Sub-annex 5: Specific conditions for sub-agreements
Some analysis methods or requirements for sample handling by the laboratory are not
described in method data sheets, Danish standards or ISO standards. The requirements for
these are described here for the individual sub-agreements.
Sub-Agreements A, B and C regarding groundwater
Groundwater, freshwater including watercourses, lakes and LOOP (the agricultural
catchment monitoring programme), as well as terrestrial samples.
The groundwater samples refer to two projects: the national monitoring programme and the
groundwater mapping funded by charges.
In connection with the national monitoring programme, groundwater samples are taken in
accordance with the groundwater monitoring programme known as GRUMO, and the
agricultural catchment monitoring programme known as LOOP.
The following specific conditions apply to both projects unless otherwise specified.
Labelling of groundwater samples
There must be clear, preprinted identification on bottle labels and order forms for
completion in the field. The samples must have identification throughout the entire system,
from sampling to data reporting.
Bottles must be labelled with information on the sample’s filtration status and the substances
(e.g. acid) that have been added to the sample bottle.
Field measurements
The parameters of pH, oxygen, conductivity, temperature and redox conditions are to be
measured in the field and applied to the overall reporting.
Reporting
The results of the analysis of the groundwater samples must be entered in the same way as
for analyses from drilling inspections at waterworks in the JUPITER joint public database
(http://www.GEUS.dk/jupiter/index-dk.htm), established by Statutory Order no. 292 of
26/03/2014.
The Danish Natural Environment Portal has further instructions (in Danish) at:
http://www.miljoeportal.dk/digital/komgodtigang/Sider/Kom-godt-igang.aspx
Pre-processing, including filtration and analysis
All necessary filtration of the groundwater samples is carried out in the field by the Danish
Nature Agency.
In some cases, it may not be possible to filter in the field. In such cases, the laboratory is
responsible for filtration by agreement with the Danish Nature Agency.
Freshwater
Alkalinity < 0.1 mmol/l (Gran titration)
The alkalinity of lakes is determined according to DS/EN ISO 9963-1. If a lake has a low
alkalinity, Gran titration is used (ECE, 1987). This produces an approximate measurement
of the concentration of strong acid (acidity). The measurement results are given in mmol ll(meq l-l). Detection limit 0.05 mmol l-1.
Excerpt from Gran titration method from Technical Report no. 21, from the Danish National
Environmental Research Institute.
Gran titration is described by Sørensen (1951), Gran (1952), Larson & Henley (1955) and
Stumm & Morgan (1981). Its practical application for analysing freshwater is described by
Mackereth et al. (1978), among others.
In the usual method of graphing potentiometric acid-base titrations, the volume of titrant
added is plotted against pH. With Gran’s titration method, 10-pH is calculated, and the titrant
volume is plotted against this value. After the equivalence point, each further addition of
hydrochloric acid will cause a proportional increase of [H+ ] = 10-pH/ fH. At a pH of
approximately 4.3, weak bases such as HCO3-, silicate etc. are fully neutralised, and
therefore titration is carried out beyond the pH value, as around four related values are noted
for the quantity of hydrochloric acid added and the pH from pH 4.3 to pH approx. 3.7. The
pH is transformed to 10-pH, and the equivalence point is then found by extrapolating the
straight line to the x-axis (figure 2). For the purposes of the transformation, it is not
necessary to take into account the activity coefficient fH since this can be regarded as
constant within this pH interval. The x-values to the right of the intersection point represent
the quantity of hydrochloric acid added in excess of that used to neutralise the base
substances (alkalinity) (Kalgård Sø in figure 2). If the sample is already acidic, the
intersection may give a negative alkalinity value. This means that the sample contains strong
acid at the concentration found on insertion into the formula for calculating alkalinity (Hund
Sø and Grane Langsø in figure 2). Water with a high aluminium, iron and humic acid
content is not immediately suitable for analysis according to Gran’s method, because its pKa
values are so low that no straight line is obtained during titration from pH 4.3 to 3.7.
Equipment and reagents
 A precision pH meter.
 A micro burette that permits reading of whole microlitres.
 Glass electrode with reference electrode or combination electrode.
 0.1 mol/l hydrochloric acid.
Method
Transfer 100.0 ml of sample to a beaker and submerge the electrodes in the sample. While
applying magnetic stirring, add 0.1 mol/l hydrochloric acid until the pH is around 4.3, then
add hydrochloric acid in portions of e.g. 20 µl. After each addition, stop the magnetic
stirring and, once the electrodes have stabilised, read the pH meter. There should be 4
readings within the pH range 4.3–3.7.
Convert the pH readings to 10-pH. Plot these values as y-values, and the number of µl of 0.1
mol/l hydrochloric acid as x-values. Draw the line of best fit between the points within the
given pH range, and extend the line to intersect with the x-axis (figure 2). Instead of drawing
the line graphically, a pocket calculator with a built-in regression analysis program can be
used to calculate the point of intersection with the x-axis.
Calculation
The alkalinity, which is defined here as the total alkalinity, is calculated using the formula:
TA = bc/v mmol/l
where
b = number of microlitres of hydrochloric acid read at the intersection with the x-axis c =
concentration of hydrochloric acid in mol/l, e.g. 0.1 mol/l
v = number of ml of sample, e.g. 100 ml.
If the concentration of hydrochloric acid is 0.1 mol/l and 100 ml of sample is used, TB is b
µmol/l or 0.001 · b mmol/l. A negative intersection with the x-axis indicates strong acidity
(SA).
In the Hund Sø example (figure 2), TA = 0.035 mmol/l, or SA = -TA = 0.035 mmol/l.
The precision and accuracy is generally approx. ±0.002 mmol/l.
Literature:
ECE, Convention on long-range transboundary air pollution 1987: International cooperative programme for assessment and monitoring of acidification in rivers and lakes.
Manual for chemical and biological monitoring. Prepared by the Programme Center,
Norwegian Institute for Water Research, NIVA, Oslo, 23 s.
Gran, G. 1952: Determination of the equivalence points in potentiometric titrations.
Part II. – Analyst 77, 661–671.
Larson, T. E. & L. Henley 1955: Determination of low alkalinity or acidity in water. – Anal.
Chem. 27, 851–852.
Mackereth, F. J. H., J. Heron & J. F. Talling 1978: Water analyses: Some revised methods
for limnologists. – Freshwater Biological Association, Scientific Publication No. 36, 120 s.
Stumm, W. & J. J. Morgan 1981: Aquatic Chemistry 2. ed. – Wiley Interscience, 780 s.
Sørensen, P. 1951: Transformation af titreringskurver til rette linier (Transformation of
titration curves into straight lines). Kemisk månedblad 32, 73–76.
