E DITORIAL M ERGER OF › upload › cms › user › Chapter2editorialmer… · EDITORIAL MERGER OF CHAPTER 2 2 of 28 2.2.6 Sub-sample A sub-sample is the portion of a sample obtained

E D I T O R I A L M E R G E R O F

C H A P T E R 2 O F

T H E I N T E R N A T I O N A L R U L E S

F O R S E E D T E S T I N G

ORDINARY MEETING 2005

Editorial merger of Chapter 2 (Bulking and Sampling) Following proposals and discussion at the 2004 meeting held in Budapest it was agreed that the Rules and Annexes of the ISTA Rules should be combined. Details of the plans for updating the rules were given in Seed Testing International (No.128, p26 to 27). The programme of amalgamation has started with Chapter 2. The first stage in this process was to merge the Rules and Annexes on an editorial basis only. This did not change the intent but removed duplication and does not require any voting by the membership. The attached file is the editorially merged version of Chapter 2. This version of Chapter 2 was sent to a two-person team of ISTA scrutineers, both ex-members of the ISTA Executive Committee. They have agreed that the editorial changes were indeed editorial so do not need to be voted on. Thanks go to Doug Ashton and Simon Cooper for volunteering to be the scrutiny team. Using the editorially merger version of Chapter 2 as the starting point, the Bulking and Sampling Committee under the leadership of Michael Kruse, have taken this opportunity to do a major revision of the structure and content of Chapter 2. These changes will be voted on during the ISTA meeting in Bangkok, Thailand. Because the changes are extensive it has not been possible to show a tracked changes as we normally do. The following editorially merged version of the file is to help understand where the changes have come from. If you have any questions please contact the ISTA Secretariat or Steve Jones. Many thanks. Steve Dr Steve Jones ISTA Rules Committee Chair Chief Officer OSTS for England & Wales ISTA Accredited Laboratory GBDL01 ISTA Designated Member Phone: +44 (0)1223 342263 Fax: +44 (0)1223 342244 E-mail: [email protected]

EDITORIAL MERGER OF CHAPTER 2 1 of 28

Chapter 2: Sampling

2.1 Object

The object of sampling is to obtain a sample of a size suitable for tests, in which the probability of a constituent being present is determined only by its level of occurrence in the seed lot.

2.2 Definitions

2.2.1 Seed Llot

A seed lot is a specified quantity of seed, physically identifiable, in respect of which an ISTA International Seed Lot Certificate may be issued.

2.2.2 Primary sample

A primary sample is a small portion taken from the seed lot during one single sampling action.

2.2.3 Composite sample

The composite sample is formed by combining and mixing all the primary samples taken from the seed lot.

2.2.4 Submitted sample

A submitted sample is a sample submitted to the testing laboratory. It must be of at least the size specified in 2.5.26.3 and may comprise either the whole or a sub-sample of the composite sample.

2.2.5 Working sample

The working sample is a sub-sample taken from the submitted sample in the laboratory, on which one of the quality tests described in these Rules is made.

Throughout the whole text consistently „seed lot“ is used


2.2.6 Sub-sample

A sub-sample is the portion of a sample obtained by reducing the sample using one of the sampling methods prescribed in 2.5.26.6 and 2.5.37.2.

2.2.7 Sealed

Sealed means that the container or individual containers in which the seed is held are closed in such a way that they cannot be opened to gain access to the seed and closed again without either destroying the seal or leaving evidence of tampering. This definition refers to the sealing of seed lots and seed samples.

2.42.3.A8 Self-sealing containers

This recommendation refers specifically to the type of bag which is commonly referred to as a ‘valve-pack’ bag. It is filled through a sleeve-shaped valve which is an integral part of the bag. The valve automatically closes when the bag is full by the sleeve collapsing and being pressed against the inner surface of the bag. If the sleeve is greater than 20% of the width of the bag, and is the only opening to the bag, it may be regarded as self- sealing in the case of seed the size of Triticum or larger. In addition, to improve the safe sealing of the valve, an additional seal such as an adhesive patch can be placed across the mouth of the valve, or the ‘lips’ of the mouth can be glued or clipped together with a suitably designed metal seal.

2.2.98 Marked/labelled

A container of a seed lot can be considered as marked or labelled when there is a unique identification mark on the container, which defines the seed lot to which the container belongs. All containers of a seed lot must be marked with the same unique seed lot designation. This designation (numbers, characters or combination of both) must be given on the ISTA International Seed Lot Certificate. Marking of working samples in the laboratory must ensure that there is always an unambiguous link between the seed lot and the working sample.

2.2.10 Coated seeds Seed pellets. More or less spherical units developed for precision sowing, usually incorporating a single seed with the size and shape of the seed no longer readily evident.

Comes from Annexe, since it defines a valve-pack it is proposed to be included here.

Throughout the whole text consistently the names as defined in chapter 17 are used

Comes from chapter 11 and contains definitions.


The pellet, in addition to the pelleting material, may contain pesticides, dyes or other additives. Encrusted seed. Units more or less retaining the shape of the seed with the size and weight changed to a greater or lesser extent. The encrusting material may contain pesticides, fungicides, dyes or other additives. Seed granules. Units more or less cylindrical, including types with more than one seed joined together. The granule, in addition to the granulating material, may contain pesticides, dyes or other additives. Seed tapes. Narrow bands of material, such as paper or other degradable material, with seeds spaced randomly, in groups or in a single row. Seed mats. Broad sheets of material, such as paper or other degradable material, with seeds placed in rows, groups or at random throughout the sheets. Treated seed. Seed to which only pesticides, dyes or other additives have been applied which have not resulted in a significant change in size, shape or addition to the weight of the original seed and which can still be tested according to the methods prescribed in other chapters.

2.3 General principles

2.1.A Object

The quantity of seed tested in the laboratory is minute compared with the size of the seed lot which it is intended to represent. To obtain uniform and accurate results in seed testing it is essential that the primary, composite and submitted samples be taken and prepared with care and in accordance with the methods prescribed in 2.65.2. No matter how accurately the laboratory work is done, the results can only show the quality of the sample submitted for analysis; consequently, every effort must be made to ensure that the sample sent to the seed testing laboratory accurately represents the composition of the seed lot in question. Likewise, in reducing the sample in the laboratory, every effort must be made to obtain a working sample that is representative of the sample submitted.

