Sub-Annex 3: 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 Eb, metals/inorganic trace elements and organic
hazardous substances in mussels and fish
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 1. 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 1. 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 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 2
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 2 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 3. 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 3 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.