Suspended solids
The sample to be analysed for suspended solids and loss on ignition must not be allowed to
stand for more than 24 hours or at a temperature higher than 4°C before analysis.
Agricultural catchment monitoring programme (LOOP) Soil water
No filtration of the water samples from soil water should take place prior to analysis, as the
water samples were subjected to filtration when passing through the suction cells.
Soil water, especially from soils with a high humic content, may be coloured. With
spectrophotometric analysis methods, care should be taken to correct for any coloured
samples by measuring the absorption resulting from mixing the sample and all reagents
except the colour reagent.
Prioritising analysis parameters in the event of an insufficient sample size for a full
programme
Common samples – listed by priority:
NO3+NO2-N, total N, PO4-P, NH4-N, pH
Extended analysis – listed by priority:
Total P, K, total Fe, SO4, Cl, conductivity
Agricultural catchment monitoring programme (LOOP) Drainage
water
With the exception of potassium, filtration/non-filtration of the samples for drainage water is
to take place in accordance with the description for freshwater in the method data sheets.
Samples for determination of potassium are to be filtered before analysis.
Terrestrial samples
Storage: The following samples are to be kept refrigerated (0–4 degrees centigrade) until analysis:
all water samples, all plant samples and soil samples to be analysed for Total Nitrogen; see the
methods described and Technical Instruction N01.
The parameters Soil Base Saturation and Soil Phosphorus Content are covered in “Common working
methods for soil analysis”, Danish Plant Directorate 1994; see below and in the annexes.
Soil Base Saturation: Method III, 12 according to “Common working methods for soil analysis”,
Danish Plant Directorate 1994.
CALCULATION OF ADSORPTION CAPACITY AND DEGREE OF BASE SATURATION
Adsorption capacity refers to the total of exchangeable K+, Na+, Mg++, Ca++ and H+ expressed as
milliequivalents per 100 g of soil. Exchangeable potassium and sodium are calculated on the basis of
Kt and Nat (see methods 15 and 16), by dividing by 39 and 23 respectively. Exchangeable
magnesium and calcium are calculated on the basis of Mgt and Cat (see methods 17 and 18A, 18B),
by dividing by 12 and 20 respectively. Exchangeable hydrogen ions are determined according to
method 10.
The adsorption capacity, T, is calculated according to the following formula:
T = (K+) + (Na+) + (Mg++) + (Ca++) + (H+)
where the letters in parentheses indicate the content in the soil of the various exchangeable cations
expressed in milliequivalents per 100 g of soil. (Ca++) + (Mg++) + (H+) generally constitutes over
90% of this total, and it is therefore possible in most cases to disregard Na+ and K+ when calculating
the T-value.
The degree of base saturation of a soil, V, expressed as a percentage is defined by:
V = S/T*100.
Here, S is the total exchangeable “bases”, (Ca + Mg + Na + K), in milliequivalents per 100
g of soil. The reaction value of a soil is approximately directly proportional to the degree of base
saturation, approx. 4 when base saturation is 0 and a little over 8 when the base saturation is 100%.
Literature
Hissink, D.J.: Base Exchange in Soils. -Transactions of the Faraday Society, Vol. XX, 1935.
Jensen, S. Tovborg: Kalkens omsætning i jordbunden (Lime conversion in the soil). - Tidsskrift for
Planteavl, Vol. 41, 1936, pp. 571-649.
Soil phosphorus content: Method III, 14 according to “Common working methods for soil
analysis”, Danish Plant Directorate 1994.
PHOSPHORUS CONTENT Pt
Extract the soil with 0.5 N sodium bicarbonate and determine the phosphate content of the extracted
soil spectrophotometrically.
A. Reagents
1) Phosphate reagent: ammonium molybdate/antimony potassium tartrate/ascorbic acid solution.
a) Ammonium molybdate/antimony potassium tartrate solution. Dissolve 19.2 g
(NH4)6Mo7O24*4H2O in 250 ml of water, and dissolve 0.47 g antimony potassium tartrate,
C4H4O7KSb, in 100 ml of water. Pour 900 ml of water into a 2 litre graduated flask, and carefully
add 225 ml of concentrated sulphuric acid while agitating. Transfer the two solutions of ammonium
molybdate and antimony potassium tartrate quantitatively to the graduated flask with the diluted
sulphuric acid. Add water to bring the amount to 2 litres. Keep the reagent cool and protected from
light.
b) To prepare the phosphate reagent, dissolve 0.80 g of ascorbic acid, C6H8O6, per 100 ml of
ammonium molybdate/antimony potassium tartrate/ascorbic acid solution. The prepared phosphate
reagent lasts only for a limited time and must be used on the same day as it is prepared.
2) Extraction solution: 0.5 M NaHCO3 solution pH 8.5. Dissolve 420 g NaHCO3 and 50 ml of 5) in
10 l of water; adjust the pH to 8.5 with 1 N NaOH. Add a layer of paraffin oil up to approximately
0.5 cm above the liquid to prevent air discharge. Check the pH of the solution at least once per
month.
3) 0.6 N sulphuric acid. Dilute 84 ml of concentrated H2SO4 to 5 litres with H2O.
4) 1 N sodium hydroxide. Dilute 40 g NaOH to 1 litre with H2O.
5) Polyacrylamide. Polyacrylamide BDH MW over 5,000,000 No. 29788 0.05% aqueous solution.
6) Standard solutions
a) Stock solution I. Dissolve 1.0533 g KH2PO4 to 1 litre in 0.2 N H2SO4. This solution contains
240 mg P/l.
b) Stock solution II. Dilute 50 ml of stock solution I to 1 litre with 0.2 N H2SO4. The solution
contains 12 mg P/l.
c) Standard solutions. Produce 10 standard solutions by diluting 2, 5, 10, 15, 20, 25, 30, 35, 40 and
45 ml of stock solution II with 0.2 N H2SO4 to 100 ml. These standard solutions contain 0.24 to
5.4 mg P/l equivalent to Pt = 0.48 to Pt = 10.8 when the procedure below is followed.
B. Special equipment.
A spectrophotometer with a photocell that is sufficiently sensitive at 890 nm.
C. Standard curve.
Pipette 15 ml of each of the 10 standard solutions into 100 ml graduated flasks. Transfer 15 ml of
0.2 N sulphuric acid into an eleventh flask. Add 15 ml of reagent 2) and 15 ml of reagent 3), and fill
up to approx. 70 ml with water. To avoid strong effervescence, the acid and base should be mixed
relatively slowly. Leave the sample to stand for approximately 10 min. Then add 10 ml of reagent 1),
and fill up to the mark with water and mix. After leaving them to stand for about 15 minutes,
measure the light absorption of the solutions. The colour remains stable for up to 24 hours.
