5. Control of Hygienic Quality of Fish from Point of View of Foreign Substances Content

(J. Máchová, Z. Svobodová, M. Hrjtmánek, M. Hrbková)

Health safety inspection of fish flesh: contaminants (Hg, pb, cd, PCB, DDT, and others) levels

The presence of contaminants, including sediments, in rivers, lakes and ponds, and generally the water quality in each particular area or place, are among the important factors that influence the health of fish and the quality (in hygiene terms) of the fish flesh.

To provide safe and good-quality food for the people, all meat, including fish flesh, must be evaluated for what is called hygienic quality.

The concept expressed by this term comprises several aspects:

From the point of view of surface water pollution, it is most important to evaluate the results of sensory assessment of the fish flesh and the result of the chemico-toxicological analysis, which is, at the same time, indicative of contamination of the aquatic medium.

The need to analyze food for contaminants arose from the tragical consequences of consumption of highly contaminated food. For example, a disease called Minimata occurred in Japan in the 1960's. The disease affected people's nervous system, induced mental disorders, paralysis of the limbs, lips and tongue, inco-ordination of motion, tremor of the hands, disturbed vision, and insomnia. The disease was found to have been caused by the consumption of fish whose flesh contained about 20 mg menthylmercury per 1 kg. Another example, again from Japan, is the disease called itai-itai (ouchi-ouchi), which caused pain in the abdominal area and limbs, deformation of the bones, decalcification and fractures. This disease was caused by the presence of cadmium used for the irrigation of rice and soya. Still another disease, called “oil diseases” or “yusho”, was caused by table oil contaminated with PCB (ca. 0.5 g per litre).

Another important factor that encouraged analysts to study food contamination was the availability of new analytical methods, which enable reliable detection of even very low contaminants concentrations in various materials, including food.

The worst food contaminants include those of a high toxicity and poor degradability (or no degradability at all). Chief among them are metals and chlorinated carbohydrates, so it is also in this report that attention is paid to them. We also mention and describe here the substances that are most frequently the cause of accidents in surface waters and which intensively change the sensory properties of the fish flesh, e.g. petroleum and its products.

Metals

Mercury

Mercury gets into water mainly with industrial effluents and atmospheric precipitation and very quickly passes into the bottom sediments. It accumulates there, usually in sulphite form. Elementary mercury and its organic and inorganic compounds are liable to methylation. The toxic products of this methylation (methylmercury) enter the food chains and accumulate in the aquatic organisms. Owing to the high accumulation capacity of mercury (the accumulation coefficient reaches the level of 10₄), its content successively increases with increasing levels in the food chain: the highest levels are recorded in the fish.

In Czechoslovakia, the maximum admissible concentration (MAC) of total mercury is 0.1 mg per kg of flesh in the non-predatory fishes and 0.6 mg per 1 kg of flesh in the predatory freshwater fishes. In the USA, Canada, Austria, Switzerland, Germany, Israel, Poland and Cyprus, the maximum admissible mercury concentration in the fish flesh is 0.5 mg Hg per 1 kg, in France 0.5 mg per 1 kg with a tolerance up to 0.7 mg, in the USSR 0.6 mg per kg, in the U.K. 0.5 to 0.7 mg per kg, in Italy 0.7 mg per kg, in Sweden, Finland and the Netherlands 1.0 mg per kg, and in Norway 1.5 mg per kg. FAO/WHO expert group on contaminants recommended that 0.3 mg per person be the still tolerable maximum mercury intake of mercury; methylmercury should not represent more than 0.2 mg within the maximum tolerable amount.

Mercury determination in biological material

Put 10 g of sample to a beaker and mineralize it by boiling for 10–20 min in a mixture of 11 ml of nitric acid and sulphuric acid (ratio 10:1) under a powerful reverse water cooler to prevent leakage of vapours. When 15–20 min elapses, cool the clear yellow solution, put it in a measuring vessel and add distilled water. The determination of the amount of mercury in the mineralization product is performed by flameless atom absorption spectrophotometry (the AAS “cold steam” method).

Mercury may also be determined on the TMA 254 instrument (trace mercury analyzer), developed at the University of Chemical Technology in Prague and produced in Czechoslovakia. The results obtained by the two methods compare well with each other.

Both the methods have a sensitivity of 0.001 mg per 1 kg.

Lead

In the aquatic medium, lead accumulates mainly in the bottom sediments where its level is usually four orders higher than in the water. Like mercury, lead is able, through the action of some micro-organisms, to produce organic methyl derivatives which accumulate in the aquatic organisms. However, as distinct from mercury, lead was not observed to accumulate in fish.

The highest admissible lead concentration in the fish flesh is 1 mg per kg in Czechoslovakia. In other European countries the limit lead concentrations in fish flesh range from 0.3 mg per kg (Denmark) to 2.0 mg per kg (the Netherlands).

