when further taxa might be excluded under certain circumstances in order to improve the sensitivity of the TDI Calculating this equation gives the weighted mean sensitivity WMS of the taxa present in
76. e verified A sample that is deemed not to have acceptable quality is returned to the original laboratory for re checking Taxonomic queries may be handled simply by a qualitative check of the slide although a partial or complete re count may be necessary under some circumstances All samples in a batch from which a sample with unacceptable quality was drawn must be considered to be suspect until the causes of rejection have been addressed This may involve qualitative or quantitative re examination of some or all samples and submission of at least one extra sample along with resubmission of the failed sample for audit It is recommended that analysts new to diatom analysis submit one in five of all samples for audit Once a satisfactory success rate has been achieved no more than 1 failure in 20 samples is the figure usually quoted in audit studies then the proportion of R amp D TECHNICAL REPORT E2 TR2 67 all samples submitted can drop to one sample in ten It has been suggested Cheeseman and Wilson 1978 that it is better to obtain 10 20 fewer results of a known accuracy rather than more results of unknown accuracy As the audit exercise forms the latter part of the learning curve for biologists involved with the TDI it is essential that possible reasons for a samples failure are passed back to the analyst The audit procedure must be seen as part of an overall QA program and as the culmination of a training exe
77. ed immediately following collection Only effective substance for both fixing and preserving samples experience shows that using alcohol preserves samples but does not fix them adequately for subsequent identification Addition to and rinsing from biological samples Road transport of samples containing Handling analysis of samples containing including by contractors Spillage spillage clean up Breakage leakage of stock solutions Combustible and incompatible with oxidising chemicals and acids storage arrangements specified in task risk assessment Contamination of waste water and atmosphere Temporary staff students have less experience of working with chemicals Others in lab e g building contractors cleaners visitors Emergency Services and other EA staff in rest of building in event of a major spill R amp D TECHNICAL REPORT E2 TR2 108 Ccv D SMALL LARGE TICK THE APPROPRIATE MEDIUM REFER TO THE TABLE ON P 94 TO FIND THE LEVEL OF CONTROL YOU NEED TICK THE APPROPRIATE BOX TO IDENTIFY THE CONTROL LEVEL 1 2 3 v 4 NEEDED LTH SAFE USE OF PPE EYE PROTECTION SKIN PROTECTION R amp D TECHNICAL REPORT E2 TR2 109 RISK CONTROL RISK CONTROL APPROACH GENERAL VENTILATION Natural or forced ventilation within the range of 5 and 15 air changes per hour to dilute the contaminant to a safe level Working procedures defined and tra
78. ed with filamentous algae these should be sampled in preference to stones lacking such growths Remove as many of the filaments as possible before brushing or scraping See 3 above Note this sampling strategy on the sample record form 5 Replace the stone in the stream and repeat the process for the other replicate stones Transfer the water which should now be brown and turbid due to the presence of diatoms from the tray into the sample bottle 6 All sample containers must be labelled and labels should be applied before the container gets wet Lids should not be labelled as they can become separated from the rest of the container when the sample is being prepared in the laboratory Labels must include watercourse name site name date of sampling and sampler s initials 7 Transfer the sample to the laboratory in a cool dark place If samples are brought to the laboratory within 24 h and these precautions are followed it is not necessary to add preservative in the field 8 If samples are to be stored add sufficient preservative for Lugol s Iodine either at this stage or immediately on return to the laboratory The sample label must include details of any preservatives that have been used Appropriate quantities of preservative IMS approximately 10 v v in sample i e 10 ml 100 m Lugol s iodine approximately 10 ml T about 6 8 drops with a Pasteur pipette Formalin approximately 2 4 v v in sample i e 2
79. emperatures and is often the dominant organism across a wide range of water qualities in late winter and early spring Fig 6 4b these rivers samples from early to mid summer may be more sensitive to nutrients In general all circumstances where diversity is low should be treated with caution and extra help sought when interpreting data The reason for low diversity may not be seasonality but this should be borne in mind The size of the dataset is an important factor influencing the confidence with which diatom based data can underpin decision making If possible more than one sample per year may be collected from each site Ideally these replicate samples should be collected during different seasons however the issues discussed above may make this difficult If very little time elapses between collection of samples from a site then the samples do not represent independent observations in statistical terms and are pseudoreplicates rather than true replicates see Hairston 1989 For this reason it is recommended that at least one month should separate replicate samples collected from the same site during the same calendar year For a statistical test to be valid all replicates must be truly independent This can never be the case in the types of pollution studies under consideration here as the upstream and downstream sites will always form two distinct blocks of observations This is overcome by the use of a pai
80. ence of many small fragments of diatoms may indicate that dead diatoms are being washed in from upstream sites so a note to this effect should be made on the count sheet lab notebook A diatom may not be identifiable for a number of reasons If overlying material obscures many valves new slides should be prepared using more dilute suspensions Some taxa are recognisable from girdle views either because the girdle view is particularly characteristic e g Rhoicosphenia abbreviata or because the girdle view can be assigned with confidence to a particular taxon by matching it with corresponding valve views of taxa found in the sample However this is not always possible and if in doubt the analyst should record the girdle views at the lowest level to which they can be assigned with confidence e g unidentified Gomphonema sp unidentified pennate girdle view This convention should also be applied to any other individuals found on the slide but not identifiable by the analyst A large number of such individuals may indicate a problem with either slide preparation or the identification skills of the analyst Not more than five per cent of the total count should be composed of unidentifiable individuals If this limit is exceeded then the analyst should seek help to remedy the problem e g by asking a more experienced analyst to examine the problem valves 4 4 Analytical procedure 1 Make a preliminary check of the microscope
81. entrifuge if trained by experienced staff Do not attempt to open the lid prior to the machine stopping See control measures given in 1 above plus Wash away supernatant with water and run tap for a few minutes afterwards in fume cupboard sink R amp D TECHNICAL REPORT E2 TR2 97 C5 Preparation of permanent microscope slides PART 1 WORK ACTIVITY TASK IDENTIFICATION 1 REGION AREA Insert as appropriate 2 TEAMS JOB TITLES Biology Team Leader Biology Biologists Student Biolo 4 TASK COMMENCES 5 TASK FINISHES Labelling of prepared microscope slides 6 TOOLS amp EQUIPMENT Vial containing chemically digested sample Microscope slides and coverslips Hotplate Distilled water Pasteur pipette NBS NAPHRAX High Resolution Diatom Mountant dissolved in toluene 7 LOCATIONS Entire task should be undertaken within defined laboratory area Steps involving Naphrax should take place in fume cupboard due to the presence of toluene 8 ASSESSORS Insert name of Team Leader Biology 11 AUTHORISATION R amp D TECHNICAL REPORT E2 TR2 98 2 DESCRIPTION OF TASK ELEMENTS Shaking of vial and removal of 2 3 drops to a cover slip Heating of coverslip over hotplate Addition of Naphrax to microscope slide and application of coverslip to slide Examination of microscope slide under compound microscope Label microscope slide and store until Naphrax has set R amp D TECHNICAL REPORT E2 TR2 3 IDENTIFIC
82. equired for spillage clean up facemask appropriate filter Restricted access to working area Laboratory CoP Laboratory induction procedure Activity undertaken by authorised personnel only Chemical inventory storage arrangements Chemical spillage procedures Eye wash facilities safety shower Use of glass dropping bottle reduces risk of spillage during chemical digestion process YES YES NO Spillage procedures are outlined in the laboratory s Chemical Spillage Procedures Correct storage arrangements are identified in the laboratorys Chemical Inventory amp Storage Arrangements R amp D TECHNICAL REPORT E2 TR2 126 IMPLEMENTATION OF FURTHER ACTIONS ACTION BY WHOM TARGET DATE No further actions required all identified risks are covered by existing procedures etc R amp D TECHNICAL REPORT E2 TR2 127 TEAMS BIOLOGY REVIEW PERIOD Decanting sulphuric acid into glass dropping bottle and subsequent use in chemical digestion of diatom samples Sulphuric acid 95 98 0 Contact with skin and eyes causes burns possible irreversible eye damage and tissue necrosis continued contact Causes gastrointestinal tract burns Possible severe and permanent damage Causes irritation and haemorrhaging of digestive tract Can be fatal Chronic exposure may cause kidney and lung damage foetal effects and cancer DESCRIBE ANY HAZARDOUS Contact with combustible materials may cause fire Conta
83. es of paired TDI values 1 e upstream and downstream in survey 1 upstream and downstream in survey 2 and so on for which the most valid statistical test is a paired sample t test which is widely available in spreadsheets and statistical packages such as Minitab This will test the null hypothesis that the mean difference between upstream downstream pairs of samples is zero If the null hypothesis is retained then this t test should be followed by a power analysis Whilst the null hypothesis is usually accepted or rejected on a criterion of 95 confidence a statistical power of 80 is usually regarded as acceptable By providing the program with the number of pairs of samples the standard deviation of the mean difference between pairs and the size of difference that you want to detect it is possible to calculate the statistical power of the analysis Alternatively by providing the program with the standard deviation of the mean difference between pairs the size of difference that you want to detect and the acceptable power i e 80 it is possible to estimate how many additional pairs of samples are required The question of what scale of difference in the TDI is acceptable is considered in example 2 below Example 1 The first Qualifying Discharge QD on the River Wear enters between diatom sampling points at Bradley 28 5 km from source and u s Gaunless 43 5 km from source Five TDI values were available from which to evaluat
84. estinal tract irritation and kidney damage Inhalation causes respiratory tract irritation Chronic exposure may cause defatting and dermatitus DESCRIBE ANY HAZARDOUS Incompatible with combustible materials and certain other PROPERTIES OTHER THAN THOSE chemicals including oxidising agents formaldehyde and CAUSING RISKS TO HEALTH AND concentrated hydrochloric acid storage conditions CONFIRM THAT CONTROLS ARE specified in task risk assessment IN THE rs aes Chemical digestion is an essential pre cursor to microscopic examination of diatom samples enviro onmenal etc 2 Chemical digestion with oxalic acid sulphuric acid and potassium permanganate has been identified as the safest option and replaces the previous use of hydrogen peroxide Breakage leakage of stock solution R amp D TECHNICAL REPORT E2 TR2 124 Biology Team Temporary staff students have less experience of working with chemicals only staff deemed competent will be permitted to undertake tasks using this substance Others in lab e g building contrators cleaners visitors access to lab restricted to authorised personnel only E 5 SMALL grams or millilitres OR LARGE kilograms or litres SMALL LARGE op vow MEDIUM R amp D TECHNICAL REPORT E2 TR2 125 Operation should be undertaken within fume cupboard Chemical resistant gloves safety spectacles amp laboratory coat Only r
