oSIST prEN ISO 23611-5:2023
(Main)Soil quality - Sampling of soil invertebrates - Part 5: Sampling and extraction of soil macro-invertebrates (ISO/DIS 23611-5:2023)
Soil quality - Sampling of soil invertebrates - Part 5: Sampling and extraction of soil macro-invertebrates (ISO/DIS 23611-5:2023)
ISO 23611-5:2011 specifies a method for sampling, extracting and preserving macro-invertebrates from soils, including the litter zone. The proposed method is a prerequisite for using these animals as bio-indicators (e.g. to assess the quality of a soil as a habitat for organisms). The main premise of this method is rapid assessment (completing the sampling of a plot in one or two days with only basic equipment and a small number of field assistants), in order to be able to address all the taxonomic groups of soil macro-invertebrates at the same time and in the same place. The Tropical Soil Biology and Fertility (TSBF) method has evolved and some modifications have been introduced in order to use it in temperate regions.
The sampling and extraction methods in ISO 23611-5:2011 are applicable to almost all types of soils, with the exception of soils in extreme climatic conditions (hard, frozen or flooded soils) and matrices other than soil, e.g. tree trunks, plants or lichens.
Sampling design is specified in ISO 23611-6.
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 5: Probenahme und Extraktion von Makroinvertebraten (Großwirbellosen) im Boden (ISO/DIS 23611-5:2023)
Qualité du sol - Prélèvement des invertébrés du sol - Partie 5: Prélèvement et extraction des macro-invertébrés du sol (ISO/DIS 23611-5:2023)
L'ISO 23611-5:2011 spécifie une méthode pour le prélèvement, l'extraction et la conservation des macro-invertébrés du sol, y compris la zone de litière. La méthode proposée est un prérequis à l'utilisation de ces animaux en tant que bio-indicateurs (par exemple pour évaluer la qualité d'un sol en tant qu'habitat pour des organismes). Les principes majeurs de cette méthode consistent à avoir une évaluation rapide (réaliser l'échantillonnage d'une zone en un ou deux jours avec un équipement de base et avec un nombre réduit d'assistants de terrain), pour être capable de traiter tous les groupes taxinomiques de macro‑invertébrés du sol en même temps et au même endroit. La méthode TSBF (biologie et fertilité des sols tropicaux) a évolué et quelques modifications ont été introduites afin de l'utiliser dans des régions tempérées.
Les méthodes de prélèvement et d'extraction de l'ISO 23611-5:2011 sont applicables à la quasi‑totalité des sols. Les sols présents sous des conditions climatiques extrêmes (sols durs, gelés ou inondés) et les matrices autres que le sol, par exemple des troncs d'arbres, des plantes ou des lichens, peuvent constituer des exceptions.
Le plan d'échantillonnage est spécifié dans l'ISO 23611-6.
Kakovost tal - Vzorčenje nevretenčarjev v tleh - 5. del: Vzorčenje in ekstrakcija velikih nevretenčarjev v tleh (ISO/DIS 23611-5:2023)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN ISO 23611-5:2023
01-julij-2023
Kakovost tal - Vzorčenje nevretenčarjev v tleh - 5. del: Vzorčenje in ekstrakcija
velikih nevretenčarjev v tleh (ISO/DIS 23611-5:2023)
Soil quality - Sampling of soil invertebrates - Part 5: Sampling and extraction of soil
macro-invertebrates (ISO/DIS 23611-5:2023)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 5: Probenahme
und Extraktion von Makroinvertebraten (Großwirbellosen) im Boden (ISO/DIS 23611-
5:2023)
Qualité du sol - Prélèvement des invertébrés du sol - Partie 5: Prélèvement et extraction
des macro-invertébrés du sol (ISO/DIS 23611-5:2023)
Ta slovenski standard je istoveten z: prEN ISO 23611-5
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 23611-5:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 23611-5:2023
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oSIST prEN ISO 23611-5:2023
DRAFT INTERNATIONAL STANDARD
ISO/DIS 23611-5
ISO/TC 190/SC 4 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2023-05-24 2023-08-16
Soil quality — Sampling of soil invertebrates —
Part 5:
Sampling and extraction of soil macro-invertebrates
Qualité du sol — Prélèvement des invertébrés du sol —
Partie 5: Prélèvement et extraction des macro-invertébrés du sol
ICS: 13.080.05; 13.080.30
This document is circulated as received from the committee secretariat.
