BRITISH STANDARD BS EN

15110:2006
BS
6068-5.41:2006

Water quality —
Guidance standard for
the sampling of
zooplankton from
standing waters

The European Standard EN 15110:2006 has the status of a

British Standard

ICS 13.060.45

12&23<,1*:,7+287%6,3(50,66,21(;&(37$63(50,77('%<&23<5,*+7/$:
BS EN 15110:2006

National foreword

This British Standard is the official English language version of

EN 15110:2006.
The UK participation in its preparation was entrusted by Technical Committee
EH/3, Water quality, to Subcommittee EH/3/5, Biological methods, which has
the responsibility to:

— aid enquirers to understand the text;

— present to the responsible international/European committee any

enquiries on the interpretation, or proposals for change, and keep
UK interests informed;
— monitor related international and European developments and
promulgate them in the UK.

A list of organizations represented on this subcommittee can be obtained on

request to its secretary.
Cross-references
The British Standards which implement international or European
publications referred to in this document may be found in the BSI Catalogue
under the section entitled “International Standards Correspondence Index”, or
by using the “Search” facility of the BSI Electronic Catalogue or of British
Standards Online.
This publication does not purport to include all the necessary provisions of a
contract. Users are responsible for its correct application.
Compliance with a British Standard does not of itself confer immunity
from legal obligations.

Summary of pages
This document comprises a front cover, an inside front cover, the EN title page,
pages 2 to 23 and a back cover.
The BSI copyright notice displayed in this document indicates when the
document was last issued.

This British Standard was Amendments issued since publication

published under the authority
of the Standards Policy and
Strategy Committee Amd. No. Date Comments
on 30 June 2006

© BSI 2006

ISBN 0 580 48602 8

EUROPEAN STANDARD EN 15110
NORME EUROPÉENNE
EUROPÄISCHE NORM May 2006

ICS 13.060.45

English Version

Water quality - Guidance standard for the sampling of

zooplankton from standing waters

Qualité de l'eau - Guide pour l'échantillonnage du Wasserbeschaffenheit - Anleitung zur Probenahme von
zooplancton dans les eaux stagnantes Zooplankton aus stehenden Gewässern

This European Standard was approved by CEN on 13 April 2006.

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. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the Central Secretariat or to any CEN member.

This European Standard exists 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 Central Secretariat has the same status as the official
versions.

CEN members are the national standards bodies of Austria, Belgium, 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.

EUROPEAN COMMITTEE FOR STANDARDIZATION

COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: rue de Stassart, 36 B-1050 Brussels

© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 15110:2006: E
worldwide for CEN national Members.
EN 15110:2006 (E)

Contents Page

Foreword..............................................................................................................................................................3
Introduction .........................................................................................................................................................4
1 Scope ......................................................................................................................................................5
2 Normative references ............................................................................................................................5
3 Terms and definitions ...........................................................................................................................5
4 Principle..................................................................................................................................................7
5 Equipment ..............................................................................................................................................7
6 Preserving solutions .......................................................................................................................... 10
7 Preliminary stages .............................................................................................................................. 11
8 Sampling procedure ........................................................................................................................... 11
9 Identification and records.................................................................................................................. 16
10 Preservation and storage of samples............................................................................................... 17
11 Quality assurance ............................................................................................................................... 18
Annex A (informative) Preservation ............................................................................................................... 19
Annex B (informative) Example of a field data sheet.................................................................................... 22
Bibliography ..................................................................................................................................................... 23

2
 EN 15110:2006 (E)

Foreword
This document (EN 15110:2006) has been prepared by Technical Committee CEN/TC 230 “Water analysis”,
the secretariat of which is held by DIN.

This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by November 2006, and conflicting national standards shall be withdrawn
at the latest by November 2006.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, 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.

3
EN 15110:2006 (E)

Introduction
Zooplankton community structure provides information on a range of physico-chemical and biotic causative
variables. These include pH- and acidification-related variables, toxic chemicals, phytoplankton structure and
abundance (i.e. lake productivity), and intensity of fish predation. The effects of size-selective predation are
well known and the size-structure of zooplankton communities can give valuable information of the fish
community.

Metazoan zooplankton (metazooplankton) constitute a large number of species within a range of total lengths
of about 0,05 mm to 20 mm, but mostly < 2 mm. The main groups are the rotifers (Rotatoria), the cladocerans
(Cladocera) and the copepods (Copepoda). Some shrimps (Natantia; e.g. Mysidae) and larvae of dipterans
(Diptera, e.g. Chaoborus) may also be considered as part of the zooplankton fauna. Rotifers and crustaceans
inhabiting the littoral of standing waters can also be grouped with the more strictly planktonic forms. Fish
larvae, hemipterans (Heteroptera, e.g. Corixidae) and coleopterans (Coleoptera) are occasionally recorded in
the plankton samples but are not considered as part of the zooplankton fauna. Procedures for sampling of
protozooplankton (Protozoa) are not included in this standard.

Surveys of zooplankton have provided valuable information for the environmental monitoring of standing
waters, because this group includes species which:

a) occur in a wide range of standing waters over a large geographical area and at the same time have
specific environmental requirements;

b) are well known with regard to their geographical distribution and environmental requirements;

c) have a generally high capacity for dispersal enabling them to respond rapidly to remedial actions;
while

d) sampling requires only a modest expenditure of time and equipment.

WARNING — Working in or around water is inherently dangerous. This standard does not purport to
address all of the safety problems, if any, 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.

NOTE According to the classification by Fryer [5] the assemblage long known as the Cladocera is split into four
orders; Ctenopoda, Anomopoda, Onychopoda and Haplopoda. Cladocera is however used in this standard as a general
descriptive term.

4
 EN 15110:2006 (E)

1 Scope
This guidance standard describes general procedures for surveying zooplankton in standing waters for the
purposes of water quality assessment and determination of ecological status.

Guidance on sampling procedures and the subsequent steps for preservation and storage are given. The
sampling procedures provide estimate for species occurrence and their abundance (relative or absolute),
including spatial distribution and temporal trends, for a given body of water. Calculation of biomass and
production is made possible.

