prEN 15708

SLOVENSKI STANDARD
01-december-2007
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Water quality - Guidance standard for the surveying, sampling and laboratory analysis of
phytobenthos in shallow running water
Wasserbeschaffenheit - Anleitung zur Beobachtung, Probenahme und Laboranalyse von
Phytobenthos in flachen Fließgewässern
Qualité de l'eau - Guide pour l'étude, l'échantillonnage et l'analyse en laboratoire du
phytobenthos dans les cours d'eau peu profonds
Ta slovenski standard je istoveten z: prEN 15708
ICS:
13.060.10 Voda iz naravnih virov Water of natural resources
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
DRAFT
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2007
ICS
English Version
Water quality - Guidance standard for the surveying, sampling
and laboratory analysis of phytobenthos in shallow running water
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee CEN/TC 230.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations which
stipulate the conditions for giving this European Standard the status of a national standard without any alteration.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other language
made by translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the
same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to
provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and
shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2007 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 15708:2007: E
worldwide for CEN national Members.

Contents Page
Foreword.3
Introduction .3
1 Scope .4
2 Normative references .4
3 Terms and definitions .4
4 Principle.6
5 Reagents.7
6 Equipment .7
7 Survey and sampling strategy.9
8 Survey and sampling procedures.11
9 Identification and basic quantification of organisms .16
10 Data processing and interpretation .17
11 Quality assurance.20
Bibliography .21

Foreword
This document (prEN 15708:2007) has been prepared by Technical Committee CEN/TC 230 “Water analysis”,
the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
Introduction
WARNING — Working in or around water is inherently dangerous. Persons using this European
Standard should be familiar with normal laboratory practice. Long periods of analysis at the
microscope can cause physical fatigue and affect eyesight. Attention should be given to the
ergonomics of the microscope and advice from a health and safety practitioner should be sought to
ensure that risks are minimized. The use of chemical products mentioned in this standard can be
hazardous and users should follow guidelines provided by the manufacturers and take necessary
specialist advice. This standard does not purport to address the safety problems associated with its
use. It is the responsibility of the user to establish appropriate health and safety practices and to
ensure compliance with any national regulatory conditions.
The phytobenthos is an important component of aquatic ecosystems and an understanding of the composition
of the phytobenthos present in a waterbody can provide useful information on the status of that waterbody,
and on appropriate management strategies. The Water Framework Directive (2000/60/EC) [2] requires
monitoring of the phytobenthos as one part of ecological status assessment, and phytobenthos assessments
have also been used in monitoring programmes associated with other European Directives (e.g. Urban
Wastewater Treatment Directive, Habitats Directive) and with national legislation (e.g. ÖNORM M6231).
This guidance standard specifically relates to the sampling of phytobenthos (other than macrophytes) in
running water. An etymologically-correct application of the term “phytobenthos” would cover all phototrophic
organisms; however, this encompasses a vast range of organisms, from microscopic unicells to macrophytes
> 2 m in length. As separate survey methods for macrophytes are available (EN 14184), this document
focuses on phototrophic algae and oxygenic cyanobacteria that live on substrata. However, the focus of this
document is shallow running waters which are generally erosive and dominated by boulders, cobbles and
pebbles and, under such circumstances, there are often competitive interactions between the larger algae and
bryophytes. Similarly, macrophyte species may, themselves, act as substrata for algae and cyanobacteria. For
these reasons, the standard provides options for including these taxa in survey and sampling procedures. The
term “periphyton” is sometimes used instead of “phytobenthos”; however, some definitions of “periphyton”
include heterotrophic organisms that live attached to substrata (protozoa, sponges, hydroides). Such
organisms fall outside the definition of “phytobenthos sensu stricto” assemblage.