A sample is obtained from the seed lot by taking small portions at random from different positions in the seed lot and combining them. From this sample, smaller samples are obtained by one or more stages. At each stage, thorough mixing is followed either by progressive sub-division or by the abstraction and combination of small portions at random.

Comes from the Annexe and describes the challenge of seed sampling and the consequences


2.54 Apparatus

Each stage in sampling the seed lot shall be performed using appropriate techniques and equipment. Such techniques and equipment for drawing primary samples and the preparation of submitted and working samples are described in 2.5.2.4.1 and 2.5.3.2.1.

2.54 ProceduresThe seed lot

2.5.1 The seed lot

For issuance of an orange or green ISTA International Seed Lot Certificate, a seed lot shall meet the following requirements.

2.54.1.1 Size of the seed lot

The seed lot shall not exceed the quantity indicated in column 2 of Table 2A, subject to a tolerance of 5% with the exception of herbage and amenity seed being transported loose in bulk containers. The conditions under which this exception may be permitted are laid down in Appendix B.

11.2.4.1 Size of lot

Providing there is satisfactory evidence that the lot is reasonably homogeneous, the maximum weight of lot may be as great as the maximum weight of lot for which sampling procedures are prescribed in Chapter 2, subject to the tolerance of 5% and subject to the seed number limitation prescribed in 11.2.4.1.A.11.2.4.1.A Size of lot

The maximum number of seeds that a seed lot of seed pellets, encrusted seeds, seed granules, seed tapes or seed mats may contain is 1,000,000,000 (10,000 units of 100,000) except that the weight of the seed lot, including the coating material may not exceed 42,000 kg (40,000 kg plus 5%)subject to a tolerance of 5%. When seed lot size is expressed in units the total weight of the seed lot must be given on the ISTA International Seed Lot cCertificate.

A consignment of seed in excess of the prescribed quantity shall be subdivided into seed lots not larger than the prescribed quantity, each of which shall be identified by a separate lotseed lot designation.

Ordering of paragraphs follows the sequence of general headings as decided in Budapest.

Comes from chapter 11 and Annexe 11. The first part is a duplication of the more detailed second part

Wording is proposed to be changed to the wording above for the non-pelleted seeds


2.45.1.2 Uniformity of the lotseed lot The quantity of seeds tested as a sample is minute compared with the size of the seed lot which it is intended to represent. When the value of a quality characteristic is reported as a single number for the entire lot, it is presumed that no kind of heterogeneity is present, i.e. there is no non-tolerable variation among different parts of the seed lot.

Therefore, aAt the time of sampling, the seed lot shall have been subjected to appropriate mixing, blending and processing techniques so that it is as uniform as practicable. Even if bulking and mixing are done carefully, it is virtually impossible to obtain a completely homogeneous seed lot, and in any case, segregation may occur within each container during filling and transport. The processor is expected to mix the seed lot with a view to achieving a random distribution of its components. He/she may not achieve p Perfect homogeneity may not be achieved, but in good seed production practice he can achieve an acceptable level of heterogeneity can be achieved. which is acceptable. If there is documentary or other evidence of heterogeneity or the seed a lot is found to be obviouslyexcessively heterogeneous when starting sampling, a submitted sample drawn according to the Rules is unlikely to be representative., tTherefore, the submission of a submitted sample sampling of such a seed lot must be refused.There shall be no documentary or other evidence of heterogeneity. In cases of doubt heterogeneity can be determined as described underin Appendix D ‘Heterogeneity tests for seed lots in multiple containers’2.5.4.

2.5.1.4.3 Containers

The seed lot shall be in containers which are self-sealing, sealed (or capable of being sealed) and labelled or marked for identification by a unique lotseed lot designation. An ISTA International Seed Lot Certificate may not be issued in respect of loose seed or seed in containers which are not self-sealing and cannot be sealed.

2.5.14.4 Marking and sealing the seed lot

At the time of sampling, all containers must be labelled or marked to show a unique seed lot identification corresponding to the seed lot identification on the certificate. The seed lot identification must be approved or allotted by the seed testing laboratory concerned.

2.4.4.A Marking the lot

The identification mark or number to appear on all containers in the lot and on the certificate to be issued, is allotted or approved by the seed testing laboratory concerned. The sampler should be notified of this mark or number with the instructions for sampling

Comes from Appendix D and describes the origin and consequences of heterogeneity.

Comes from the Annexe 2, the first sentence is a duplication of the text above.

Should be gender neutral


the lotseed lot and is responsible for ensuring that it appears on every container in the lotseed lot.

The containers shall be sealed, or seen to be sealed by the sampler, or be of a self-sealing type approved by the seed testing laboratory responsible for the sampling operation (see 2.2.8). A container shall be regarded as sealed if it is apparently impossible to open without either destroying the seal or leaving evidence of tampering. If the containers are not of an approved self-sealing type there shall be affixed to each container, under the control of the sampler, an officially recognised seal, or indelible mark, or non-removable label. No sampled lotseed lot, or part of a seed lot, shall be left unsealed.

2.65.2 Procedure for sampling the lotseed lot

2.5.26.1 General directions

Sampling for an ISTA International Seed Lot Certificate shall be carried out only by persons trained and experienced in seed sampling, and, either recognised individually by the seed testing laboratory concerned, or employed by other official or semi-official organisations recognised by the head of the laboratory for this purpose. The sampler shall hold a formal certificate of proficiency in seed sampling recognised by the head of the seed testing laboratory concerned. The sampling prescriptions laid down in these Rules shall be followed when seed samples are drawn for the issue of ISTA International Seed Lot Certificates.

The seed lot shall be so arranged that each individual container or part of the seed lot is conveniently accessible. The sampling rules are intended to be appropriate for most practical situations. If the nature of the presentation of the seed lot, or the type of container makes it impossible to adequately apply these procedures, then sampling shall not be undertaken, and an alternative presentation of the seed lot be sought. Upon request by the sampler, the owner shall provide full information regarding the bulking and mixing of the seed lot. When there is definite evidence of heterogeneity, either physical or documentary, sampling shall be refused.