This method is restricted to the sampling of multicellular zooplankton that inhabit the pelagic and littoral
regions of lakes, reservoirs and ponds. The sampling procedure may be also employed in slow running waters
and canals.

NOTE The field methods described are suitable for the collection of open-water plankton and littoral plankton species.
They are inappropriate for the collection of littoral species that primarily live on or in the surface of sediments and on the
surface of aquatic plants.

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.

prEN 14996, Water quality — Guidance on assuring the quality of biological and ecological assessments in
the aquatic environment.

EN 25667-1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes
(ISO 5667-1:1980).

EN ISO 5667-3, Water quality - Sampling - Part 3: Guidance on the preservation and handling of water
samples (ISO 5667-3:2003)

3 Terms and definitions

For the purposes of this document, the following terms and definitions apply.

3.1
anoxic
condition in which the concentration of dissolved oxygen is so low that certain groups of micro-organisms
prefer oxidized forms of nitrogen, sulphur, or carbon as an electron acceptor

NOTE As the oxygen concentration approach zero the concentration of hydrogensulfide (H2S), released by bacterial
anaerobic (no oxygen present) activity, is increasing. The anoxic conditions markedly affect the cycling of other nutrients,
ecosystem productivity, and the distribution of biota.

3.2
body of surface water
discrete and significant element of surface water such as a lake, reservoir, stream, river or canal, part of a
stream, river or canal, a transitional water or a stretch of coastal water [EC Directive 2000/60/EC]

3.3
dimictic lake
lake with spring and autumn turnovers (temperate lake)

5
EN 15110:2006 (E)

3.4
epilimnion
water above the thermocline in a stratified body of water

3.5
fixation
protection from disintegration of the morphological structure of organisms

3.6
impact study
investigation of the physical, physico-chemical and biological consequences of a given intervention in a body
of water

NOTE A study of consequences should be capable of forming a basis for the subsequent remedial measures.

3.7
habitat
locality in which a plant or animal naturally grows or lives

NOTE It can be either the geographical area over which it extends, or the particular station in which a specimen is
found.

3.8
hypolimnion
water below the thermocline in a stratified body of water

3.9
littoral zone
shallow marginal zone of a body of water within which light penetrates to the bottom; usually colonised by
rooted vegetation

3.10
metazoan
multicellular animals that develop from embryos

3.11
metazooplankton
multicellular zooplankton (see 3.21)

3.12
pelagic zone
free body of water beyond the littoral zone

3.13
plankton
organisms drifting or suspended in water, consisting chiefly of minute plants or animals, but including larger
forms having only weak powers of locomotion

3.14
preservation
protection from (bio)chemical degradation of organic matter

3.15
sampling site (sampling station)
general area within a body of water from which samples are taken

NOTE A station is defined in terms of its location (geographical position, depth) and invariant conditions (e.g. type of
bottom in shallow-water areas) and is delimited on the basis of the accuracy with which these are given. In cases of doubt
when sampling stations have to be re-identified, most weight should be placed on depth and type of bottom.

6
 EN 15110:2006 (E)

3.16
stratified water
standing water with temperature gradients resulting in an upper, warmer, isothermal layer floating on cooler,
denser, usually also isothermal water

NOTE Between the upper layer, the epilimnion, and the lower layer, the hypolimnion, is a transitional zone, the
metalimnion (see thermocline). The thermal stratification may be very short-lived or persist for all of the warmer part of the
year. Lakes with ice-cover during the cold season may show inverse stratification; an upper, cooler (< 4 ˚C) layer floating
on warmer water. Water has its highest density at 4 ˚C and during stratification and inverse stratification the deeper water
has a temperature of approximately 4 ˚C.

3.17
subsampling
collection of a sub-sample that consists of a known fraction of the total sample and that is representative of
the quantity and species composition of the latter

3.18
thermocline (metalimnion)
layer in a thermally stratified body of water in which the temperature gradient is at a maximum

3.19
trend monitoring
study intended to reveal any changes in the ecological status of a body of water over time

3.20
turbidity
reduction of transparency of water caused by the presence of undissolved matter

3.21
zooplankton
animals present in plankton

4 Principle
The sampling strategy adopted provides information on the current status of the metazooplankton community.
The selection of sampling sites (numbers and location), sampling depth, time and frequency of sampling, size
of samples and type of sampling gear is of great importance for the evaluation of the data collected. As a
general guidance EN 25667-1 should be consulted.

5 Equipment
There exist several overviews of the most widely used zooplankton sampling techniques and their advantages
and drawbacks (e.g. [1], [2], [3], [7], [8] and [13]). This standard provides some general recommendations.

7
EN 15110:2006 (E)

Key

a conical plankton net

b Schindler trap
c modified Ramberg sampler
d Clarke-Bumpus sampler

Figure 1 — Examples of different zooplankton sampling equipment

5.1 Qualitative sampling

5.1.1 Nets

Nylon plankton nets of various dimensions and mesh sizes can be used for sampling (Figure 1a). It is
important that nets should have a large filtering surface relative to their opening in order to ensure that filtering
is as efficient as possible. A net with an opening diameter of 30 cm, for example, should have a length of
about one metre as a minimum. A cylindrical net section above the conical part increase the filtering area
compared with a conical plankton net with the same opening diameter and length.

If both rotifers and crustaceans are to be analysed, a net with a mesh of about 40 µm to 50 µm should be
utilised. Nets with meshes smaller than 40 µm will readily become clogged and their use should normally be
avoided, although they may be useful in oligotrophic waters. If only crustacean plankton are to be analysed a
mesh of 90 µm (max. 100 µm) can be used. If both rotifers and crustaceans, including large predatory species,
are to be sampled with a reasonable degree of efficiency, the use of three nets with different mesh sizes are

8
 EN 15110:2006 (E)

recommended: 45 µm for rotifers, 90 µm for most of the crustaceans, and 150 µm or more for the predatory
species.

