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5.1 General principles for preventing fish poisoning

Everyone who manufactures, handles, or uses substances that can pollute the environment should regularly check their equipment and take the appropriate measures to prevent accidents; this is a moral obligation, which is also contained in the legislation of many countries. A strict discipline and a proper responsibility exercised by all those who are involved in every stage from manufacture to disposal provides the most effective, the most readily available and the cheapest means of pollution prevention.

The main method for preventing the chronic pollution of surface water is the installation of treatment plants, as are used for domestic sewage and for industrial effluents. In principle, all waste waters must be treated before they are discharged into the aquatic environment. These treatment processes may be simple; for example, the so-called biological oxidation ponds are used to intercept and biodegrade the organic wastes derived from agricultural production (livestock fattening and rearing facilities, including those for waterfowl) and from the food industries (slaughterhouses, poultry processing plants, dairy products, etc.). To a lesser extent these oxidation ponds may be used to intercept and degrade the sewage from residential areas, so long as such waste waters are not polluted with petroleum products, PCB, pesticides and other dangerous chemicals that are resistant to degradation.

However, oxidation ponds need to be extensive because of the large surface area required to allow sufficient oxygen to diffuse into the water. The amount of land required can be reduced by increasing the water surface area either by the use of stones or other media in trickling filters or by artificial aeration (the activated sludge process), both widely used in sewage treatment works. Substances that are not degraded by biological oxidation should be removed from waste waters by specific processes.

The removal of soluble harmful substances from waste waters usually results in the production of a precipitate or sludge. The disposal of these solids can cause problems if they are spread or contained on land and harmful substances (including by-products) can leach from them into surface waters. The environmental fate of such waste products needs to be carefully examined and appropriate steps taken to prevent aquatic pollution from their disposal.

At industrial sites, especially at those factories where there is a danger of leakage of oils and refined products or other very toxic substances into drains that discharge directly to surface waters, it may be necessary to build special leak-proof pits or containment walls to protect the nearby aquatic environment from contamination.

Apart from these direct discharges to water, considerable attention should be paid to some of the technological processes used in agriculture, in particular the spray application of chemicals, including fertilizers and pesticides, onto fields. Any direct drift of the chemicals to water areas during their application should be avoided and in particular precautions should be taken to prevent the subsequent leaching of the chemicals by rainfall into rivers and ponds. Such precautions are incorporated in the regulations set to control such applications (no chemical spraying in wet or windy weather, or 24 hours before rain is expected etc.). They are also reflected in some principles of land management; the use of grassy strips around water reservoirs, the use of appropriate crops (e.g. those not needing excessive pesticide application) and the proper tillage of sprayed fields. However, the improper disposal of surplus or unused pesticides, or the careless disposal of containers, is probably responsible for more pollution incidents than the proper use of pesticides on land.

It is required by current regulations that any new chemical must be subjected to a programme of toxicological testing and evaluation before it can be placed on the market. In particular, its potential for biological degradability, acute and chronic toxicity and/or teratogenicity and mutagenicity should be established. For pesticides, priority should be given to chemicals with a high target selectivity, a low active concentration, a low toxicity to non-target species and a rapid degradability. As stated earlier, the natural environment should not be allowed to become contaminated with toxic substances of low degradability.

The toxicity of substances, formulations and effluents to fish depends, first of all, on their chemical properties (e.g. their composition, water solubility, and pH), then on the sensitivity of the fish exposed to these substances. Within species, salmonids are generally the most sensitive, and cyprinids tend to be somewhat more resistant; within life stages, older fish tend to be more resistant than younger fish. Also important is the general state of health of the fish including the state of feeding of early fry and, finally the influence on toxicity of the water quality characteristics of the aquatic medium (temperature, pH, dissolved oxygen concentration, hardness, etc.).

5.2 Evaluation of the preparations and effluents

5.2.1 General principles

For fisheries management and fish culture, the most important property of any new product or chemical is its toxicity to aquatic organisms. Toxicity tests are designed to provide information on the potential harmfulness of the chemical at three levels of biological organisation:

  1. cells and tissues
  2. organisms (individuals)
  3. biocoenoses (communities).