85. esults from TDI surveys alongside chemical data from the same sites Do not just consider annual means but concentrate on data from the main growing season Y Consider N P ratios as well as P concentrations in isolation Use N P gt 15 as an approximate indication of a P limited rather than an N limited system Consider output from modelling programs such as AARDVARK to estimate the likelihood of post stripping conditions fulfilling the criteria when a change in TDI should be expected Bear in mind the influence of non nutrient factors on diatom assemblages downstream of sewage discharges Use information from other sources chemical analyses invertebrate surveys and assume that other stress factors will be significant unless the water course falls into GQA biological and chemical classes A or B The issue of spatial variation is illustrated in Fig 6 4 A strict interpretation of the UWWTD would involve a decision based on samples from sites B and C only as these would indicate the effect of the discharge on the receiving water However there are three practical problems with a sampling scheme based on sites B and C only e If site B has nutrient concentrations that are high enough to place it in zone I or II of Fig 6 1 then nutrient control at the STW is unlikely to have a noticeable impact on the ecology of the river at site C If nutrient concentrations here are high enough to place site B in zone III of Fig 6 1 then the
86. et al 1998 Shallow fast flowing water with a distinctly disturbed surface forming upstream facing unbroken standing waves usually over gravel substrate Raven et al 1998 Generally fast moving water with a rippled surface Often associated with a rapid or riffle just upstream or where the channel narrows and therefore speeds up the flow Raven et al 1998 A waterbody which is found to be eutrophic or which in the near future may become eutrophic if protective action is not taken European Community 1991 A biotic index of eutrophication based on the percentages of benthic diatom taxa in a sample A structural component of the diatom frustule Two valves fit together like the two halves of a date box or petri dish to form along with associated girdle bands a frustule R amp D TECHNICAL REPORT E2 TR2 74 REFERENCES Allan J D 1995 Stream Ecology Structure and Function of Running Waters London Chapman amp Hall Anderson N J amp Rippey B 1994 Monitoring lake recovery from point source eutrophication the use of diatom inferred epilimnetic total phosphorus and sediment chemistry Freshwater Biology 32 625 639 Armitage P D Moss D Wright J F amp Furse M T 1983 The performance of a new biological water quality score system based on macroinvertebrates over a wide range of unpolluted running water sites Water Research 17 333 347 Barber H G amp Haworth E Y 1981 A Guide to the
87. f benthic diatoms using the Open Learning course which takes approximately 30 hours to complete although some of the time is spent collecting and analysing samples relevant to the individual s work 2 Attend one day on site training towards end of Open Learning course to iron out any particular problems and queries R amp D TECHNICAL REPORT E2 TR2 63 3 Start routine use of TDI submitting 1 in 5 slides for external quality audit until a satisfactory standard is reached section 7 3 3 dropping to 1 in 10 once a satisfactory standard 1 reached Periodic ring tests are organised by Bowburn Consultancy and are recommended as refresher exercises for staff who use the TDI only sporadically These tests are also useful pre qualification exercises for staff with some experience of diatom taxonomy or for potential contractors Periodic ring tests are organised by Bowburn Consultancy and are recommended as refresher exercises for staff who use the TDI only sporadically These tests are also useful pre qualification exercises for staff with some experience of diatom taxonomy or for potential contractors All staff who are not able to analyse at least 30 audited samples per year should perform this test of competence Note that the emphasis here is on training rather than qualifications The ultimate demonstration of quality is a satisfactory success rate in the external quality audit More importantly
88. g scarer Risk Reminders For Fieldwork Appropriate clothing inc waterproofs Waders with high grip soles Double manning Field worker monitoring procedure Water Safety training course Risk Reminders For Fieldwork Double manning Life jacket Waders with high grip soles Field worker monitoring procedure Water Safety training course Risk Reminders For Fieldwork Waders with high grip soles Life jackets Heavy duty rubber gloves Immunisation against tetanus hepatitus A amp B Leptospirosis card letter to GP Double manning Anti bacterial hand wipes Field worker monitoring procedure Heavy duty rubber gloves Immunisation against tetanus hepatitus A Leptospirosis card letter to GP Anti bacterial hand wipes RISK CONTROL MEASURES ADEQUATE YES NO 9 OPTIONS FOR IMPROVED RISK CONTROL 10 PRIORITY OF ACTIONS REQUIRED H M L C2 Addition of preservative to diatom sample PART 1 WORK ACTIVITY TASK IDENTIFICATION REGION AREA Insert as appropriate 1 TEAMS JOB TITLES Biology Team Leader Biology Biologists Student Biologists Temporary Biologists 2 TASK SUMMARY Addition of preservative to diatom sample 3 TASK COMMENCES Opening sample container 4 TASK FINISHES Sealing sample container following addition of preservative 5 TOOLS amp EQUIPMENT Sample container sample Preservative Pipette 6 LOCATIONS Entire task should be undertaken in a fume cupboard unless form
89. gua Navicula lanceolata N gregaria Hints for data interpretation 1 use of motile valves This criterion should be used as a guide to factors controlling the composition of the diatom assemblage and not as an absolute property of the assemblage Use the criterion as a guide to inform interpretation and comment on those taxa which are responsible for the differences Use a difference of 20 as a threshold when considering whether the change in 96 motile valves between two sites is significant If the difference is 2046 assume that a comparison between two TDI values is valid bearing in mind issues considered elsewhere in this chapter V motile taxa usually increases downstream of a sewage discharge Exclude planktonic taxa from the sum before calculating motile taxa but do not exclude any benthic taxa even if using a modified TDI see 6 2 3 quality remained unchanged As it is not possible at this stage to eliminate fully the effect of such a change on the TDI it is important that users are aware of this possibility when interpreting results It is recommended that motile valves is used to make broad comparisons between samples which may highlight non nutrient factors influencing the flora rather than as an absolute measure of a community characteristic 6 2 3 Modification of TDI calculation Where one or two taxa dominate a sample for reasons other than water quality there is a good case for excluding t
90. haded 2 Artificial and natural substrates must be submerged for at least four weeks prior to sampling All depths that can be easily sampled wearing waders are suitable so long as they remain in the euphotic zone Within these limits the precise depth is unimportant so long as the sampler is sure that the surfaces have not been exposed to air 3 Workers must collect samples from similar substrates upstream and downstream of discharges in order to minimise the effect that substrate has on community structure Ideally a single type of substratum should be used at all sites included in a survey 4 Notes on the prevailing conditions must also be made to aid subsequent interpretation of the data a model field record form is given in Appendix B A segment of river that has suitable substrates for sampling should be selected and defined in relation to permanent physical features so that it can be revisited in the future As a general rule it should be at least 10 m long but longer lengths may be appropriate depending upon the nature of the river are recommended as these tend to have a good variety of natural hard surfaces but shallow runs and glides are also suitable In deeper rivers or where no hard substrates are available consideration should be given to using artificial substrates within the euphotic zone R amp D TECHNICAL REPORT E2 TR2 7 2 2 Choice of substrate Diatoms can be found growing on most subme
91. hem from the TDI calculation but bear in mind provisos given in 4 3 2 This practice was recommended in the first edition of this manual for samples with a large proportion of phytoplankton In this revised version the same principle is extended to include weedy species such as Cocconeis placentula and Navicula lanceolata The justification for this comes from theoretical studies on species diversity which suggest that unpredictable unstable and severe environments will be dominated by one or a few very common species and a smaller number of less abundant species May 1975 Sanders 1968 By contrast greater diversity may be expected in situations where one or a few environmental resources control the relative abundance of species Magurran 1988 May 1975 diatom assemblage developing under low flow conditions in summer with intense grazing is a plausible scenario though not as yet tested for the development of dominance by Cocconeis placentula Similarly an assemblage from early spring dominated by Navicula lanceolata may reflect this taxon s ability to thrive at relatively low temperatures Interpretation of the TDI under circumstances such as these should proceed with caution as the primary assumption that nutrients are the main variable influencing community structure has been violated There are two possibilities either adjust the sample date or modify the TDI calculation The former is preferable but is not always possible
92. here in this manual chapters 2 4 in this chapter other elements of the quality system will be explained This is one area where the protocols outlined in the previous version of the manual have been improved and updated In particular the audit protocol has been replaced by a new one based on the Bray Curtis similarity measure No Europe wide harmonisation of methods has yet been attempted so these protocols may have to change again as this process continues within CEN 7 2 Training requirements During the original evaluation of the TDI open learning materials were developed to enable Agency staff to learn enough about diatoms to use the TDI These proved highly successful and have subsequently been used by many Agency and SEPA staff They are recommended as a cost effective means for non specialists to develop a basic awareness of aspects of diatom taxonomy Individuals can learn at their own pace and on the job and the prepared slides supplied with them can act as a reference collection for future use However some form of additional help is also recommended particularly for some of the more difficult groups As users have already gained some basic awareness through the open learning materials this can take the form of a highly focussed one or two day tutorial to an individual or small group A suggested training protocol for complete beginners is as follows 1 Gain familiarity with genera and common species o
93. here statistical power analysis can play a role The standard deviation of the difference between the two sites was 7 32 Under these circumstances and using a one sided t test because it is very unlikely on theoretical grounds that the TDI will fall downstream of a discharge a sample size of four has a 21 chance of detecting a 10 change in TDI i e 4 units 35 chance of detecting a 15 change 6 units and a 51 chance of detecting a 20 change 8 units The effect of increasing sample size is illustrated in Fig 6 5 statistical power of 80 is usually considered to be acceptable Fairweather 1991 Peterman amp M Gonigle 1992 R amp D TECHNICAL REPORT E2 TR2 57 100 ee e 9 md o mw bd P ad 9 e Me m 60 a 2 4 5 _ _ __ _ 10 change _ 15 change 20 e 20 change 0 2 4 6 8 10 12 14 Number of samples Fig 6 5 Statistical power analysis as applied to the difference in TDI values between Wolsingham and Bradley River Wear Co Durham This in turn raises the question of what constitutes a significant change in the TDI This will vary from case to case and it will also depend upon the level of the TDI upstream For this reason the scale of difference that should be considered to be significant is better quoted as a percentage rather than as an absolute number The standard approach to interpretation of MTR results suggests a down
94. hes other Kolbesia Z ew taxon Split from Achnanthes other Z Luticola ew taxon Genus split from Navicula other Motile Martyana ormerly freshwater species of Opephora R amp D TECHNICAL REPORT E2 TR2 34 Taxon 33 New taxon Motile Melosira varians 4 Meridion circulare Navicula capitoradiata Navicula cryptotenella type Navicula gregaria 2 Motile Includes N menisculus N reichardtiana uA 1 5 4 Navicula lanceolata 2 2 excluding small species see below Including related genera such as Cavinula etc Motile Navicula tripunctata igttur wm Navicula other Navicula small species nominally lt 12 um Including small species of Sellaphora Diadesmis and related genera Neidium Nitzschia acicularis Nitzschia amphibia Nitzschia dissipata Nitzschia fonticola New taxon Motile New taxon Motile New taxon Motile 2 generally large gt 100 forms Motile includes Psammodictyon Motile see Martyana New taxon Formerly Achnanthes lanceolata type but now including P Nitzschia inconspicua Nitzschia palea Nitzschia pusilla Nitzschia section Sigmoideae j lt Nitzschia other Opephora Peronia fibula Pinnularia Planothidium delicatulum formerly A delicatula Pseudostaurosira brevistriata New taxon Split from Achnanthes other New taxon Split from Ach