NOTE All the mesh sizes mentioned in this standard should be regarded as for guidance only. Mesh sizes will also
vary somewhat from manufacturer to manufacturer.

5.1.2 Other field equipment

Winch with line-length counter or line with length markings fitted with a shackle or similar device to enable the
line to be joined to the net.

Draining cup with nylon netting, which is capable of being attached to the net either by means of a tightening
strip or tape sewn into the net. The netting of the draining cup should have the same mesh size as the net. A
draining cup with hose and hose clamp can also be utilised.

Weight, e.g. a standard sounding lead weight.

Spray bottle with water for rinsing out the net and draining cup.

A small plastic funnel may be needed to transfer the sampled material to the sample bottle.

5.1.3 Optional equipment

Portable echo sounder or depth meter to estimate the water depth at a sampling site. The former may also be
used to quantify large zooplankton species.

Global Positioning System (GPS) to define site location.

5.2 Quantitative sampling

5.2.1 Sampling equipment

For this purpose various types of volume samplers (bottles/traps/tubes) can be used (e.g. Schindler-Patalas
trap or modified Ramberg sampler) (Figure 1b and 1c). Plankton nets with a closure mechanism and built-in
water flow-through meter (e.g. Clarke-Bumpus sampler) (Figure 1d), plankton pumps (e.g. [10] or [14]) and
flexible tubes (e.g. [9], [11]) can be used for obtaining vertically or horizontally integrated quantitative
zooplankton samples. Echo sounder records can be used in the field to quantify large zooplankton species
(e.g. Chaoborus). Several of the most widely used quantitative zooplankton samplers have been described in
detail by [2] and [3].

Where volume samplers are concerned, a sampler should be chosen that allows a free flow of water when the
sampler is not closed. It should also be possible to close the sampler rapidly, and the sampler should be as
transparent as possible (plexiglas walls) in order to prevent avoidance by large plankton species with good
vision and mobility. For the same reason, it is advantageous to use a sampler that is not too small (minimum
5 l). In locations with large populations of algae (nutrient-rich lakes), however, it may be advantageous to use
a smaller model of volume sampler (e.g. a tube sampler of 3 l). Such samplers may also be suitable for use in
lakes in locations that require equipment to be carried over long distances.

Motorised plankton pumps with continuous flow-through are recommended rather than hand-powered
plankton pumps, because motorised pumps provide a regular flow, thus providing better estimates of the
quantity and composition of the plankton. Large active plankton species are liable to be sampled less
efficiently using a plankton pump than by other types of quantitative samplers. The opposite can be the case
for small species. A large plastic funnel (diameter about 50 cm) at the end of the sampling tube may be useful
in order to prevent escape of “jumping” copepods.

For practical and safety reasons, when deep lakes are being sampled, it may be more appropriate to use
sampling equipment that allows efficient sampling of the whole water column (e.g. a plankton net with a
closure mechanism or a plankton pump) rather than a volume sampler.

9
EN 15110:2006 (E)

When shallow lakes or littoral areas with a great deal of vegetation are being quantitatively sampled the use of
a volume sampler, plankton pump or flexible tube is recommended.

5.2.2 Other field equipment

The equipment listed in 5.1.2 (excluding the lead weight) and in addition a mixing vessel (e.g. plastic bucket or
similar) to combine a number of individual samples into a single sample in the field. Conducting mixed
samples may be necessary to reducing analysis times and costs.

If a volume sampler is being used (with the exception of a Schindler-Patalas trap) filtration equipment is also
required to concentrate the samples. This may take the form of either a plankton net (mesh size 45 µm, or
90 µm if only crustacean plankton are being collected) or a large funnel with draining cup fitted with a netting.

5.3 Storage

Bottles (100 ml, 200 ml or 250 ml brown bottles with screw-tops) or glass vials for storing samples.

Labels or tape to attach to the outside of the sample bottles. Waterproof paper for labels to put inside the
sample bottles.

Marker pen. If ethanol is being used, an alcohol-proof pen or pencil is recommended for both internal and
external marking.

NOTE Plastic vials are not suitable for storing samples if Lugol’s Iodine is used as preserving solution.

6 Preserving solutions

6.1 General

A number of different preserving solutions with different areas of application are available. The advantages
and disadvantages of each of these solutions are defined in Annex A.

6.1.1 Formaldehyde, 37 % by volume.

This is neutralised, e.g. with hexamethyl tetramine (C6H12N4). Dilute the formaldehyde with water to 20 % (v/v)
to avoid precipitation, and then add 100 g of hexamethyl tetramine and 40 g to 80 g sucrose ([6]) per litre of
20 % formaldehyde.

WARNING — Formaldehyde may trigger allergies or cancers, and should therefore be handled with
care.

6.1.2 Ethanol, 96 % or 99 % C2H5OH.

6.1.3 Lugol’s Iondine

Acidified 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 this solution is
close to saturation, any precipitate should be removed by decanting the solution before use.

Most preservatives are also commercially available. For more details on the use of different preservatives the
reader is referred to Annex A.

Preserving solutions for field use should be kept in small stoppered bottles and should be accompanied by a
pipette for transferring the solution to the plankton samples. The bottles should be kept in a plastic box or
container with lid during transportation.

10
 EN 15110:2006 (E)

7 Preliminary stages

7.1 Documentation of strategies and methods

The following documentation should be available before the start of field work:

 description of objectives and strategy;

 description of methods;

 safety instructions;

 personnel plan;

 overview of equipment and instruments;

 registration forms;

 procedures for the maintenance of records and samples;

 quality assurance requirements according to prEN 14996.

7.2 Preparation of sampling equipment

After each day of sampling, the net should be washed in warm freshwater with detergent or in an ultrasonic
water-bath in order to reduce clogging and ensure optimum filtration capacity.

Check that the netting in the plankton net and draining cups is completely free of holes and tears.

Check that the line is securely attached to the plankton net/volume sampler.

Check that the plankton sampler’s closing mechanism is functioning well and that any seals are in order.