Methods using phytobenthos to assess water quality in running water have been developed in several
European countries and in the USA [1]. Recent work is summarised in the proceedings of three symposia [6],
[9], [10]. Methods for the sampling and analysis of one group of phytobenthos, the diatoms, have already been
the subject of harmonisation (EN 13946, EN 14407). However, these standards are concerned with only a
single group of the phytobenthos and there are situations where other phototrophs are more obvious and can
contribute additional ecological information.
According to the precise usage to which this standard is to be put it is essential for specifiers and users to
mutually agree on any necessary variations or optional procedural details prior to use.
1 Scope
This European Standard provides guidelines for the survey/sampling, identification and basic quantification of
phytobenthos (other than macrophytes) in running waters. It is applicable to rivers where benthic algae and
bryophytes are the main phototrophs. This method encompasses all phytobenthic growth forms and enables
biological responses to environmental events over one or more years to be monitored. In this respect it
provides an alternative to methods based on benthic diatoms (EN 13946; EN 14407) and macrophytes
(EN 14184). Data obtained for the phytobenthos growth forms are suitable for pilot surveys, water quality
assessment and trend monitoring. This European Standard encompasses all aspects from the design of
survey and sampling programmes to the identification and basic quantification of the phytobenthos.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
EN 13946, Water quality — Guidance standard for the routine sampling and pretreatment of benthic diatoms
from rivers
EN 14184, Water quality — Guidance standard for the surveying of aquatic macrophytes in running waters
EN 14407, Water quality — Guidance standard for the identification, enumeration and identification of benthic
diatom samples from running waters
EN 14996, Water quality — Guidance on assuring the quality of biological and ecological assessments in the
aquatic environment
EN 15204, Water quality — Guidance standard for the routine analysis of phytoplankton abundance and
composition using inverted microscopy (Utermöhl technique)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
aquatic macrophytes
larger plants of fresh water which are easily seen with the naked eye, including all aquatic vascular plants,
bryophytes, stoneworts (Characeae) and macro-algal growths
3.2
assemblage
the organisms that share a habitat or microhabitat
NOTE This term is preferred to “community”, as the latter implies a level of ecological integration of the organisms;
whereas sampling may inadvertently combine representatives from more than one true “community” that are not distinct to
the naked eye.
3.3
belt transect
defined band across a river or stream at right angles to the bank, along which the area covered by
phytobenthos is estimated
3.4
benthic algae
algae and oxygenic cyanobacteria living attached to substrata (rather than suspended in the water column)
3.5
biofilm
mucilagineous polysaccharide matrix on submerged stable surfaces consisting of photo(auto)trophic and
heterotrophic organisms
3.6
boulder
stones with a diameter > 256 mm
3.7
bryophytes
a collective term for liverworts, mosses and hornworts – plants which are often abundant on boulders and
bedrock of upland streams
3.8
cobble
stones with a diameter from 64 mm to 256 mm
3.9
degree of cover
percent of substratum at the sampling site covered (by the organism)
3.10
epilithic algae
algae living attached to or in close association with stony substrata
3.11
epiphytic algae
algae living attached to or in close association with macrophytes or other algae
3.12
epipelic algae
algae that live in or on fine sediments
3.13
epipsammic algae
algae that live attached to or in close association with sand
3.14
habitat
the type of environment where individuals of a species live
3.15
macroscopic benthic algae
multicellular algae and aggregations (units/groupings) of unicellular algae living attached to substrata (as
opposed to those suspended in the water column) that are visible to the naked eye
3.16
nuisance biomass
accumulations of benthic algae that are a nuisance to users of the watercourse and/or that detrimentally affect
its ecology
3.17
periphyton
a group of organisms (principally algae, but also including fungi, bacteria and protozoa) living on or in close
contact with surfaces in aquatic environments
NOTE 1 Bryophytes have an intermediate position. They are often regarded as a component of the macrophytes,
particularly in slow flowing rivers where macrophytes are common.
NOTE 2 The term “periphyton” is often used as a synonym for benthic algae in recent literature.