2.5.26.2 Sampling intensity

For seed lots in containers of 15 kg to 100 kg capacity (inclusively), the following sampling intensity according to Table 2.1 shall be regarded as the minimum requirement.:

The inclusion of the sampling scheme into a table is proposed for easier cross -referencing


Table 2.1: Sampling intensity for seed lots in multiple containers of 15 kg to 100 kg capacity (inclusively).

Number of containers Number of primary samples to be taken

1 - 4 containers: 3 primary samples from each container

5 - 8 containers: 2 primary samples from each container

9 - 15 containers: 1 primary sample from each container

16 - 30 containers: 15 primary samples from the seed lot

31 - 59 containers: 20 primary samples from the seed lot

60 or more containers: 30 primary samples from the seed lot

For seed lots in containers smaller than 15 kg capacity, containers shall be combined into sampling units not exceeding 100 kg, e.g. 20 containers of 5 kg, 33 containers of 3 kg or 100 containers of 1 kg. and tThe sampling units shall be regarded as containers in the sampling scheme above.

2.6.2.A Sampling intensity for small containers

If the seed is in small containers such as tins, cartons, or packets as used in the retail trade, the following procedure is recommended:

A 100 kg weight of seed is taken as the basic unit and the small containers are combined to form sampling units not exceeding this weight. For sampling purposes, each unit is regarded as one ‘container’ and the sampling intensity prescribed in 2.6.2 is applied.

When sampling seed in containers of more than 100 kg, or from streams of seed entering containers the sampling intensity according to Table 2.2 following shall be regarded as the minimum requirement.

Examples come from the Annexe 2 and were found to be helpful

Comes from the Annexe 2 and is a duplication of the text below.

Tables within the chapter are numbered as 2.1 2.2 2.3 … whereas the tables at the end of the chapters are numbered as 2A 2B 2C ….. This allow that table 2A and later on also 5A and 6A can keep their number.


Table 2.2: Sampling intensity for seed lots in containers of more than 100 kg, or from streams of seed entering containers.:

Seed Llot size Number of primary samples to be taken

Up to 500 kg At least five primary samples.

501-3,000 kg One primary sample for each 300 kg, but not less than five.

3,001-20,000 kg One primary sample for each 500 kg, but not less than 10.

20,001 kg and aboveOne primary sample for each 700 kg, but not less than 40.

When sampling a seed lot of up to 15 containers, regardless of their size, the same number of primary samples shall be taken from each container. 11.2.6.2.A Sampling intensity

Sampling a the lot of seed pellets should be done according to the intensity appropriate to the particular lot as laid down in Chapter 2as described above. Sampling the lots of seed tapes should be done analogously by taking packets or (from reels) pieces of tape at random analogously following the prescriptions of 2.6.2 and 2.6.2.A,. provided that pPackets or reels containing up to 2,000,000 (20 units of 100,000) seeds, may be combined as a basic unit considered as one container in Table 2.1.

2.65.2.3 Weight of submitted sample

Minimum weights of submitted samples are as follows:

– For moisture determination - 100 g for species that have to be ground (see Table 9A) and 50 g for all other species.

– For verification of species and cultivar - As prescribed in Chapter 8.

– For all other tests - At least the weight prescribed in column 3 of Table 2A. Provided that no determination of other seeds by number is requested, the submitted sample shall weigh at least the amount indicated for the working sample for purity analysis in column 4 of Table 2A. 11.2.6.3 Size of submitted sample In the case of coated seeds, the Ssubmitted samples shall contain not less than the number of pellets or seeds indicated in column 2 of Table 11A2B, Part 1 and Part 2. If a smaller

The inclusion of the sampling scheme into a table is proposed for easier cross -referencing

Comes from Annexe 11 and is only changed to fit into the paragraph.

Comes from Chapter 11, the last sentence is included in the last sentence of the paragraph below.


sample is used the following statement must be inserted on the certificate: “The sample submitted and is not in accordance with the International Rules for Seed Testing.”

In case the submitted sample is smaller than prescribed, the sampler shall be notified accordingly and analysis withheld until sufficient seed is received in a single submitted sample; except that in the case of very expensive seed, the analysis may be completed to the extent possible and the following statement inserted on the certificate: “The sample submitted weighed only ..... g [or in the case of pelleted seeds ‘contained only .... pellets (seeds)’] and is not in accordance with the International Rules for Seed Testing.”

2.5.26.4 Taking primary samples

Primary samples of approximately equal size shall be taken from each container sampled, or from each place sampled in such container, or from each location sampled in a bulk. Suitable methods are described in 2.5.26.4.A1. The instruments being used must neither damage the seed nor select according to seed size, shape, density, chaffiness or any other quality trait.

When the seed lot is in containers (including bags), the containers to be sampled shall be selected at random or according to a systematic plan throughout the seed lot and primary samples drawn from top, middle and bottom of containers, but not necessarily from more than one position in any container, unless so specified in the sampling frequency tables.

When the seed is in bulk or in large containers, the primary samples shall be drawn from random positions and depths.

In the case of chaffy seeds that have not been rendered free-flowing or seeds that may be damaged if sampled by instruments, the primary samples may be drawn by hand.

When seed is to be packed in small containers (see 2.5.26.2) or in moisture-proof containers (e.g. tins or plastic bags), it should be sampled, if possible, either before or during the filling of the small containers provided that

- the large containers are sealed and marked according to 2.5.14.4,

- opening of the large containers and moving the seed into the small or moisture proof containers is done under the supervision of the seed sampler,

- there is no processing of the seed during filling of the small or moisture proof containers.

It is only in this case that the ISTA International Seed Lot Certificate can be issued for the small or moisture proof containers with the results based on the sample drawn from the large seed lot. When this has not been done, a sufficient number of containers shall be


opened or pierced for abstraction of primary samples. The sampled containers shall then be closed or the contents transferred to new containers.

Seed may be sampled as it enters the containers, provided that the instrument uniformly samples the cross section of the seed stream and seeds entering the instrument do not bounce out again; it may be operated either under manual or automatic control. The intervals between taking primary samples should be constant but may also vary randomly.