It may be advantageous to label the sample bottles and to add the requisite amounts of preserving solution to
them before the start of fieldwork.

In order to prevent spreading of flora and fauna between water bodies, the sampling equipment should be
disinfected between uses in the different waters.

7.3 Safety instructions

Before initiating the survey notify a contact of which localities and areas are to be surveyed on a specific day.
If the samples are being collected from a boat, always have a shore-based contact in case of emergencies.
Check the weather forecast in order to ensure safe and effective surveying conditions.

For safety reasons, it is recommended that surveys should not be undertaken by lone workers but by a
minimum of two people.

8 Sampling procedure

8.1 Investigation program

An investigation programme shall be developed according to the investigation aims, required precision of
results, hydromorphological conditions in the area, prior knowledge of local pollution sources, results of

11
EN 15110:2006 (E)

previous investigations and any other factors that may be of significance. The choice between qualitative and
quantitative methods is made according to the aims of the investigation and the required precision of results.

Qualitative samples (net hauls) provide information about the species composition, number of species, size
distribution and relative dominance of species and groups of zooplankton.

Quantitative samples also provide information regarding the quantity of zooplankton (individual density) per
unit volume. Quantitative samples also allow calculation of biomass and production for the zooplankton
assemblage as a whole as well as for the individual species.

In production studies (estimates of biomass and secondary productivity) it is essential to obtain quantitative
samples. If the objective is to give a species list as complete as possible, net hauls are also recommended.

The vertical and horizontal distribution of zooplankton is uneven. Some species are found regularly in the
shallow-water regions with stands of aquatic vegetation whereas others perform horizontal migration in the
course of the day. Vertical patterns and vertical migration is also common among zooplankton species. To
obtain whole-lake estimates of species composition and abundances, samples should be taken from both
pelagic and littoral areas as well as from different depths.

NOTE Standard plankton nets are not suitable for quantitative sampling and usually give a less accurate estimate on
species composition than quantitative zooplankton sampler.

8.2 Number and location of sampling sites

8.2.1 General consideration

The number and location of the sampling sites should be determined according to the aim of the study, the
morphology of the water body and the level of accuracy in the provided estimates. In general, sites selected
should be representative of the area under consideration.

8.2.2 Pelagic samples

The samples are normally collected at the same site as used for other observations (temperature, Secchi
depth, water chemistry, phytoplankton, etc.).

The deepest area near the centre of the main basin of the lake is usually preferred as the sampling location, if
a single location is regarded as sufficient for the purposes of the study. If this is not known, the maximum
depth of the lake should be estimated, by use of a portable echo sounder or a depth-meter, prior to the
sampling.

In large lakes, and lakes with several more or less separate basins or with a complex morphology, it will often
be desirable to have several sampling locations in order to obtain an impression of any intra- or inter-basin
differences. It is recommended that a minimum of one station should be established in each basin.

If, for example, the effects of point discharges are to be assessed, it may also be appropriate to select a
location, which is not near the centre of the main basin of the lake, and/or to set up more than one sampling
station. Generally, samples of strictly pelagic species should be collected at a good distance from the shore in
order to avoid as far as possible any influence from the littoral fauna.

Zooplankton are often irregularly distributed in lakes, i.e. there is often a horizontal and a vertical variation. If it
is important, to the aims of the study, to obtain information regarding the horizontal distribution of the
zooplankton, samples should be obtained from several locations along a gradient that represents the
dominant wind direction. Other horizontal gradients may also be considered. The vertical variation is
discussed in 8.3. If information regarding spatial variation or a high level of precision in the estimates is
required, it may be necessary to draw up a sampling programme adapted to the lake morphology and vertical
stratification (see 8.4).

12
 EN 15110:2006 (E)

8.2.3 Littoral samples

Such samples are collected from the lake’s shallow-water regions (usually at depths of < 1 m). Samples are
taken from areas containing aquatic vegetation (protected shores) and from areas with little vegetation
(exposed shores). In large lakes with a wider range of aquatic vegetation, it is recommended that separate
samples should be collected from areas with different types of aquatic vegetation, prioritising the dominant
types.

8.3 Sampling depth

8.3.1 General consideration

Samples may be collected individually from fixed depths, or as combined samples where samples from
different depths are mixed together to form a single sample. A third option is to use integrated samples, where
sampling encompasses the entire water column within fixed depth intervals.

It can be difficult to decide upon the appropriate sampling approach or exact sampling depths in each
individual case. Zooplankton usually are distributed unevenly. More information on how to deal with spatial
variation is given in 8.4.

8.3.2 Pelagic samples

The samples are collected from specific depths or from the whole watercolumn, depending on the objectives
of the study and the depth of the sampling station. In general, it is most important to have a relatively dense
network of observations from the surface and down to the thermocline, as the majority of the plankton is
usually to be found at these depths in the water column (epilimnion). In clear lakes, zooplankton is usually to
be found at greater depths than in humic (“brown-water”) lakes or water with high turbidity.

Vertical net hauls should cover all depths in order to include deepwater species. In locations with large
quantities of algae and other particles the net will easily become clogged and an additional sample should
therefore be taken from the uppermost 1 m to 5 m.

In order to avoid stirring up sediments and contaminating the samples, the deepest samples should be taken
no lower than 0,5 m from the sediment surface. If the greatest depths of the lake are anoxic, the samples
should be collected down to this zone.

If the objectives of the study make it important to obtain information regarding the vertical distribution of the
zooplankton, it is recommended to collect samples at a number of depths. The samples should be taken at
intervals of no more than 2 m in the epilimnion and metalimnion. In the hypolimnion samples should be taken
at less frequent intervals. In deep lakes the samples taken should cover at least the 0 m to 20 m stratum.

If mixed samples (i.e. integrated samples taken from a given range of depths) are to be taken, separate mixed
samples should also be taken from the epilimnion and the hypolimnion.

8.3.3 Littoral samples

Samples are collected from the shallow-water region of the lake at depths of between 0,2 m and maximum
depth of rooted vegetation. These samples should be collected just above the sediment surface within areas
of vegetation and areas with little vegetation.