95. in Fliessgewassern 211 Umweltforschungsplan des Bundesministers f r Umwelt Naturschutz und Reaktorsicherheit Wasserwirtschaft Forschungsbericht 102 01 504 M nchen Bayerisches Landesamt f r Wasserwirtschaft Simpson E H 1949 Measurement of diversity Nature London 163 688 Spellerberg LF 1991 Monitoring Ecological Change Cambridge Cambridge University Press Stevenson R J 1990 Benthic algal community dynamics in a stream during and after a spate Journal of the North American Benthological Society 9 277 288 Whitton B A amp Kelly M G 1995 Use of algae and other plants for monitoring rivers Australian Journal of Ecology 20 45 56 Whitton B A John D M Johnson L R Boulton P N G Kelly M G amp Haworth E Y 1998 A Coded List of Freshwater Algae of the British Isles iv 274pp LOIS publication number 222 Institute of Hydrology Wallingford Wyatt R J Ellis J C Milne I amp Gunby J A 1998 Improved Environmental Monitoring Manual of Best Practice for the Design of Water Quality Monitoring Programmes R amp D Technical Report E59 Environment Agency Bristol Zelinka M amp Marvan P 1961 Zur Prazisierung der biologischen Klassifikation des fliessender Gewasser Archiv fiir Hydrobiologie 7 389 407 R amp D TECHNICAL REPORT E2 TR2 78 APPENDIX CHANGES TO THIS EDITION OF TDI MANUAL Change Chapter 1 Purpose and scope added Performance characteristi
96. indicate a problem with the sample beyond the control of the analyst and is not necessarily grounds for rejection whilst slides in Class 5 should be rejected as counts based on very dense slides may be prone to human errors 7 3 3 Ensuring the quality of analyses The audit is based on a comparison between the original analysis termed the primary count and a replicate analysis by an independent auditor termed the audit count Results of the two analyses are compared using the Bray Curtis similarity measure The equation for this 15 Di 2 2 qj where D is the similarity between samples 1 and 2 and q is the smaller of the two relative abundances of species comparison between two identical samples will result in a Bray Curtis value of 100 whereas two totally different samples will result in a value of 0 Samples that share several common taxa in roughly the same proportions will have high scores and can be treated as replicates The question of what constitutes a replicate will vary depending upon the system being examined but a Bray Curtis score of gt 60 is often quoted Engelberg 1987 Spellerberg 1991 and evidence suggests that this also applies to benthic diatoms Kelly 2001 However Bray Curtis similarity values also depend on the diversity of the sample with low diversity i e where one species is strongly dominant giving either very low or very high similarity values depending upon whether the dominant s
97. ing may differ by several weeks between years depending on R amp D TECHNICAL REPORT E2 TR2 8 Abundant cobbles Hard man made surfaces available gt 25 of cobbles boulders free from filamentous algae Possible to introduce artificial substrata such as frayed ropes Fig 2 1 Flow chart for selecting appropriate sampling strategies for collecting benthic diatoms for pollution monitoring studies Under some circumstances e g chalk streams large pebbles may be substituted for cobbles Increase the number of pebbles sampled accordingly R amp D TECHNICAL REPORT E2 TR2 9 3 the intensity of late winter or spring storms However as a general rule repeat sampling should take place in the same season each year and not within four weeks of a storm or within two weeks of other periods of high flow 4 The ability to detect a change in TDI between two sites is dependent upon the size of the dataset see Chapter 6 and the collection of two or three samples per year from a site should be considered in order to increase the sensitivity 5 The first survey at a particular site should be treated as exploratory and strategies may need to be modified in future surveys in order to maximise sensitivity See Chapter 6 for more details 6 Although diatom samples can be collected at any of time of the year sampling in winter 1s not recommended as cell growth rates are lower Because of this the diatom communities will have les
98. ining and information provided to employees Restricted access to working ENGINEERING CONTROL Local exhaust ventilation applied at source to capture contaminants CONTAINMENT Closed systems possibly under negative pressure with safe discharge arrangements for extracted air Typically requiring permit to work maintenance procedures SPECIAL CONTROLS Controls designed by experts in occupational hygiene to provide site specific solutions for cases where an extreme risk to health exists PROTECTION OF SKIN amp EYES Special attention to control measures protecting the individual and ensuring the effective use of items of personal protective equipment Restricted access to working area Laboratory Code of Practice Laboratory induction procedure Activity undertaken by authorised personnel only Chemical inventory storage arrangements Chemical spillage procedures GIVE DETAILS OF THE LEV facility annual testing certification CURRENT OR PROPOSED Equipment maintenance record METHODS OP CONTROL Personal exposure monitoring PPE heavy duty rubber gloves safety spectacles laboratory coat Information requested on safety precautions in contractor s _ laboratories Field applications undertaken in open air wearing heavy duty rubber gloves and safety spectacles R amp D TECHNICAL REPORT E2 TR2 110 IMPLEMENTATION OF FURTHER ACTIONS OF FURTHER ACTIONS ACTION BY WHOM TARGET DATE R amp D TECHNICAL REPORT E2 TR
99. ion is an essential pre cursor to microscopic _ examination of diatom samples DESCRIBE WHY IT IS NOT Chemical digestion process requires use of acid and strong REASONABLY PRACTICABLE TO oxidising agents Chemicals used are the only ones which USE A SAFER SUBSTITUTE are suitable for polypropylene rope samples LIST OF ASSOCIATED ACTIVITIES Spillage spillage clean up AND POSSIBLE CAUSES OF Breakage leakage of stock solution EXPOSURE DESCRIBE ANY HAZARDOUS Storage away from heat flame and direct sunlight and in PROPERTIES OTHER THAN THOSE cool dry and well ventilated area specified in task risk CAUSING RISKS TO HEALTH AND assessment CONFIRM THAT CONTROLS ARE SPECIFIED IN THE TASK RISK ASSESSMENT None identified Biology Team Temporary staff students have less experience of working with chemicals Others in lab e g building contractors cleaners visitors R amp D TECHNICAL REPORT E2 TR2 116 Ccv D SMALL LARGE TICK THE APPROPRIATE MEDIUM REFER TO THE TABLE ON P 94 TO FIND THE LEVEL OF CONTROL YOU NEED TICK THE APPROPRIATE BOX TO IDENTIFY THE CONTROL LEVEL NEEDED SAFE USE OF PPE EYE PROTECTION SKIN PROTECTION R amp D TECHNICAL REPORT E2 TR2 117 RISK CONTROL RISK CONTROL APPROACH GENERAL VENTILATION Natural or forced ventilation within the range of 5 and 15 air changes per hour to dilute the contaminant to a safe level Working proced
100. ions of Cocconeis placentula and Navicula lanceolata in benthic diatom samples from the UK Shaded area indicates the seasonal Statistical power analysis as applied to the difference in TDI values between Wolsingham and Bradley River Wear Co Durham Decision tree for interpreting TDI values above and below major ST Ws Longitudinal distribution of TWINSPAN end groups in the River Wear based on sampling by Northumbria Area between 1993 and 1997 R amp D TECHNICAL REPORT E2 TR2 70 LIST OF TABLES Table 1 1 Table 4 1 Table 5 1 Table 5 2 Table 5 3 Table 6 1 Table 6 3 Table 6 4 Effects of organic and nutrient pollution on plants and the ecophysiological adaptations required for tolerance Keys and identification guides suitable for use with the TDI Many older keys are also useful this table lists those that are currently in print Nutrient sensitivities s and indicator values v for taxa included in the TDI No entry in comment column indicates no change since previous version of index Summary of changes to the updated version of the TDI Data used in calculation of TDI and DQI for site on River Browney on 5 July 1994 Some non nutrient factors which can influence the composition of the diatom assemblage A look up chart for interpreting results of the Trophic Diatom Index Example of look up chart as applied to the situation described in Table 6 3 R amp D TECHNICAL REPORT E2 TR2 71
101. iphyton episammon and epipelon communities in practice these tend to represent extremes of continua Thus an epilithic community might contain filaments of green algae such as Oedogonium or Cladophora that bear epiphytic Rhoicosphenia abbreviata The epilithic diatoms themselves produce mucus that traps sediment permitting epipelic taxa such as Navicula gregaria to invade as well as phytoplankton cells such as Cyclotella mene ghiniana In general communities in the upper reaches of rivers where current velocity is high tend to consist of diatoms closely adpressed to rocks Further downstream upright and stalked taxa become more common along with motile taxa However within a reach community composition can vary depending upon current velocity and boulder size related to susceptibility to movement during storms Other factors such as heavy shade may also be important Similar processes govern community composition on other substrates There is a widespread assumption that water quality overrides physical factors however physical variability between sampling sites should be avoided wherever possible by adhering strictly to these guidelines and by a consistent approach to sampling at all sites Four principles provide a framework 1 Samples should be collected as far as possible from cobbles and small boulders that are free from filamentous algae and found within the main flow of the river in reaches that are not heavily s
102. irements for environmental monitoring Australian Journal of Marine amp Freshwater Research 42 555 567 Hairston N G 1989 Ecological Experiments Purpose Design and Execution 370pp Cambridge Cambridge University Press Harding J P C amp Kelly M G 1999 Recent developments in the use of algae to monitor rivers in the U K In Use of Algae for Monitoring Rivers III ed J Prygiel B A Whitton amp J Bukowska pp 26 34 Agence de l Eau Artois Picardie Douai Harper D M 1992 Eutrophication of Freshwaters Chapman amp Hall London Hartley B Barber H G amp Carter J R 1996 An Atlas of British Diatoms ed P A Sims Biopress Bristol Hendey N I 1974 The permanganate method for cleaning freshly gathered diatoms Microscopy 32 423 426 Hill M O 1973 Diversity and evenness a unifying notation and its consequences Ecology 54 427 432 Holmes N T H Newman J R Chadd S Rouen KJ Saint L amp Dawson F H 1999 Mean Trophic Rank User s Manual R amp D Technical Report E38 Environment Agency Bristol Hustedt F 1930 Die S sswasserflora Deutschlands sterreich und der Sweiz 10 Bacillariophyta Diatomeae Fischer Jena Jacoby J M 1987 Alterations in periphyton characteristics due to grazing in a Cascade foothill stream Freshwater Biology 18 495 508 Kelly M G 1998 Use of community based indices to monitor eutrophication in rivers Environmental Conservation 25 22 29
103. ites and a separate technique statistical power analysis is R amp D TECHNICAL REPORT E2 TR2 55 necessary to ensure that the latter two options are not responsible for the observed results This manual does not include a detailed description of how to perform the necessary calculations but provides users with a broad overview of the potential of power analysis They should then be in a position to seek specialist help to perform the necessary analyses The most useful role of power analysis for UWWTD related monitoring is to estimate the number of samples required to make a reliable statistical inference This can take several forms 1 Estimating the required number of samples in advance of collecting any data 2 Testing the validity of a statistical analysis based on existing data and 3 Using existing data to estimate how many additional samples are required In practice the first option is difficult at present because this requires knowledge of the scale of within site variation over time This characteristic is itself highly variable with some sites having a very predictable flora whilst others have a range of possible assemblages depending upon prevailing conditions The recommendation at this stage is that the number of samples required is predicted on the basis of existing data The recommendation to collect all samples for a particular survey on the same day section 2 3 means that repeated sampling will result in a seri
104. ittle influence on benthic diatom ecology Stevenson 1990 or the value of diatom indices Kelly et al 1995 these observations must be placed into a broader hydrological chemical and ecological context Thus high flows and low temperatures in late spring one year might favour Navicula lanceolata to dominate in April or May whilst warmer drier conditions the following year might lead to a different dominant taxon on the same date High flows will also influence nutrient concentrations and the development of macroalgal macrophyte and invertebrate assemblages all of which may in turn interact with the diatom assemblage From the point of view of a monitoring biologist providing information to underpin decisions on water management the problem lies in separating this noise driven by non nutrient factors from genuine spatial or temporal changes in nutrient concentrations Interpretation of diatom based data for UWWTD designations should always be based on more than one set of samples sections 2 3 6 5 1 Additional samples will improve the precision of any estimates however the required level of precision will vary from case to case and will depend upon 1 the difference in the TDI before and after nutrient removal or above and below the discharge 2 the reliability of the TDI as an indicator of eutrophication see Fig 6 6 and 3 the variance of the data Point 1 presents a problem The magnitude of the TDI before can be es