2.65.2.4.A1 Instruments and methods for sampling the seed lot

2.65.2.4.A1.1 Stick or sleeve type trier and its use

One of the most commonly used instruments is known as the stick or sleeve-type trier which consists of a hollow brass tube inside a closely fitting outer shell or sleeve which has a solid pointed end. The tube and sleeve have open slots in their walls so that when the tube is turned until the slots in the tube and sleeve are in line, seeds can flow into the cavity of the tube, and when the tube is given a half turn the openings are closed. The tubes vary in length and diameter, being designed for different kinds of seed and various sizes of containers, and are made with or without partitions. In sampling seed in bags the following sizes of triers have been found suitable: for clovers and other small free-flowing seeds, 762 mm trier with outside diameter of 12.7 mm and nine slots; for cereals, 762 mm trier with outside diameter of 25.4 mm and six slots.

Bin samplers are constructed on the same principle as bag triers but are much larger, ranging up to 1600 mm in length and 38 mm in diameter with six or nine slots.

This trier may be used horizontally or vertically. However, when used vertically the trier must have partitions dividing the instrument into a number of compartments. Otherwise, the seed will drop into the sampler from the upper layers when the trier is opened, leading to an over-representation of seed from these layers. When using a stick sampler vertically a certain drag of the seeds from the top towards the bottom is unavoidable. This drag is reduced if the trier is made as smooth and with as few ribs as possible.

Using it either vertically or horizontally, one should insert the trier diagonally into the bag or container. For seed in bulk, vertical insertion is more practicable. The trier is thrust into the bag in the closed position, then opened and turned a couple of times or gently agitated to allow it to fill completely. Thereafter it is closed again, withdrawn and emptied into a suitable seed pan, or onto a piece of waxed paper or similar material. Care should be exercised in closing the trier so that seeds are not damaged.

The stick trier may be used for most seeds with the exception, perhaps, of some very chaffy species. Up to a certain diameter of tube it may be used through sack walls of

Comes from Annexe 2 and is taken over without any changes of the text.


coarsely woven jute or other similar material. When the trier is removed the point should be run across the hole a couple of times in opposite directions to pull the threads together and close the hole. Closed paper bags may also be sampled by puncturing the bag and afterwards sealing the hole with a special adhesive patch.

2.5.26.4.A1.2 Nobbe trier and its use

This trier is made in different dimensions to suit various kinds of seeds. It is a pointed tube, long enough to reach the centre of the bag, with an oval hole near the pointed end. The total length of the instrument should be approximately 500 mm, including a handle of about 100 mm and a point of about 60 mm, leaving about 340 mm to penetrate into the bag, which should be sufficient to reach the centre of all types of bags. For cereals the internal diameter of the tube should be about 14 mm, but for clovers and similar seeds 10 mm is sufficient.

The Nobbe trier is suitable for sampling seed in bags but not in bulk. The trier should be inserted gently into the bag, pointing upwards at an angle of about 30° to the horizontal, with the hole facing downwards until it reaches the centre of the bag. The trier is then revolved through 180°, bringing the hole to face upwards, and is withdrawn with decreasing speed so that the quantity of seed obtained from successive locations increases progressively from centre to side of the bag. Alternatively, a trier long enough to penetrate to the farther side of the bag should be withdrawn at a relatively constant speed. While the trier is being withdrawn it should be gently agitated so that an even flow of seed is maintained. The more polished the inner surface of the trier is, the more freely the seed will flow.

The sampling should be varied from top, middle and bottom of the bags. To sample the bottom of standing bags they may be raised off the floor and placed on top of other bags. The holes made in bags by the trier may be closed as described for the stick trier.


2.5.26.4.A1.3 Sampling by hand

In certain cases and for certain species, especially chaffy, non-freeflowingfree flowing species, hand sampling is sometimes the most satisfactory method. Examples are:

Agropyron Cynosurus Melinis

Agrostis Dactylis Panicum

Alopecurus Deschampsia Pascopyrum

Anthoxanthum Digitaria Paspalum

Arrhenatherum Elymus Poa

Axonopus Elytrigia Psathyrostachys

Bromus Festuca Pseudoroegneria

Chloris Holcus Trisetum

Cynodon Lolium Zoysia

Sampling by hand is also the most suitable method for seed that may be damaged by the use of triers e.g. in seed lots of large seeded legumes, seeds with wings or seeds which have a low moisture content.

Using this method samples can be drawn only from open containers. It is difficult by this method to sample deeper than about 400 mm. This will mean that it is impossible to obtain samples from the lower layers in bags and bins. In such cases the seed sampler may take special precautions such as requesting that some bags be emptied or partly emptied to facilitate sampling, and then be refilled. When sampling is done by hand great care should be taken to keep the fingers tightly closed about the seeds so none may escape.

2.5.26.5 Obtaining the composite sample

If the primary samples appear uniform they shall be combined to form the composite sample. In some cases (e.g. when sampling the seed stream) primary samples may be collected directly into only one container. The content of this container shall be regarded as the composite sample only if it appears uniform. If it is not uniform, the seed lot shall not be accepted for testing.

Species are arranged in a table as in the other list below for better reading


2.5.26.6 Obtaining the submitted sample

The submitted sample shall be obtained by reducing the composite sample to an appropriate size by one of the methods referred to in 2.5.37.2, using larger equipment if necessary.

If it is difficult to mix and reduce the composite sample properly under warehouse conditions, the entire composite sample shall be forwarded to the seed testing laboratory for reduction.

If the composite sample is of appropriate size it may be regarded as the submitted sample without reduction.

Additional samples requested not later than at the time of sampling, by the owner of the lotseed lot, if granted, shall be prepared in the same way as the submitted sample and marked ‘Duplicate’.

2.5.26.7 Dispatch of submitted sample

Each submitted sample shall be marked in a way that establishes the connection between lot and sample. The submitted sample shall be marked with the same identification mark as the seed lot. For an ISTA International Seed Lot Certificate, the sample shall be sealed.

It is recommended that if labels are already prepared for the seed lot, an additional label be attached to, or included in the submitted sample. The sample packet should be provided with a printed form on which the necessary information can be filled in - see 17.4.2 and 17.4.3. Ordinarily, samples should be packed in bags of jute, other cloth material, or paper.

Samples shall be packed so as to prevent damage during transit. Submitted samples for moisture test, and samples from seed lots which have been dried to low moisture content, shall be packed in moisture proof containers. From the moisture proof containers as much air as possible shall be excluded.