8.4 How to deal with spatial variation

The spatial distribution of zooplankton in a lake is uneven and patchy. Both systematic and random variations
are common.

Systematic variations often take the form of sharp gradients or patterns (vertical and horizontal) and are best
mapped by sampling from a large number of depths and stations along dominant gradients (see 8.2 and 8.3).

13
EN 15110:2006 (E)

If the objectives of the study require information to be collected regarding spatial variation in general, or a high
level of accuracy in the estimates, it will be necessary to draw up a sampling programme that is adapted to the
morphometry and depth conditions of the lake. Stratified, randomised sampling (see [4]) of zooplankton is
based on dividing the lake horizontally and vertically into a series of sampling units, from which a random
selection of units to be sampled is selected. The optimum number of samples per stratum will depend upon
three factors: the size of the stratum, the variability within the stratum and the cost of sampling in the stratum
[4].

8.5 Sampling season and sampling frequency

For studies that involve comparisons of several lakes or changes over time (trend monitoring), it is
recommended that samples should be collected at comparable points in time in order to reduce variability due
to the natural population dynamics.

Studies whose primary objective is to provide information regarding species composition and dominance
should include sampling times that cover the population maxima of as many species as possible. It is
recommended that samples should be collected on three occasions in the course of the season: spring/early
in the summer (in dimictic lakes: immediately after the spring turnover; in lakes with ice-cover during winter:
within the first month following the spring melt); high summer; and late summer. If, for practical reasons,
samples can be collected only twice in the course of the season, they should be taken in spring/early summer
and high or late summer. Similarly, if only one sampling occasion per season is carried out, the samples
should be taken in high or late summer.

Studies that have the objective of providing information about individual density/biomass and productivity
should at least include monthly samples during the growth season, e.g. five or six times, if the growth season
lasts from May until September/October.

NOTE The composition and biomass of the zooplankton community varies widely in the course of the year. To a
great extent, zooplankton overwinters in the form of resting eggs and dormant instars, and to a lesser extent as active
individuals. In the course of the summer, populations grow, normally reaching a maximum in terms of density and biomass
in August until September in low-nutrient and/or cold temperate, alpine, and polar lakes. In nutrient-rich and/or warm
temperate lakes populations will normally reach a maximum somewhat earlier in the summer, to be followed by a new
maximum in the autumn. Rotifers reach their maximum density in the spring and early summer. The highest total species
diversity is usually found in the mid-summer and late summer, while samples collected soon after the spring melt can
provide important information about individual species (copepods) which have adult individuals early in the season. Adult
individuals are often more easy to determine to species level than young individuals.

8.6 Diurnal sampling period

Most zooplankton species perform vertical migrations in the course of the day. They are normally found higher
in the water column at night than during the day. For studies that involve comparisons of several water bodies
or changes over time (trend monitoring), it is recommended that samples should be collected at the same time
of day in order to reduce variability caused by the animals’ daily migrations.

Zooplankton should normally be sampled between 10 a.m. and 4 p.m. In studies of vertical migration,
sampling every 6 h is recommended; as a minimum, sampling should be performed at midday and midnight.

8.7 Sample size

Sample size will depend on the lake productivity (Table 1) and the aims of the study. Estimating the total
density/biomass of zooplankton will require relatively few animals in comparison with studies of the total
species diversity (studies of biological diversity).

Samples should contain a minimum of 200 animals (for crustaceans: exclusive nauplii) in order to provide a
good estimate of numbers and species composition. If both crustaceans and rotifers are included in the
analysis, the samples should contain a minimum of 200 animals of the group, which has the fewest individuals.

14
 EN 15110:2006 (E)

Table 1 — Recommendations of total sample size (may consist of several smaller samples)

Qualitative samples Quantitative samples

Lake productivity Vertical net hauls Littoral net hauls Volume samplers
Ultraoligotrophic Whole water column Up to 30 m Up to 50 l
Oligo-/Mesotrophic Whole water column 15 m to 20 m 20 l to 25 l
Eutrophic Whole water column 2 m to 10 m 5 l to 15 l

NOTE 1 Certain species are normally found in such small numbers (e.g. predators and other large species) that it is
necessary to collect a larger sample (approximately 10 times the volume of an ordinary zooplankton sample) in order to
obtain a good estimate of their numbers.

NOTE 2 A net-haul will normally contain many more animals than are required for analysis. Before counting and
estimating the occurrence of the more typical species, a representative sub-sample may be taken out (see [4]).

8.8 Operating the sampling device

8.8.1 Qualitative samples

If the samples are being collected from a boat, it is important to prevent the boat from drifting rapidly with the
result that the net moves too rapidly through the water, as this will reduce the filtration efficiency (“sea-anchor”
effect). This can be done either by anchoring or mooring to a buoy.

8.8.2 Vertical net hauls

The net is sunk vertically to the desired depth and is hauled up again slowly and smoothly (approximately
0,15 m/s to 0,2 m/s). Since the samples are to be analysed only qualitatively, it is important that sufficient
material should be obtained. It may be necessary to make several vertical hauls per sample site. If this is done,
the net should be emptied between each haul.

8.8.3 Littoral net hauls

The net is hauled slowly (approximately 0,2 m/s to 0,5 m/s) just above the bottom in such a way as to avoid
too much sediment-/plant debris entering the sample. Since the samples are going to be analysed only
qualitatively, it is important that sufficient material should be obtained. It may be necessary to make several
horizontal hauls per sample. If this is done, the net should be emptied between each haul.

When the plankton net has been taken out of the water, all the material hanging on the inside of the net is
rinsed into the draining cup. The simplest way of doing this is to raise and lower the net several times through
the water surface. In order to empty out animals that remain at the bottom of the net, water is poured or hosed
over it from the outside, from the top and downwards, by use of a spray bottle. The material is then transferred
to the sample bottle.