The colour of the solution is determined using the spectrophotometer; measurement should be done
at 890 nm. Then, on the basis of the galvanometer readings, construct a standard curve by plotting
the values of Pt corresponding to the samples on the x-axis and the galvanometer readings on the yaxis.
Which of the standard solutions can be used depends on the spectrophotometer available. Only use
measurements taken with volumes of solution such that 15–65% light absorption is achieved.
Outside this range, the determination of phosphorus content is too uncertain. If the
spectrophotometer is stable, the standard curve produced can be used for a reasonably long period.
However, it will be necessary to check continuously whether the standard curve applies, i.e. whether
the galvanometer readings have changed.
D. Execution of the analysis.
Transfer 5 g of soil to a bottle or flask with a volume of 250 ml. With a pipette, add 100 ml of
reagent 2), and shake the flask for 30 minutes in a rotary shaker.
Filter through a 15 cm phosphorus-free filter. Transfer 15 ml of the filtrate into a 100 ml graduated
flask, add 15 ml of reagent 3) and fill with water to around 70 ml. Then proceed as described for the
standard solutions. Read the phosphorus content off from the standard curve using the galvanometer
reading obtained. If the phosphorus content is too high, dilution is required. If it is estimated, for
example, that 7.5 ml of filtrate will be sufficient, extract 50 ml and dilute with reagent 2) to 100 ml.
Extract 15 ml of this diluted solution, corresponding to 7.5 ml of the original soil extract.
The extraction process is highly dependent on temperature, pH and time. It is absolutely critical that
the extraction time is observed, that the pH in the extraction solution is 8.5 and that the temperature
is kept under control. An extraction temperature of 22"1oC should be aimed for. Moreover, the
temperature affects the colour intensity obtained when measuring. It is therefore important that the
standard curve is produced under the same conditions as those under which the analysis is carried
out.
E. Calculation
Read the results from the standard curve as Pt. The unit corresponds to 1 mg P per 100 g of soil or
approximately 25 kg P per hectare in the topsoil (20 cm) on normal arable land.
Literature
Olsen, S. R.; C. V. Cole; F. S. Watanabe & L. A. Dean (1954): Estimation of available phosphorus
in soils by extraction with bicarbonate. U.S. Dept. Agric. Circular, 939, pp. 1–29.
Banderis, A.; D. H. Barter & K. Henderson (1976): The use of polyacrylamide to replace carbon in
the determination of “Olsen’s” extractable phosphate in soil. Journal of Soil Science, 27, pp. 71 -74.
Nielsen, J. Dissing (1980): Sammenligning af nogle analysemetoder til vurdering af
fosfortilstanden i forskellige jordtyper (Comparison of some analysis methods for the assessment of
the phosphorus status of different soil types). Tidsskr. Planteavl, Vol. 85 (1981) pp. 31–38.
Sub-Agreements Ea and Eb, metals/inorganic trace elements and
organic hazardous substances in sediment and biota
Environmentally hazardous substances in mussels
Storage and transport of dissected material
The samples must be transported deep-frozen or freeze-dried. The dissected samples must
be stored deep-frozen (-20°C) or freeze-dried in suitable containers; see Table. Samples for
metal, PCB, dioxin and organotin analysis can be stored deep-frozen for up to a year.
Samples for PAH and brominated flame retardants must be analysed as quickly as possible.
Empty sample vessels must be checked with relevant blank samples for any contamination
problems.
Table. List of suitable samples containers for storing tissue samples.
Parameter/substance groups
Organic matter
Container and lid
Glass/aluminium
(PCB, PAH, brominated flame
retardant, dioxins and organotin)
Metals
Glass/polyethylene
Cleaning procedure
The container must be washed with a
detergent, rinsed, heated and,
immediately before use, rinsed with
an organic solvent
(e.g. hexane/acetone). For further
details, see the annex to technical
annex 2 for organic chlorine
compounds.
The container must be washed in
10%
v/v HNO3 and then rinsed three
times with demineralised
water.
Environmentally hazardous substances in sediment
Pre-process (sift, homogenise) and dissolve the sample, or alternatively extract it and
determine (quantify) the concentration of environmentally hazardous substances in the
solution/extract in accordance with the specific guidelines drawn up for the relevant
substances.
Storage and transport
Extract one sediment sample (also called a mixture sample) as a mixture of at least five
individual inserts to a depth of 1 cm in the surface sediment, mixing equal quantities from
each insert. The analysis laboratory must inform the sampler of the required sample size in
grams.
Keep the sediment samples refrigerated (approx. 4°C) during storage and transport, and
freeze them within 24 hours of sampling if they are not sent directly to the analysis
laboratory. Remove a subsample for sieve analysis before freezing. The samples may be
stored and transported for up to 24 hours, either in a refrigerator or in a cooler bag with
freezer packs. When storing for longer periods, the samples must be kept frozen.
Equipment used for sampling or for storing the samples must be cleaned as specified and
must be supplied by the laboratory carrying out the analysis.
Organic environmentally harmful substances
A determination of the concentration of organic environmentally harmful substances in
sediment generally covers the following points:




sifting (2 mm sieves)
homogenisation
(freeze-drying where applicable)
extraction with an organic solvent
o removal or destruction of substances that could interfere with the analysis,
e.g. sulphur compounds and hydrogen sulphide
o purification


separation by gas or liquid chromatography
detection, where the detector type will depend on the substance group to be analysed
for. Depending on whether the detector is specific or not, one column or two
columns with different polarities should be used. For example, PCB and other
chlorinated compounds should be determined either in a column with a mass
spectrophotometer (iSIM mode) (GC-MS) as a detector or in two columns with
electron capture detection (GC-ECD).
Metals – Method
Determination of the concentration of metals in sediment generally covers the following
points:






sifting (2 mm sieves)
homogenisation
drying, preferably freeze-drying • digestion
dilution
(a matrix separation where applicable)
detection with an element-specific detector (e.g. AAS, ICP-MS, ICP-AES)
Several methods are used for digesting a sediment sample: firstly those methods where the
metal content in the entire sample including the silicate matrix is analysed, referred to here
as a total method, and secondly those methods where only a certain fraction of the metal
content is determined, referred to here as a partial method.
The result will depend on the digestion method. It is therefore essential to the comparability
of the data that the sediment samples are digested using the same method.
The metal content of the entire sample must be determined here. An acid digestion method
or a non-destructive method that ensures this must therefore be used. In the case of acid
digestion, an acid mixture containing hydrofluoric acid must therefore be used.
For additional information on the acid digestion method, refer to Loring and Rantala (1992),
who provide a detailed description of a suitable method, including specific problems with
the method and possible interferences in the method.