The cell- and tissue-level tests are often used to provide an explanation for the findings obtained from experiments conducted at the organism level. Their advantage is their small scale and good reproducibility but unfortunately the results obtained in vivo often differ considerably from those found in vitro. At the other end of the scale, tests on biocoenoses have the advantage that the toxic actions are studied in either the natural environment or with a model which can simulate reality with reasonable accuracy. However, such tests have their drawbacks; the changes in community composition may not always be a consequence of direct toxic action on a given species but may be due to a disturbance in the food chain or other natural factors. The reproducibility of such tests is often very limited because their wide natural variability makes it difficult to obtain exactly the same conditions as those of preceding tests.

For these reasons, most of the studies are performed at the organism level, especially for acute toxicity tests. Though there are still problems of reproducibility, and errors can be made when extrapolating from these results to actual natural conditions, such tests represent a practical compromise and are generally acceptable to the fish culturist, industrialist and economist, and to a lesser extent the conservationist.

A distinction is drawn between acute toxicity and chronic toxicity; hence, substances, preparations and/or waste waters (effluents) which have been subjected to acute toxicity tests and shown to have harmful properties are then tested for chronic toxicity. Almost all these tests are carried out at the organism level.

5.2.2 Acute toxicity tests

Determination of the acute toxicity of chemical substances, formulations and effluents to aquatic organisms is one of the main duties of those responsible for their production. The majority of the acute toxicity tests are performed on fish and selected aquatic invertebrates. A variety of methods of toxicity test methods have been standardized in different countries. Some of these procedures are recognized internationally, notably the standard methods published by the International Standards Organization (ISO), and the guidelines given by the OECD. For determining the acute toxicity of substances to aquatic organisms, the following ISO methods are now widely used:

-    ISO 6341 Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) issued in 1982

-    ISO 7346/1 Water quality - Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) - Part 1: Static method of 1984

-    ISO 7346/3 Water quality - Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 2: Semi-static method of 1984

-    ISO 7346/3 Water quality - Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 3: Flow-through method of 1984

-    ISO 5664 - Inhibition of algal growth.

These standard acute toxicity tests use the highly sensitive water flea Daphnia magna and the aquarium fish Brachydanio rerio; the procedures also allow for the use of some other fish species, e.g. Poecilia reticulata, Pimephales promelas, and Oryzias latipes. The basic data obtained is the LC50 for periods of 24, 48, 72 and 96 hours; for Daphnia the usual maximum exposure period is 48 hours. In order to complete the trophic levels of primary producers, herbivores and carnivores, acute toxicity tests are carried out with algae.

Apart from these standard methods of toxicity testing, laboratories worldwide have developed many modifications of these toxicological methods. In doing so, the laboratories have taken into account the prevailing natural and economic conditions in their country and in each particular region. Some countries have standardized the methods of determining the tests of acute toxicity of waste waters to freshwater fish (e.g. the USA, UK), others have introduced special tests for the determination of the toxicity of pesticides to fish (e.g. Japan, the USA). The national methods differ in the test organisms used, in the time of exposure, and in the type and temperature of the water used for dilution, but in the majority of cases the main output is the LC50 value as the most accurate of the measurements that can be made.

The standard methods used in the former Czechoslovakia, ON 46 6807 Acute Toxicity Test on Fish and Other Aquatic Organisms, have been derived from the above-mentioned ISO standards and OECD guidelines. In these methods (which include a “range-finding” test to precede the definitive test), the basic test organisms are Daphnia magna and Poecilia reticulata, and the auxiliary species include common carp, rainbow trout, Brachydanio rerio, Rasbora heteromorpha, Cyclopidae, Tubificidae; also, the larvae of the amphibians Xenopus laevis and Rana temporaria are used in special tests.

The selection of these aquatic organisms for acute toxicity tests should be matched to the purpose for which the tested substances are being evaluated. For example, when evaluating a substance or preparation for use in fish culture or for direct application to the aquatic environment, toxicity tests will have to be performed on a wide range of representative aquatic fauna. On the other hand, for classification of substances into categories, e.g. newly developed products for general use but on a limited scale, the basic compulsory tests on Daphnia magna and Poecilia reticulata may be sufficient. However, tests on other species, e.g. Rasbora heteromorpha may be required if the chemicals are to be exported.