Submitted samples for germination tests, viability tests and health tests may only be packed in moisture proof containers if suitable storage conditions can be assured.

As submitted samples of coated seeds normally contain fewer seeds than corresponding samples of uncoated seeds, special care is necessary in drawing the sample to ensure that it is representative of the seed lot. Precautions are necessary to avoid damage to or change in the seed pellets or seed tape during drawing, handling and transport, and samples must be submitted in suitable containers.

Samples shall be packed so as to prevent damage during transit. Submitted samples shall be dispatched by the sampler to the seed testing laboratory without delay. The samplers are

Comes from the Annexe 2 and text from Chapter 2 is deleted because of duplication.

Comes from Chapter 11

Sentence was moved from 3 paragraphs above to here since it is valid also for coated seeds.

Comes from the Annexe 2 and partly deleted because of duplication


personally responsible for the seals, labels and bags supplied to them and it is their duty to ensure that no unauthorised person has access to them. andprimary, composite or unsealed submitted samples shall never be left in the hands of persons not authorised by the sampling agency or by the seed testing laboratory unless they are sealed in such a way that they cannot be tampered with (see 2.6.7.A). Where the seed has been chemically treated the name of the treatment shall be given to the seed testing laboratory.

2.6.7.A Marking, sealing and packing the sample

On no account shall come into the hands of unauthorised persons.

2.5.2.8.1 Storage of submitted samples Bbefore testing Every effort must be made to start testing a sample on the day of receipt. If delay is unavoidable, the sample shall be stored in a cool well-ventilated room in such a way that changes in the quality of the seed are minimised.

Analysts should appreciate the importance of testing samples as soon as possible after receipt. For instance, the moisture content may rise or fall considerably during storage under laboratory conditions depending on room temperature and humidity. Storage may also modify dormancy, which in certain instances it might be important to discover and report, or it may increase the number of hard seeds in species of Fabaceae (Leguminosae). Storage, when necessary, should therefore be in a cool, well-ventilated room.

Every effort must be made to start testing a sample on the day of receipt.

If delay is unavoidable, the sample shall be stored in a cool well-ventilated room in such a way that changes in the quality of the seed are minimised.

2.75.3 Procedure for Oobtaining the working sample

2.5.37.1 Minimum weight of working sample Minimum weights of working samples are prescribed in the appropriate chapter for each test. The working sample weights for purity analyses given in Table 2A are calculated to contain at least 2500 seeds. These weights are recommended for normal use in purity tests, see 3.5.1. The sample weights in column 5 of Table 2A, Part 1, for counts of other species are 10 times the weights in column 4, subject to a maximum of 1000 g.

To follow the sequence of the various procedures during sampling, the storage of samples before testing is proposed to move to here.


11.2.7.1 Size of working sample Working samples of coated seeds shall contain not less than the number of pellets or seeds indicated in column 3 of Table 11A2B, Part 1 and Part 2. If a smaller sample of pelleted seed is used the actual number of pellets or seeds in the sample shall be reported on the ISTA International Seed Analysis Certificate.

2.5.37.2 Sample reduction methods

If the seed sample needs to be reduced to a size equal to or greater than the size prescribed, the seed sample shall first be thoroughly mixed. The submitted/working sample shall then be obtained either by repeated halving or by abstracting and subsequently combining small random portions. The apparatus and methods to be used are described in 2.75.3.2.A1. 11.2.7.2.A Sampling in the laboratory When using one of the dividers described in 2.7.2.A(i)there, for pelleted seed pellets the distance of fall must never exceed 250 mm.

Except in the case of seed health, the method of hand halving shall be restricted to certain genera listed in 2.75.3.2.A1.4. Only the spoon method and hand halving method may be used in the laboratory to obtain working samples for seed health testing where other samples or equipment may be contaminated by spores or other propagative material. For seed tapes take pieces of tape at random, to provide sufficient seeds for the test.

Duplicate samples shall be drawn independently; after drawing the first working sample or half-working sample the remainder of the submitted sample shall be re-mixed before the second sample or half-sample is drawn. 11.2.7.2 Obtaining the working sample

For pelleted seeds use one of the dividers described in 2.7.2.A(i). However the distance of fall must never exceed that indicated in 11.2.7.2.A.

2.75.3.2.1A Sampling in the laboratory

In using all sample reduction methods the sampler/analyst should aim at taking out a quantity slightly more than the required weight. One of the following methods must be used.

2.5.37.2.A1.1 Mechanical divider method

This method is suitable for all kinds of seeds except the extremely chaffy types. The apparatus divides a sample passed through it into two or more approximately equal parts.

Comes from Chapter 11, the (big) tables were added as Tables 2B and 2C at the end of Chapter 2.

Comes from Annexe 11

Comes from Chapter 11

Comes from Chapter 11 and is deleted because of duplication

Comes from Annexe 2 and is included without changes of the text.


The submitted sample can be mixed by passing it through the divider, recombining the parts and passing the whole sample through a second time, and similarly, a third time if necessary. The sample is reduced by passing the seed through repeatedly and removing parts on each occasion. This process of reduction is continued until a working sample of approximately, but not less than, the required size is obtained.

The dividers described below are examples of suitable equipment.

(a) Conical divider. The conical divider (Boerner type) is produced in two sizes, the smaller size for small-seeded species and the larger one for large-seeded species (the size of wheat and larger). The essential parts consist of a hopper, cone, and series of baffles directing the seed into two spouts. The baffles form alternate channels and spaces of equal width. They are arranged in a circle at their summit and are directed inward and downward, the channels leading to one spout and the spaces to an opposite spout. A valve or gate at the base of the hopper retains the seed. When the valve is opened the seed falls by gravity over the cone where it is evenly distributed to the channels and spaces, then passes through the spouts into the seed pans.

The following dimensions have been found suitable. In the large divider, designed for large seeds and grains, there are 19 channels and 19 spaces, each 25.4 mm wide. In the small divider designed for small free-flowing seeds there are 22 channels and 22 spaces, each 7.9 mm wide. The overall dimensions of the dividers are as follows: large divider, 812.8 mm high and 368.3 mm in diameter; small divider, 406.4 mm high and 152.4 mm in diameter.