For mercury determination, it is not necessary to digest the sediment sample with a
hydrofluoric acid mixture. For example, nitric acid diluted 1:1 with good quality
demineralised water may be used here. Experience has shown that the boric acid solution
that is used to neutralise the hydrofluoric acid often contains too high a concentration of
mercury.
Normalisation factors etc. (Al/Li, dry matter determination, TOC, clay/silt content,
loss on ignition and salt correction) Al and Li (only when analysing for metals)
Aluminium (Al) and lithium (Li) are determined in the same way as described for the other
metals.
Dry matter
The dry matter content is determined as loss of weight after drying a subsample of the
sediment to be analysed to a constant weight at 105°C; see Method Data Sheet M029.
The dry matter content is given as a percentage.
Loss on ignition content
The loss on ignition content is determined in accordance with Method Data Sheet M029, i.e.
as loss of weight after igniting a subsample of the dried sediment at 550°C.
The loss on ignition is given in mg loss on ignition/g dry weight.
Salt correction
The benthic water in the marine areas may contain quantities of salt large enough to affect
dry matter determination. As the concentration of environmentally harmful substances must
be stated per kg of dry matter, a correction must be made for salt content when calculating
the dry matter content.
The salt correction is calculated from the dry matter content and the salinity of the benthic
water, assuming as much salt per litre of evaporated water as the salinity indicates when
measured as PSU.
Particle size distribution
In addition, it is recommended that the particle size distribution is determined – at least the
proportion that is < 63 µm. Sifting must be done as wet sifting, as drying may alter the
particle size distribution. If possible, the particle curve may be established more precisely by
sifting the fraction > 63 µm and Coulter counter size fractioning of the fraction < 63 µm or
sedimentation methods (e.g. DS/ISO 11277:2001).
Environmentally hazardous substances in fish
For fish, liver and muscle tissue in particular are used for analysis, since many substances
are concentrated in the liver and muscle tissue. The liver functions as a “purification organ”
and retains most organic substances, but for mercury, for example, higher concentrations
may be found in the muscle tissue than in the liver.
The species of fish that are relevant to the marine monitoring programme can be seen in
Table 1 below. These species have been selected because they meet the basic requirements
described above. Due to climactic conditions, there may be changes in populations during
the programme period, such that the size interval cannot be observed each year. If it is not
possible to obtain enough samples, the laboratory must be informed and an assessment made
of whether the detection limit can be raised or whether the number of analysis parameters
should be cut in relation to the sample material available.
Table 1 Species in the marine monitoring programme.
Species
Number#
Flounder:
Platichthys min. 20
flesus
Pleuronectes min. 20
platessa
Eelpout:
Zoarces
viviparus
min. 25
Size
Age*
Sex
250–300
mm ##
250–300
mm
2–3 years
Female (preferred) Muscle and
liver
Female
Muscle and
(preferred)
liver
230–280
mm
2–3 years
2–3 years
Male
(preferred)
Tissue type#
Muscle and
liver
#
For fish, 10 livers and associated muscles should be analysed for metals (Hg analysed in
muscle), and 10 livers are analysed for PCB. Other organic subjects should be analysed in
pooled samples of fillets from 25 fish of the same size. Note that it is necessary to collect
more than the required 20 fish for the analysis if the sex of the fish cannot be determined
before dissecting. If the liver is large enough for all analyses to be carried out in the same
liver, this is preferable.
## Approx. 150 mm in the Wadden Sea, where experience shows that only this size can be caught
every year.
* Calendar year, i.e. a fish born on 1 December becomes one year old on 1 January.
Determined using the otoliths.
Procedure for dissecting the liver and otoliths in the marine programme
Non-dissected fish should be stored frozen (< -20°C), packaged individually in a suitable
material.
A frozen fish must be dissected before it is fully thawed, as this is significantly easier than if
the fish is fully thawed and avoids disintegration of internal organs such as the liver.
If the fish thaws, liquid containing contaminants may leak out, for example from the liver or
gall bladder, to the surrounding tissue and thus compromise the analysis.
Species of fish that may have a very high liver fat content, such as cod, must be dissected
straight away and not frozen first, so that the liver does not begin to disintegrate.
It is important that the dissection be carried out under conditions that are as clean as possible
in order to avoid contamination of the sample, preferably in a so-called clean cabinet
(laminar flow cabinet), in which the air is filtered for particles through a filter. The work
should therefore be carried out in the laboratory that will be carrying out the analysis.
When dissecting, first remove the otoliths (for flounder), then the internal organs (e.g. liver),
then determine the sex by checking whether there are sperm or roe in the abdomen, and
finally take a muscle sample, after drying the scales for residual viscera if necessary.
In order to comply with the detection limits, guiding minimum requirements for sample
sizes are given in Table 2. The analysis laboratory will indicate the required quantity. There
will be a certain loss during homogenisation (approx. 5 g), which is accounted for in the
pooled weight.
If it is unlikely there will be enough sample material after dissection for fish under about
10 cm, an entire homogenised fish should be used. Experience shows that it is best to
homogenise the frozen fish with an Ultra-Turrax homogeniser or a glass and stainless steel
blender, rather than homogenising dried fish. Larger discrepancies are likely in the double
determinations on whole fish and on organs, due to the greater inhomogeneity at the outset.
Table 2 Sampling quantity and preferred organs for different analyses to achieve NOVANA
detection limits (min. quantity for individual determinations in parentheses)
Matrix
Liver
Hg including TS
Fillet
10 g (5 g)
Other metals including TS
10 g (2 g)
TBT
10 g (5 g)
PCB including Fat
10 g (5 g)
BDE
20 g (10 g)*
Dioxin
50 g (30 g)
PFOS
10 g (5 g)
Total quantity
65 g (25 g)
100 g (40 g)
*where both BDE and PCB are measured, this quantity may be halved
Dissection of otoliths
The calendar age of flounder is determined by counting the growth rings in the fish’s
otoliths. Remove the otolith carefully with a knife by cutting down in a straight line from the
gills and cutting the ear canal to reveal the otoliths. The otolith is placed in a labelled
container, e.g. in designated paper envelopes or in a glass or plastic container. It may be
difficult to find the otoliths, and there is a risk of cutting into them while attempting to
locate them.
Dissection of the liver
When dissecting the liver, ensure that it is not contaminated by other organs such as the gall
bladder, which may contain higher concentrations of certain substances. The entire liver
must be homogenised (see next section) before any subsamples can be extracted.
For metal analyses, the liver may also be freeze-dried before it is homogenised (see next
section) and before any subsamples can be extracted.
