ASTM D4638-95a(1999)

Standard Guide for Preparation of Biological Samples for Inorganic Chemical Analysis

SCOPE
1.1 This guide describes procedures for the preparation of test samples collected from such locations as streams, rivers, ponds, lakes, estuaries, oceans, and toxicity tests and is applicable to such organisms as plankton, mollusks, fish, and plants.  
1.2 The procedures are applicable to the determination of volatile, semivolatile, and nonvolatile inorganic constituents of biological materials. Analyses may be carried out or reported on either a dry or wet basis.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. For a specific hazard statement, see 9.3.3.

General Information

• Status: Historical
• Publication Date: 09-Dec-1999
• ICS: 71.040.40 - Chemical analysis
• Technical Committee: D19 - Water
• Drafting Committee: D19.05 - Inorganic Constituents in Water

Relations

• Replaced By: ASTM D4638-03 - Standard Guide for Preparation of Biological Samples for Inorganic Chemical Analysis
• Effective Date: 01-Dec-2003

Standards Content (Sample)

NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
Designation: D 4638 – 95a (Reapproved 1999)
Standard Guide for
Preparation of Biological Samples for Inorganic Chemical
Analysis
This standard is issued under the fixed designation D 4638; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope to remove most of the occluded water. Larger organisms, such
as fish, should be patted dry, using paper towels.
1.1 This guide describes procedures for the preparation of
4.4 Where less than a whole organism is to be analyzed,
test samples collected from such locations as streams, rivers,
tissue excisions are made with nonmetallic tools such as plastic
ponds, lakes, estuaries, oceans, and toxicity tests and is
knives or TFE-fluorocarbon-coated scalpels.
applicable to such organisms as plankton, mollusks, fish, and
4.5 Moisture determinations are made on separate samples
plants.
from those analyzed for volatile or semivolatile constituents.
1.2 The procedures are applicable to the determination of
4.6 Analyses for volatile constituents are made using wet
volatile, semivolatile, and nonvolatile inorganic constituents of
samples from which supernatant liquid or occluded water has
biological materials. Analyses may be carried out or reported
been removed (see 4.3). The results may be calculated to the
on either a dry or wet basis.
dry, original-sample basis, using the results of a moisture
1.3 This standard does not purport to address all of the
determination carried out on a separate sample.
safety concerns, if any, associated with its use. It is the
4.7 Analyses for semivolatile constituents are made on wet
responsibility of the user of this standard to establish appro-
samples or samples previously dried at a temperature (depen-
priate safety and health practices and determine the applica-
dent on constituents of interest), or using a procedure, found to
bility of regulatory limitations prior to use. For a specific
be adequate for the purpose, and specified in the corresponding
hazard statement, see 9.3.3.
analytical procedure.
2. Referenced Documents
4.8 Analyses for nonvolatile constituents are made on
samples previously dried at a temperature (dependent on
2.1 ASTM Standards:
constituents of interest), or using a procedure found to be
D 1129 Terminology Relating to Water
adequate for the purpose, and specified in the corresponding
D 1193 Specification for Reagent Water
analytical procedure.
3. Terminology
4.9 Digest the samples according to the procedures outlined
in Section 9.
3.1 Definitions—For definitions of terms used in this guide,
4.10 A flow diagram outlining typical procedures is shown
refer to Terminology D 1129.
in Fig. 1.
4. Summary of Guide
5. Significance and Use
4.1 Samples are collected, where possible, with nonmetallic
5.1 The chemical analysis of biological material, collected
or TFE-fluorocarbon-coated sampling equipment to prevent
from such locations as streams, rivers, lakes, and oceans can
contamination, stored in plastic containers, and kept either at
provide information of environmental significance. The chemi-
4°C or frozen until returned to an adequate facility for analysis.
cal analysis of biological material used in toxicity tests may be
4.2 Before analysis, samples are allowed to return to room
useful to better interpret the toxicological results.
temperature. Large foreign objects are mechanically removed
5.2 Many aquatic biological samples, either as a result of
from the samples based upon visual examination; smaller
their size, or their method of collection, are inherently hetero-
foreign objects are also removed mechanically, with the aid of
geneous in that they may contain occluded water in varying
a low-power microscope.
and unpredictable amounts and may contain foreign objects or
4.3 Wet samples of small organisms such as plankton, are
material (for example, sediment) not ordinarily intended for
mixed for preliminary homogenization, then allowed to settle,
analysis, the inclusion of which would result in inaccurate