The following construction features should be observed when purchasing a conical divider: (1) the sliding valve or gate should move with ease but not allow seeds to pass along its edges when closed; (2) sharp angles should be reduced to a minimum and there should be no small openings or rough edges on surfaces over which seeds flow because seeds may lodge in these angles and crevices and be carried into other samples. A disadvantage of this divider is that it is difficult to check for cleanliness.

(b) Soil divider. A simpler divider, built on the same principle as the conical divider, is the so-called soil divider. The channels are here arranged in a straight row instead of a circle as in the conical divider. The soil divider consists of a hopper with attached channels or ducts, a frame to hold the hopper, two receiving pans and a pouring pan.

The following dimensions have been found suitable. Ducts or channels, 12.7 mm wide, lead from the hopper to the collecting pans. There are 18 of these channels, alternate ones leading to opposite sides. The maximum dimensions are: 355.6 mm long; 254 mm wide; and 279.4 mm high.

In using the divider the seed is scattered fairly evenly in a pouring pan the length of the hopper and poured in at approximately equal rates along the entire length of the hopper.


This divider is suitable for large-seeded and chaffy species, but suitable types for small-seeded species can also be made.

(c) Centrifugal divider. The centrifugal divider (Gamet type) makes use of centrifugal force to mix and scatter the seeds over the dividing surface. In this divider the seed flows downward through a hopper onto a shallow rubber cup or spinner. Upon rotation of the spinner by an electric motor the seeds are thrown out by centrifugal force and fall downward. The circle or area where the seeds fall is equally divided into two parts by a stationary baffle so that approximately half the seeds fall in one spout and half in the other spout.

The centrifugal divider tends to give variable results when not carefully operated. However, satisfactory results have been obtained when this divider is operated as described below.

Preparation of apparatus:

(i) The divider is levelled by means of the adjustable feet.

(ii) The divider and four containers are checked for cleanliness.

Sample mixing:

(iii) A container is placed under each spout.

(iv) The whole sample is fed into the hopper; when filling the hopper, seed must always be poured centrally.

(v) The spinner is operated and the seed passes into the containers.

(vi) Full containers are replaced by empty containers. The contents of the two full containers are fed into the hopper together, the seed being allowed to blend as it flows in. The spinner is operated.

(vii) The procedure described in (vi) above is repeated at least once more.

Sample reduction:

(viii) Full containers are replaced by empty containers. The contents of one full container are set aside and the contents of the other are fed into the hopper. The spinner is operated.

(ix) This procedure is repeated until the appropriate size of working sample is reached.

(d) Rotary divider. The rotary divider comprises a rotating crown unit with 6 to 10 attached sub-sample containers, a vibration chute and a hopper. In using the divider the seed is poured into the hopper and the rotary divider is switched on so that the crown unit with the containers rotates with approx. 100 r. p. m. and the vibration chute starts to feed the seed into the inlet cylinder of the rotating crown. The feeding rate and therefore the duration of


the dividing operation can be adjusted by the distance between the funnel of the hopper and the chute and the vibration intensity of the chute. The inlet cylinder feeds the seed either centrally onto a distributor within the rotating crown distributing the seed to all containers simultaneously or de-centrally into the inlets of the containers rotating below the inlet cylinder so that the seed stream is subdivided into a lot of sub-samples. Both principles can achieve sufficient accuracy for seed testing purposes.

The divider is suitable for small seeded species and also for most of the chaffy seeded species e. g. grass, flower or herb species. Only very chaffy seeds (e.g. Trisetum flavescens) cannot be divided by this type due to getting stuck in the hopper. It gives accurate results when the duration of the dividing operation is at least 1 minute to achieve at minimum 100 sub-samples in each container. The working sample can be regarded as the content of one or more preferably opposite containers or their content is used for repetitions of the dividing process.

(e) Variable sample divider. The variable sample divider allows a variation of the dividing ratio depending on the seed size that may be between 1:2 and 1:15 and by this to reduce a sample of known size to a predetermined sub-sample size in one operation. It consists of a pouring hopper and a tube underneath that rotates with about 40 revolutions per minute. The tube distributes the seed stream from the pouring hopper onto the inner surface of a further hopper, which is well fitted into a third hopper all being concentric. In the second and the third hopper there are slots which comprise 50% of the perimeter of the hoppers. 50% of the seed will pass through the two hoppers into a collecting pan. The other 50% will stay within the hoppers and will then go into a second collecting pan. The two hoppers can be twisted against each other resulting in more narrow slots. The effect is that a smaller percentage will pass the slots. Either the smaller sample outside the hoppers or the bigger sample inside the hoppers can be used as the required sample. The position of the two hoppers in relation to each other can be adjusted accurately, resulting in pre-determined sub-sample sizes.

2.5.37.2.A1.2 Modified halving method

The apparatus comprises a tray into which fits a grid of equal-sized cubical cells, open at the top and every alternate one having no bottom. After preliminary mixing, the seed is poured evenly over the grid. When the grid is lifted, approximately half the sample remains on the tray. The submitted sample is successively halved in this way until a working sample of approximately, but not less than, the required size is obtained.


2.5.37.2.A1.3 Spoon method

It is permissible to use this method only for samples of a single small-seeded species and for sample reduction for moisture determination or seed health testing (7.4.1). A tray, a spatula and a spoon with a straight edge are required. After preliminary mixing, pour the seed evenly over the tray; do not shake the tray thereafter. Only in the case of sample reduction for moisture determination, the sample must not be poured over a tray but can be sampled from the container. With the spoon in one hand, the spatula in the other, and using both, remove small portions of seed from not less than five random places. Sufficient portions of seed are taken to constitute a sub-sample of approximately, but not less than, the required size.

2.5.37.2.A1.4 The hand halving method

This method is restricted to the following genera of chaffy seeds:

Agrimonia Cenchrus Melinis

Andropogon Chloris Oryza

Anthoxanthum Dichanthium Pennisetum (non glaucum)

Arrhenatherum Digitaria Psathyrostachys

Astrebla Echinochloa Scabiosa

Beckmannia Ehrharta Sorghastrum

Bouteloua Elymus Stylosanthes (non guianensis)

Brachiaria Eragrostis Taeniatherum

Briza Gomphrena Trisetum

and to the following genera of tree and shrub seeds:

Acer Corylus Populus

Aesculus Fraxinus Quercus

Ailanthus Juglans Salix

Castanea Liriodendron Tectona

Cedrela Platanus Ulmus


For all other species it can be used only to obtain working samples in the laboratory for seed health tests (7.4.1).

