Determination of toxicity to an invertebrate population.
Determination of Toxicity to an Invertebrate Population
Aim
The aim of this experiment is to observe how increasing concentrations of copper (II) sulphate pentahydrate solution affect the mortality of Daphnia (freshwater zooplankton).
Introduction
Toxicity is deemed to mean 'the inherent poisonous potency of a substance' (lecture notes). Freshwater pollutants are extremely diverse and are described by Jeffries and Mills (1990, p135) as "anything in the wrong place, in damaging quantities". Toxic pollutants cover a wide range of substances, substances that have no normal place in natural systems and many of which are man made (Jeffries & Mills, 1990). These pollutants include metals, organic compounds, gases, anions, radioactive material and acids and alkalis and can enter freshwater systems by means of domestic, industrial or agricultural effluents. Metals are often leached from mineworkings (Mason, 1991). Toxicity has been described to have two main effects on living organisms, acute toxicity, which has been defined as a dose of poison administered in a short period of time, being generally lethal and chronic toxicity, which refers to a relatively low dose being administered over a prolonged period, being lethal or sub-lethal (Gilbertson, Kent and Pyatt, 1989). The toxicity of most poisons can be affected by the environment with factors involved being, temperature, oxygen content, pH and dissolved salts (Haynes, 1971).
Results of toxicology studies on organisms can be expresses as lethal dose (LD) or lethal concentration (LC), where death is the criterion of toxicity. Results are shown, for example, as LC50 that indicates 50% mortality of the organisms at a certain concentration. Time is also an important factor in these studies so it must be indicated in results (Mason 1991). Organisms such as Daphnia are often used in laboratory experiments to determine the LC50 of a substance. The damage done as the dose changes in a given time determines the results of the experiment. Most commonly, the threshold of 50% mortality, of varying concentrations in a specified time, is used and is known as the median lethal concentration (Jeffries & Mills, 1990).
Method
Copper (II) sulphate pentahydrate (CuSO4.5H2O) solution was provided at 6 different concentrations, 0.1M, 0.05M, 0.01M, 0.005M, 0.001M and 0.0005M, of which 20cm3 at each concentration was dispensed into separate universal bottles, using separate pipettes for each concentration to reduce contamination. Each concentration was replicated 5 times. Distilled water was also dispensed at 20cm3 into 5 universal bottles, as the control. Room Temperature was recorded at 20oC. From a large bottle of solution containing Daphnia, 5 individual Daphnia were extracted repeatedly using a pipette and placed into each of the above bottles. The time the Daphnia were placed in the bottles was noted and 30 minutes later the number of dead Daphnia in each bottle was recorded.
Results
The results from the experiment were recorded and are shown in Table 1.
Table 1 - Number of dead Daphnia in given concentrations of copper (II) sulphate, at room temperature (20oC), after a time period of 30 minutes.
Concentration of copper (II) sulphate
(M)
Number of dead Daphnia
Total number
of dead
Daphnia
Mean of total no. dead Daphnia
% Mortality
± Standard deviation on mean % mortality
Replicates
2
3
4
5
0.1
5
5
4
...
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Results
The results from the experiment were recorded and are shown in Table 1.
Table 1 - Number of dead Daphnia in given concentrations of copper (II) sulphate, at room temperature (20oC), after a time period of 30 minutes.
Concentration of copper (II) sulphate
(M)
Number of dead Daphnia
Total number
of dead
Daphnia
Mean of total no. dead Daphnia
% Mortality
± Standard deviation on mean % mortality
Replicates
2
3
4
5
0.1
5
5
4
5
5
24
4.8
96
8.9
0.05
0
2
0
4
0.8
6
6.7
0.01
2
0
2
6
.2
24
6.7
0.005
0
0
0
2
0.4
8
1.0
0.001
2
3
0
0
6
.2
24
26.1
0.0005
3
3
2
2
1
2.2
44
30.3
Distilled water - control
Replicates, number of dead Daphnia
Total
2
3
4
5
0
0
0
0
0
0
The results for this experiment were very inaccurate. According to data, the relationship between mortality and concentration should be proportionate, the higher the concentration, the higher the mortality. From the graph shown in figure 1 (attached), the 50% mortality rate, and therefore the LC50 , at 20oC over a period of 30 minutes, has been calculated at 0.68M. The error bars are so large that it was very difficult to draw a curve as samples taken were too few at each concentration.
The results did indicate that the higher the concentration of copper (II) sulphate, the higher the mortality rate of the specimens. Also, comparing the number of deaths in any concentration of copper (II) sulphate, there were no deaths of Daphnia in the control solution.
Discussion
To accurately predict the effect pollutants have on freshwater organisms, it is very difficult to extrapolate these results from laboratory experiments. The conditions in the laboratory do not have the variety of other factors that occur in aquatic conditions such as rain and weather patterns. Also, for example, there is never usually just one poison in effluents and usually rivers receive several effluents. In the laboratory experiment using Daphnia, only one toxin was present, therefore not reflecting real life conditions. It has been shown that many poisons have been known to increase each other's toxicity, so the LC50 of copper (II) sulphate in the laboratory may not be correct for freshwater purposes (Haynes, 1971). Another difficulty is the interpretation of results. It is relatively simple to find out how many Daphnia die in different concentrations of copper (II) sulphate when testing in the laboratory, but when in 'real-life' conditions, it is possible that the Daphnia would survive longer than 30 minutes with the dilution occurring in the water in their natural environment.