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oSIST prEN ISO 23611-5:2023
DRAFT INTERNATIONAL STANDARD
ISO/DIS 23611-5
ISO/TC 190/SC 4 Secretariat: AFNOR
Voting begins on: Voting terminates on:
Soil quality — Sampling of soil invertebrates —
Part 5:
Sampling and extraction of soil macro-invertebrates
Qualité du sol — Prélèvement des invertébrés du sol —
Partie 5: Prélèvement et extraction des macro-invertébrés du sol
ICS: 13.080.05; 13.080.30
This document is circulated as received from the committee secretariat.
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PROVIDE SUPPORTING DOCUMENTATION. © ISO 2023
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oSIST prEN ISO 23611-5:2023
ISO/DIS 23611-5:2023(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principe. 2
5 Reagents . 2
6 Apparatus . 2
7 Field procedure .3
7.1 General . 3
7.2 Collecting macro-invertebrates from the litter zone . 3
7.3 Collecting macro-invertebrates from soil . 4
7.3.1 General . 4
7.3.2 Temperate regions . 4
7.3.3 Tropical regions. 4
8 Laboratory procedure .5
8.1 Treatment of collected samples . 5
8.2 Preservation of specimens . 5
8.3 Biomass determination . 6
9 Assessment of results . 6
10 Test report . 6
Annex A (normative) Background information . 8
Annex B Sampling soil macro-fauna using Pitfall Traps . 9
[68]
Annex C Monitoring example with Pitfall Traps (taken from Silva et al. 2015 ) .11
Bibliography .16
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ISO/DIS 23611-5:2023(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: www.iso.org/iso/foreword.html.
ISO 23611-5 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological methods.
ISO 23611 consists of the following parts, under the general title Soil quality — Sampling of soil
invertebrates:
— Part 1: Hand-sorting and formalin extraction of earthworms
— Part 2: Sampling and extraction of micro-arthropods (Collembola and Acarina)
— Part 3: Sampling and soil extraction of enchytraeids
— Part 4: Sampling, extraction and identification of soil-inhabiting nematodes
— Part 5: Sampling and extraction of soil macro-invertebrates
— Part 6: Guidance for the design of sampling programmes with soil invertebrates
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Introduction
This part of ISO 23611 was prepared in response to a need to standardize sampling and extraction
methods for soil macro-invertebrates in several European (temperate) and tropical countries. These
methods are needed for the following purposes:
— biological classification of soils, including soil quality assessment (e.g. References [14], [28] and [37]);
— terrestrial bio-indication and long-term monitoring (e.g. References [65], [74], [75] and [76]).
Data collected using standardized methods can be evaluated more accurately as it allows more reliable
comparison between sites (e.g. polluted vs non-polluted sites, changes in land-use practices).
Soils of the world host an abundance of highly diverse macro-invertebrate communities. Their biology
and ecology have been widely studied. Soil invertebrates are irreplaceable actors of soil formation and
conservation in natural ecosystems. Their relevance to the soil system comes from their abundance
and diversity, and also from their role in key biological processes. They are sensitive indicators of soil
quality and recognized agents of its fertility (e.g. References [58] and [52]). Among the wide diversity of
species, adaptive strategies and size ranges represented, one specific group, also called "soil ecosystem
engineers", includes large invertebrates that actually determine the activities of other smaller
organisms through the mechanical activities they produce in soil (e.g. References [18] and [46]).
Soil macro-invertebrates span a wide range of ecological functions in soil: decomposition of organic
matter, through their own activity and by stimulating the soil's microbiological activity (e.g.
References [2], [3] and [36]), predation that plays an important part in food webs (e.g. References [9],
[51], [56], [59] and [63]), soil aggregation by the production of organo-mineral structures (e.g. nests,
galleries, casts) that can last for days, months or years, and soil bioturbation (e.g. Reference [28]),
etc. These characteristics, coupled with in-depth taxonomic knowledge, has enabled their use as
study organisms in several research programmes dealing with the impacts of forest practices (e.g.
References [11], [36], [47], [57], [60] and [70]) or crop management practices (e.g. References [8], [19],
[27], [29], [30], [33], [38], [55] and [62]). These features make them suitable organisms for use as
bio-indicators of changes in soil quality, especially with respect to land-use practices and pollution (e.g.