Cadmium

In waters, cadmium is accompanied by zinc; it is also contained in industrial effluents. Waters that wash phosphate fertilizers from farm land are also a significant source of cadmium contamination. Like lead, cadmium was not found to significantly accumulate in aquatic organisms. In Czechoslovakia the maximum admissible concentration in fish is 0.05 mg per kg of fish flesh and 0.5 mg per kg in fish entrails. In other European countries this level ranges from 0.05 mg per kg (Denmark, the Netherlands, Great Britain) to 0.2 mg per kg of fish flesh (USSR). According to the FAO/WHO Expert Commission on Contaminants, the still tolerable cadmium intake per one person should not be higher than 400 to 500 microgrammes.

Determination of metals in biological materials

Sample mineralization: The so-called “dry method” is used. Dry a weighed porcelain or quartz dish to a constant temperature and weigh fresh tissue (about 10 g of muscle or 5 g of liver) in the dish. Dry the samples to constant weight at 105°C and determine dry matter content. Moisten the dry sample with 5 % NH₄NO₃ p.a., dry it at 105°C and leave the dry sample to incinerate on an electrically heated plate. Use a platinum rod to carefully crush the carbonized sample and incinerate it in the Mufle oven at a temperature of 45°C (for cadmium or lead determination) or 550°C (chromium determination) at the maximum. The ash obtained should be white to greyish in colour. If it is not, moisten it again with NH₄NO₃, dry it and incinerate repeatedly until the ash is white or greyish. Dissolve the ash in a mixture of HCl and HNO₃ and evaporate it on water bath until it is almost dry. Dissolve the evaporation residue in 10 ml HNO₃ and put the solution into measuring beakers (volume 25 to 10 ml), using distilled water. If part of the evaporation residue remains on the dish, warm it carefully and then put all the sample into the measuring vessel.

Blank determination is performed in the same way as with all the other samples (incineration in the Mufle oven for about and hour).

The concentration of metal in the mineralization product is determined by atom absorption spectrophotometry.

A quicker and simpler method of sample mineralization is to use a mineralizer. The Research Institute of Fish Culture and Hydrobiology at Vodňany has a mineralizer made by the Austrian company PAAR, which breaks the sample by means of a microwave system at a high pressure (80 barr) and at a temperature of about 300°C.

Chlorinated carbohydrates

Pesticides based on DDT and HCH

Pesticides based on chlorinated carbohydrates, including for example DDT, HCH and HCB, are among substances of a high cumulation capacity and low biological degradability. This is the reason why their use is strictly limited today; the use of DDT, for example, has been forbidden in Czechoslovakia since 1975. Nevertheless, residues of these substances and their metabolites are still often recorded in biological materials, owing to these pesticides poor degradability. Contents of these substances must be continuously monitored in various materials. They are lipophilous, accumulating in fats. Owing to this, the highest levels of residues occur in the high-fat fishes, e.g. eel.

The following hygienic limits, applied in Germany, are used for evaluation of the residues of these substances recorded in freshwater fish:

Polychlorinated biphenyls

Polychlorinated biephenyls are among the highest-stability compounds. They are used as part of the filling of power condensers, fillings in hydraulic equipments, are contained in lubricants, synthetic lacquers, dyes and plastics. The common brand names include Delor, Aroclor, Clophen, Kaneclor, Savol, Softol and others.

As a rule, numbers are added to the brand names to indicate the number of chlorine atoms (e.g. Delor 103 is a low-chlorine substance, Delor 106 is a high-chlorine substance). High-chlorine substances have a higher stability in natural environments than are PCBs with a low content of chlorine.

Owing to the warning data on the harmful effect of chlorinated biphenyls, the production of these substances was considerably restricted in Czechoslovakia in 1971 and in 1984 it was copletely forbidden. Nevertheless, like in the case of chlorinated carbohydrates, residues of polychlorinated biphenyls are still encountered. In each particular locality, their content in the fish depends mainly on the species of the fish, especially on the content of fat in their flesh. Therefore, high concentrations of PCBs residues occur in high-fat fishes such as the eel in which the fat content is as great as 20–30 %, unlike in the low-fat fishes, e.g. pike or perch having less than 1 % of fat in their muscle.

The hygienic concentration limit of PCBs level in fish is 0.5 mg per kg in the edible portion in Czechoslovakia, 2.0 mg per kg of flesh in the USA, Canada and Germany. The tolerated maximum daily PCBs intake is 1 μ of PCBs per 1 kg of human body weight.

Determination of polychlorinated biphenyls (PCBs) and chlorinated pesticides in fish flesh, sediments and water

Chlorinated carbohydrates are extracted by organic solvents, the extracts are purified on Florisil column and with sulphuric acid. Gas chromatography with an EC detector is used for the determination itself. The results of PCBs determination are expressed as the sum of Delor 103 and Delor 106 and the results of determination of chlorinated carbohydrates are determined as the sum of HCB, α-HCH, β-HCH, τ-HCH, p,p'-DDE, p,p'-DDD, p,p'-DDT.