105. l 1996 6 7 Use of the TDI for other purposes In addition to UWWTD designations the TDI has also been used to investigate eutrophication and general water quality problems in a wide variety of situations The criteria discussed above apply in general to these situations the main difference is that the focus of analyses is not a large qualifying discharge In the case of small rivers the TDI can be used to examine the influence of STWs smaller than those covered by the UWWTPD as well as the influence of fish farms and other sources of nutrients Diffuse nutrient pollution from agricultural runoff is a major problem in some areas The TDI can be used to study the impact of diffuse pollution however the manifestation of this problem necessitates a more liberal sampling policy When water quality in the upper Hampshire Avon was examined samples were collected from above and below numerous small village STWs and fish farms in order to eliminate each of these individually as causes of step changes in the TDI Under such circumstances the TDI can provide evidence to demonstrate the impact of diffuse pollution but this should complement other sources of data e g direct chemical analyses In addition to studies on nutrients benthic diatoms have also been used to investigate a range of other water quality problems Examples of recent uses in the UK include R amp D TECHNICAL REPORT E2 TR2 59 Fig 6 6 Decision tree for interpreting TDI
106. lean diatom frustules All diatomists have their own particular method All agree in the use of one or more oxidizing agents which are by their very nature highly reactive and or explosive For this reason those preparing diatoms for microscopy must be fully conversant with the appropriate health and safety guidelines before they start see Appendix C and Good Laboratory Practice must be followed at all times Methods are not inherently dangerous if proper precautions are followed however many Agency biologists will have had little experience of using strong mineral acids and oxidising agents Appropriate safety training must take this into account Two methods are described here however other methods based on strong oxidising agents are also suitable References for the two methods included are Hendey 1974 and Battarbee 1986 whilst Barber and Howarth 1981 Round 1993 and the forthcoming CEN standard draft BS EN 13946 provide alternative approaches Note that samples rich in organic matter in addition to diatoms need stronger oxidation than samples from relatively clean sites and the optimum ratio of sample to oxidants may need to be tested first Solid carbonates can cause problems and may influence the type of treatment and the sequence of oxidants used for example in carbonate rich waters the carbonates have to be removed by before using strong sulphuric acid in order to prevent formation of gypsum Treatment with dil
107. llection Locality place name not code Substratum from which sample obtained i e rock plant sediment artificial substrate Date the mount was made Name of mountant Nphx for naphrax the preferred mountant storage of suspensions permits additional slides to be prepared in the future and for further analyses e g by scanning electron microscope to be performed For long term storage diatom material should be allowed to settle the supernatant decanted and the material resuspended in methanol or ethanol Suspensions should be stored in airtight glass vials R amp D TECHNICAL REPORT E2 TR2 24 4 ENUMERATION OF DIATOM SAMPLES 4 1 Introduction and scope This Chapter describes a protocol for the identification and enumeration of relative proportions of diatom taxa on prepared slides with the intention of calculating the TDI A high power light microscope is used to identify benthic diatoms cleaned of cell contents and mounted in a medium with a high refractive index until an appropriate sample size has been obtained The technique is in principle very straightforward The analyst puts a slide on the microscope stage focuses finds the edge of the patch of diatoms and then slowly moves the stage horizontally underneath the objective recording every valve seen If the far side of the patch of diatoms is reached before the required sum is obtained the analyst moves down or up the slide and starts again The results for
108. longside the TDI value to indicate the reliability of the TDI as a measure of eutrophication This feature has proved to be very useful but is not without problems Firstly the term percent pollution tolerant valves PTV focussed attention on organic pollution whereas experience from the subsequent five years has indicated that a range of other water quality issues can lead to elevated PTV values upstream as well as downstream of STWs and consequently interfere with interpretation of the TDI Secondly the term PTV leads to R amp D TECHNICAL REPORT E2 TR2 3 erroneous use of the measure as an index of organic pollution rather than strictly as a measure of the reliability of the TDI The first version of the TDI was then subjected to an evaluation exercise by the Environment Agency as a result of which a number of changes were made to the original TDI The most notable of these was the change in the scale of the index so that it now extends from O indicating very low nutrient concentrations to 100 indicating very high nutrient concentrations This change was made to aid interpretation by biologists familiar with other biological indices in use in the UK In addition taxa such as Cyclotella and Stephanodiscus which were principally planktonic were removed from the calculation see Chapter 4 for explanation A final development recommended by the Regional Biologists of the Environment Agency was to adopt a common scale for
109. mediately on return to the laboratory the preservative should be omitted however as most samples are likely to be collected from lowland rivers with high bacterial loads some form of preservative is recommended whenever samples are to be stored before preparation Note that Lugol s Iodine can cause distortion of delicate frustules such as those of Nitzschia acicularis R amp D TECHNICAL REPORT E2 TR2 10 Fine mesh netting Plastic cable tie to attach sample bottle Fig 2 2 Photograph of sampler for vertical hard surfaces To prepare buffered formalin 40 formaldehyde add 200 g of the organic base examine hexamethylene tetramine to each litre of undiluted formaldehyde The buffer is necessary to prevent dissolution of the silica frustules Lugol s Iodine can be prepared by dissolving 2g potassium iodide and 1g iodine crystals in 300 ml water The resulting liquid should be straw coloured Some other recipes also include acetic acid or glutaraldehyde to prevent the loss of flagella These reagents are not necessary when the solution is only to be used for diatoms Note that iodine sublimates from most types of container so should not be used for long term storage Samples should be stored in air tight containers in a cool dark place such as a refrigerator until they can be cleaned 2 5 Method for sampling cobbles and boulders 1 At least five cobbles gt 64 lt 256 mm or small boulders gt 256 mm should be colle
110. nanthes other New taxon Split from Achnanthes other R amp D TECHNICAL REPORT E2 TR2 35 Psammothidium other Psammothidium lauenburgianum MU wp M ae ume Psammothidium subatomoides 2 Taxon v Comment Psammodictyon see Nitzschia other Reimeria sinuata Rhizosolenia see Urosolenia Rhoicosphenia abbreviata Rhopalodia Rossithidium See Achnanthes other Sellaphora Included with small Navicula Semiorbis 3 Skeletonema Stauroneis New taxon Previously included with Fragilaria other Staurosira Staurosirella 4 1 Stephanodiscus fo E i Serre SE Mae Serpia peCmeseu dida Synedra ulna 31 Synedra other Tabellaria 23 Tabularia 52 Tetracyclus agr Thalassiosira 00 Tryblionella P Formerly included within Nitzschia other Motile Urosolenia 00 2 69 I j lt A KO R amp D TECHNICAL REPORT E2 TR2 36 Table 5 2 Summary of changes to the updated version of the TDI Taxon Change Achnanthes Achnanthidium Moved to Planothidium lanceolata type Achnanthes Achnanthidium Renamed Achnanthidium minutissima type Achnanthes conspicua New taxon added Achnanthes oblongella New taxon added Fragilaria crotonensis New taxon added Psammothidium other Genus split from Achnanthes other This broad category has the same values as Achnanthes other Psammothidium lauenburgianum New taxon added Ps
111. nd streams R amp D TECHNICAL REPORT E2 TR2 iii This page has been left blank intentionally R amp D TECHNICAL REPORT E2 TR2 CONTENTS INTRODUCTION 1 1 Purpose and scope 1 2 Performance characteristics 1 3 Background 1 4 Changes since the first edition of this manual SAMPLE COLLECTION 2 1 Overview of sampling strategy 2 2 Choice of substrate 2 3 Timing of surveys 2 4 Apparatus 2 5 Methods for sampling cobbles and boulders 2 6 Method for sampling vertical man made surfaces in situ 2 7 Method for installing and using frayed polypropylene rope as an artificial substrate 2 8 Method for sampling emergent macrophytes 2 9 Method for sampling submerged macrophytes PREPARATION OF DIATOMS FOR MICROSCOPY 3 1 Introduction 3 2 Cold acid permanganate method 3 3 Hot peroxide method 3 4 Preparation of permanent slides 3 5 Archiving diatom slides and samples ENUMERATION OF DIATOM SAMPLES 4 1 Introduction and scope 4 2 Apparatus 4 3 Preliminary steps 4 4 Analytical procedure CALCULATING THE TROPHIC DIATOM INDEX 5 1 Introduction 5 2 Nutrient sensitivities and indicator values 5 3 Calculation of the TDI and DQI 5 4 Worked example INTERPRETING THE RESULTS 6 1 Introduction 6 2 Influence of non nutrient factors 6 3 Threshold of sensitivity 6 4 Variation in space 6 5 Variation in time 6 6 Use of other techniques in UWWTD evaluations 6 7 Use of the TDI for other purposes R amp D TECHNICAL REPORT E2 T
112. ng varies between different eco regions These all involve changes in wording from 1 edition in order to provide a clearer explanation of what is required 79 Chapter 3 Inclusion of hot peroxide method as alternative to cold acid method Several minor changes in wording Added new section on archiving samples Chapter 4 Was Chapter 5 but 1s now Chapter 4 Comments suitable floras for calculation of the TDI now included here Inclusion of health and safety note Recommendations on count size changed stratified counting procedure included for use under certain conditions Various minor changes Chapter 5 Reference to Diatom Quality Index deleted from chapter title Chapter 6 Rewritten from scratch Chapter 7 Training requirements rewritten and expanded to cover situations when external contractors are involved R amp D TECHNICAL REPORT E2 TR2 Widely used alternative to method in 1 edition Included in CEN standard Reflecting experience over the past 5 years and the CEN standard Mainly based on material in 1 edition but emphasising the importance of diatom slides as a long term record of conditions Chapter 4 from the first edition Identification is now available as a separate publication published by Field Studies Council To provide users with information on appropriate floras Modified from protocol used at Blandford Forum Experience since first edition To overcome
113. ods of using plants to monitor rivers in light of the existing and anticipated needs of the NRA Whitton amp Kelly 1995 Early on in this work several NRA staff mentioned concerns about increasing levels of eutrophication in rivers mainly from diffuse sources coupled with an inability to monitor eutrophication using existing invertebrate based tools These concerns were given a sharper focus as the implications of the UWWTD became clear In particular Article 5 of the Directive calls for more stringent treatment usually interpreted as phosphorus stripping of wastewaters that discharge into sensitive areas These are defined as freshwater bodies which are found to be eutrophic or which in the near future may become eutrophic if protective action is not taken Annex UWWTD European Community 1991 Sewage discharges into such areas of greater than 10 000 population equivalent require nutrient stripping unless it can be demonstrated that removal of phosphorus will have no effect on the level of eutrophication workshop organised in Durham as part of the Fellowship highlighted the need for new monitoring tools to identify sites that were vulnerable to R amp D TECHNICAL REPORT E2 TR2 2 eutrophication and to monitor the effectiveness of nutrient stripping where this was installed Kelly and Whitton 1995 Diatoms were one group identified during the Fellowship as having particular potential for monitoring eutrophicati