3.18
phototroph
organism whose main source of carbon is obtained through photosynthesis
NOTE For the purpose of this standard, facultative phototrophs such as many Euglenophyta are included within this
definition.
3.19
phytobenthos
all phototrophic algae and oxygenic cyanobacteria that live on substrata, rather than suspended in the water
column
3.20
reach
length of a watercourse forming a major sub-division of a river basin and defined by physical, chemical or
hydrological characteristics (or any combination of these) that distinguishes it from the watercourse upstream
and downstream
NOTE The boundaries between reaches mark the principal points of transition where the overall character of the
watercourse changes.
3.21
reference conditions
conditions reflecting a totally undisturbed state, lacking human impact, or near-natural with only minor
evidence of distortion
3.22
riffle
fast-flowing shallow water with distinctly broken or disturbed surface over gravel/pebble or cobble substratum
3.23
survey unit
length of river from which data are collected during field survey; this may be a fixed length (e.g. 10 m) or
variable, according to the methods used, but must always be defined and recorded
3.24
taxon (pl. taxa)
taxonomic unit, such as family, genus or species
4 Principle
Phototrophs associated with submerged surfaces in running water are surveyed and / or sampled. Specimens
of those taxa that cannot be identified in the field are taken back to the laboratory for identification. Three
different options are provided within the standard, suitable for different circumstances. Outcomes of the
survey / sampling process may include
a) a list of all macroscopic algae (and, optionally, non-vascular plants) observed in the survey unit;
b) a list of all macroscopic and microscopic algae (and, optionally, non-vascular plants) observed in the
survey unit;
or
c) all microscopic algae found on a single substratum. Semi-quantitative estimates of the abundance of
each taxon are also made. These data can be used to give an integrated picture of ecological status
and/or water quality.
5 Reagents
Preservatives are necessary if samples are to be stored prior to analysis. If treated with care, many algal
samples can be stored in a refrigerator or cool room for several days without deterioration. However, where
longer-term storage is necessary, then one or more of the following reagents may be necessary (see 8.4).
5.1 Acid Lugol’s iodine
Dissolve 100 g KI (Potassium Iodide) in 1 l of distilled or demineralised water; then add 50 g iodine
(crystalline), shake until it is dissolved and add 100 ml of glacial acetic acid. As the solution is near saturation,
any possible precipitate should be removed by decanting the solution before use. Lugol’s solution can be
stored in a dark bottle at room temperature for at least 1 year.
5.2 Alkaline Lugol’s iodine
Dissolve 10 g of KI (Potassium Iodide) in 1 l of distilled or demineralised water; then add 50 g iodine
(crystalline), shake until it is dissolved and add 100 g NaAc (Sodium Acetate, CH COO-Na). As the solution is
near saturation, any possible precipitate should be removed by decanting the solution before use.
Lugol’s iodine can be stored in a dark bottle at room temperature for at least 1 year.
5.3 Buffered 30 % formalin (formaldehyde)
Formaldehyde (37 %) is diluted to 30 % with distilled water and 100 g hexamethylen-tetramin (hexamin)
-1
added per litre. The inclusion of 1 g l copper nitrate in a formalin solution helps to maintain the colour of
chloroplasts of green algae.
Lugol’s iodine and formalin for field use should be stored in small bottles with a tight screw cap and pipette
dispenser. The bottle should be kept in a box or solid plastic bag during transport. They should be used in a
cup ventilator or fume cupboard in the laboratory.
6 Equipment
6.1 Field equipment
Necessary:
6.1.1 Appropriate water safety equipment
6.1.2 A means of locating sampling reaches on repeat visits, if there is no permanent landmark
adjacent. Options include iron bolts, fast drying paint, waterproof tape or similar to delimit the sampling
reaches;
6.1.3 Waders
6.1.4 Aqua-scope, or bucket with clear Perspex base, for scanning the river bottom in turbulent water.
6.1.5 Stainless steel knife or other suitable blade, forceps and stiff toothbrush
6.1.6 Hand lens
6.1.7 White plastic or enamel tray, volume 2 l to 3 l, for sorting material and sub-sampling.

6.1.8 Sample vials with tight fitting lids
Recommended sizes are 5 ml and 125 ml, to encompass both singular macroscopic units and composite
samples.
