The diagnosis of fish poisoning is a difficult and complicated task because there may be a delay in the discovery of the mortality, and the fish and water are then not sampled at the time when the pollution occurred. In such cases the patho-anatomic changes in the fish are obscured by the onset of post-mortem changes and the toxic conditions that caused the fish to be poisoned may have been carried away from the affected area with the water flow or, in the case of natural events, reverted to normal. Hence, it is necessary to use all the available information and all possible and relevant analytical methods to detect the cause of the harm to fish and, where appropriate, to aquatic invertebrates.
The analytical study should begin with an assessment of previous records of factors that might influence natural changes, and of recent discharges that may have been made, and then performing the necessary physico-chemical and hydrobiological analyses of the water. If necessary, the bottom sediments, the periphytes and then the fish themselves should be examined. Bioassays to measure whether the water has an acute toxicity is an important tool in the diagnosis of fish poisoning.
If the fish are observed to be behaving strangely or are dying, the following important actions should be taken at the site.
Define the area within which the fish are seen to die or change their behaviour.
Catch some of the affected or newly dead fish and submit them to veterinary examination as soon as possible. Recognized procedures should be used for their storage and/or preservation.
Record the status of the zooplankton, phytoplankton and benthic communities.
Take water samples for hydrochemical analysis (some of the analyses and measurements must be carried out at the site e.g. O2, temperature, pH, transparency, smell, colour etc), and for the bioassay for acute toxicity.
Draw a map of the affected area (and, if appropriate the surrounding areas) and record the site where water and sediment samples were taken. Fill in a form (an example is given below) for the in-situ examination.
If it is suspected that the fish might have died or changed their behaviour because of a discharge or use of a chemical in the vicinity of the affected water course or pond or lake, detailed information should be obtained of the time and method of the use and disposal, and of the identification and amount of the chemical applied.
The following checklist is an example of the information necessary to document a local investigation into the cause of abrupt changes in the behaviour or of mortality of fish.
| 1. | Day, hour and place of the investigation | ||||||
| 2. | Persons involved in investigation | ||||||
| 3. | Name and address of the organization that suffered the loss (e.g. the owner of the fishery) | ||||||
| 4. | Name or other identification of the watercourse or reservoir where fish were killed | ||||||
| 5. | Species and age of the fish that occur in the affected watercourse or reservoir | ||||||
| 6. | The number, species and age of fish killed | ||||||
| 7. | Clinical signs and macroscopic changes in those fish with a changed behaviour pattern | ||||||
| 8. | Present state of health of the fish in the watercourse or reservoir being investigated | ||||||
| 9. | Status of zooplankton | killed | yes | no | |||
| phytoplankton | killed | yes | no | ||||
| benthos | killed | yes | no | ||||
| aquatic plants | killed | yes | no | ||||
| 10. | Possible sources of pollution that might be associated with mortality of the fish | ||||||
| 11. | Preliminary estimate of the extent of the damage (length or area of watercourse or reservoir affected) | ||||||
| 12. | Total weight of the dead of each species of fish | ||||||
| 13. | Place, hour of sampling, description of samples and their purpose | ||||||
| water | |||||||
| sediments | |||||||
| periphyton | |||||||
| fish for examination: | |||||||
| - newly killed | |||||||
| - with clinical symptoms | |||||||
| - with no signs of intoxication or disease | |||||||
| 14. | Measurement of water quality in situ (including water temperature, pH, colour, transparency, smell, concentration of dissolved oxygen, and ammonia concentration) | ||||||
| 15. | Opinion of the participants in the site investigation on the cause of the damage | ||||||
| 16. | Signatures of local investigation participants | ||||||
If the fish are thought to have been killed by the use of a chemical in the vicinity of a water course or reservoir (e.g. a pesticide), the following data should also be included in the report of the site investigation:
Map: A sketch map of the location, indicating the area where fish had died and the places where water samples were taken.