In line with the internationally agreed methods, the Czechoslovak standard methods in ON 46 6807 recommend the use of artificially prepared dilution water. Further, they include the use of a reference substance, i.e. a control preparation, within the test. Deviations in the LC50s obtained for the reference substance will reflect the variability in the conditions under which the tests are performed and in the condition of the organisms tested. Large deviations may indicate that the test laboratory has not achieved the necessary accuracy and precision required for carrying out a standard method. Potassium dichromate (K2Cr2O7) is the most frequently used reference substance.

From the lowest value obtained for the 48h LC50, determined on the basis of ON 46 6807, a test substance or product is classified (in Czech Republic) into one of the following toxicity categories:

0-substances of almost no toxicity: substances in which the 48h LC50 is higher than 10 000 mg per litre,
1-substances of very low toxicity: substances in which the 48h LC50 is between 1 000 and 10 000 mg per litre.
2-substances of low toxicity: substances in which the 48h LC50 is between 100 and 1 000 mg per litre,
3-substances of medium toxicity: substances in which the 48h LC50 is between 10 and 100 mg per litre,
4-substances of high toxicity: substances in which the 48h LC50 is between 1 and 10 mg per litre,
5-substances of very high toxicity: substances in which the 48h LC50 is between 0.1 to 1 mg per litre,
6-substances of extreme toxicity: substances in which the 48h LC50 is less than 0.1 mg per litre.

The test substance or preparation is placed in the appropriate toxicity category on the basis of the 48h LC50 obtained for the most sensitive organism tested.

The acute toxicity test can also yield further data which should be reported: - LC5 (concentration that kills 5% of the individuals within the given period of time), formerly also referred to as the minimum lethal concentration;

-    LT50 (the time within which half of the individuals are killed at the given concentration of the substance), also referred to as the mean or median survival time;

-    Relation between the LT50 and the concentrations of the substance tested (the toxicity curve);

-    Description of changes in the behaviour of fish or other organisms during the test, obvious changes found in the fish from the patho-anatomic dissection, and visible effects on the aquatic invertebrates. Poecilia reticulata, Brachydanio rerio and Rasbora heteromorpha are examined for their ability to consume normal quantities of food at the end of the test.

5.2.3 Chronic toxicity tests

The maximum concentration of a substance in water which still allows the normal growth and reproduction of fish, (MAC, maximum admissible concentration) forms the basis for assessing the quality of the water supplied to fish culture facilities, for investigating the causes of fish damage or mortality, or for giving permits for the discharge of effluents (which are likely to contain several significant contaminants) to surface waters. The MACs are usually derived from the results of chronic toxicity tests.

A standard procedure for the use of chronic test data in the assessment of limiting toxic concentrations of substances, products and effluents has been proposed by Lesnikov (1976). Lesnikov defines the MAC as the maximum concentration of a substance or its metabolites in waters at which a continuous exposure has no adverse effect on:

  1. the hydrochemical regime of water courses, lakes and ponds and on the microorganisms,

  2. primary production in the above-mentioned water bodies,

  3. planktonic food organisms,

  4. the fish (including the eggs and fry during larval development as well as fish in higher age categories) and also the marketable value of the fish (the hygienic requirements).

The recommended procedure for determining the MAC is first to perform acute toxicity tests, then test for the rate of detoxification (e.g. by degradation) of the substances and their metabolites into less toxic products, and finally, longer-term observations based on the results of acute toxicity tests and detoxification tests. The MAC should then be derived on the basis of the parameter (e.g. the response of an organism) which is adversely affected by the lowest concentration of the substance or its metabolite. The three major hazardous properties of a chemical or product are its potential for toxicity, persistence and bioaccumulation. High values for any one of these alone may not cause the substance to be hazardous in practice; for example, a very high toxicity combined with a half-life of a few minutes will not present a high environmental risk. It is clear, however, that substances of high stability (i.e. a very slow decomposition into non-toxic compounds even at summer temperatures over six months), with a high bioaccumulation capacity (>1000 times) and with a MAC below 0.0001 mg per litre will be regarded as particularly dangerous.