8.8.4 Quantitative samples

The following description applies only to volume samplers. Use of other types of sampling equipment (e.g.
Clarke-Bumpus sampler, plankton pumps, flexible tubes) for obtaining quantitative samples of zooplankton
should follow the description given for the specific sampler.

When samples are being collected from a boat, drifting should be reduced to a minimum, and care should be
taken to avoid slack on the line or wire when the sampler is being lowered. This is in order to avoid losing
control of the sampling depth and to ensure free water flow-through all the way down. The use of an anchor or
mooring buoy is recommended.

15
EN 15110:2006 (E)

When the sampler is being sent out a check should be made that the lid or closure mechanism is open in
order to ensure free water flow-through. The equipment is lowered vertically to the required depth at an even
and moderate speed (approximately 1 m/s to 2 m/s). This eliminates most of the pressure wave ahead of the
equipment, and the water that is taken up in the sampler will come from the correct depth. The sampler is then
drawn up again smoothly and at a moderate speed (1 m/s to 2 m/s). It is important to ensure that the water
pressure on the upper surface of the sampler is regular, in order to avoid it being blown open and the inside
water being replaced.

When the sampler has been hauled up, it is opened and its contents are filtered through a plankton net or
other straining equipment. Ensure that all the material hanging on the inside of the net/straining equipment is
rinsed into the draining cup (see 8.8.1). The material is then transferred to the sample bottle.

8.9 Filling sample bottles

It is important to fill sample bottles almost to the top. An insufficient sample volume relative to bottle size may
leave specimens stuck to the walls of the bottle, where they may dry out in storage. However, to facilitate
homogenisation, for instance as a part of sub-sampling procedures (see [4]), bottles should not be filled
completely.

9 Identification and records

9.1 Labelling

Unambiguous identification of the samples is important. Therefore, each sample should be labelled in such a
way that information on the source of the sample and the condition under which it was collected could be
tracked back to the exact sample. The sample bottles should be labelled on the outside using a waterproof
marker pen or pencil on a label or tape. In addition, as a back-up, it is recommended to put a label, using
waterproof paper, inside the sample bottles. As a minimum, the following information should follow each
sample (included on the label or in the sampling protocol):

 date of collection;

 name of locality (or locality ID);

 name of sampling site (or locality number or GPS position);

 sampling depth (or mixed-sample interval);

 type of sampling gear;

 sample volume / length of net haul;

 mesh size of plankton net / draining cup;

 weather and water conditions;

 initials of the surveyor(s);

 and any other relevant information that will assist the interpretation of the results.

Any deviations from this standard should be noted on the label and/or in the sampling protocol.

If mixed (integrated) samples are being used, it is important to note the number of individual samples, or the
total volume that the mixed sample represents.

16
 EN 15110:2006 (E)

9.2 Field data recording

A field data sheet should be drawn up in order to ensure that information regarding sampling location,
environmental data (weather conditions, water flow, water level, etc.) and data regarding the sample itself are
kept for future reference. An example of a field data sheet is illustrated in Annex B. All data records relating to
the specific survey should be individually identifiable and retained for a period usually not less than 5 years.

9.3 Geographical localisation of sampling sites

Sampling stations should be unambiguously located and in such a way that they can be found subsequently
by other workers. The location of sites should be carried out by means of geographical coordinates based on
a grid reference system (e.g. European Datum: ED-50, World Geodetic System: WGS-84), or based on the
UTM system. Site location should be performed in accordance with relevant guidelines. The Global
Positioning System (GPS) should be used as far as possible for defining site location.

In addition to its geo-references, the study site should be identified with reference to characteristic landmarks
and at least one fixed reference point at an easily identifiable place close to the sampling station.

The location of littoral stations should also be recorded by means of photographs.

10 Preservation and storage of samples

10.1 General

General procedures for handling of samples are described in EN ISO 5667-3.

10.2 Preservation

Zooplankton organisms may be anaesthetized before preservation, e.g. with carbon dioxide in mineral water.

Preserving fluid can be added to the sampling bottles in advance. If this has not been done, preserving fluid
should be added immediately after the samples have been taken.

Lugol’s Iodine is added in the amount of 0,5 ml to 1,0 ml per 100 ml sample volume. Samples containing large
quantities of animals and other organic material (particularly samples collected in littoral regions) require the
addition of more Lugol’s Iodine than samples that contain little organic material; e.g. 3 ml to 5 ml per 100 ml
sample volume.

If ethanol is used, the preserved sample should have an ethanol content of at least 70 % to 75 %. If the
samples are used for genetic analysis based on DNA extraction or if reproduction parameters are to be
measured, an ethanol content higher than 90 % is recommended. The best method of achieving this is to filter
off as much as possible of the sampling water before the preserving solution is added. A high ethanol
concentration helps to maintain body shape (Cladocerans) as well as preventing the animals from releasing
their eggs.

When formaldehyde is used, the preserved sample should have a formalin concentration of around 4 %, i.e.
20 ml aqueous 20 % formalin should be added per 100 ml sample volume. Preservation in a cold solution of
formalin (6 ˚C) with added sucrose reduces the chances of the animals releasing their eggs.

NOTE Samples for genetic analysis based on electrophoresis should not have any preserving solution added to them.
Such analysis should be performed on living or frozen samples.

17
EN 15110:2006 (E)

10.3 Storage

The samples should preferably be preserved immediately after collection. Non-preserved samples should be
kept in the dark at temperatures between 2 °C and 5 °C. A maximum storage time of 48 h under such
conditions should be observed. Samples for quantitative analyses should be immediately preserved.

Samples preserved with Lugol’s Iodine should always be stored in the dark and preferably chilled to below
5 °C, unless they are to be analysed within a week, in which case they can be stored in the dark at room
temperature. The reason for cooled storage is to slow down the rate of physical and chemical processes that
lead to reduced sample quality. Storage in the dark is always necessary in order to prevent photo-oxidation.
Samples that have been preserved with Lugol’s Iodine should be straw coloured and should be checked after
a couple of days for oxidation. For storage for periods of years it is recommended to preserve the sample with
formaldehyde or ethanol. If Lugol’s Iodine is used, it is necessary to check the sample periodically in order to
determine whether additional preserving solution is needed.