For flounder/plaice, use the livers from 10 individuals to analyse for PCB and chlorinated
pesticides, and analyse a pooled sample of 10 livers from fish in the same size interval for
metals, BDE and perfluorinated compounds. BDE may be analysed in a blend of the 10
individual samples extracted for PCB. As far as possible, extract female fish for analysis for
PCB and fish of the same sex for the blend. This may require significantly more than 20
fish; if there are not enough females, some of the analyses may be carried out on a pool of
males. The prioritised substance groups to be analysed in males and females are determined
on the basis of the sample size collected for each sex. The results are to be delivered for
each sex.
For eelpout, analyse only the pooled samples from at least 25 males, and analyse the livers
for metals, PCB and other organochlorides, brominated flame retardants (BDE) and
perfluorinated compounds such as PFOS. If there are not enough males, some of the
analyses may be carried out on a pool of females. The prioritised substance groups to be
analysed in males and females are determined on the basis of the sample size collected for
each sex. The results are to be delivered for each sex.
For the pooled samples, there must be approximately 25 g of liver in total, which should be
divided after homogenisation into 5 g each for the PFOS, TBT and metal analyses and 10 g
each for the BDE and lipid analyses.
Dissection of muscle tissue from marine and freshwater fish
This procedure is common to marine and freshwater fish. For fish from which other organs
also need to be extracted, the muscle tissue is extracted after extracting samples from the
internal organs to avoid contamination of the internal organs. It is therefore necessary to
prevent the skin around the sampling area from coming into contact with these as far as
possible.
Extraction of muscle samples from larger fish
If analyses are to be carried out for anything other than mercury in the fish, check in the
relevant technical instructions whether there are any additional special precautions that need
to be taken before beginning dissection.
Dissection
The fish should be dissected in a partially frozen (not fully thawed) state.
This makes dissection easier and also prevents the internal organs (such as the liver and gall
bladder) from breaking and starting to disintegrate, which may contaminate the muscle
tissue and affect the results of the analyses.
Extract a sample (minimum 10 g) of muscle tissue from the right dorsal muscle directly
below the first dorsal fin. Take care as far as possible to extract the tissue from the same part
of the dorsal muscle on each fish. This ensures optimum uniformity, since water and fat
content may vary significantly in different parts of the muscle tissue and this can therefore
affect the concentration of the substances to be measured. Avoid getting epidermal or
subcutaneous fat into the sample (OSPAR 2012, HELCOM COMBINE 2008), as the
concentration in this may differ from that in the muscle tissue. The sample should therefore
be extracted below the dark-coloured outer part of the muscle.
Freeze-dry and homogenise the extracted muscle tissue samples. Keep the samples from
each fish separate. Freeze a subsample for possible lipid analysis for normalisation, without
freeze-drying. If there is not enough muscle tissue on the right-hand side, the left-hand side
may also be extracted; note this for the sample. Alternatively, for smaller fish, the entire fish
may be used.
Supplement the mercury analysis by measuring the dry matter percentage. Since the analysis
is often carried out on a dried sample, it is important to check for loss of mercury, which is
volatile.
For marine fish, 10 livers and associated muscles are to be analysed for metals (Hg
analysed in muscle), and 10 livers are to be analysed for PCB. Other organic subjects should
be analysed in pooled samples of fillets from 25 fish of the same size. Note that it is
necessary to collect more than the required 20 fish for the analysis if the sex of the fish
cannot be determined before dissecting. If the liver is large enough for all analyses to be
carried out in the same liver, this is preferable.
Avoid contamination of the tissue samples
The dissection must be carried out under conditions that are as clean as possible to avoid
contamination of the sample, preferably in a so-called clean cabinet (laminar flow cabinet),
in which the air is filtered for particles through a filter. The work should therefore be carried
out in the laboratory that will be carrying out the analysis.
Use a clean stainless steel scalpel and colourless forceps made from polyethylene or teflon.
Wear talc-free gloves (talc can contain metals); use AnsellEdmont nitrile gloves, for
example.
Rinse the scalpel/forceps between each sample as follows: wash in acetone or 96% ethanol
and then rinse with demineralised water (Milli-Q water or equivalent quality).
New instruments made from stainless steel may be coated with an adhesive layer. To
remove this, they must be treated either in a furnace at 460°C for a few hours or at 250°C
for 24 hours. If this is not possible, clean the instrument carefully using washing-up liquid,
then rinse it in plenty of demineralised water (Milli-Q water or equivalent quality). The use
of sterile scalpels is recommended, as they are packaged individually without adhesive. Do
not use acid bath as this will corrode the stainless steel and produce metal contamination
when used.
Analysis of whole small fish (sticklebacks etc.)
Dry the fish or rinse it with Milli-Q water to ensure that there is no dust on the fish before
homogenising. Carry out the homogenisation itself in a blender or with an Ultra-Turrax
homogeniser before freeze-drying. Ball milling may be carried out after freeze-drying of the
entire fish in order to re-homogenise it before extracting samples for analysis.
Storing samples before analysis
The dissected samples or whole fish must be stored in a dark place and deep-frozen (at 20°C) or freeze-dried in ordinary plastic bags. Samples for mercury analysis can be stored
deep-frozen or freeze-dried for up to a year. In the case of frozen samples, check that the
internal organs are intact during dissection; if they are not, the sample may be compromised
and the result must be marked as such.
Annex 1 to Specific conditions Supplemental description of method for the analysis parameter Soil Base
Saturation in accordance with the “Common working methods for soil analysis” (“Fælles arbejdsmetoder for
jordbrugsanalyser”), the Danish Plant Directorate 1994.
METHOD 10
EXCHANGEABLE
HYDROGEN IONS
“Exchangeable hydrogen ions” means the number of meq H+ released in 100 g of soil when
brought into equilibrium with a 0.06 M m-Nitrophenol solution with so much Ca(OH)2 added
that, in its equilibrium state, it is 0.023 N in terms of CA-m-Nitrophenolate and has a pH
value of approx. 8.1.
This figure is determined as the difference in acid consumption on titration with HCl of the
pure phenolate solution and an aliquot part of the filtrate from the soil suspension to a pH of
approx. 4. The difference thus found is a measure of the amount of H + released by the soil
suspension.
A.
Reagents
1) Calcium hydroxide solution (hydrated lime), 0.030
and 0.025 N
100 g Ca(OH)2 are poured into a 2-litre glass-stoppered flask. The flask is filled with water,
shaken a few times and left for any solids to settle. The clear solution of Ca(OH) 2
which is approx.
0.04 N, is drawn off by pipette, and its normality is determined by titration with
0.05 N HCl with bromocresol green (reagent 5) as the indicator. The solution is
then diluted with enough water for normality to be exactly 0.030. Another
solution of 0.025 N is obtained in a similar manner.