114. of the key species This was the situation when the first edition of the TDI was published in 1996 subsequently the TDI has been used by the Environment Agency throughout England and Wales and by the Scottish Environment Protection Agency in Scotland and diatom evidence was included in submissions for the second tranche of UWWTD sensitive area designations in 1997 Harding amp Kelly 1999 and for the third tranche in 2001 In addition there have been developments elsewhere in Europe with new trophic indices being developed in Germany Coring et al 1999 and Austria Rott et al 1999 along with a combined pollution eutrophication index developed in Italy Dell Uomo 1999 Furthermore there has been collaboration at a European level to harmonise R amp D TECHNICAL REPORT E2 TR2 4 methods with the first stage being harmonisation of sampling techniques Kelly et al 1998 The output from these discussions and workshops is a draft European standard BS EN 13946 for sampling with further standards covering other aspects of methodology available soon 1 4 Changes since the first edition of this manual The combination of the extensive experience gained in using diatoms in the UK along with the additional European perspectives provided the impetus to revise and update the TDI manual Changes compared to the first edition include Sample collection Chapter 2 modifications to bring chapter in line with draft European Standa
115. oint is that this is linked to a house rule that ensures that no diatom 1 counted more than once Two options for enumeration are 1 Recommended for routine use A slow vertical or horizontal traverse is performed with each diatom identified and added to the total as it passes one of the lines on the eyepiece graticule or 2 All diatoms visible in a field of view or within the grid of a graticule are identified and counted before either moving along a horizontal or vertical traverse to the next field or selecting a new field of view at random R amp D TECHNICAL REPORT E2 TR2 27 In both cases the procedure is repeated until the total has been reached A further house rule is needed to cover situations where a diatom is only partially inside a defined counting area Such rule might include taxa that are only partially visible at the upper but not the lower margin in the case of horizontal traverses or the left but not the right margin in the case of vertical traverses The precise form of this rule is less important than consistency in its use when analysing samples The field of view visible at each magnification must be calibrated against the Vernier scale on the microscope s mechanical stage Whether a horizontal or vertical traverse is used it is important that each subsequent traverse does not overlap with the previous one The distance that the stage is moved on each occasion must also account for any diatoms
116. on in running waters The development of a Trophic Diatom Index TDI started with an evaluation of a number of existing techniques including a zoning system Round 1993 along with general pollution indices developed in France Indice de polluosensibilit IPS Indice Diatomique Generique GDI Coste et al 1991 Rumeau and Coste 1988 and two earlier TDIs developed in Germany Schiefele and Kohmann 1993 An important point that emerged was that there was a high correlation between the IPS based on identification to species and the GDI which required identification only to genus Coste et al 1991 Kelly et al 1995 Examination of the relationship between IPS and GDI suggested that part of the scatter was caused by occasional differential responses of a species e g Amphora pediculus from other members of a genus By expanding a generic index to include these species it was hoped to improve the sensitivity of the index further A further observation was that neither of the two German TDIs were particularly effective in the UK and for this reason a new TDI was developed specifically for UK conditions Kelly amp Whitton 1995a The first version of the TDI included 86 taxa genera plus indicator species and produced numerical values from 1 indicating low nutrient concentrations to 5 indicating high nutrient concentrations Kelly and Whitton 1995b The relative effectiveness of generic based indices was significant because monito
117. ot available about 5 ml per sample Concentrated sulphuric acid 5 ml per sample A saturated solution of potassium permanganate a few drops approximately 1 2 ml per sample Saturated oxalic acid dissolve approx 10 g oxalic acid in approx 100 ml distilled water over gentle heat whilst stirring Allow to cool Crystals of oxalic acid should precipitate out If not add a further 10 g approximately of oxalic acid and reheat 10 ml per sample Distilled water Ultra high purity water can also be used but is not necessary Centrifuge and appropriate tubes these must be large enough to contain all the reagents listed above plus space for dilution with distilled water 30 50 ml capacity is recommended Check if using plastic tubes that they are resistant to the reagents involved before starting A further word of advice if you do use a centrifuge don t leave it in the fume cupboard when you are not preparing diatoms and periodically strip it down and clean and grease all moving parts Exposure to corrosive vapours can shorten its life significantly A means of pipetting 5 10 ml volumes of acids an automatic pipette is suitable if it is periodically stripped and cleaned in order to prevent corrosion Glass stirring rods one per sample you can also use disposable Pasteur pipettes Disposable Pasteur pipettes Labelled sample vials with secure lids A capacity of 10 20 ml is recommended The label must be water
118. pecies was identified correctly whilst replicate analyses of diverse samples rarely achieve very high similarity values In addition to the Bray Curtis similarity value the following parameters should be calculated e Hill 1973 s measure of diversity should be calculated This is the reciprocal of simpson 1949 s index and measures the extent to which a sample is dominated by asingle taxon The calculation is 1 gt where is the relative abundance of the ith species 1973 species diversity measure is preferred to a measure of species richness as the latter would be skewed by the large number of rare taxa that are frequently encountered when analysing diatom samples e The difference in TDI between primary and audit counts ATDI R amp D TECHNICAL REPORT E2 TR2 66 The numbers of gains and losses gain is defined as a species recorded in the audit count that was not recorded in the primary count whilst a loss is a taxon recorded in the primary count but not in the audit count Only taxa that comprise more than 2 of either the primary or audit count are included in the number of gains or losses Procedure l One in ten of all samples per analyst one in five during the early stages selected at random are submitted for audit These ten samples represent a batch that must be formally linked in some means by a numbering system for example to the audit sample
119. ping bottle and subsequent use in chemical digestion of diatom samples 8 Use of hydrogen peroxide in chemical digestion of diatom samples R amp D TECHNICAL REPORT E2 TR2 86 C1 Fieldwork PART 1 WORK ACTIVITY TASK IDENTIFICATION 1 1 to 1 3 1 REGION AREA Insert name of Region and Area 2 TEAMS JOB TITLES Biology Team Leader Biology Biologists Student Biologists Temporary Biologists 3 TASK SUMMARY Collection of diatom samples from freshwaters including deployment of polypropylene rope for diatom colonisation 4 TASK COMMENCES Loading vehicle 5 TASK FINISHES Unloading vehicle 6 TOOLS amp EQUIPMENT Polypropylene rope artificial colonisation samples only scissors White tray and toothbrush samples from natural substrates only Mobile telephone Life jacket throwing line hand wipes first aid kit Waterproof clothing thigh chest waders heavy duty rubber gloves Anti bacterial hand wipes Warrant card Leptospirosis card 7 LOCATIONS Streams rivers and ditches 8 ASSESSORS Insert name of Team Leader Biology 11 AUTHORISATION R amp D TECHNICAL REPORT E2 TR2 87 PART 2 RISK ASSESSMENT SHEET Driving to from sampling sites Walking from vehicle to sampling site Accessing sampling site Deploying collecting polypropylene rope natural substrates collecting only in from watercourse Scrubbing of polypropylene rope natural substrates to remove diatoms R amp D TECHNICAL REPOR
120. principles of sampling preparation and enumeration have been included into CEN Guidance Standards that are currently under development However after five years it was time to take a new look at the manual and to incorporate as much new experience as possible One major change of this updated manual is that the number of authors has increased from one to nine indicating the much wider pool of experience on which to draw compared with the situation when the first edition of the manual was published This bodes well for the future not just for monitoring eutrophication but also looking towards the monitoring requirements of the Water Framework Directive R amp D TECHNICAL REPORT E2 TR2 i This page has been left blank intentionally R amp D TECHNICAL REPORT E2 TR2 ii EXECUTIVE SUMMARY The Trophic Diatom Index TDI is a new index developed in response to the monitoring needs of the Urban Wastewater Treatment Directive UWWTD This document guides users of the TDI in appropriate methods of collection preparation identification and enumeration of benthic diatom samples and calculation and interpretation of the index It is based on an earlier manual R amp D Technical Report E2 but updated to include more recent experiences and incorporating elements of Guidance Standards under development by CEN The TDI is recommended for widespread use by the Environment Agency and other regulatory bodies to assess the trophic status of rivers a
121. proof and any writing on the label must use a waterproof pen Basic laboratory safety equipment fume cupboard gloves lab coats etc R amp D TECHNICAL REPORT E2 TR2 18 3 2 2 Preliminary sample preparation l Scan material and remove large pieces of silt and sand If necessary pass sample through 1 mm sieve to remove coarse particles Homogenise the sample by shaking and transfer 5 10 ml of the suspension into a centrifuge tube Always keep some of the sample in reserve in case of disaster If calcareous material is present add a few drops of dilute HCl and shake gently The material should effervesce as the carbonates are reduced to CO Continue adding dilute and agitate the tube gently until effervescence stops This stage can be omitted if you are sure that the sample does not come from a site with any calcareous rock in the catchment Add distilled water to 1 cm below the rim of the centrifuge tube and centrifuge to remove the acid The purpose of centrifugation is to separate solid and liquid phases The speed and time are not critical A speed of 3000 5000 rpm for five minutes has been used successfully in past but the efficiency depends upon the gravitational force exerted which is a function of the rotor properties as well as the speed and users must satisfy themselves that these conditions are suitable for their own equipment before proceeding 3 2 3 Sample oxidation 1 Pour off
122. ratory procedures diatom samples preserved in iodine solution Necessary to preserve samples between time of collection and analysis dictated by seasonality of sampling DESCRIBE WHY IT IS NOT None identified REASONABLY PRACTICABLE TO USE A SAFER SUBSTITUTE LIST OF ASSOCIATED ACTIVITIES Spillage spillage clean up AND POSSIBLE CAUSES OF Breakage leakage of stock solutions EXPOSURE DESCRIBE ANY HAZARDOUS None identified PROPERTIES OTHER THAN THOSE CAUSING RISKS TO HEALTH AND CONFIRM THAT CONTROLS ARE SPECIFIED IN THE TASK RISK ASSESSMENT Temporary staff students have less experience of working with chemicals Others in lab e g building contractors cleaners visitors R amp D TECHNICAL REPORT E2 TR2 104 Ccv D SMALL LARGE wow REFER TO THE TABLE ON P 94 TO FIND THE LEVEL OF CONTROL YOU NEED TICK THE APPROPRIATE BOX TO IDENTIFY THE CONTROL LEVEL NEEDED SAFE USE OF PPE EYE PROTECTION SKIN PROTECTION R amp D TECHNICAL REPORT E2 TR2 105 RISK CONTROL RISK CONTROL APPROACH GENERAL VENTILATION Natural or forced ventilation within the range of 5 and 15 air changes per hour to dilute the contaminant to a safe level Working procedures defined and training and information provided to employees Restricted access to working area ENGINEERING CONTROL Local exhaust ventilation applied at source to capture contaminants CONTAINMENT Closed systems possibly under neg
123. rcise rather than as a post hoc check on performance The experience from the evaluation exercise was that running the audit program in this way engendered a spirit of friendly co operation which made it easy for problems to be accepted and ironed out It is the experience from invertebrate audit procedures that audit failures decrease as more confidence and experience is gained 7 3 4 Ensuring the quality of computer records At least ten percent of all computer based records must be selected at random and compared to the original count sheet Any discrepancies should be noted 7 4 Use of contractors QA procedures must be modified for any diatom analyses performed by contractors rather than Agency staff Selection of contractors As diatom identification skills are less common than skills in identifying other taxonomic groups e g invertebrates and macrophytes a pre selection stage is recommended before tender documents are issued Potential contractors must be able to demonstrate that the analysts concerned have experience of working with river diatom assemblages This information can be obtained from other Regions or Areas that have let diatom analysis contracts or by asking potential contractors to perform a pre qualification exercise 7 2 Management of contract The contract should be organised in such a way that the contractor can supply slides and results in regular batches i e 20 slides per batch so that audits can detect and