Salmonids, usually up to one-year-old rainbow trout (about 12 cm length), are most frequently used for the chronic toxicity tests. The main parameters examined at the end of such tests are the physical condition or condition factor (ratio between length and weight) of the fish, the individual and total weight gain of the fish, the change in the odour and taste assessment of the flesh, and the extent to which toxic substances are accumulated in the fish body or specific organs. The supplementary parameters include the behaviour of the fish during the test, the patho-anatomical and histo-pathological picture, and the physiological, biochemical and haematological changes in the fish when the test is terminated. Together with the basic parameters studied, the histological examination of the organs and tissues at the end of long-term tests is one of the most important items for the evaluation of the results, because its findings usually represent the most sensitive response, and therefore form the basis for setting the MAC. Also, specific effects found can assist in the diagnosis of the cause of harm to fish in natural populations.

These principles are incorporated in the manual of methods by Svobodová and Vykusová (1991). This manual recommends that the duration of the chronic toxicity test be 90–100 days and that common carp and rainbow trout are used as experimental fish. A reasonably stable concentration of the test substance is maintained by transferring the fish into a fresh solution of the toxic substance every day, usually after they have been fed. The concentration at which there were no significant effects on the experimental fish, when compared with the control group, is taken as the maximum admissible concentration (MAC).

Among the aquatic invertebrates, chronic toxicity tests are mainly conducted with the water flea Daphnia magna. All the individuals of this species used in the test must be of the same age (3–7 days), which can be achieved by synchronized culture. The main test parameters are survival of the adults, release of the young from the brood sac, viability of the juvenile stages, and change in the biomass. Subsidiary parameters and criteria recorded during the toxicity tests are behaviour before and during mortality, condition of the gonads, contents of the brood sac, feeding (as shown by the content and colour of the intestine), body colour, and abundance and colour of fat droplets. A detailed description of this method is given in the OECD guidelines.

Water fleas can also be used for a reproduction toxicity test, based on the capacity of several successive generations to reproduce in a range of concentrations of the test substance. The parameters examined include the release of the young, the number of the young, their survival, and potential for parthenogenetic reproduction.

5.2.4 Other toxicity tests

Considerable efforts have been made in recent years to replace the lengthy and therefore costly chronic toxicity tests by other tests, which would be as sensitive but considerably shorter. The use of cell cultures is one of the promising methods. These tests are based on the observation of the direct toxic action of chemicals on primary cell cultures from different fish tissues or on stable cell lines (e.g. FMH, PG, RTG-2 etc). These tests are still being developed; at present, their main value would seem to be for screening tests, rather than for the identification of long-term effects.

The use of embryo-larval toxicity tests is also being examined (the technique developed by Birge et al., 1977, is regarded as an ISO method). Exposure of the embryo to the toxic substance is continued until the stage when the yolk sac is completely absorbed. Unfortunately, the initial results of experiments conducted in our laboratories do not confirm the claims for a high sensitivity of this technique; toxic concentrations are not much lower that those obtained with the acute toxicity test. To obtain a greater sensitivity, the duration was extended to include a period of starvation, in which the mortality rate of the experimental fish is compared to that of the control fish. This provided a slight improvement in sensitivity but even so the technique cannot completely replace the traditional chronic toxicity test.

It is clear, however, that no single chronic test technique will be appropriate for all types of toxic substances. Embryo-larval tests may be sufficient for those substances that can be readily detoxified by the fish (for which the MAC may be 1½ orders of magnitude lower than the 96 hour LC50), but they will be inappropriate for substances that are persistent and are highly bioaccumulated. For these substances, tests of long duration are required, perhaps with the feeding of contaminated food, and with fish of a reasonable size so that the accumulation and effects in different tissues and organs can be studied.

Another method which is becoming increasingly used particularly in marine toxicity tests is the bacterial bioluminescence inhibition technique (the Microtox test; ISO Standard N110/1988, a draft being prepared in France). Of course, this technique also has to be adapted to local conditions.