2) Buffer solution I
8.34 g m-Nitrophenol, C6H5O3N are diluted in a 1-litre graduated flask in 0.030 N Ca(OH)2.
The flask is filled to the mark with 0.030 N Ca(OH)2. The solution will have a
pH value of approx. 8.33 and be 0.06 N in terms of m-Nitrophenol.
3) Buffer solution II
8.34 g m-Nitrophenol, C6H5O3N are diluted in a 1-litre graduated flask in 0.025 N Ca(OH)2.
The flask is filled to the mark with 0.025 N Ca(OH)2. The solution will have a
pH value of approx. 8.16 and be 0.06 N in terms of m-Nitrophenol.
4) Hydrochloric acid, 0.05 N
5) Bromocresol green, 0.1% solution
0.1 bromocresol green is diluted in 100 ml of 96%
ethanol. B.
Performing the analysis
Transfer two 5 g portions of soil to two 300 ml glass-stoppered conical flasks marked 1 and 2
respectively. Add 100 ml of buffer solution I to flask
1, and add 100 ml of buffer solution II to flask 2. The flasks are shaken overnight in a
mechanical rotating shaker.
The next day the suspensions are filtered. Filter immediately after removing the samples from
the shaker, placing them in plastic flasks that are hermetically sealed until titration can
be completed. Please note: Titration must be done as soon as possible after filtering, as
any delay causes the samples to change.
Extract 25 ml of each filtrate and add to this 3 drops of bromocresol green (reagent 5) and
titrate with
0.05 N HCl (reagent 4). The colour of the solution will change during titration from
brown to yellow and green to a strong emerald green shortly before reaching the
equivalence point. At the equivalence point, the solution turns pale yellow with a green
hue, and the colour does not change if more acid is added. The acid consumption may
also be determined precisely using electrometric pH determination and plotting a
titration curve. The quantity in ml of 0.05
N HCl used for titration is termed b1 and b2 respectively. Then titrate 25 ml of each of
the two buffer solutions in the same way. The quantity in ml of 0.05 N HCl used is
termed a1 and a2 respectively.
C. Calculation
Two approximate values, H1 and H2, emerge from the titration results pertaining to the soil
sample’s content of exchangeable hydrogen. The values are calculated using the
following equations:
H1 = 4 (a1 - b1)
H2 = 4 (a2 - b2).
The actual content, H 0, of the sample is determined using the two samples as follows: Plot the
calculated H values as a function of the b values in a rectangular coordinate system. Plot
the two points (b1H1) and (b2H2), and connect the points by a straight line. The ordinate
for the point on the line with the abscissa 11.5 will then indicate H 0, i.e. the sample’s
content of exchangeable hydrogen expressed in terms of meq per 100 g of soil.
When analysing soil with little or unusually high exchangeable hydrogen content, b 1 and b2
may end up on the same side of the abscissa value 11.5 and far enough away from this as
to make extrapolation uncertain. A third determination will then be needed, using 10
and 3 g of soil respectively. The factors to be used when calculating the H values will
then be 2 and 6.67 respectively.
March 1994
Common working
methods for soil analysis III,
15 page1
Literature
Piper, C. S.: Soil and Plant Analysis. – Adelaine 1950, pp. 98–112.
Piper, C. S.: Exchangeable Hydrogen in Soils. – Journal of the Council for Scientific and
Industrial Research, vol. 9, 1936, pp. 113–124.
METHOD 15
POTASSIUM CONTENT
The exchangeable potassium ions in the soil are released by extraction with ammonium
acetate solution. The potassium content of the extract is determined using flame photometry.
A. Reagents
1) 150 mM LiCl
6.3585 g LiCl filled up to 1 litre.
2) 1 M ammonium acetate solution, 6 mM LiCl
77 g CH3COONH4 are diluted in water. Add to this 40 ml of reagent 1 and fill up to 1
litre.
3) 0.5 M ammonium acetate solution, 3 mM LiCl
192.5 g CH3COONH4 and 100 ml of reagent 1 are diluted with water up to 5 litres.
4) 0.01 M calcium acetate solution
3.2 g Ca(C2H3O2)2 are diluted in water up to 2 litres.
5) Standard solutions
a) Stock solution
0.1907 g KCl are diluted in 0.01 M calcium acetate (reagent 4) up to 1 litre. The
solution contains 100 mg K/litre. The solution forms the basis of the standard
solutions according to the table below using 200 ml graduated flasks.
March 1994
Common working
methods for soil analysis III,
15 page2
b) Standard solutions
No.
1
2
3
4
5
Stock solution Reagent 4
ml
ml
0
100
10
90
20
80
30
70
40
60
Reagent 2
ml
100
100
100
100
100
mg K/l
Kt
0
5
10
15
20
0
5
10
15
20
B. Specialist equipment
One flame photometer.
C. Standard curve
The 5 standard solutions are measured using the flame photometer at 768 nm. By plotting mg
K/litre as the abscissa and the galvanometer reading as the ordinate, a standard curve is
described that indicates the correlation between the galvanometer reading and the
potassium content of the solutions in mg K/litre. Once the analysis set out below is
performed, it also indicates the correlation between the reading and potassium content,
where the various standard solutions then correspond to the potassium content figures
above.
The standard curve must be checked regularly and tested often during analysis with a single
standard solution to ascertain whether the reading used in designing the standard curve
is obtained.
D. Performing the analysis
Transfer 10 g of soil to a 300 ml glass-stoppered conical flask to which 100 ml of reagent 3 are
added. The flask is shaken for half an hour using a mechanical shaker. After leaving
the flask to stand overnight, shake the flask by hand and filter the contents using a 15
cm potassium-free filter.
Then measure the filtrates using the flame photometer. Take a minimum of 2 independent
measurements from each extract.
E. Calculation
The potassium content, Kt, indicating in mg the exchangeable K per 100 g of soil, is read from the
standard curve.
March 1994
Common working
methods for soil analysis III,
15 page3
The unit corresponds to 1 mg K/100 g of soil or approx. 25 kg K per hectare of topsoil (20 cm)
on standard arable land.
As calcium ions affect the intensity of the “potassium colour”, and given that this effect
increases as flame temperature increases, calculations should be made using as low a
flame temperature as possible (preferably using a propane/air flame).
Literature
Aslyng, H.C.: Ombytteligt kalium i jorden. – Tidsskrift for Landøkonomi, 1953.
Mogensen, Th.: Kaliumtallet (Ombytteligt kalium i jorden). – Hedeselskabets tidsskrift 1953.
March 1994
Common working
methods for soil analysis III,
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METHOD 16
SODIUM CONTENT
The exchangeable sodium ions in the soil are released by extraction with ammonium acetate
solution. The sodium content of the extract is determined using flame photometry.