References [21], [35], [45], [48], [49], [54], [60] and [74]).
The method proposed in this part of ISO 23611 covers the sampling of all soil macro-invertebrates.
However, the sampling of earthworms is already covered in ISO 23611-1. This method is described
in ISO 23611-1:2006, Annex C, as an alternative sampling method for earthworms.
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oSIST prEN ISO 23611-5:2023
DRAFT INTERNATIONAL STANDARD ISO/DIS 23611-5:2023(E)
Soil quality — Sampling of soil invertebrates —
Part 5:
Sampling and extraction of soil macro-invertebrates
1 Scope
This part of ISO 23611 specifies a method for sampling, extracting and preserving macro-invertebrates
from soils, including the litter zone. The proposed method is a prerequisite for using these animals
as bio-indicators (e.g. to assess the quality of a soil as a habitat for organisms). The main premise of
this method is rapid assessment (completing the sampling of a plot in one or two days with only basic
equipment and a small number of field assistants) in order to be able to address all the taxonomic
groups of soil macro-invertebrates at the same time and in the same place. The Tropical Soil Biology and
Fertility (TSBF) method has evolved and some modifications have been introduced in order to use it in
temperate regions.
The sampling and extraction methods in this part of ISO 23611 are applicable to almost all types of soil,
with the exception of soils in extreme climatic conditions (hard, frozen or flooded soils) and matrices
other than soil, e.g. tree trunks, plants or lichens.
A sampling design is specified in ISO 23611-6.
NOTE 1 The method specified in this part of ISO 23611 is based on guidelines developed under the Tropical
[1]
Soil Biology and Fertility Program (TSBF method) .
NOTE 2 Basic information on the ecology of macro-invertebrates and their use can be found in the references
listed in the Bibliography.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
macro-invertebrates
soil organism whose longest dimension is greater than 10 mm
Note 1 to entry: See Annex A for further details.
EXAMPLE These include especially the following groups: Oligochaeta, Gastropoda, Chilopoda, Diplopoda,
Isopoda, Arachnida, plus various insects: Coleoptera, Orthoptera, Hymenoptera, Hemiptera, Dermaptera,
Lepidoptera (larvae) and Diptera (larvae).
1
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3.2
blotted mass
mass of individuals after preservation in formalin or ethanol (when the substance used for preservation
has been absorbed by the tissues)
4 Principe
Soil macro-invertebrates are collected in the field using a metallic frame to delimit the soil surface of
the sampling point. Macro-invertebrates present in litter and soil are picked up separately. In temperate
regions, a reagent is used to extract macro-invertebrates from soil. The sampling is completed by
hand-sorting. Animals are preserved and transported to the laboratory for further identifications
(e.g. References [4], [5], [6], [7], [10], [12], [13], [16], [17], [22], [24], [25], [26], [31], [32], [34], [42], [43],
[44], [50], [53], [64], [66], [67], [71], [72], [73] and [77]). Abundance values are usually recalculated
2
relative to area (1 m ).
5 Reagents
5.1 Ethanol, (70 % volume fraction).
5.2 Formalin [formaldehyde solution], 4 % (volume fraction).
Both 70 % ethanol and 4 % formalin should be available for the preservation of specimens (4 % formalin
is more suitable for taxa with soft body parts, which can be transferred to ethanol after about 4 d
fixation).
5.3 Formalin, 0,2 % (volume fraction), prepared by diluting 25 ml of formalin (39 %) in 5 l of water,
for soil macro-invertebrate extraction.
6 Apparatus
Use standard laboratory equipment and the following.
6.1 Petri dishes.
6.2 Stereo-microscope.
6.3 Plastic vials.
6.4 Entomological forceps.
6.5 Pencil, notebook, water-resistant marker, labels.
6.6 Tape measures.
6.7 Knife (cut glass).
6.8 Spade.
6.9 Plastic-weave produce sacks, for spreading on the ground.
6.10 Precision balance.
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6.11 Large flat plastic trays (500 mm × 400 mm × 100 mm), for sorting the soil and litter.