6.1.9 Waterproof labels for sample vials, or a marker pen with waterproof ink.
6.1.10 Waterproof fieldbook, or standardised recording sheets plus pencil or indelible pen.
6.1.11 Preservative, buffered formalin, Lugol’s iodine, or other.
Optional:
6.1.12 Global position satellite (GPS) receiver
6.1.13 Rake with attached net or hoe attached to a long handle, to facilitate sampling at high flow.
6.1.14 Bucket, to transfer large substrata to laboratory.
6.1.15 Camera or video-camera
6.1.16 Portable refrigerator or ice box
6.1.17 Boxes with room, to store all sample vials from one locality, to facilitate storage.
6.2 Laboratory equipment
6.2.1 Binocular microscope, equipped with a mechanical stage and at least 40× magnification for sorting
of samples.
6.2.2 Compound light microscope, equipped with a mechanical stage and medium (e.g. 40×) and high
power (e.g. 100×) objectives. The microscope should incorporate facilities for measurements (e.g. an
eyepiece graticule) with a resolution of at least 1 µm. Use of a phase contrast or differential interference
(Nomarski) condenser may be useful.
6.2.3 Microscope slides and cover glasses
6.2.4 Immersion oil, dispenser, lens papers and absorbent tissues
6.2.5 Floras, identification guides and iconographs (illustrations), appropriate to the habitats under
consideration.
6.2.6 Facility for recording data, as they are collected.
This can be a pro forma sheet with a list of taxa and space beside each on which the abundance estimation
can be made or a laboratory notebook organised in such a way that taxon identities and abundance can be
clearly recorded.
Optional
6.2.7 Apparatus for photo-microscopy or digital image capture
6.2.8 Tissue homogenizer or blender
6.2.9 Magnetic stirrer and stir bar, forceps
6.2.10 Tally counter, for species proportional count.
7 Survey and sampling strategy
7.1 Approaches
Any reach within a river or stream is likely to contain a number of different substrata on which phytobenthos
can grow, and some of these substrata may, in turn, be colonised by more than one type of phytobenthic
growth form. In many cases, these growth forms will be visible to the naked eye, even if the constituent
organisms are microscopic. In some cases, the growth form will contain one, or a few, dominant organism(s)
along with epiphytes and loosely-associated taxa. The dominant organism(s) may be identified in the field,
although it may be necessary to confirm the identity in the laboratory. In other cases (e.g. epilithic biofilms),
the dominant organism may be too small to be identified in the field. The result is that any survey unit will
contain a wide variety of growth forms that may need to be recorded and/or sampled.
Analysis of the phytobenthos at a site / survey unit consists of three stages, which can be combined in various
ways to give a number of survey / sampling strategies, each applicable to different purposes. These stages
are:
 Survey: a detailed inspection of a defined length of the river or stream, recording the nature of the stream
environment, the substrata available for phytobenthos and the nature and abundance of any phytobenthic
growth forms present.
 Sampling: removal of small quantities of some or all the phytobenthic growth forms for subsequent
examination in the laboratory.
 Laboratory analysis: identification and abundance assessment of the organisms present in the growth
forms.
In a few cases (e.g. Hildenbrandia rivularis), species-level determinations can be made in the field but in most
cases, the identities of macroscopic algae and bryophytes should be checked in the laboratory unless the
surveyors have proven competence in field identification of these organisms.