124. rd Preparation of permanent slides Chapter 3 inclusion of method for using hot hydrogen peroxide alongside the sulphuric oxalic potassium permanganate method recommended in the first edition Identification of common diatoms Chapter 4 in the first edition was included to enable Agency biologists to use the TDI without recourse to standard floras The chapter has been revised and extensively tested before publication in 1999 as a Field Studies Council AIDGAP key Kelly 2000 As this key is now available separately Chapter 4 is not included in this revised version Enumeration Chapter 5 in 1 edition Chapter 4 in revised edition modified to include guidelines for handling circumstances where diversity is very low New procedures include a stratified counting protocol More detailed guidelines on sample size are also given Calculation of the TDI Chapter 6 in 1 edition Chapter 5 in revised version includes TDI values for a few taxa e g Achnanthes oblongella A conspicua whose previous inclusion in generic categories of the index could obscure potentially interesting patterns Also the PTV measure has been replaced by a broader index of reliability based on the percent of valves that belong to motile taxa Interpretation of the TDI Chapter 7 in 1 edition Chapter 6 in revised version extended and rewritten in light of experience gained over the past five years Quality assurance Chapter 8 in 1 edition Chapter 7
125. re also applicable to rivers Fig 6 1 insofar as thresholds are recognized above which the system is saturated with nutrients and further ecological changes will not occur Kelly 1998 The effort required to produce an observable change will be in direct proportion to the concentration of phosphorus before nutrient reduction starts An obvious conclusion from Fig 6 1 is that a community based index of eutrophication will have an upper limit of responsiveness to P the zone I zone II boundary on Fig 6 1 although it is possible that the apparent relationship with P will continue above this threshold because of cross correlations between P and other limiting factors The nutrient concentrations corresponding to this threshold has not been established yet in a manner that is widely applicable However preliminary data suggest that it may be as low 0 3 mg I available P i e as orthophosphate or soluble reactive P It is important however that the threshold is considered on a case by case basis Two useful indicators that the threshold might be exceeded are e Concentrations of other nutrients in particular N This can be evaluated by examining the N P ratio based on available forms Although N P ratios are relatively crude values gt 15 can generally be regarded as indicating P limited conditions whilst values lt 10 typically indicate N limited conditions An example from a lowland river is given in Fig 6
126. reault 1992 Kelly et al 1998 1 Artificial substrates must be left in the river for a minimum of four weeks Longer periods of exposure may be appropriate under some circumstances i e very oligotrophic conditions low temperatures heavy shade 2 At least five replicate substrates should be sampled However extra replicate substrata should be deployed to allow for potential losses due to spates or vandalism Care should be taken with the design and deployment of artificial substrates to ensure that they do not interfere with the activities of legitimate river users and to minimise risks of vandalism 3 Where artificial substrates are to be used for comparative studies in the same watercourse e g upstream and downstream of a sewage outfall it is important that all are exposed to identical conditions Both the length of exposure and the start date must be the same to allow for the impact of spates upon the developing diatom community 4 Atthe end of the exposure period the final 5 cm of the rope should be removed with a pair of scissors and placed in a sample container 5 See section 2 5 points 6 to 9 for guidance on labelling preservatives transport and storage 6 There are two options for treatment prior to analysis If samples are prepared using the hot hydrogen peroxide method then entire rope samples should be placed directly in flasks and hydrogen peroxide added directly Alternatively the ropes should be either
127. red sample test A further assumption is that all the samples within each block are independent of one another There are sufficient catastrophic events for this assumption to be valid between years i e surfaces are scoured back to colonisation stages after each winter and probably between seasons as well The shorter the gap between samples the greater the probability that the composition of the first sample influences that of the second sample so violating the assumptions underpinning the statistical analysis Hints for data interpretation 5 timing of surveys See comments on timing of surveys in 2 3 x Do not base decisions on data from a single survey Treat the first survey on a new stream reach as exploratory and be prepared to adjust the sampling program in subsequent years in order to maximise sensitivity Minimise the influence of seasonality by always sampling at approximately the same time of year If more than one sample is collected during a calendar year try to collect these from different seasons and ensure that at least one month elapses between sample dates R amp D TECHNICAL REPORT E2 TR2 51 Table 6 3 look up chart for interpreting results of the Trophic Diatom Index Enter values for sites above and below discharge into appropriate boxes and join adjacent sites by arrows A vertical movement on the chart indicates a change due to nutrients horizontal movement indicates change due to other f
128. results of this calibration must be displayed in a position where users of the microscope can easily consult them R amp D TECHNICAL REPORT E2 TR2 26 Table 4 1 Keys and identification guides suitable for use with the TDI Many older keys are also useful this table lists those that are currently in print Hartley et al 1996 Drawings of most species of diatom recorded from the British Isles No keys but a very useful compendium of illustrations Cox 1996 Keys are designed for use with live rather than cleaned material However it 1s still a useful adjunct to publications adopting a more traditional approach Kelly 2000 Includes all TDI taxa and subjected to Field Studies Council s stringent tests prior to publication Krammer and Lange Bertalot 1986 1991 Standard European flora in four volumes Written in German but well illustrated Many nomenclatural differences to the TDI Prygiel and Coste 2000 Well illustrated key to most common taxa designed for use with French Indice Biologique Diatom es Includes a CD ROM with an electronic version of the key Hustedt 1930 An old key with line illustrations of most of the common taxa Available in facsimile edition from Koeltz Scientific Books Box 1360 D 61453 K nigstein Germany The second eyepiece may be equipped with a second graticule to aid enumeration This can take several forms including a square grid H shape Whipple field etc The important p
129. rged surfaces however the composition of the community varies according to the nature of the substrate Ideally a single type of substrate should be used at all sites included in a survey this is not possible then the use of artificial substrates 1s recommended see below Cobbles are the recommended substrate as these balance stability allowing diatom communities to develop with manoeuvrability Pebbles and small boulders can also be used if cobbles are not available If these are not available then vertical faces of man made structures such as quays and bridge supports should be sampled so long as these are not made from wood Other man made hard surfaces such as bricks can also be sampled if these have been submerged for at least four weeks prior to sampling In many lowland reaches the majority of hard surfaces are smothered with growths of Cladophora or other filamentous algae Under these circumstances those few cobbles and boulders that lack Cladophora are clearly not representative and a modified sampling protocol should be adopted under such circumstances see below Further options include the use of introduced artificial substrates and sampling from macrophytes The disadvantage of introduced substrates is that two visits to each site are required in order to deploy and collect the substrates and there are often losses due to interference by river users However they do enable a consistent sampling policy to
130. ring of river eutrophication was likely to be performed largely by biologists already employed in the water industry few of whom were trained in diatom taxonomy The Biological Monitoring Working Party BMWP score used routinely in the UK for assessment of organic pollution using macroinvertebrates Chesters 1980 Armitage ef al 1983 was used as a benchmark for development of a practical index that balanced scientific integrity with ease of use Kelly et al 1996 No attempt was made at this stage to differentiate between the response of P and As trophic variables are often highly correlated it was considered to be better to model a broad response to nutrients or eutrophication using a single variable e g molybdate reactive P filtrable reactive phosphorus FRP orthophosphate as a proxy The underlying assumption was that P rather than N was the limiting nutrient in most rivers The validity of this assumption is discussed in Chapter 6 A particularly important feature of the first version was a facility to separate nutrient rich waters from those that are organically polluted Organic pollution is frequently associated with high nutrient concentrations but under such conditions nutrients were not necessarily the primary factor influencing community composition Table 1 1 Kelly et al 1996 For this reason the sum of taxa tolerant to organic pollution e g Nitzschia palea Gomphonema parvulum was used a
131. rking on any practical work all readers of the manual must familiarise themselves with the procedures laid down in Appendix C Risk assessments for Activities associated the Trophic Diatom Index 1 2 Performance characteristics Biota sampled Benthic diatom communities Habitats sampled Streams and rivers Basis of operation Collection of benthic diatom films from natural or artificial substrates within a 10 metre study reach Preparation of permanent slides from this material Identification and estimate of relative proportion of each taxon present on slide Calculation of index of eutrophication and associated measures of reliability for each sample Form of data Lists of relative proportions of taxa present plus index showing degree of eutrophication Trophic Diatom Index TDI and reliability of this measure for each sample TDI indicates floristic changes in response to increased nutrient concentrations In addition permanent slides are lodged with national herbaria R amp D TECHNICAL REPORT E2 TR2 1 Limitations of method Availability of suitable substrates or positions to leave artificial substrates Water depth low light may limit diatom growth in deep water River flow high flows may limit access to sites and scour away diatom films Seasonal factors can override water quality in determining community composition These factors vary in intensity between sites and are described in more detail in chapter 6 Performanc
132. rtaken by authorised personnel only Chemical inventory storage arrangements Chemical spillage procedures Eye wash facilities safety shower Use of glass dropping bottle reduces risk of spillage during chemical digestion process YES YES NO Spillage procedures are outlined in the laboratory s Chemical Spillage Procedures Correct storage arrangements are identified in the laboratorys Chemical Inventory amp Storage Arrangements R amp D TECHNICAL REPORT E2 TR2 130 IMPLEMENTATION OF FURTHER ACTIONS ACTION BY WHOM TARGET DATE No further actions required all identified risks are covered by existing procedures etc R amp D TECHNICAL REPORT E2 TR2 131 TEAMS BIOLOGY REVIEW PERIOD Use of hydrogen peroxide in chemical digestion of diatom samples Hydrogen peroxide 30 v v PROPERTIES OTHER THAN THOSE Chemical digestion is an essential pre cursor to microscopic examination of diatom samples R amp D TECHNICAL REPORT E2 TR2 132 Biology Team Temporary staff students have less experience of working with chemicals only staff deemed competent will be permitted to undertake tasks using this substance Others in lab e g building contrators cleaners visitors access to lab restricted to authorised personnel only al e Jel 1 jsi raas or millilitres SMALL LARGE MEDIUM ma IDENTIFY HOW DUSTY OR VOLATILE