Water sampling sites
1 - place where the greatest kill was observed - 10.00 h September 20th
2 - 60 m downstream of the sewer outlet - 10.15 h
3 - at the sewer outlet - 10.30 h
4 - 40 m upstream of the sewer outlet - 10.45 h
5 - about 3 km downstream of the sewer outlet - 11.45 h
6 - about 6 km downstream of the sewer outlet - 12.30 h
The choice of the right sampling sites and the correct water sampling method is the main prerequisite for a successful diagnosis, so this must be given maximum attention. In flowing waters, water sampling sites should be distributed as follows:
- 50 to 100 m downstream of a possible pollution source(s)
- at the place where the pollution source joins a watercourse stocked with fish
- 30 to 50 m upstream of the possible pollution source
In reservoirs and fish ponds, the places where water should be sampled should be located on the basis of each specific situation; in some cases the samples may have to be taken from different depths within the water column. Special sampling equipment (e.g. the Hrbáček displacement bottle, Fig. 6) are used to take water samples from different depths and from just above the bottom.
The samples are poured into clean 1- to 2-litre bottles. It is not recommended that bottles are filled with water taken from near the bank or shore; it is usual to take the samples from 1–2 m away from the water's edge. Water samples from near to the bottom must be taken with care in order not to include mud and other sediment. To obtain the maximum value in terms of analytical accuracy and usefulness of the data, the time between sampling and analysis must be as short as possible. Ideally, the samples should be transported in thermally insulated containers. In the laboratory the samples should be stored in refrigerators and kept at 3–4°C. However, these procedures may not be adequate to ensure the stability of some water parameters. In such cases, the samples must be analyzed as soon as they are collected, or they may be stabilized with a small amount of preservative. Detailed data on the preservation and treatment of the samples are given in Table 3.
Fig. 6: Hrbáček's displacement-type bottle for water sampling to determine the concentration of dissolved oxygen (Hrbáček et al. 1974)
Table 3: Preservation and treatment of water samples
| Water characteristics | Preservative (amount per 1 litre of sample) | Method of sample treatment |
|---|---|---|
| Temperature | - | Measure during sampling |
| Colour | - | 1. Determine during sampling 2. Store at 4°C - determine within 24 h |
| Transparency | - | 1. Determine during sampling (field determination) 2. Determine within 24 hours (laboratory determination) |
| Odour | - | Identify some smells during sampling (e.g. chlorophenol), others within 24 h at the maximum |
| pH | - | 1. Determine during sampling 2. Take sample in oxygen bottle - see Fig. 20, determine within 24 h |
| Oxygen capacity | - | 1. Determine during sampling 2. Take sample in oxygen bottle determine within 24 h |
| Dissolved oxygen (if DO probe is not used) | - | Take sample in oxygen bottle, fix after collecting winkler |
| Chemical oxygen demand CODMn, CODCr | a) 1 ml H2SO4 b) store at 4°C | Determine as soon as possible after sampling, within 24 h at the maximum |
| Biochemical oxygen demand (BOD5) | - | Maintain at 4°C, process within 24 h |
| Ammonia | a) 1 ml H2SO4 b) 3 ml CHCI3 | 1. Determine during sampling 2. Store at 4°C - determine within 24 h 3. Determine after preservation |
| Nitrates, nitrites | a) 1 ml H2SO4 | 1. Determine on the day of sampling 2. Maintain at 4°C - determine within 24 h 3. Determine after preservation |
| Chlorine | - | Collect into brown bottle, determine immediately after sampling |
| Cyanides | solid NaOH up to pH 11 at least | Determine immediately, or at least within several hours after sampling |
| Copper, zinc | 5 ml HNO3 | Cannot be preserved if sample contains cyanides |
| Iron (total) | 5 ml HNO3 | Completely filled bottles: prevent contact with air |
| Phenols | NaOH added to obtain pH 11 (about 4 g per litre) | 1. Determine within 24 h 2. Determine after preservation 3. No phenol preservation needed at phenol levels above 150 mg per litre |
| Tensides: anion active cation active nonionic | 3 ml CHCI3 | Collect to glass bottles, determine within 72 h |
| Petroleum and its products | The sample volume is 1 to 5 litres, depending on the pollution situation. Use glass bottles (never use polythene bottles). Avoid sampling the surface film of oil. Determine as soon after sampling as possible. | |
The basic chemical analysis of the water includes the determination of the colour, odour, pH, acid capacity (alkalinity), concentration of dissolved oxygen, chemical oxygen demand (COD), biochemical oxygen demand (BOD5), ammonia, nitrites, nitrates, phosphates and total phosphorus. The need to analyze for any other chemicals depends on the outcome of the local investigation into possible sources of pollution; the aim is to obtain chemical data which, together with ecotoxicological data, will identify the causes of the mortality or damage of the fish. When assessing the results of the physico-chemical analysis of water samples in order to identify causes of mortality, the parameters should not be evaluated in isolation; possible interactions have to be taken into account. The toxicity of the different chemicals and products to fish and aquatic invertebrates is influenced by the natural quality characteristics of the aquatic environment.