These three stages are combined, in different ways, to give the following sampling strategies:
 Macroscopic Phytobenthos Survey (MPS): detailed survey of all, or a selection, of the phytobenthic
growth forms that are visible with the naked eye, with sampling and laboratory analysis confined to
checking the identities of macroscopic algae and bryophytes. MPS provides semi-quantitative (or, with
slight modification, quantitative) estimates of the abundance of those taxa that are visible to the naked
eye. It is recommended for trend monitoring and, particularly, for detecting changes in abundance of
“nuisance” algae such as Cladophora and Hydrodictyon.
 Multi Habitat Sampling (MHS): survey and sampling of all available habitats / substrata in order to
compile a list, with semi-quantitative estimates of abundance, of all phytobenthic taxa present at a survey
unit. MHS best characterises the phytobenthos in the reach, but results may not be sensitive to subtle
water quality differences because of habitat differences between reaches.
 Single Habitat Sampling (SHS): a single type of habitat / substratum is sampled at each survey unit and
examined in the laboratory. The output is, as for MHS, a list, with abundance estimates, of all taxa
present. SHS should reflect water quality differences between streams more precisely than MHS,
provided that the same type of habitat / substratum is sampled at all sites. Impacts on other habitats /
substrata in the reach may however be missed. It is identical, in principle, to methods described in
EN 13946, except that groups other than the diatoms are included in the subsequent analysis.
Figure 1 is a diagrammatic representation of how these strategies can be applied to a survey unit.

Figure 1 — Relationships between field and laboratory stages for the three types of analysis described
in this standard
7.2 Number and location of sampling sites
The number and location of sampling or survey sites should be determined according to the aim of the study.
In general, sites selected should be representative of the reach of river under consideration, avoiding heavily-
shaded sites unless these are characteristic of the reach. Where possible, sites selected should have similar
conditions of light, current velocity, substratum etc. to facilitate comparability.
Reaches should be identified within a watercourse with respect to points where marked changes in quality are
likely to occur or where there are important river uses, for example major discharges or abstractions. Sufficient
sample sites should be included within each reach to differentiate naturally occurring changes (geology,
climate etc.) from changes caused by human impact. If sampling is intended to monitor the effects of a
discharge, sample sites upstream and downstream (beyond the mixing zone) of the discharge should be
included. Additional sites should be added at distances downstream to assess the extent of the influence of
the impact, and any possible recovery.
The location of the selected survey unit within a given site should be defined in field notes by reference to map
co-ordinates and location relative to permanent bankside objects (e.g. bridges). GPS (global positioning
satellite) receivers may be useful for obtaining accurate co-ordinates. It may also be appropriate to indicate
the location of a site by the use of permanent markers such as iron bolts or paint on large stones.
The survey units should all be the same length: 10 m is recommended, but longer lengths may be appropriate
under some circumstances. The samples should be collected from the main channel of the river (i.e. the zone
that is normally submerged). Rivers with naturally fluctuating discharge have specific algal associations in the
flood zone that may be of interest to sample.

7.3 Time of sampling
The time of sampling depends on the scope of the survey and the local conditions. Seasonal runoff patterns
should be considered. It is recommended that surveys are performed and samples collected during periods of
stable water flow, preferably at low discharge conditions. In Scandinavia, the phytobenthos is at peak
development in late summer/autumn before the onset of the decomposition of the vegetation; however,
timings elsewhere in Europe may be different. For consistency, it is recommended that surveys are performed
or samples collected at approximately the same time every year. When periodic discharges are surveyed,
sampling should be undertaken in conjunction with such episodes (preferably both before and after). It is,
however, still recommended that sampling is undertaken at low river discharge levels.
The composition of stream phytobenthos varies throughout the year. A single sample or survey may not be
sufficient to characterise fully the diversity in a reach. One sample or survey may, however, be sufficient for
spatial and temporal comparisons, as long as all the samples are collected, or surveys performed, at the same
time of year.
8 Survey and sampling procedures
8.1 Field survey method: MPS - Macroscopic phytobenthos survey
A detailed examination of
...