133. rtunity for rigorous analysis of situations before and after the installation of nutrient removal This chapter whilst benefiting from the experience of the past five years should therefore be regarded as providing guidance rather than authoritative advice The aim of biological monitoring is to aid decision making Typically data are used to infer aspects of river quality that cannot be obtained by direct physical and chemical measurements Thus when monitoring eutrophication biological data are not just concerned with the amount concentration or load of nutrients available but also with how the impact of those nutrients are manifested in the biota We assume a positive relationship between the index and nutrient concentrations but also understand that part of the variation in the TDI is due to other factors see 6 2 These factors must be taken into account when interpreting the data This chapter 1 based on the assumption that the TDI is being used to provide evidence to support a designation of a eutrophic sensitive area under the Urban Wastewater Treatment Directive European Community 1991 following guidelines issued by DETR and the Welsh Office DETR Welsh Office 2000 This means that the emphasis of the chapter is on assessing large STWs gt 10 000 population equivalent discharging predominately to lowland rivers The assumption underlying this chapter is that temporal trends in eutrophication can be inferred from predomina
134. s manual 6 6 Use of other techniques in UWWTD evaluations Several multivariate statistical techniques such as Two way Indicator Species Analysis TWINSPAN and Canonical Correspondence Analysis CCA may play valuable roles alongside the TDI in demonstrating temporal as well as spatial changes The longitudinal distribution of end groups resulting from a TWINSPAN classification of samples collected from the River Wear candidate Sensitive Area between 1993 and 1997 1 given in Fig 6 7 End group A was found at sites all along the river and closer analysis revealed this to be primarily associated with samples collected in September 1995 following a prolonged period of unusually low flow The other end groups are divided into those found primarily upstream of 40 km and those found primarily downstream of 50 km the 10 km in the middle have a mixture of end groups Interestingly the sensitive area designation for the River Wear starts close to km 40 Nutrient stripping has not yet been installed on the River Wear but we can predict on the basis of these data that the range of end groups E F G and H should extend further downstream in the years following In this particular case TWINSPAN is used alongside the TDI to highlight spatial patterns and in time to demonstrate temporal change as the effects of nutrient removal are manifest A good examples of how ordination techniques can be used in a similar manner can be found in Lancaster et a
135. s opportunity to reflect prevailing environmental conditions 2 4 Apparatus e Thigh waders and associated water safety equipment see Procedures for Collecting and Analysing Macroinvertebrate Samples for more details e Toothbrush this must be cleaned in stream water before and after each sample is collected to minimise cross sample contamination e Plastic tray large enough to contain a small boulder and at least 2 cm in depth e Wide mouthed plastic sample bottles with watertight lids approximately 100 ml or greater e Waterproof marker pen or other means of labelling samples e Special equipment for scraping hard surfaces A small funnel of net 8 cm width at mouth 100 um mesh size attached to a metal frame with a bottle to collect diatom material attached to the bottom using a Jubilee clip This allows the bottle to be removed in order to decant the material or to be replaced with another sample bottle Fig 2 2 The net is attached to a wooden or metal handle approximately 45 cm in length e Preservative The type of preservative is less critical than for many groups of algae where it is important to preserve delicate structures such as flagella Formalin alcohol industrial methylated spirits IMS or Lugol s Iodine are all suitable although formalin is not recommended for health and safety reasons Read Health and Safety Appendix before using any of these preservatives If samples are to be prepared im
136. should be identified This will provide greater flexibility in data analysis A further potential use that is being explored is to use existing and new data to track the impact of changing flow regimes complementing the LIFE index Extence et al 1999 Finally the Framework Directive on Water Resources WFD 2000 60 EC requires good ecological status to be defined in terms of the status of a number of separate components of the biota one of which is the phytobenthos for which the diatoms are an obvious candidate Development of appropriate methods applicable across Europe is hoped to start soon R amp D TECHNICAL REPORT E2 TR2 61 90 This page has been left blank intentionally R amp D TECHNICAL REPORT E2 TR2 62 7 QUALITY ASSURANCE 7 1 Introduction The TDI should only be used within the context of a quality assurance QA system so that managers and users of the data can be assured of its integrity A robust analytical quality control AQC procedure is central to this but it must be emphasised this is only a guarantee that the quality assurance procedure as a whole is working Definitions of relevant terminology audit analytical quality control and quality assurance are given in the glossary The QA system includes e specifications for training e standard operating procedures including specifications for apparatus e systems of internal and external audit Standard operating procedures are dealt with elsew
137. stream change of 15 Holmes et al 1999 and this is probably a reasonable criterion for most TDI surveys although in some circumstances such as example 2 a difference of 20 might be more realistic Fig 6 5 However any rule of thumb needs to be tested for each individual case and supported by appropriate power analyses 6 5 3 Deciding whether to continue monitoring at a site e If the upstream downstream difference is not significant and the statistical power is gt 80 then there is no benefit for monitoring to continue if the UWWTD is the sole motivation If designation goes ahead despite the diatom evidence e g on the basis of MTR or chemical evidence then continued diatom monitoring might be of interest to see if any changes occur once nutrient removal is installed e If the upstream downstream difference is not significant and the statistical power is 8046 then continued diatom monitoring is recommended until sufficient samples are available to make a robust analysis e If the upstream downstream difference is significant then continued monitoring should continue in order to strengthen the baseline dataset against which changes post stripping can be measured see also section 6 6 R amp D TECHNICAL REPORT E2 TR2 58 Fig 6 6 summarises the steps involved in interpreting TDI results for the purposes of UWWTD designations Methods for detecting changes once stripping has been installed are beyond the scope of thi
138. t contribute to the target of 200 include phytoplankton as in the first edition but also some other taxa with broad ecological tolerances such as Cocconeis placentula and Navicula lanceolata that can dominate the flora under some conditions This strategy 1 necessary because such taxa can obscure patterns of less common taxa that may convey useful environmental information When these taxa are overwhelmingly dominant then either the total should be increased or a stratified counting procedure should be adopted see 4 4 Any such steps must be noted and explained in reports Chapters 5 and 6 explain how such counts should be treated and interpreted Planktonic taxa are a particular problem downstream of impoundments and in some lowland rivers For the purposes of this study planktonic taxa are Acanthoceras Asterionella Aulacosira Chaetoceras Cyclostephanos Cyclotella Skeletonema Stephanodiscus Thalassiosira and Urosolenia Under some conditions Fragilaria crotonensis some Tabellaria spp and Nitzschia acicularis can also fall into this category In practice only Cyclostephanos Cyclotella and Stephanodiscus are likely to be of quantitative importance in rivers although other taxa can be abundant immediately downstream of lakes and impoundments at certain times of the year 4 3 3 Preparation of microscope The eyepiece graticule or other measuring equipment must be calibrated against a stage micrometer prior to the analysis The
139. ted monitoring whilst those dominated by small Naviculas and relatives represent typically nutrient rich conditions Under such circumstances a stratified count and modified TDI calculation may not be necessary If in doubt seek expert advice Hints for data interpretation 2 influence of non nutrient factors v When examining changes between sites consider not just the change in the TDI but also changes in the dominant growth forms at the two sites Y Always compare like with like valid spatial comparisons can only be made on samples from similar substrates collected at the same time of year Y Be aware of any known sources of other pollutants at sites included in the survey Ensure that sample record forms include estimates of the abundance of substrate types and filamentous algae Consider modifying the TDI calculation in order to increase sensitivity particularly when the TDI shows little longitudinal variation despite the presence of known point sources of nutrients 6 3 Threshold of sensitivity Those attempting to control nutrients in rivers can learn from the experience of nutrient control in lakes As a result of observations on a large number of lakes where efforts to reduce nutrient loading had been made four distinct zones were recognized within which the response to reductions in P loading could be evaluated Sas 1989 The R amp D TECHNICAL REPORT E2 TR2 45 principles of this descriptive model a
140. tely spatial data i e changes in eutrophication over time are measured by longitudinal differences in TDI This assumption is valid so long as the limitations of the TDI described in this chapter are recognised However issues such as the scale of within site variation and the influence of non nutrient factors including seasonality and substrate also need to be considered This interpretation in turn requires an understanding of basic river and algal ecology Further details of survey design and data interpretation are given in Wyatt ef al 1998 which is based around software packages available within the Environment Agency and SEPA Section 6 7 considers other situations where the TDI may be useful In addition some of the broader issues covered in the chapter may also be relevant when using other indices such as the MTR R amp D TECHNICAL REPORT E2 TR2 41 6 2 Influence of non nutrient factors 6 2 1 General comments Each organism has a unique niche controlled by various factors Some factors are related directly to water quality e g nutrients ammonia whilst others e g current speed grazing are not Similarly it is also important to distinguish between factors that are related directly to the variable of interest and those caused by other variables which might be auto correlated with that variable Some examples of variables that might influence the diatom assemblage and as a result the TDI are listed in Table 6
141. the TDI represents the minimum level of taxonomy that is required Additional taxonomic information should also be recorded where possible as it may be useful in the future Beginners and less experienced analysts may find it useful to scan the slide carefully and make a list of all taxa encountered before they start the analysis They should take particular care to finding both raphe and non raphe valves of Achnanthes and Cocconeis species and their relatives Occasional filaments should be recorded as the corresponding number of valves If large numbers of valves are found in filaments a new preparation using a more aggressive mix of oxidising agents should be considered 5 If a diatom unit cannot be identified for any reason follow conventions outlined in 6 4 Photographs captured digital images or detailed drawings should be made Notes should also be taken of shape and dimensions of the diatom unit striae density and arrangement at centre and poles shape and size of central area number and position of punctae and arrangement of raphe endings The position of the specimen on the microscope slide should also be recorded using the microscope s Vernier scale or an alternative means 6 Once all taxa within the first field of view have been recorded the count should continue until the preliminary target of 300 valves has been reached At this point the analyst must decide whether this target is sufficient or if a larger count