There are many other factors that have an effect on freshwater organisms that are difficult to replicate in a laboratory. The toxicity of most poisons is largely affected by environmental factors. Temperature has a great effect on toxicity as, in general, "at a given concentration of poison, a rise of 10oC halves the survival time" (Haynes, 1971, p75). The increase in temperature can affect the metabolic activity and behaviour of an organism and it can also alter the physical and chemical state of the pollutant (Mason, 1991). It can, therefore, be suggested that many poisons may become more poisonous during summer. This would be very difficult to replicate in a laboratory. The decrease in the content of oxygen in the water can have an effect on the toxicity of substances. It is understood that several poisons become more toxic at low oxygen concentrations. This is because an increase in respiratory rate occurs, and the organism is exposed to more of the poisonous substance. The Daphnia in the above experiment were not exposed to decreased levels of oxygen concentration, therefore, it would be inaccurate to state whether the LC50 would apply in certain conditions. The pH of water and whether it is hard or soft water can have an effect on the toxicity of pollutants. It is suggested that in metals, the ionic forms involved are altered, making changes to the toxins, and that many toxins are less poisonous in hard water than in soft (Jeffries & Mills, 1990).
Waterways are often used to dispose effluents in by domestic users, industry and agriculture. When it is summer, or when there has been a dry spell, water in rivers and streams have a decreased volume of water so that the pollutants become more concentrated and are more damaging to wildlife (Mason, 1991). Pollution from mines can be acidic and is usually discharged as metals and heavy metals. The pollutants, such as lead and zinc, run into rivers poisoning aquatic organisms. Pesticides used in agriculture, although having an effect on the organisms they are aimed at, can be transported, by rain or under foot, to other areas, be it rivers or woodland or even residues in foods, thereby harming a variety of species unintentionally (Clegg and Mackean, 1998). Oil, petrol and detergent pollution is common in freshwaters, which can be leaked from industrial plants, urban areas and roads and waste tips. These factors can result in fires breaking out, covering the surface of the water, or oils smothering mammals and birds which leads to "fatal cooling and drowning and suffocation of submerged insects that need to penetrate the surface film to obtain air", (Jeffries & Mills, 1990, p177). Another area for concern is thermal pollution. Many industries use water as a coolant. This is discharged into waterways but is usually much warmer than the water it is entering thereby overpowering the tolerance limits animals and plants have.
Sources of Error
The sources of error for this experiment were vast. The results obtained from the experiment indicated that more Daphnia died at the lowest concentration of copper (II) sulphate - 0.0005M - at 11 out of 25, than all the other concentrations, except the highest concentration - 0.1M at which 24 out of 25 Daphnia died. Possible reasons for this include: some of the specimens deposited in the concentrations may have been already near death, thereby not surviving for another 30 minutes of experiment time, also, there was no way of knowing the maturity of each individual Daphnia, therefore some may have survived longer because of their level of development whereas some may have died almost immediately due to their physical immaturity. Another reason for error is that it was almost impossible to obtain 5 Daphnia from the general solution, therefore 7 or 8 Daphnia could have been collected, some of these may have already been dead, then these dead were being counted for mortality purposes in the results. The conditions the Daphnia were kept in before the experiment took place can also have an effect on their mortality.
The error bars shown on the graph in figure 1 were so large because only 5 replicates were tested,
and the specimens were of a poor quality (not many were active in the general solution and those that were, were very difficult to obtain using a pipette without getting already dead specimens as well). I would not say that this experiment was very successful, although it does indicate why experiments like this are so difficult to replicate in a laboratory.
Conclusion
In conclusion, it is possible to see from the results and from general written text that the higher the concentration of toxin, the greater effect is has on the mortality of, in this case, Daphnia.
Pollution is a general concern in the threat to wildlife, humans and the planet. Man's resources are being damaged by pollutants. Careful observation and preventative measures need to be undertaken to prevent further damage. Pollutants being released from petrol stations, oil refineries, chemical works, industries and household solvents all have an effect on life. These pollutants are being released into, and trapped in the atmosphere in the form of 'acid rain'. Acid rain has an effect on the soil, it lowers its pH levels therefore the soil begins to leach minerals and trace elements necessary for plant nutrition. At very low pH levels, most valuable nutrients have leached away and "aluminium ions may appear in solution at poisonous concentrations", (Clegg & Mackean, 1998). Acid rain also has an impact on rivers and lakes, in that aluminium ions may reach poisonous concentrations resulting in fish ceasing to live in certain lakes and rivers.
In order to prevent further pollution to our water systems, and to prioritise the components of our environment that we control, a classification system for water uses has been constructed and the water quality criteria for these uses has been formulated. (Mason, 1991).
Bibliography
Clegg, C.J and Mackean, D.G. (1998) Advanced Biology, Principles and Applications. John Murray (Publishers) Ltd. London.
Gilbertson, D.D., Kent, M. and Pyatt, F.B. (1989) Practical Ecology, for Geography and Biology Survey, Mapping and Data Analysis. Unwin Hyman Ltd. London.
Hynes, H.B.N. (1971) The Biology of Polluted Waters. Liverpool University Press. Liverpool.
Jeffries, M. and Mills, D. (1990) Freshwater Ecology, Principles and Applications. Belhaven Press. London.
Mason, C.F. (1991) Biology of Freshwater Pollution. 2nd ed. Longman Group UK Ltd.
Catherine Wilcock