Technique:

1. The seed is poured evenly onto a smooth clean surface.

2. Thoroughly mix the seed into a mound with a flat-edged spatula.

3. The mound is divided into half and each half is halved again, giving four portions. Each of the four portions is halved again giving eight portions which should be arranged in two rows of four.

4. Combine and retain alternate portions: e.g. combine the first and third portions in the first row with the second and fourth in the second row. Remove the remaining four portions.

5. Steps two, three and four are repeated using the retained portions from step four until the weight of sample required is obtained.

2.5.4 Heterogeneity testing for seed lots in multiple containers

Appendix D: Heterogeneity Tests for Seed Lots in Multiple Containers 1 Object In practice, the Rules require that sampling be refused if the lot is so heterogeneous that differences between containers or primary samples are visible to the sampler. For doubtful cases, the methods described below may be used.

The aim object of heterogeneity testing is to detect the presence of heterogeneity which makes the seed lot technically unacceptable. 2.5.4.1 Heterogeneity of the seed lot 3 The H-value test 2.5.4.13.1 Definitions of terms and symbols The testing of predominantly in-range heterogeneity of an attribute adopted as indicator involves a comparison between the observed variance and the acceptable variance of that attribute. The container-samples of a seed lot are samples drawn independently of each

Since heterogeneity testing is a procedure applied within the framework of sampling the former Appendix D is proposed to be included here. Since it is now no longer a separate chapter, there is not such a long introduction required. Part of the former introduction went to “general principles”


other from different containers. The examinations of container-samples for the indicating attribute must also be mutually independent. Since there is only one source of information for each container, heterogeneity within containers is not directly involved. The acceptable variance is calculated by multiplying the theoretical variance caused by random variation with a factor f for additional variation, taking into account the level of heterogeneity which is achievable in good seed production practice. The theoretical variance can be calculated from the respective probability distributions, which is the binomial distribution in the case of purity and germination, and the Poisson distribution in the case of the other seed count.

No number of containers in the lot

N number of independent container-samples n number of seeds tested from each container-sample (1000 for purity, 100 for germination and 10 000 for other seed count, see 3.3) X test result of the adopted attribute in a container-sample � symbol for sum of all values f factor for multiplying the theoretical variance to obtain the acceptable variance (see Table D2.1)

= å xX

N mean of all X-values determined for the lot in respect of the adopted attribute

(100 )× -= ×X XW fn acceptable variance of independent container-samples in respect

of purity or germination percentages

= ×W X f acceptable variance of independent container-samples in respect

of number of other seeds

( )22

( 1)

-=

-å åN X X

VN N observed variance of independent container-samples based on

all X-values in respect of the adopted attribute

H-value: = -VH f

W Negative H-values are reported as zero

Formulas and symbols are not corrected here but will appear in the final version as at present in Appendix D


Table 2D.13: Factors for additional variation in seed lots to be used for calculating W and finally the H-value Attributes Non-chaffy seeds Chaffy seeds Purity 1.1 1.2 Other seed count 1.4 2.2 Germination 1.1 1.2 Remarks: - For purity and germination calculate to two decimal places if N is less than 10 and

to three decimal places if N is 10 or more. - For the number of other seeds, calculate to one decimal place if N is less than 10,

and two decimal places if N is 10 or more. - For definition of non-chaffy and chaffy seeds see 3.6.A.3 of the International

Rules for Seed Testing. The chaffiness of various genera is listed in Table 3.2.1.A. 2.5.4.1.3.2 Sampling the lot The number of independent container-samples shall be not less than presented in the following Appendix D TTable D.2C.: 2.5.4.1.3.4 Use of Table D.2C and reporting results Table D.2C shows the critical H-values which would be exceeded in only 1% of tests from seed lots with an acceptable distribution of the attribute adopted as indicator. If the calculated H-value exceeds the critical H-value belonging to the sample number N, the attribute and the chaffiness in Table D.2C, then the lot is considered to show significant heterogeneity in the in-range, or possibly also the off-range sense. If, however, the calculated H-value is less than or equal to the tabulated critical H-value, then the lot is considered to show no heterogeneity in the in-range, or possibly off-range sense with respect to the attribute being tested. The results of the H-value test shall be reported as follows: X , N, No, calculated H-value and the statement that “This H-value does/does not indicate significant heterogeneity.”

If X is outside of the following limits, the H-value shall not be calculated or reported:

purity components: above 99.8% or below 0.2% germination above: 99.0% or below 1.0% number of specified seeds: below two per sample


2.5.4.24 The R-value Test The object of this test is to detect off-range heterogeneity of the seed lot using the attribute adopted as an indicator. The test for off-range heterogeneity involves comparing the maximum difference found between samples of similar size drawn from the lot with a tolerated range. This tolerated range is based on the acceptable standard deviation, which is achievable in good seed production practice.

Each independent container-sample is taken from a different container, so that

heterogeneity within containers is not directly involved. Information about heterogeneity

within containers is contained, however, in the acceptable standard deviation which is in

fact incorporated into the tabulation of tolerated ranges. The acceptable standard deviation

was calculated by the standard deviation due to random variation according to the binomial

distribution in the case of purity and germination, and to the Poisson distribution in the

case of the other seed count, multiplied by the square root of the factor f given in Table

D2.1, respectively. The spread between containers is characterised by the calculated range

to be compared with the corresponding tolerated range.

2.5.4.2.4.1 Definitions of terms and symbols No number of containers in the lot N number of independent container-samples n number of seeds tested from each container-sample (1000 for purity, 100 for germination and 10 000 for other seed count, see 3.3) X test result of the adopted attribute in a container-sample � symbol for sum of all values

= å XX

N mean of all X-values determined for the lot in respect of the adopted attribute

max min= -R X X the range found as maximum difference between independent container samples of the lot in respect of the adopted attribute

Remark: For precision of X for the R-value test see 2.5.4.1.1D.3.1 ‘Remarks’ to the H-value test.