analysis.
This guide is under the jurisdiction of ASTM Committee D-19 on Water and is
5.3 Standard methods for separating foreign objects, to
the direct responsibility of Subcommittee D19.05 on Inorganic Constituents in
facilitate homogenization, will minimize errors due to poor
Water.
Current edition approved Dec. 10, 1995. Published February 1996. Originally
mixing and inclusion of extraneous material.
published as D 4638 – 86. Last previous edition D 4638 – 95.
5.4 Standardized procedures for drying provide a means for
Annual Book of ASTM Standards, Vol 11.01.
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NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
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D 4638
FIG. 1 Flow Diagram for the Preparation of Biological Samples for Inorganic Analysis
reporting analytical values to a common dry weight basis, if number of small portions (at least 5) from random locations in
desired. Analyses may also be carried out or reported on a wet the beaker, and composite them to obtain a representative
weight basis. sample of a size sufficient for the analysis. Using a tissue
disrupter, blender, or equivalent, homogenize the sample, or
6. Preliminary Treatment of Samples
composite (to ensure lack of contamination, carry a standard or
6.1 Treat small heterogeneous samples, such as plankton, as blank, or both, through this procedure), and proceed to Section
follows: 7.
6.1.1 Allow for the sample to return to room temperature.
6.2 Treat large samples such as fish as follows:
6.1.2 Remove foreign objects, such as leaves and twigs,
6.2.1 Allow the sample to return to room temperature.
mechanically, using nonmetallic instruments. Use a low-power
6.2.2 Pat the sample dry with paper toweling to remove as
microscope to facilitate removal of smaller foreign objects
much water as possible.
such as paint chips.
6.2.3 Transfer the sample to a nonmetallic surface, such as
6.1.3 Transfer the sample to a beaker and thoroughly mix it
a flat glass plate, and excise a sufficient quantity of material, or
with a glass stirring rod or equivalent, and allow it to settle so
specific organs, to obtain sufficient material for analysis. Make
that most or all of the occluded water can be decanted.
excisions with plastic knives or TFE-fluorocarbon-coated scal-
6.1.4 If chemical analyses are to be carried out on a wet
pels.
sample, and a large amount of material is available, remove a
6.2.4 If chemical analyses are to be carried out on a wet
number of small portions (at least 5) from random locations in
sample, use a tissue disrupter, blender, or equivalent, to
the beaker, and composite them to obtain a representative
homogenize the material (to ensure lack of contamination,
sample of a size sufficient for chemical analysis and a separate
carry a standard or blank, or both, through this procedure).
moisture determination. Using a tissue disrupter, blender, or
Remove a subsample for moisture determination and proceed
equivalent, homogenize the sample or composite (to ensure
to Section 7. Retain the remainder and proceed to Section 9.
lack of contamination, carry a standard or blank, or both,
through this procedure). Remove a subsample for moisture 6.2.5 If chemical analyses are to be carried out on a dry
determination and proceed to Section 7. Retain the remainder sample, use a tissue disrupter, blender, or equivalent, to
and proceed to Section 9. homogenize the material (to ensure lack of contamination,
6.1.5 If chemical analyses are to be carried out on a dry carry a standard or blank, or both, through this procedure) and
sample, and a large amount of material is available, remove a proceed to Section 7.
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
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D 4638
7. Drying Procedures 7.5.2.3 Repeat weighings at 4-h intervals, to attain a con-
stant weight (see Note 1).
7.1 Use a sample or subsample prepared in accordance with
the directions given in Section 6.
NOTE 3—Air-drying in the open is strongly discouraged unless it is
7.2 Treat subsamples from biological materials which are to
carried out in a clean room, where possible contamination from airborne
undergo chemical analysis without drying for moisture deter-
particulates can be controlled.
minations as follows:
7.6 If a moisture determination (or sample drying) is to be
7.2.1 Accurately weigh 5 to 10 g 6 1mgor10to25g 6
made using a freeze dryer, treat the determination as follows:
10 mg of material into a nonmetallic container which has been
previously tared, and weighed with the same accuracy. 7.6.1 Transfer the containers holding the material to the
7.2.2 When a limited amount of material is available,
freeze dryer.
determine the moisture ona1to2-g sample, and weigh with an
7.6.2 Follow the manufacturer’s instructions for the particu-
accuracy of6 0.1 mg. The use of samples smaller than1gis
lar unit in use. Make certain that a trap is placed between the
not recommended for moisture determination.
vacuum pump and the drying chamber to prevent pump oil