Samples for genetic analysis based on DNA extraction should be stored in a refridgerator or freezer, but in
such a way that the sample itself does not freeze (this is ensured by preservation in high concentrations of
ethanol). Samples for genetic analysis based on electrophoresis should be stored in a bio-freezer
(approximately -75 °C).

11 Quality assurance
The project manager is responsible for informing the field personnel about the purposes of the study and for
training them in the practical implementation of the fieldwork. The project manager is also responsible for
adherence with the sampling manual and protocol.

A fieldwork leader with the overall responsibility for performing the fieldwork should be appointed. The leader
should ensure that all sampling, labelling and preservation procedures are followed. General procedures for
quality assurance are described in prEN 14996.

18
 EN 15110:2006 (E)

Annex A
(informative)

Preservation

A.1 Preservation
Preserving solutions are toxic by definition. Their addition leads to the death of living organisms, preceded by
a strong chemical reaction. As a result of this excitation the most delicate organisms that lack strong cell walls
and rigid lorica (e.g. several rotifers) may collapse before fixation is complete. All types of preserving solutions
result in the loss of organic matter. A consequence of this is that estimates of biomass based on
measurements of weight of wet or dry matter will underestimate true biomass values. For this reason, only
material that has not been preserved should be weighed.

Preservatives should meet the following requirements:

 The effect of the agent on the loss of organisms by chemical shock or otherwise should be known
beforehand;

 The preservative should effectively prevent the microbial degradation of organic matter for at least the
storage period of the samples;

 The preservative should guarantee a good recognition of taxa for at least the storage period of the
samples.

 The most frequently used preservatives in zooplankton research are formaldehyde and Lugol’s Iodine.
For genetic analysis samples are preserved in ethanol.

A.2 Formaldehyde (formalin)

A.2.1 General

Formaldehyde has a fixating effect that is based on the principle of protein-cross-linking (by the formation of
hydrogen bridges between protein molecules). As a result organic tissues and cellular structures are durably
preserved. However, formaldehyde is slowly transformed into formic acid and methanol (Cannizzaro reaction),
negatively affecting fixation and preservation. For this reason formaldehyde should not be kept in stock too
long and should preferably be applied with an added buffer, for instance hexamethylene tetramine. The
process of transformation into formic acid and methanol can be counteracted if the solution is prepared from
paraformaldehyde, a polymer of formaldehyde. If the concentration of formaldehyde is not more than 20 %,
precipitation will not be produced.

A.2.2 Advantages of formaldehyde

 Effectively prevents the microbial degradation of organic matter;

 Organic structures and other morphological characteristics remain visible;

 When stored properly in suitable bottles, samples will stay in good condition for many years without
attention.

19
EN 15110:2006 (E)

A.2.3 Disadvantages of formaldehyde

 Distortion of the body structure often takes place in soft-bodied organisms (e.g. several rotifers);

 Carapaces (cladocerans) may balloon followed by the loss of brood-pouch contents (eggs and embryos).
This can be prevented by using a cold (6 ˚C) solution of formalin with added sucrose;

 Formaldehyde is irritating at even very low concentrations. Formaldehyde can also be carcinogenic. This
preserving solution should therefore be handled with care, and should be “washed out” of samples within
an air extraction hood before they are analysed.

A.3 Lugol’s Iodine

A.3.1 General

Lugol’s Iodine is a mixture of iodine (I2), potassium iodide (KI) and acetic acid. The iodine compounds are
oxidised in the air and by light. The solution should therefore be stored in the dark or in brown glass bottles,
with a minimum of headspace. Samples preserved with Lugol’s Iodine should also be stored in the dark.
Lugol’s Iodine does not fix organic biomass. In the long term the inner cell structure may disintegrate. In order
to prevent this formaldehyde can be added. This should be done as soon as possible but preferably after
counting. Samples preserved with Lugol’s Iodine can be cleared completely by reducing the iodine with
sodium thiosulphate.

A.3.2 Advantages of Lugol’s Iodine (over formaldehyde)

 It enters the cell more quickly than formaldehyde, leaving shock-sensitive organisms better preserved in
the sample;

 Detection of small zooplankton is easier, thanks to the enhanced contrast between organisms and the
surrounding fluid;

 Only slightly toxic in normal conditions of use and offers minimal discomfort to laboratory personnel.

A.3.3 Disadvantages of Lugol’s Iodine

 The intense staining may obscure certain cellular structures that need to be observed for proper
identification (e.g. surface structures). Overstaining can be cleared up by adding sodium thiosulphate,
which reduces the iodine;

 Organic matter is not fixed, and soft materials may lose their characteristic structure during storage of the
sample. This alteration can be prevented by the addition of formaldehyde;

 Iodine is oxidised over a period of time; therefore, when storage time is long or samples contain a large
amount of organic matter, samples need attention to prevent them from decay.

A.4 Ethanol

A.4.1 Advantages of ethanol

 It provides good preservation of organic material;

 Organic structures remain visible;

20
 EN 15110:2006 (E)

 Samples preserved in ethanol retain their quality for long periods of time if they have been stored
correctly. Such samples thus require little attention;

 The use of 96 % ethanol prevents carapace distortion and loss of eggs and embryos due to ballooning
(cladocerans);

 DNA is retained in a form which can subsequently be extracted for genetic analysis (for this purpose,
should be stored in fridge or freezer).

A.4.2 Disadvantages of ethanol

 Can cause cell shrinkage, which will result in underestimation of dimensions;

 May be unpleasant for laboratory personnel (dizziness, headaches). For this reason, this preserving
agent should be diluted before samples are analysed.

21
EN 15110:2006 (E)

Annex B
(informative)

Example of a field data sheet

Information which should follow the sample (the locations of sampling sites are also marked on a lake map).