2.5.4.2.4.2 Sampling the lot Sampling for the R-value test is the same as for the H-value test (see D.3.2.5.4.1.2), the same samples must be used. 2.5.4.2.3 Testing procedure The same testing procedures of purity, germination and the other seed count are used for the R-value test as are used for the H-value test (see D.3.2.5.4.1.3). For calculations, the same set of data must be used. 2.5.4.2.4 Use of tables and reporting of results Seed lot off-range heterogeneity is tested by using the appropriate table for tolerated, i.e. critical range. Table 2DD.3 for components of pure seed analyses, Table 2ED.4 for germination determinations and

Table 2FD.5 for numbers of other seeds.

Find the value X in the ‘Average’ columns of the appropriate table. When entering the

table, round averages following the usual procedure; read off the tolerated range which would be exceeded in only 1% of tests from seed lots with an acceptable distribution of the attribute

in Column 5-9 for cases when N = 5 to 9, in Column 10-19 for cases when N = 10 to 19, or in Column 20 when N = 20.

If the calculated R-value exceeds this tolerated range, then the lot is considered to show significant heterogeneity in the off-range sense. If, however, the calculated R-value is less than or equal to the tabulated tolerated range, then the lot is considered to show no heterogeneity in the off-range sense with respect to the attribute being tested.

When using the tables, round averages to the next tabulated value (if in the middle, then downwards). 2.5.4.53 Interpretation of results Whenever either of the two tests, the H-value test or the R-value test, indicates significant heterogeneity, then the lot must be declared heterogeneous. When, however, neither of the two tests indicates significant heterogeneity, then the lot must be adopted as non-heterogeneous, having a non-significant level of heterogeneity.


2.5.5 8 Storage of samples after testing

2.8.A Storing samples

2.8.2 After testing

To provide for re-testing by the original or by another seed testing laboratory, submitted samples on which ISTA International Seed Analysis Certificates have been issued shall be stored for one year from the date of issue of the certificate. Only in the case of very expensive seed, the remainder of the submitted sample, except 25 seeds for assurance of identity, may be sent back to the applicant. Storage after testing is for a long period and should be in special rooms with temperature and humidity control. Storage of the sample shall be done in conditions calculated to minimise any change in quality. Protection against insects and rodents may be necessary. The seed testing laboratory cannot be held responsible for any deterioration of the sample during storage. Nevertheless, the testing laboratory shall not be held responsible for any deterioration that occurs.

When a re-test is required, a portion shall be drawn from the submitted sample in accordance with 2.75.3.2, sealed and submitted to the designated testing laboratory. The remainder shall be retained in store.

2.6. Calculation and expression of results

See under 2.5.4 for calculating the results of heterogeneity tests.

2.7 Reporting of results

See under 2.5.4 for reporting the results of heterogeneity tests.

2.8 Tables

Table 2A

Storage of samples after testing is the last step and stays at the end. Text comes from the Annexe 2 and is partly deleted because of duplication.

These two paragraphs appear as a consequence of the general headings as agreed in Budapest. Since the only place in chapter two describing a procedure for reporting results is the heterogeneity test, cross references are made. The text was not moved to here since this would split the heterogeneity test.

Table 2A remains unchanged. Heading and content is not reproduced here.


Table 11A 2B Part 1 Sample sizes of pelleted seeds in number of pellets Determinations Submitted Working samples not samples not less than less than 1 2 3 Purity analysis (including verification of species) 7500 2500 Weight determination 7500 Pure pellet fraction Germination 7500 400 Determination of other seeds 10000 7500 Determination of other seeds (encrusted seeds and seed granules) 25000 25000 Size grading 10000 2000

Table 2B Part 2 Sample sizes of seed tapes Determinations Submitted samples Working samples not less than not less than 1 2 3 Verification of species 2500 seeds 100 seeds Germination 2500 seeds 400 seeds Purity analysis (if required) 2500 seeds 2500 seeds Determination of other seeds 10000 seeds 7500 seeds

These tables come from Chapter 11 and remain unchained.


Table D.2C Sampling intensity and critical H-values Number of independent container-samples to be drawn as depending on the number of containers in the lot and critical H-values for seed lot heterogeneity at a significance level of 1% probability. Number of containers in the lot (No)

Number of independent container-samples (N)

Critical H-value for purity and germination attributes

Critical H-value for other seed count attributes

non-chaffy seeds

chaffy seeds non-chaffy seeds

chaffy seeds

5 5 2.55 2.78 3.25 5.10 6 6 2.22 2.42 2.83 4.44 7 7 1.98 2.17 2.52 3.98 8 8 1.80 1.97 2.30 3.61 9 9 1.66 1.81 2.11 3.32 10 10 1.55 1.69 1.97 3.10 11-15 11 1.45 1.58 1.85 2.90 16-25 15 1.19 1.31 1.51 2.40 26-35 17 1.10 1.20 1.40 2.20 36-49 18 1.07 1.16 1.36 2.13 50 or more 20 0.99 1.09 1.26 2.00

Table 2DD.3.A Part 1Maximum tolerated ranges for the R-value test at a significance

level of 1% probability using components of purity analyses as indicating attribute in

non-chaffy seeds.

Table 2DD.3.B Part 2 Maximum tolerated ranges for the R-value test at a significance

level of 1% probability using components of purity analyses as indicating attribute in

chaffy seeds.

Table 2ED.4.A Part 1 Maximum tolerated ranges for the R-value test at a significance level of 1% probability using components of germination tests as indicating attribute in non-chaffy seeds.

Table 2ED.4.B Part 2 Maximum tolerated ranges for the R-value test at a significance level of 1% probability using components of germination tests as indicating attribute in chaffy seeds.

Table 2FD.5.A Part 1 Maximum tolerated ranges for the R-value test at a significance

These tables come from Appendix D and remain unchained. For the tables below the number are not reproduced here.


level of 1% probability using components of other seed count analyses as indicating

attribute in non-chaffy seeds.

Table 2FD.5.B Part 2 Maximum tolerated ranges for the R-value test at a significance level of 1% probability using components of other seed count analyses as indicating attribute in chaffy seeds.

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E DITORIAL M ERGER OF › upload › cms › user › Chapter2editorialmer… · EDITORIAL MERGER OF CHAPTER 2 2 of 28 2.2.6 Sub-sample A sub-sample is the portion of a sample obtained