7.3 When an entire sample is to be dried prior to chemical
fumes from possibly contaminating the sample. Drying is
analysis, a moisture determination is also required. Transfer the
usually complete when the internal pressure in the drying
accurately weighed material (1 to 2 g 6 0.1 mg,5to10g 6
chamber reaches 50 millitorrs or less.
1 mg, >10 g 6 10 mg) into a dry nonmetallic container which
7.6.3 Transfer the freeze-dried samples to a desiccator for
has been previously tared, and weigh with the same accuracy.
storage, and weigh them with the same accuracy as the wet
7.4 If a moisture determination (or sample drying) is to be
samples (see Note 1).
made using an oven, treat as follows:
7.4.1 Transfer the containers holding the material to an oven
NOTE 4—Because freeze drying occurs under vacuum, this may cause
and dry for2hatone of the following temperatures:
the loss of volatile or semivolatile inorganics such as mercury, or both, if
7.4.1.1 For the determination of semivolatile constituents,
the dried sample is to be subjected to chemical analysis.
use the temperature specified in the analytical procedure for the
7.7 The possibility of loss of volatile constituents dictates
constituents(s).
the drying procedure to be used, prior to chemical analysis.
7.4.1.2 For determination of nonvolatile constituents use
Determine volatile constituents using undried samples. Deter-
105 6 2°C.
mine semivolatile constituents using samples dried at a tem-
7.4.2 Cool in a desiccator, then weigh the dried samples
perature at which no significant losses occur.
with the same accuracy as the wet samples.
7.8 Analytical data reported on a dry weight basis should
NOTE 1—Biological materials tend to be very hygroscopic. Keep
include percent moisture so that wet weight values can be
weighing times to a minimum.
obtained. Likewise, wet weight analytical data should include
7.4.3 Repeat drying at hourly intervals, to attain a constant
percent moisture to permit recalculation to a dry weight basis.
weight.
7.9 Use the following equations to calculate percent mois-
7.5 If a moisture determination (or sample drying) is to be
ture and to correct analytical results from samples analyzed
made at room temperature, treat as follows:
when wet.
7.5.1 If drying is to be done in a desiccator, ensure that the
desiccant in the bottom is fresh, and some means is available to 7.9.1 Calculate percent moisture as follows:
indicate when the desiccant loses its drying capacity (for
moisture, % 5 ~W /W !100 (1)
w d
example, color change). A vacuum desiccator may also be
used. where:
W 5 wet weight, g, and
w
NOTE 2—If a vacuum desiccator is used, bear in mind that this may
W 5 dry weight, g
d
cause the loss of volatile or semivolatile inorganics such as mercury, if the
7.9.2 To calculate concentrations on a dry weight basis,
dried sample is to be subjected to chemical analysis.
when determinations have been made on an undried sample,
7.5.1.1 Transfer the containers holding the material to a
use the following equation:
desiccator.
C ~100!
7.5.1.2 Leave the material in the desiccator for 48 h, then
w
C 5 (2)
d
100 2 % moisture
weigh the dried samples with the same accuracy as the wet
sample.
where:
7.5.1.3 Repeat weighings at 4-h intervals, to attain a con-
C 5 concentration on a dry weight basis, and
d
stant weight (see Note 1).
C 5 concentration on a wet weight basis.
w
7.5.2 Alternatively, sample drying or moisture determina-
tions may be carried out in a laminar flow hood; treat as
8. Reagents
follows:
8.1 Purity of Reagents—Reagent grade chemicals shall be
7.5.2.1 Transfer the containers holding the material to an
used in all tests. Unless otherwise indicated, it is intended that
appropriate hood and turn it on.
all reagents conform to the specifications of the Committee on
7.5.2.2 Leave the material in the hood for 48 h, then weigh
the dried samples with the same accuracy as the wet sample. Analytical Reagents of the American Chemical Society where
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
D 4638
such specifications are available. Other grades may be used, 9.2 Dry ashing entails procedures in which organic matter
provided it is first ascertained that the reagent is of sufficiently is oxidized by reaction with gaseous oxygen, generally with the
high purity to permit its use without lessening the accuracy of application of energy in some form. Included in this general
the determination. term are the methods in which the sample is heated to a
8.2 Purity of Water— Unless otherwise indicated, refer- relatively high temperature in a stream of air or oxygen and the
ences to water shall be understood to mean reagent water related low-temperature technique where excited oxygen is
conforming to Specification D 1193, Type I. Other reagent used.
water types may be used, provided it is first ascertained that the 9.2.1 For high temperature ashing, digest as follows:
water is of sufficiently high purity to permit its use without 9.2.1.1 Place a weighed 2 to 10-g sample, prepared accord-
adversely affecting the bias and precision of the test metho
...