142. the sample This varies from 1 for sites with very low nutrient concentrations to 5 for sites with very high nutrient concentrations TDI is the WMS expressed on a scale from 0 to 100 It is calculated as follows TDI WMS x 25 25 The WMS can be calculated using either the number of valves of each taxon recorded or the percent that each taxon contributes to the total The former is quicker but the latter allows differences between sites to be more readily compared It is recommended that raw numbers are entered directly into databases or spreadsheets used for calculations in order to avoid calculation errors R amp D TECHNICAL REPORT E2 TR2 3 5 2 Nutrient sensitivities and indicator values The list of sensitivities and indicator values for each taxon included in the TDI is given in Table 5 1 based on Kelly and Whitton 1995a Changes from the previous version of the TDI are printed in bold These include incorporation of recent nomenclatural changes in order to harmonise the TDI with the latest taxonomic checklist Whitton et al 1998 and splitting off some species from broad generic categories in order to increase sensitivity under certain circumstances A pragmatic approach has been adopted in order to balance the sensitivity of the index with ease of use The genus Rossithidium for example was split from Achnanthes Round amp Bukhtiyarova 1996 but contains no representatives that are common in rivers so rather than add
143. the value of the training falls unless the skill is exercised on a regular basis The Open Learning materials and slides for the tests of competence are available from Bowburn Consultancy 11 Monteigne Drive Bowburn Durham DH6 5QB tel 0191 377 2077 e mail Bowburn_Consultancy compuserve com 7 3 Quality control and audit procedures 7 3 1 Introduction The frequency of diatom analyses performed in most Agency laboratories is too small to justify formal quality control measures as used for benthic invertebrate samples and the boundaries between internal quality control and external audit are consequently blurred However because diatom samples are routinely made into permanent mounts replicate analyses either on site or elsewhere are relatively straightforward and these are recommended as the basis of quality control for the TDI The method described here is appropriate for independent evaluation of the quality of the analysis of a diatom slide by a third party It has evolved gradually over the past five years and further explanation can be found in Kelly 1999 and Kelly 2001 It consists of a preliminary analysis of the quality of the slide preparation followed by a duplicate count which is then compared with the original count by various statistics These provide an objective comparison between the two samples and are supplemented by notes made by the auditor to help the analyst address any problems encountered with the
144. thority Percent of valves belonging to motile taxa Percent Pollution Tolerant Valves Quality Assurance Qualifying Discharge Sensitivity of taxon to nutrients variable in weighted average equation Scottish Environment Protection Agency Sewage Treatment Works Trophic Diatom Index Two way Indicator Species Analysis Urban Wastewater Treatment Directive Indicator value variable in weighted average equation Water Framework Directive Framework Directive on Water Resources Weighted Mean Sensitivity R amp D TECHNICAL REPORT E2 TR2 69 LIST OF FIGURES Figure 2 1 Figure 2 2 Figure 4 1 Figure 6 1 Figure 6 2 Figure 6 3 Figure 6 4 Figure 6 5 Figure 6 6 Figure 6 7 Flow chart for selecting appropriate sampling strategies for collecting benthic diatoms for pollution monitoring studies Under some circumstances e g chalk streams large pebbles may be substituted for cobbles Increase the number of pebbles sampled accordingly Photograph of sampler for vertical hard surfaces Two examples of how to treat broken valves in TDI analyses Schematic representation of stages in response of photosynthetic community to increase in nutrient loading Modified from Sas 1989 Relationship between ortho P concentration and N P as TON and ortho P respectively in the River Stour above and below Ashford STW Kent A catchment based approach to detecting major sources of nutrients Seasonal occurrence of high proport
145. timated relatively easily at sampling sites both upstream and downstream of the STW The magnitude after at a site upstream of STW may be assumed to be the same as that at the site before the change but the magnitude after downstream of the discharge is harder to predict and at this stage it is not possible to model an expected diatom assemblage for a given nutrient concentration Point 2 is discussed in more detail in section 6 2 2 number of samples required is linked to points 1 and 3 A consistently large difference in TDI between two sites is more likely assuming that other conditions are fulfilled to generate a statistically valid difference based on a small number of samples than if the relative difference in TDI was small This is related to the concept of statistical power The null hypothesis implicit in studies of the impact of point sources on rivers is that there is no change between upstream and downstream sampling stations However if no statistically significant difference was found between the upstream control and downstream treatment sites then this may either mean that there is genuinely no difference between the two sites or it may mean that the sample size was too small or that the methods were too coarse for the difference to be detected Peterman amp M Gonigle 1992 Conventional statistics test only the first of these possibilities i e that there is genuinely no difference between the two s
146. tom es 134pp NF T 90 354 Etude Agences de l Eau Cemagref Bordeaux March 2000 Agences de l Eau Rosemund A D Mulholland P J amp Ellwood J W 1993 Top down and bottom up control of stream periphyton effects of nutrients and herbivores Ecology 74 1264 1280 R amp D TECHNICAL REPORT E2 TR2 77 Rott E Pipp E Pfister P van Dam H Ortler K Binder N amp Pall K 1999 Indikationslisten fiir Aufwuchsalgen in Osterreichischen Fliessgewassern Teil 2 Trophieindikation 248pp Bundesministerium fuer Land und Forstwirtschaft Wien Austria Round F E 1993 A review and methods for the use of epilithic diatoms for detecting and monitoring changes in river water quality 1995 65 Methods for the Examination of Waters and Associated Materials Her Majesty s Stationary Office London Round F E amp Bukhtiyarova L 1996 Four new genera based on Achnanthes Achnanthidium together with a re definition of Achnanthidium Diatom Research 11 345 361 Round F E Crawford R M amp Mann D G 1990 The Diatoms Biology and Morphology of the Genera 747pp Cambridge University Press Cambridge Sanders H L 1968 Marine benthic diversity a comparative study American Naturalist 102 243 282 sas H 1989 Lake Restoration by Reduction of Nutrient Loading Expectations Experiences Extrapolations St Augustin Academia Verlag Richarz GmbH Schiefele 5 amp Kohmann F 1993 Bioindikation der Trophie
147. ure of chemicals refer to COSHH assessment Splashing spillage chemical mixture within beaker refer to COSHH assessments Physical injury from incorrect operation of centrifuge Electric shock 93 6 SUGGESTED RISK CONTROL 7 LEVEL 8 RISK 9 OPTIONS 10 MEASURES OF RISK CONTROL FOR PRIORITY H M L MEASURES IMPROVED OF ADEQUATE RISK ACTIONS YES NO CONTROL REQUIRED H M L Restricted access to working area Laboratory CoP Laboratory induction procedure Activity undertaken by authorised personnel only Chemical inventory storage arrangements Chemical spillage procedures Hot plate operating instructions Electrical testing Fume cupboard Equipment maintenance record Oxalic acid COSHH Risk Assessment Latex gloves heat resistant gloves safety spectacles laboratory coat Eye wash facilities safety shower 2 See control measures given in 1 above hydrochloric acid sulphuric acid and oxalic acid COSHH Risk Assessments 3 See control measures given in 1 above COSHH Risk Assessment for appropriate preservative 4 See control measures given in 1 2 and 3 above Hydrochloric acid COSHH Risk Assessment and 3 above See control measures given in 1 2 and 3 above See control measures given in 1 2 3 and 7 above Potassium permanganate COSHH Risk Assessment 7 a 2 3 and 7 above See control measures given in 1 2 3 and 7 above centrifuge operating instru
148. ures defined and training and information provided to employees Restricted access to working ENGINEERING CONTROL Local exhaust ventilation applied at source to capture contaminants CONTAINMENT Closed systems possibly under negative pressure with safe discharge arrangements for extracted air Typically requiring permit to work maintenance procedures SPECIAL CONTROLS Controls designed by experts in occupational hygiene to provide site specific solutions for cases where an extreme risk to health exists PROTECTION OF SKIN amp EYES Special attention to control measures protecting the individual and ensuring the effective use of items of personal protective equipment Restricted access to working area Laboratory CoP Laboratory induction procedure Activity undertaken by authorised personnel only Chemical inventory storage arrangements Chemical spillage procedures GIVE DETAILS OF THE CURRENT OR PROPOSED METHODS OF CONTROL Fume cupboard Eye wash facilities safety shower PPE heavy duty rubber latex gloves safety spectacles laboratory coat Use of glass dropping bottles reduces risk and volume of any spillage during chemical digestion R amp D TECHNICAL REPORT E2 TR2 118 IMPLEMENTATION OF FURTHER ACTIONS OF FURTHER ACTIONS ACTION BY WHOM TARGET DATE R amp D TECHNICAL REPORT E2 TR2 119 TEAMS BIOLOGY DESCRIBE ANY HAZARDOUS PROPERTIES OTHER THAN THOSE 8 DIN THE T R amp D TECHNICAL REPORT E
149. ute will also be necessary for samples that are rich in iron Note that it is extremely important to avoid contaminating samples with diatoms from elsewhere For this reason glassware must be scrupulously clean and if glass rods are used for stirring samples these must not be used in more than one sample in order to prevent diatoms being transferred from one tube to another Workers must follow a strict protocol to ensure that no cross contamination of samples occurs 3 2 Cold acid permanganate method This method is suitable for all types of sample described in Chapter 2 with the exception of circumstances where entire rope or macrophytes samples need to be digested R amp D TECHNICAL REPORT E2 TR2 17 3 2 1 Reagents and apparatus required Figures in square brackets give some indication of the quantities of reagents required for typical sample All of these reagents are stable under normal conditions and if you are planning to look at lots of diatom samples in the next few months then it is worth preparing large batches of the reagents The limiting step in terms of efficient use of time is centrifugation A batch size that is equal to or divisible by the number of buckets available on the centrifuge is recommended Use of centrifuge tubes as digestion vessels 1 recommended Analytical grade reagents are not necessary for this procedure Dilute e g 0 1 hydrochloric acid another mineral acid will do if HCI is n
150. values above and below major STWs Is the comparison valid e P likely to be the limiting nutrient e GQA chemistry or biology class A or B e Reliability index generally lt 20 fultilled Stop gt 3 samples Continue sample collection Perform paired significant Benthic diatoms sample t test support case for designation Not significant STOP Perform power Detection power analysis gt 80 Detection power Benthic diatoms do not support case fo designation lt 80 SLOE Continue sample collection R amp D TECHNICAL REPORT E2 TR2 60 End group 000 001 010 011 100 101 110 111 momnmoow 30 40 50 60 70 80 Distance from source km Fig 6 7 Longitudinal distribution of TWINSPAN end groups in the River Wear based on sampling by Northumbria Area between 1993 and 1997 The river is divided into 10 km lengths and the horizontal lines indicate the occurrence of a TWINSPAN end group within that length The sensitive area designation starts at approx km 40 and there are 9 Qualifying Discharges between here and the tidal limit e Investigations of the impact of mining and quarrying including the provision of baselines against which future changes due to remediation work may be measured e Investigation of the impact of other types of land use including forestry and military training For situations such as these all species not just TDI taxa in a sample
151. waste noxious fumes and burns 6 EXISTING PLANNED RISK CONTROL 8 RISK 9 OPTIONS 10 MEASURES CONTROL FOR PRIORITY MEASURES IMPROVED OF ADEQUATE RISK ACTIONS YES NO CONTROL REQUIRED Restricted access to working area Laboratory CoP Laboratory induction procedure Activity undertaken by authorised personnel only Chemical inventory storage arrangements Chemical spillage procedures Hot plate operating instructions Electrical testing Fume cupboard Equipment maintenance record Latex gloves heat resistant gloves safety spectacles laboratory coat Eye wash facilities satety shower Carry out preparation in active fume cupboard COSHH risk assessments for hydrochloric acid and hydrogen peroxide See control measures given in 1 above plus Ensure bench area is clear See control measures given in 1 above plus Have ready supply of water swab and dilute any spillage R amp D TECHNICAL REPORT E2 TR2 96 6 EXISTING PLANNED RISK CONTROL 8 9 OPTIONS 10 MEASURES CONTROL FOR PRIORITY MEASURES IMPROVED OF ADEQUATE RISK ACTIONS YES NO CONTROL REQUIRED See control measures given in above plus Clear bench area around and work away from hot plate Place and retrieve sample glassware plate when hot plate is cool See control measures given in 1 above plus Place warning notice by hot plate and inform others of hazard See control measures given in 1 above plus Only use c

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