Chemical examination of the water is carried out on site during the field investigation and in the laboratory. For field analyses, a portable chemical laboratory, such as Hach or the Combi kit, produced by the Central Laboratories of the Fish Culture and Hydrobiology Research institute at Vodňany, Czech Republic, can be used. The Combi kit can be used for the following determinations: Secchi disc water transparency, concentration of dissolved oxygen, pH, alkalinity, ammonia and phosphates.
Concentrations of metals in the water are measured by the atomic absorption spectrophotometry (AAS). The gas and liquid chromatography methods are used for the determination of organic compounds, e.g. the active ingredients of pesticides, surfactants, organic dyes, PCBs.
Where required, benthic sediments may be sampled in addition to collecting samples of water. This is the case, for example, when there is suspicion of watercourse or reservoir pollution with petroleum products, metals, pesticides and other persistent substances which can accumulate in sediments.
There is no standardized method for sediment sampling; local conditions must always be taken into account. The main principle is to take the samples mainly from the top layer of the sediments, to take them at several sites within the locality investigated, and to thoroughly mix each sample before analysis. Benthic sediments are analyzed using modifications of the methods used for determination of pollutants in water and other (mainly biological) materials.
Hydrobiological examination of water is very important for a diagnosis of the poisoning of fish and lower aquatic organisms. This examination includes an evaluation of the qualitative and quantitative structure (at the individual, population and community levels) of the lower aquatic organisms in order to assess the extent to which they are damaged, and to record changes in behaviour of the fish, or the extent and duration of the mortality in their populations. Evidence that a specific group of poisons was responsible for the pollution can be obtained from the changes in the composition of the aquatic community after the incident. For example, crustaceans and insect larvae are very sensitive to insecticides, aquatic plants are sensitive to herbicides, algae to algicides etc. In cases of accidental pollution of watercourses and in reservoirs, effects on aquatic invertebrates are usually the first indicator of pollution of the aquatic environment, and the effects on fish are seen later. This is especially characteristic of the pollution of watercourses and reservoirs with pesticides and some metals. However, surface active compounds (e.g. surfactants) have a similar toxicity to fish and to aquatic invertebrates. On the other hand, fish are the main indicators of pollution where organic substances are accidentally discharged to watercourses or to reservoirs.
Periphyton form a mat consisting of an association of aquatic organisms (bacteria, fungi, algae, protozoans, bryozoans, rotifers and others) growing on, or attached to, surfaces such as the stems of higher aquatic vegetation, stones, structures built in water, and submerged logs. In surface waters, periphyton is an important source of food for many aquatic animals, including fish. It makes a significant contribution to the self-purification processes in rivers and lakes, and its quality and quantity provides an indication of the average quality of water at a specific site; short-term and minor changes in water quality usually exert an influence on the community structure of organisms which make up the periphytic mat. This is of great importance in water analyses. After an accidental discharge into a watercourse, mainly characterized as a volume of toxic water killing the free-living organisms and carrying them downstream, the damaged periphyton may provide information about the length of watercourse affected, and those upstream on the quality of water during the preceding period. Periphyton analyses are an important part of the general examination of water quality, particularly in places with flowing water where the analyses of single samples taken from single sampling sites fail to provide a true indication of the water quality over a period of time.