5.3 Persistence of substances in aquatic environment

In addition to toxicity, another important measure of the potential hazard of substances and products is their degradability in the aquatic environment. Such degradation may be by physical, chemical or biological processes; only biological degradation is considered here. Biological degradation involves a sequence of processes by which organic substances are broken down, metabolized or assimilated by micro-organisms. This may be measured by analyzing the processes involved in biodegradation (oxygen consumption, CO2 production (i.e. non-specific method similar to the 5 day BOD test), or by measuring directly the rate of loss of the test substances from the aquatic medium over a period of time (a specific method).

Czech and Slovak ichthyotoxicologists, conservationists and water management experts generally use the technique proposed by Pitter (1974) as a standard test to measure the biological degradability of organic substances. This is a single-step kinetic test performed in an open system using dilutions of test substance with a mixed culture of bacteria. The decrease of the amount of the test substance is measured in terms of the reduction of the chemical oxygen demand (COD), of the total organic carbon (TOC), or by other more specific reactions. The results are compared with a blank test and with the degradability of a standard substance. Environmental importance is attached not only to the extent to which the substance is broken down but also to the rate at which it is degraded. For practical reasons it is recommended that biological degradability should be expressed as the percentage of removal of COD or TOC during the course of the incubation period.

When the results of biological degradability tests are used to predict what will occur in natural conditions, it should be noted that the tests were originally developed to simulate the conditions existing in a sewage treatment plant. A number of other factors such as temperature, bacteria, pH, dissolved oxygen concentration in water, can influence the rate of degradation of the test substance under natural conditions away from the sewage treated plants. Nevertheless, it is generally true that a substance that is readily biodegradable in a non-adapted activated sludge from a sewage treatment works is also very likely to degrade rapidly under natural conditions; as stated earlier, a sewage treatment plant concentrates the natural biodegradation processes into a small area.

The techniques for measuring the residues of the various pollutants and their metabolites in the various compartments of the aquatic environment are not easy to perform except in highly specialized laboratories; for this reason, attention has been focused on the highly toxic substances and those which are not readily degradable (i.e. persistent). The residues can be measured directly by chemical analysis (e.g. for metals, DDT and its metabolites, HCH, PCB, triazines and others) and to a much lesser extent by bioassays (e.g. toxicity tests with Daphnia magna can be used to measure residues of organo-phosphate pesticides).

5.4 Legislation

Special legislation is now being developed in those countries where efforts are being made to maintain and improve the state of the aquatic environment. In addition, international conventions are being prepared and nationally adopted to promote the conservation of aquatic resources and the natural environment as a whole, especially for international rivers and marine areas.

The legislation of every country should include measures designed to prevent damage being caused to the aquatic environment by the action of various chemicals, products, waste waters and solid wastes. There are two types of legislation which can be used. The simplest is that based on liability; the person who causes the damage has to pay compensation to the person(s) affected by the damage, together with an extra financial penalty or even imprisonment to act as a deterrent. This legislation requires that the cause of the damage should be established, and much of the information in this document is given to show how this can be done in the case of pollution damage to fish. This is seldom an easy task as there may be several possible explanations for the cause of the damage. Nevertheless, the combination of careful research and the collection and analysis of case histories will provide a steady improvement in the ability to provide an accurate diagnosis.

The second type of legislation is that based on regulations. In the context of aquatic pollution, this includes codes of practice for the handling and disposal of chemicals, and the setting of MACs for specific chemicals in the water. These regulations require a comprehensive system of monitoring to ensure that they are properly enforced, and this continuing effort can be costly. However, the presumption is that the aquatic environment will have been damaged if the MAC is exceeded; therefore, the MAC must be accurately set and information given in this document shows how this can be done. The advantage of regulations is that they are preventative; the disadvantage is that they are costly to enforce, and are likely not to be enforced if the MACs are shown or thought to be too stringent.

In practice, most countries have a mixture of both types of legislation, the balance being determined by national, political and economic factors. However, whatever the type of legislation in force, it is essential that the duties of the producers and users of substances as well as those of the producers of wastes must be clearly defined. The penalties for violation or circumventing the legislation must be a sufficient deterrent, strict and regular inspections must be carried out, and there should be a strong emphasis on personal responsibilities.

The ultimate goal is clear: to reduce the number of pollution incidents in watercourses, to eliminate the sources of pollution, and to minimize the consequences of accidental discharges on aquatic life.

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