6.12 Trowel.
6.13 Small plastic trays.
6.14 Fine forceps (or entomological forceps), pipette, fine paint brushes.
6.15 Sample vials, in various sizes with secure alcohol-tight caps (plastic throw away or plastic/glass
reusable vials)
6.16 Indian-ink pen (waterproof).
6.17 Stiff card for labels, ranging compass.
6.18 Large strong plastic bags (sealable).
6.19 Table and plastic chairs, for sorting.
6.20 Cover, for protection from heavy rain.
6.21 Polyvinyl gloves, to protect hands from formalin.
6.22 Metallic frame, preferably 250 mm × 250 mm.
Sample frame (250 mm × 250 mm × 50 mm) made of stainless steel and with sharpened edges to delimit
the sampling point where animals are sampled from the litter layer and soil.
6.23 Watering can.
6.24 Pair of scissors, to cut vegetation inside the frame.
6.25 Field balances.
7 Field procedure
7.1 General
Sampling should take place when accessible biodiversity is thought to be largest. In temperate regions,
it corresponds to spring or autumn, and in the tropics, it should take place towards the end of the rainy
season.
When sampling soil invertebrates, it is strongly recommended that the site be physico-chemically
characterized. In particular, pH, particle size distribution, C/N ratio, organic carbon content and water-
holding capacity should be measured using ISO 10390, ISO 10694, ISO 11274, ISO 11277, ISO 11461,
ISO 11465. Natural minerals present in the site soil should be also described.
7.2 Collecting macro-invertebrates from the litter zone
At each sampling point (= monolith) (previously defined according to sampling design rules), a litter
sample is collected using a metallic frame (5.22). The metallic frame is pressed into the litter by hand.
The litter inside the frame is removed and checked manually in the field using a large tray (5.11). Litter
invertebrates are preserved in 4 % formalin (4.2).
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7.3 Collecting macro-invertebrates from soil
7.3.1 General
In temperate countries, the extraction of soil macro-invertebrates is carried out in two steps (see 6.3.2.1
and 6.3.2.2), while in tropical countries only the second step shall be performed (see 6.3.3). In both
cases, extraction of macro-invertebrates may be complemented by the use of pitfall traps (see Annexes B
and C for further details)
7.3.2 Temperate regions
7.3.2.1 Formalin extraction
The soil surface delimited by the metallic frame (5.22) is sprayed with 0,2 % formalin (4.3) using a
watering can (5.23). Two applications of 1,5 l of formalin are performed at intervals of about 10 min.
Soil invertebrates coming up to the surface are collected and preserved in vials (5.3) containing
formalin (4.2).
7.3.2.2 Hand-sorting of "passive" macro-invertebrates
At the end of the formalin extraction, the metallic frame (5.22) is removed and the upper 150 mm of
soil is excavated within the frame area (250 mm × 250 mm). The excavated soil is placed in a plastic
bag (5.18) that can be closed with a cover to prevent animals from escaping from the soil sample.
Appropriate sub-samples of soil are taken from the container and spread on a large tray (5.11).
Macro-invertebrates are collected and preserved in vials (5.3) with formalin (4.2). When hand-sorting
is finished, the excavated soil is replaced to avoid creating holes on the sampling site.
7.3.3 Tropical regions
In tropical countries, soil macro-invertebrates are sampled using a 250 mm × 250 mm × 300 mm
deep soil monolith. The monolith is isolated by cutting with a spade (5.8) a few centimetres outside
the quadrate (metallic frame) and then digging a 20 mm wide by 300 mm deep trench around it. This
facilitates cutting of the sample into horizontal strata and collecting animals escaping from the block.
The delimited block is divided into three layers, 0 mm to 100 mm, 100 mm to 200 mm and 200 mm
to 300 mm, and the soil and litter material is hand-sorted in trays (5.11). Since formalin is not applied in
tropical regions, the sampling depth should be doubled in order to be sure to collect endogenic species
of earthworms.
For social insects, it is recommended that special measures be considered that take account of their high
abundance and marked patchiness; a nest can contain millions of individuals, of which none are sampled
by a short transect, and the contribution of the species concerned to a macrofaunal assemblage can
thus be completely missed. On the other hand, a highly populated nest sampled directly by a monolith
can lead to a large overestimation of the overall numerical or biomass density. In general, the TSBF
transect should be placed so as to avoid direct contact with termite and ant nests. For discussions, see
References [35] and [36]. The protocol for a 100 m × 2 m transect designed to assess termite biodiversity
(and feeding group representation) is given in Reference [48]. In suitable circumstances, this protocol
can also be deployed in parallel with the TSBF transect.