Project: Project leader: Fieldwork leader/team: Diurnal sampling period

(start–end):

Locality (e.g. lake ID)/watershed Water body type (lake/ Date (yy-mm-dd):
(drainage area code): reservoir/pond):

County/community: Co-ordinates (mid-lake position): Weather cond.;

Objective of the sampling:

Sampling site Position (GPS): Depth: Sample size Equipment (incl. Comments:
(loc. no etc): mesh size):

(# litre/net haul in m)

22
 EN 15110:2006 (E)

Bibliography

[1] ANON, 1968. Zooplankton sampling. Monographs on oceanographic methodology 2. UNESCO, Paris,
174 pp.

[2] BOTTRELL H.H., DUNCAN A., GLIWICS Z.M., GRYGIEREK E., HERZIG A., HILLBRICHT-ILKOWSKA A.,
KURASAWA H., LARSSON P. & WEGLENSKA T. 1976. A review of some problems in zooplankton
production studies. Norw. J. Zool., 24, 419-456.

[3] DE BERNARDI R. 1984. Methods for the estimation of zooplankton abundance. In: DOWNING J.A. &
ROGLER F.H. (eds.), A Manual of methods for the assessment of secondary productivity in fresh waters.
Blackwell Scientific, Oxford, pp. 59-86.

[4] EDMONDSON W.T. and WINBERG G. G. (eds.) 1971. A manual of methods for the assessment of
secondary productivity in fresh waters. IBP-Handbook No. 17. Blackwell Scientific, Oxford, 358 pp.

[5] FRYER G. 1987. A new classification of the branchiopod Crustacea. Zool. J. Linnean Soc. 91, 357-383.

[6] HANEY I.F. & HALL D.J. 1973. Sugar coated Daphnia: A preservation technique for Cladocera. Limnol.
Oceanogr., 18, 331-333

[7] HORN W. 1999. Metazooplankton. In: TÜMPLING W.V. & FRIEDRICH G. (eds.), Biologische
Gewässeruntersuchung, G. Fischer, Jena, pp. 53-76.

[8] JOHANNSSON O.E., SHAW M.A., YAN N.D, FILION J.-E. & MALLEY D.F. 1992. A comparison of freshwater
zooplankton sampling gear: Nets, Traps and Submersible Pump. Can. Tech. Rep. Fish. Aquat. Sci.
1894, 29 pp.

[9] KNOECHEL R. & CAMPBELL C.E. 1992. A simple, inexpensive device for obtaining vertically integrated,
quantitative samples of pelagic zooplankton. Limnol. Oceanogr., 37, 675-680.

[10] NAYAR S., GOH B.P.L. & CHOW L.M. 2002. A portable, low-cost, multipurpose, surface-subsurface
plankton sampler. J. Plank. Res., 24, 1097-1105.

[11] PENNAK R.W. 1962. Quantitative zooplankton sampling in littoral vegetation areas. Limnol. Oceanogr.,
7, 487-489.

[12] SCHINDLER D.W. 1969. Two useful devices for vertical plankton and water sampling. J. Fish. Res.
Board Can., 26, 1948-1955.

[13] SCHWOERBEL J. 1966. Methoden der Hydrobiologie. Kosmos Verlag, Stuttgart.

[14] WAITE S.W. & O’GRADY S.M. 1980. Description of a new submersible filter-pump apparatus for
sampling plankton. Hydrobiologia, 73, 187-191.

23
BS EN
15110:2006
BS 6068-5.41:2006 BSI — British Standards Institution
BSI is the independent national body responsible for preparing
British Standards. It presents the UK view on standards in Europe and at the
international level. It is incorporated by Royal Charter.

Revisions
British Standards are updated by amendment or revision. Users of
British Standards should make sure that they possess the latest amendments or
editions.
It is the constant aim of BSI to improve the quality of our products and services.
We would be grateful if anyone finding an inaccuracy or ambiguity while using
this British Standard would inform the Secretary of the technical committee
responsible, the identity of which can be found on the inside front cover.
Tel: +44 (0)20 8996 9000. Fax: +44 (0)20 8996 7400.
BSI offers members an individual updating service called PLUS which ensures
that subscribers automatically receive the latest editions of standards.

Buying standards
Orders for all BSI, international and foreign standards publications should be
addressed to Customer Services. Tel: +44 (0)20 8996 9001.
Fax: +44 (0)20 8996 7001. Email: orders@bsi-global.com. Standards are also
available from the BSI website at http://www.bsi-global.com.
In response to orders for international standards, it is BSI policy to supply the
BSI implementation of those that have been published as British Standards,
unless otherwise requested.

Information on standards
BSI provides a wide range of information on national, European and
international standards through its Library and its Technical Help to Exporters
Service. Various BSI electronic information services are also available which give
details on all its products and services. Contact the Information Centre.
Tel: +44 (0)20 8996 7111. Fax: +44 (0)20 8996 7048. Email: info@bsi-global.com.
Subscribing members of BSI are kept up to date with standards developments
and receive substantial discounts on the purchase price of standards. For details
of these and other benefits contact Membership Administration.
Tel: +44 (0)20 8996 7002. Fax: +44 (0)20 8996 7001.
Email: membership@bsi-global.com.
Information regarding online access to British Standards via British Standards
Online can be found at http://www.bsi-global.com/bsonline.
Further information about BSI is available on the BSI website at
http://www.bsi-global.com.

Copyright
Copyright subsists in all BSI publications. BSI also holds the copyright, in the
UK, of the publications of the international standardization bodies. Except as
permitted under the Copyright, Designs and Patents Act 1988 no extract may be
reproduced, stored in a retrieval system or transmitted in any form or by any
means – electronic, photocopying, recording or otherwise – without prior written
permission from BSI.
This does not preclude the free use, in the course of implementing the standard,
of necessary details such as symbols, and size, type or grade designations. If these
details are to be used for any other purpose than implementation then the prior
BSI written permission of BSI must be obtained.
389 Chiswick High Road Details and advice can be obtained from the Copyright & Licensing Manager.
London Tel: +44 (0)20 8996 7070. Fax: +44 (0)20 8996 7553.
Email: copyright@bsi-global.com.
W4 4AL