Periphyton samples are usually obtained by removing them from the different submerged surfaces with a pair of long tweezers. A scraper attached to a long handle will be of help in water of greater depths. The periphyton attached to underwater structures of concrete or other materials can be easily scraped by means of a stiff brush (such as used for washing laboratory glassware) attached to a long pole; the material attached to the bristles of the brush is then transferred into the sampling phial, using a pair of tweezers. In the laboratory, the periphyton samples are subjected to analysis by a microscope; the organisms present in the samples are identified and their abundance is recorded using a qualitative scale.
An investigation in the cause and tracing the source of a pollution incident in a water course or reservoir can include, besides the hydrochemical and hydrobiological analysis, a bioassay for water toxicity. The water used for the bioassay is taken from the water sample (free of preservatives) sent to the laboratory for physico-chemical analysis. The aquatic organisms used for the assay include aquarium fish, especially Poecilia reticulata, and aquatic invertebrates, especially the water flea (the genus Daphnia) which is one of the most sensitive of aquatic organisms to the majority of pollutants. Although Poecilia reticulata is not an extremely sensitive fish, its advantage is that like the majority of aquarium fish it is easily available all the year round.
The following procedures for the bioassay of water toxicity are generally used. Ten water fleas (Daphnia) or two or three aquarium fish (Poecilia reticulata) are put in 100–200 ml of the water sample. If a sufficient water sample volume is available, the assay is carried out simultaneously on both organisms. At the same time, the same number of organisms are put in uncontaminated water to act as controls. The best test vessels are, in our experience, crystallizing dishes where the water depth is shallow, allowing sufficient oxygen to diffuse from the air into the water. The behaviour and state of the organisms are monitored during the period of the bioassay, which may be from 24 to 48 hours.
If the result of the assay is negative (the behaviour patterns of the fish or water flea do not differ from the controls), it may be assumed with some confidence that the water sample tested does not contain toxic substances at acutely harmful or lethal concentrations. If the test organisms show changes in their behaviour or die, physico-chemical and/or other analytical methods should be carried out as soon as possible to find the cause of the poisoning.
Although a bioassay for water toxicity testing cannot provide a positive identification of the causative agents of the mortality of fish and other aquatic organisms, it can provide useful information for the diagnostic process, including locating the source of the pollution. Other aquatic organisms, e.g. rainbow trout, common carp or cyclops (an aquatic microcrustacean), may also be used for the bioassays, but these may not be available all the year round or, if they are, not at a convenient size. Rainbow trout and common carp have the additional disadvantage of requiring a large volume of sample for the bioassay and the water has to be aerated. It is a common principle for all water toxicity bioassays that the methodology can be varied: in specific cases they may reflect the actual field conditions at the time of the investigation, within the restraints of the facilities available at the laboratory.
The number of the fish that have to be examined and sent for diagnostic examination is not always the same. If there is any suspicion that fish might have been poisoned or suffocated by water pollution, samples of 3 to 5 fish are taken from each species that is common amongst those that are the dead or dying. If such a situation occurs in a reservoir (pond) with a single-species stock, the number of fish sampled should be between 5 and 20, depending on their weight, age and other particular circumstances.
The success of the examination depends on the state of the fish. It is useless to send fish that have started to decompose or are decayed. Ideally, the sampled fish should be showing clinical symptoms of damage and be delivered alive. If this is impossible, it is absolutely essential that the fish be fresh and intact when they reach the examining laboratory. They should never be sent in the water in which they have died. The fish sent for general examination should be free of preservatives (formalin, alcohol etc.) because these make the diagnosis impossible. In those cases when the fish are only sent for chemico-toxicological examination (i.e. for the tissue concentration of metals, residues of pesticides, and other pollutants), it is recommended that the samples should be frozen.