NOTE Besides the general characterization of the site, it is useful to determine the actual moisture of the soil
to be sampled.
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8 Laboratory procedure
8.1 Treatment of collected samples
In the laboratory, samples are cleaned with either distilled or tap water in a Petri dish with the help of
a brush or placing the organisms on a 0,5 mm to 1 mm sieve under the tap. Afterwards, the animals are
placed in new vials (5.15) with ethanol (70 % volume fraction) (4.1). Organisms with soft body parts
are kept in formalin for at least 4 d, or forever if possible.
For taxonomic identification, specimens are placed on petri dishes (5.1) and observed under the
stereo-microscope (5.2). A practical way to identify macro-invertebrates is to group them first into
orders. Each order is then identified into families and each family into species using taxonomy keys
(examples of taxonomy keys are given in the Bibliography (see References [4], [5], [6], [7], [10], [12],
[13], [17], [22], [24], [25], [26], [31], [32], [34], [42], [43], [44], [53], [66], [67], [72], [73] and [77]).
Ideally, taxonomic determination should be based on the species level. If identification of species levels
fails due to time constraints, taxonomic expertise or missing taxonomic keys, e.g. mainly in tropical
regions, sorting to genus (and some higher taxonomic units) represents a good compromise between the
morphospecies and ordinal level approaches, especially as this allows most specimens to be assigned to
a functional group.
WARNING — Appropriate precautions (i.e. gloves, mask) should be taken when dealing with
formalin to avoid danger from inhalation or skin exposure. According to the "Material Safety
Data Sheet" for formaldehyde 37 % solution published by producing companies, the compound
is a skin sensitizer and is considered to be carcinogenic (humans: limited evidence; animals:
sufficient evidence). It is legally notified in industrialized countries for scientific use.
8.2 Preservation of specimens
From any mixed soil sample of macrofauna, the following steps should be followed in order to obtain
standardized preserved specimens.
a) If the animal has no soft body parts, the organisms should be preserved in 70 % ethanol (commercial
ethanol should be diluted).
b) If the animal has soft body parts, the organism should be fixed in 4 % formalin and should,
if possible, be preserved in the same solution. Alternatively, 80 % ethanol could be used (if the
organism has been fixed during at least 4 d with 4 % formalin).
c) In all cases, samples should be stored separately in different vials, according to the smallest unit of
analysis (i.e. a monolith if the data is compared at that level).
d) Every vial should be labelled without using code numbers and should at least be written using
permanent ink, like Indian or Chinese ink, and using sturdy paper like goatskin parchment. Every
label should contain the following information:
— country;
— region;
— locality;
— collector’s name;
— date of collection.
e) For storing specimens:
— use vials (or glass tubes) that are not degraded by the ethanol or formalin, with screw caps;
— monitor levels of ethanol and formalin in order to keep them constant;
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— store vials away from direct sunlight;
— change the preserving solution of each vial once every five years.
8.3 Biomass determination
Determination of biomass is performed using the preserved material. The animal's surface should be
gently dried with filter paper, then weighed using a precision balance (0,001 g).
It is virtually impossible to keep invertebrates alive after their capture in order to measure fresh
masses. In most cases, invertebrates are conserved in 70 % (volume fraction) ethanol or 4 % (volume
fraction) formalin. The latter is recommended for earthworms that should at least be fixed in formalin
before being kept in 70 % ethanol. Preservation always involves a decrease in mass, as body water is
extracted by osmotic forces. The amount lost can vary between 15 % and 40 %, depending on the water
content of the animal and its physiological state. Since most studies only aim to compare different sites
and/or situations, mass loss is not likely to distort the result. If accurate fresh mass data are necessary,
it is easy to keep an aliquot of each group and compare the mass, alive and fixed, a few days after
fixation.
9 Assessment of results
The following measurement end points can be used for the bioclassification of a soil, including bio-
indication or biomonitoring (e.g. anthropogenic stress-like chemicals or land-use changes):
— abundance (number of individuals per area);
— biomass;
— number of species or other taxonomically or ecologically defined groups;
— diversity indices (alpha, beta and gamma diversity).
Firstly, the number of individuals (total number by species or group) is counted and expressed as
individuals per sample. Secon
...
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