Chlorinated carbohydrates are extracted from fish flesh and bottom sediment samples as follows: Weigh about 5 g of homogenized sample on analytical balance and extract it with acetone. Put the extract in distilled water and shake it into petrolether. Dry the petrolether layer with sodium sulphate, concentrate it and clean it on a Florisil column.

Depending on the nature of the sample, chlorinated carbohydrates are extracted from water samples by shaking 1 to 3 litre of water with petrolether. Shake the petrolether extract with anhydrous sodium sulphate, discharge it via an anhydrous sodium sulphate column, evaporate it to a small volume and clean it on a Florisil column. Use acetone to displace the intercepted chlorinated carbohydrates. Evaporate the acetone until the extract is dry and purify the extract again on a Florisil column or by means of concentrated sulphuric acid. The use of PCBs sorption on solid sorbent, e.g. small PRESEP columns (Separon S6 × 18), followed by washing with acetone, is also applicable for PCBs determination in water.

To determine chlorinated carbohydrates on gas chromatograph, the following conditions have to be provided:

1.PCBs:

The column: length 2 m, inner diameter 3 mm, filling 5 % DOW 200 on Varaport 30 carrier.
Carrier gas: nitrogen.
Operational temperatures: injector 240°C, column 225°C, detector 280°C.
Limits of determination:
fish flesh and sediment: 0.004 mg per kg D 103 and D 106; water: 0.01 μg per 1 litre D 103 and D 106.

2. Chlorinated pesticides:

The column: length 1.5 – 2 m, inner diameter 2–3 mm, filling: mixture of phases 3 % OV 17, 7.5 % OF 1, 3 % XE 60 at a ratio of 2:2:1, on Chromaton N-AW-DMCS as carrier.
Carrier gas: nitrogen.
Operational temperatures: injector 235°C, column 210°C, detector 250°C.
Limits of determination:
fish flesh and sediment: HCB, α-HCH, τ-HCH: 0.05 μg per 1 kg, β-HCH, DDE, DDD, DDT: 0.1μg per 1 kg;
water: HCB, α-HCH, τ-HCH: 0.0002 μg per 1 litre, β- HCH, DDE, DDD, DDT: 0.0005 μg per 1 litre.

Petroleum and the petroleum products

Petroleum and the products of its processing (gasoline, kerosene, diesel oil, mineral oils) are a group of substances responsible for an increasing extent and proportion of environment pollution. They are dangerous because of their toxicity to the aquatic organisms, but may also badly affect fish farming because even the presence of a very small amount of these substances in water (petroleum 0.025 mg per 1 litre, gas oil 0.01 mg per 1 litre, kerosene 0.01 mg per 1 litre, gasoline 0.002 mg per 1 litre) can spoil the sensory characteristics of fish flesh. The flesh gets a disagreeable "petrol smell" and its taste is spoiled. To remove the bad taste and smell, the fish must have been kept in clean water for several weeks before consumption; some sources even assert that these substances' half-life in the fish body is 400 to 700 days.

Phenols

Rivers, lakes and ponds may be polluted with phenols carried to the water with industrial effluents, especially the waste waters generated during the thermal processing of coal, effluents from petroleum refineries, from synthetic fabrics production plants and from other industrial operations.

When petroleum and its products get into contact with chlorine, offensively smelling chlorophenols develop, which induce sensory changes in the fish flesh even at very low concentration (0.02 mg per 1 litre). Phenols themselves cause sensory changes in fish flesh at and above a concentration of 0.1 mg per 1 litre.

Sensoric evaluation of fish flesh

Fish flesh is sensorically assessed according to Czechoslovak Standard CSN 46 6802 Marketed Freshwater Fish. The smell and taste of the fish are assessed in cases of fish flesh contamination with petroleum, its products and phenols. The flavour (smell) is first assessed in raw flesh as soon as the viscera are removed. Then the samples are boiled in steam (not by immersing them in water) with no ingredients, in a closed container on a water bath for 30 to 40 min. Every sample is boiled separately. When the required time elapses, the samples are evaluated as to their flavour and taste.

The sensory assessment should be performed by at least three persons with good senses of taste and smell. During work, the assessors are not allowed to smoke or eat other kinds of food.

Recommended literature

Cibulka et al. (1991): Lead, cadmium and mercury transfer in biosphere.Academia, Praha, in press (In Czech)

Halm J. (1980): Blei-, Cadmium-, Arsen- and Zinkgehalte von Fischen aus unbelasten and belasten Binnengewassern. Fleischwirtschaft, 60, 1076–1083.

Matyas Z. (1989): Hygiene and technology of frozen and fish products. SPN, Praha, pp. 53 (In Czech).

Nuorteva p. (1991): Bioaccumulation of mercury. Department of Environmental Conservation University of Helsinky, pp. 30

Schuler W., Brunn H., Manz D. (1985): Pesticides and PCBs in fish from the Lahn river. Bull. Environ. Contam. Toxicol., 34, 608–616.

Svobodová Z. et al. (1987): Toxicology of water animals. SZN, Praha, pp. 231 (In Czech).