Fish that are sampled at the point of death, or those with clinical symptoms of damage, are subjected to a detailed health examination in order to eliminate infective or invasive diseases as a cause of the harm. If such diseases are eliminated, it is then necessary to identify those environmental factors which changed abruptly and caused the death of the fish or a change in their behaviour. The clinical symptoms given in the report of the local investigation are evaluated as the first stage of the examination; this is followed by the patho-anatomic dissection, and where necessary the organs and tissues of the fish are subjected to chemico-toxicological analysis. Further examinations can be performed if the information obtained shows that these are necessary.
Chemico-toxicological examination of the organs and tissues is one of the most definitive, and also the most difficult, of the methods used in the diagnosis of poisoning. Where there is a possibility that the poisoning has been caused by metals, or for the monitoring of metals in fish for human consumption, the chemico-toxicological examination is a justifiable requirement. Such examinations may also be carried out when phenol and pesticide pollution may be involved and, if certain conditions are met, chemicotoxicological analysis may help in the diagnosis of ammonia poisoning in fish. On the other hand, there are some substances (e.g. chlorine, hydrogen sulphide) for which appropriate analytical methods are not available.
The concentration of metals in fish organs and tissues is determined by atomic absorption spectrophotometry (AAS). An increase in the concentration of metals is most frequently recorded in the parenchymatous organs and in the gills of the fish. For example, acute copper poisoning can be diagnosed on the basis of chemical analysis of the gills, if the metal concentration has been significantly (several-fold) increased. The copper content in gills of fish in uncontaminated water is up to 10 mg per kg dry weight.
Fish poisoning by organo-phosphorus pesticides can be diagnosed either by direct measurement of these substances or their metabolites in the organs and tissues, or by indirect determination, based on the inhibition of acetylcholine hydrolase mainly in the brain of the fish. This method is applicable also to the diagnosis of fish poisoning by carbamate pesticides. Diagnosis of fish poisoning by other pesticides and also by other organic compounds may be based on the determination of these substances in the organs and tissues of the fish using gas chromatography.
It must be stressed at this point that most of the ecotoxicological information for chemicals relate to harmful concentrations in the water; there is very little information on harmful tissue concentrations. Therefore, the identification of a chemical in fish tissue does not prove that it was responsible for the damage, unless it can be shown that the concentration present has been associated with harm in carefully conducted laboratory experiments.
One of the chemico-toxicological methods that has been used in the diagnosis of poisoning is the detection of phenols in fish. The chemicals are determined in the flesh and skin of the fish by a photometric method using 4-amino-antipyrine after reflux distillation of the tissue sample.
Ammonia poisoning of fish can be diagnosed, if certain conditions are met (fish blood and brain sampled during toxic exposure or immediately after death, and analyses made within 48 hours at the latest on deep-frozen samples), by determining the ammonia nitrogen levels in the blood serum and brain homogenate. These levels can be readily determined using the Blood Ammonia Test kit, made by Hyland. However, the ammonia nitrogen level in the serum and brain of fish varies with their metabolic rate and will increase in response to various adverse factors, especially O2, deficiency. Because of this variability it is impossible to diagnose ammonia poisoning of fish merely by determining the ammonia N level in these specific tissues; the definitive diagnosis must be based on a thorough examination of the water quality and a detailed examination of the state of health of the fish.
The presence of petroleum and its products in fish can be easily detected by characteristic changes in odour and taint; the “petroleum smell” can be detected at concentrations as low as 0.01 mg per litre of water. Contamination of water by phenols and chlorophenols can be detected in the same way at or above concentrations of 0.1 mg per litre and 0.02 mg per litre respectively. This sensory method of detection needs no sophisticated laboratory equipment, and its sensitivity compares favourably with that of chemico-toxicological examination.