A. Reagents
1) 150 mM LiCl
6.3585 g LiCl filled up to 1 litre.
2) 1 M ammonium acetate solution, 6 mM LiCl
77 g CH3COONH4 are diluted in water. Add to this 40 ml of reagent 1 and fill up to 1
litre.
3) 0.5 M ammonium acetate solution, 3 mM LiCl
192.5 g CH3COONH4 and 100 ml of reagent 1 are diluted with water up to 5 litres.
4) 0.01 M calcium acetate solution
3.2 g Ca(C2H3O2)2 are diluted in water up to 2 litres.
5) Standard solutions
a) Stock solution
0.1271 g NaCl are diluted in 0.01 M calcium acetate (reagent 4) up to 1 litre. The
solution contains 50 mg Na/litre. The solution forms the basis of the standard
solutions according to the table below using 200 ml graduated flasks.
March 1994
Common working
methods for soil analysis III,
16 page2
b) Standard solutions
No.
1
2
3
4
5
Stock solution Reagent 4
ml
ml
0
100
10
90
20
80
30
70
40
60
Reagent 2
ml
100
100
100
100
100
mg Na/l
Nat
0
2.5
5
7.5
10
2.5
5
7.5
10.5
B. Specialist equipment
One flame photometer.
C. Standard curve
The 5 standard solutions are measured using the flame photometer at 589 nm. By plotting mg
Na/litre as the abscissa and the galvanometer reading as the ordinate, a standard curve is
described that indicates the correlation between the galvanometer reading and the
sodium content of the solutions. Once the analysis set out below is performed, it also
indicates the correlation between the reading and sodium content, where the various
standard solutions then correspond to the sodium content figures above.
The standard curve must be checked regularly and tested often during analysis with a single
standard solution, to ascertain whether the reading found when designing the standard
curve is obtained.
D. Performing the analysis
Transfer 10 g of soil to a 300 ml glass-stoppered conical flask to which 100 ml of reagent 3 are
added. The flask is shaken for half an hour using a mechanical shaker. After leaving
the flask to stand overnight, shake the flask by hand and filter the contents using a
sodium-free filter.
Then measure the filtrates using the flame photometer. Take a minimum of 2 independent
measurements from each extract.
E. Calculation
The sodium content, Nat, indicating in mg the exchangeable Na per 100 g of soil, is read from the standard
curve.
March 1994
Common working
methods for soil analysis III,
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The unit corresponds to 1 mg Na/100 g of soil or approx. 25 kg Na per hectare of topsoil (20
cm) on standard arable land.
As calcium ions affect the intensity of the "sodium colour", calculations should be made using
as low a flame temperature as possible (preferably using a propane/air flame).
Sodium determination is often performed in soils that have previously been subject to
saltwater flooding, with potentially very high sodium content as a result. Therefore,
when analysing such soils, it is appropriate to use stronger standard solutions (as
opposed to diluting the soil extractions) and, if necessary, reduce the flame photometry
sensitivity level.
Determination of the sodium content alone is only of interest in exceptional cases. Usually,
the main concern is to establish the proportion of exchangeable sodium ions as a
percentage of the soil’s exchangeable metal cations.
When determining the sodium content of the soil, the
extent of relative sodium content =
(Na)
x 100 (exchangeable
metal cations) should therefore
also be calculated.
In the formula above, (Na) means the soil’s content of exchangeable sodium measured in
meq/100 g of soil, i.e. Nat divided by 23. The denominator is the sum of exchangeable
metal cations, i.e. Ca + Mg + K in meq/100 g of soil, cf. method 12.
Literature
Benjaminsen, J. & Jens Jensen: Flammefotometrisk bestemmelse af det ombyttelige sodium i
jord. – Tidsskrift for Planteavl, bd. 60, 1956, pp. 43–58.
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METHOD 17
MAGNESIUM CONTENT
The exchangeable magnesium ions in the soil are released by extraction with ammonium
acetate solution. The magnesium content of the extract is determined using atomic absorption
spectrophotometry.
A. Reagents
1) Ammonium acetate solution, 1 M
77 g CH3COONH4 are diluted in water up to 1 litre. The solution pH must be between 6.5
and 7.3.
2) Ammonium acetate solution, 0.5 M
1 litre of 1 molar ammonium acetate solution (reagent 1) is diluted with water up to 2
litres.
3) Lanthanum chloride solution
6.0 g LaCl3,7H2O are diluted in water up to 1 litre.
4) Standard solutions
a) Stock solution
0.507 g MgSO4,7 H2O are diluted in 0.5 M ammonium acetate (reagent 2) up to 1
litre. This solution contains 50 ppm Mg.
b) Standard solutions
Pipette portions of 10, 20, 30 and 40 ml respectively from the standard solution and
transfer these to separate 100 ml graduated flasks, diluting them with the
ammonium acetate solution (reagent 2) to the mark. The solutions contain 5, 10,
15 and 20 ppm Mg. Pipette 10 ml from each of the 4 solutions and transfer these
to individual 100 ml graduated flasks. Transfer 10 ml of the ammonium acetate
solution (reagent 2) to a fifth 100 ml graduated flask. Fill the 5 graduated flasks
to the mark with the lanthanum chloride solution (reagent
3). The 5 standard solutions produced contain 0, 0.5, 1.0, 1.5 and 2.0 ppm Mg
respectively.
March 1994
Common working
methods for soil analysis III,
17 page 2
B. Specialist equipment
One atomic absorption spectrophotometer.
C. Standard curve
The standard solutions are atomized using an air/acetylene flame in an atomic absorption
spectrophotometer, determining the absorption of the magnesium content at 285 nm.
D. Performing the analysis
a) Producing soil extract
Transfer 10 g of soil to a 300 ml glass-stoppered conical flask. Add 100 ml of ammonium
acetate solution (reagent 2). A 0.5 ammonium acetate solution, which is also
0.003 M in terms of lithium chloride, may be used instead of reagent 2 (see
method 15, reagent 3). The flask is placed in a mechanical shaker and rotated
every half hour. After leaving the flask to stand overnight, filter the contents
using a dense magnesium-free filter.
b) Measuring the magnesium content of the extract
Transfer 5 ml of filtrate to a 50 ml graduated flask. Fill the flask to the mark with
lanthanum chloride solution (reagent 3) and mix. Then measure the absorption
of the solution as indicated for the standard solutions. Using the measured
absorption and the standard curve, read off the magnesium content of the
solution in ppm.
E. Calculation
The soil sample’s magnesium content, Mgt, which indicates exchangeable Mg/100 g of soil in
mg, is calculated by multiplying the magnesium content in ppm, as read off above, by 10.
The unit corresponds to approx. 25 kg Mg per hectare of topsoil (20 cm) on standard
arable land.
Literature
Henriksen, Aage: Sammenlignende kompleksometriske og atomabsorptiometriske
magnesiumbestemmelser i jord. – Tidsskrift for Planteavl, vol. 69, 1965, pp. 328-333.
Jensen, Jens: Bestemmelse af ombytteligt Ca og Mg. Beretning nr. s. 1675 fra Statens
Planteavlsforsøg pp. 1–18, 1983.
March 1994
Common working
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METHOD 18A
CALCIUM CONTENT IN CALCIUM-FREE SOIL
The exchangeable calcium ions in the soil are released by extraction with ammonium acetate
solution. The calcium content of the extract is determined using atomic absorption
spectrophotometry.
NB: The method cannot be used where the soil contains identifiable amounts of calcium
carbonate
(effervesces clearly with acid).
A. Reagents
1) Ammonium chloride solution, 1 M
53.5 g NH4Cl are diluted in water up to 1 litre.
2) Lanthanum chloride solution
6.0 g LaCl3,7H2O are diluted in water up to 1 litre.
3) Standard solutions
a) Stock solution
1.2485 g CaCO3 are diluted in 30 ml 1 N HCl and filled up to 1 litre with 1 M
ammonium chloride (reagent 1). This solution contains 500 ppm Ca.
b) Standard solutions
Pipette portions of 10, 20, 30 and 40 ml respectively from the standard solution and
transfer these to separate 100 ml graduated flasks diluting them with the
ammonium acetate solution (reagent 1) to the mark. The solutions contain 50,
100, 150 and 200 ppm Ca. Pipette 10 ml from each of the
4 solutions and transfer these to individual 100 ml graduated flasks. Transfer 10
ml of the ammonium chloride solution (reagent1) to a fifth 100 ml graduated
flask.
Fill the 5 graduated flasks to the mark with the lanthanum chloride solution
(reagent 2). The 5 standard solutions produced contain 0, 5, 10, 15 and 20 ppm
Ca respectively.
B. Specialist equipment
One atomic absorption
spectrophotometer. C. Standard
curve
March 1994
Common working
methods for soil analysis III,
17 page 2
The standard solutions are atomized using an air/acetylene flame in an atomic absorption
March 1994
Common working
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17 page 3
spectrophotometer, determining the absorption of the calcium content at 422.7 nm.
A standard curve is described using the calcium content of the solution in ppm as abscissa and
the absorption as ordinate.
D. Performing the analysis
a) Producing soil extract
Transfer 10 g of soil to a 300 ml glass-stoppered conical flask. Add 100 ml of ammonium
chloride solution (reagent 1), and place the flask in a mechanical shaker
rotating it for an hour.
After leaving the flask to stand overnight, filter the contents using a dense
calcium-free filter.
b) Measuring the calcium content of the extract
Transfer 5 ml of filtrate to a 50 ml graduated flask. Fill the flask to the mark with
lanthanum chloride solution (reagent 2) and mix. Then measure the absorption
of the solution as indicated for the standard solutions. Using the measured
absorption and the standard curve, read off the calcium content of the solution
in ppm.
E. Calculation
The soil sample’s calcium content, Cat, which indicates exchangeable Ca/100 g of soil in mg, is
calculated by multiplying the calcium content in ppm, as read off above, by 10.
The unit corresponds to approx. 25 kg Ca per hectare of topsoil (20 cm) on standard
arable land. Calcium content greater than 100 is indicated without decimals.
METHOD 18B
CALCIUM
CONTENT
CALCAREOUS SOIL
IN
Calcareous soil is extracted twice using sodium chloride solution. The calcium content of the
extracts is determined using atomic absorption spectrophotometry. The first extract contains
the exchangeable calcium ions (now released). Both extracts contain calcium ions from the
diluted calcium carbonate. The amount of exchangeable calcium is found as the difference.
NB: This method should only be used if the soil effervesces clearly
with acid
A. Reagents
1) Sodium chloride solution, 1 M
58.5 g NaCl are diluted in water up to 1 litre.
2) Lanthanum chloride solution
6.0 g LaCl3,7H2O are diluted in water up to 1 litre.
3) Standard solutions
a) Stock solution
0.6243 g CaCO3 are diluted in 15 ml 1 N HCl and filled up to 1 litre with 1 m sodium
chloride
(reagent 1). The solution contains 250 ppm Ca. b)
Standard solution
Pipette portions of 10, 20, 30 and 40 ml respectively from the standard solution and
transfer these to separate 100 ml graduated flasks, diluting them with the
sodium chloride solution to the mark. The solutions contain 25, 50, 75 and 100
ppm Ca. Pipette 20 ml from each of the 4 solutions and transfer these to
individual 100 ml graduated flasks. Transfer 20 ml of the sodium chloride
solution (reagent 1) to a fifth 100 ml graduated flask. Fill the 5 graduated flasks
to the mark with the lanthanum chloride solution (reagent 2). The 5 standard
solutions produced contain 0, 5, 10, 15 and 20 ppm Ca.
B. Specialist equipment
One atomic absorption spectrophotometer.
C. Standard curve
The standard solutions are atomized using an air/acetylene flame in an atomic absorption
spectrophotometer, determining the absorption of the magnesium content at 422.7 nm.
A standard curve is described using the calcium content of the solution in ppm as abscissa and the
absorption as ordinate.
D. Performing the analysis
a) Producing soil extract
10 g of soil are placed in a glass-stoppered conical flask with 75 ml of sodium chloride solution
(reagent
1). After leaving the flask for 2 hours and shaking it regularly, filter the soil using
filtration at reduced pressure and leaching with a further 225 ml of NaCl solution,
divided into portions of 15 ml each. This yields the “first filtrate”, to be collected
separately. Then the soil on the filter is rinsed using 300 ml of the NaCl solution – also
divided into portions of 15 ml each – yielding the “second filtrate”.
b) Measuring the calcium content of the extracts
Transfer 10 ml of each of the two filtrates to individual 50 ml graduated flasks. Fill the flask to
the mark with lanthanum chloride solution (reagent 2) and mix. Then measure the
absorption of the solution as indicated for the standard solutions. Using the measured
absorption and the standard curve, read off the calcium content of the solutions in ppm.
E. Calculation
The calcium content of the soil sample, Cat, indicating mg of exchangeable Ca/100 g of soil, is
calculated by multiplying the difference between the calcium contents in ppm, as identified in
the first and second filtrates, by 15.
The unit corresponds to approx. 25 kg Ca per hectare of topsoil (20 cm) on standard arable land.
Calcium readings greater than 100 are indicated without decimals.