Exp. Biol. (1961), 38, 447-455 447 With 5 text-figures Printed in Great Britain EFFECT OF DISSOLVED OXYGEN CONCENTRATIONS ON THE TOXICITY OF SEVERAL POISONS TO RAINBOW TROUT (SALMO GAIRDNERII RICHARDSON) BY R. LLOYD Water Pollution Research Laboratory, Stevenage {Received 20 January 1961) INTRODUCTION The low dissolved oxygen concentrations which are characteristic of many polluted rivers have been shown by several authors to increase the toxicity of poisons to fish. Since most toxicity tests are made in well aerated water, it is important to know what factor to apply to the results when predicting the effect of a reduced dissolved oxygen concentration on toxicity. Evidence presented in this paper suggests that there may be a common relation between the dissolved oxygen concentration and the toxicity of poisons, and this is supported by a theoretical consideration of the problem. METHODS Toxicity of monohydric phenols, and zinc, lead and copper salts Details have been published of determinations of the effect of various levels of dissolved oxygen concentration on the toxicity of a mixture of monohydric phenols (Department of Scientific and Industrial Research, 1958), zinc sulphate (Lloyd, 1960), lead nitrate and copper sulphate (Department of Scientific and Industrial Research, i960). Toxicity of ammonium chloride The effect of low dissolved oxygen concentrations on the toxicity of ammonium chloride to rainbow trout was determined in fixed volumes of solution; pH values and oxygen concentrations were controlled by aeration with known mixtures of air, nitrogen, and carbon dioxide. The trout were fed before they were acclimatized for 18 hr. to the temperature (17-5° C.) and carbon dioxide concentration used in the tests. Other details of procedure were similar to those described by Lloyd & Herbert (i960). Oxygen consumption of rainbow trout Measurements of the oxygen consumption of rainbow trout at i7-5° C. were made in a simple continuous flow respirometer, similar in essentials to that illustrated by Fry (1957) in his figure 10B. Values for oxygen consumption were first obtained in water saturated with air and the same fish were immediately used again to obtain oxygen-consumption values at a lower oxygen level. The rainbow trout used weighed between 1 and 11 g. and were acclimatized to the temperature and free carbon concentration (about 80 mg./l.) before the test. 448 R. LLOYD RESULTS Monohydric phenols, and zinc, lead and copper salts When the log. survival times of rainbow trout are plotted against the corresponding log. concentrations of these poisons in well aerated water, a curvilinear relation is obtained, and at those concentrations of the poisons in which periods of survival are long the line is nearly vertical, so that a further slight decrease in concentration is associated with a prolonged period of survival. It is these slightly toxic concentrations of poisons which are important for predicting safe concentrations in a river. If the dissolved oxygen concentration of the water is reduced, the survival time/concentration curve is displaced towards lower concentrations of poison, and a value for this increase in toxicity can be obtained by comparing concentrations of poison which are equitoxic at prolonged periods of survival. This can be expressed as the factor X8jX, where Xs is the concentration of poison at ioo % of the air-saturation value of oxygen, Cg, and X is the equitoxic concentration at a lower value of dissolved oxygen, C. Values of X8/X at different levels of dissolved oxygen concentration were derived from the experimental data for monohydric phenols, and zinc, lead, and copper salts for median periods of survival between iooo and 2000 min. Values of XsjX for these four poisons are shown in Fig. 1, where it appears that the relation between increase in toxicity and dissolved oxygen concentration is similar for these poisons. 30 40 50 60 70 80 90 100 Dlsiolved oxygen (^ of air-saturation value) Fig. 1. Relation between the factor Xs/X for several poisons and the dissolved oxygen concentration of the water. For explanation of XS\X see text. Ammonium salts Batches of ten rainbow trout were exposed to various concentrations of ammonium chloride at two levels of free carbon dioxide (3-4 and 19-8 mg./l.) and three levels of dissolved oxygen (37-5, 66-0 and 100% of the air-saturation value); concentrations of ammonia corresponding to 500-minute median periods of survival (at which time the survival time/concentration curve has become practically vertical) were calculated by probit analysis for each series. The experimental values of XafX for ammonia Dissolved oxygen concentration and toxicity of poisons 449 (Fig. 2) are higher than those for the previous four poisons, and are affected by the free carbon dioxide concentration of the water; these differences can be explained by the following hypothesis. •40 SO 60 70 80 90 100 Dissolved oxygen (% alr-saturatlon value) Fig. 3. Relation between the factor Xs/XfoT ammonia and the dissolved oxygen concentration of the water at two levels of free carbon dioxide. Continuous lines are theoretical curves (see text). Limits to experimental points are 95 % fiducial limits. It is well established that the toxicity of ammonia solutions is due to the un-ionized ammonia molecule, and that the ionized fraction is not toxic; the un-ionized pro- portion of an ammonia solution increases with a rise in pH value. However, it has been shown by Lloyd & Herbert (i960) that the toxicity of ammonium salts is dependent, not on the pH value of the bulk of the solution, but on that of the water at the gill surface. This latter value can be calculated from the bicarbonate alkalinity, temperature, and free carbon dioxide concentration in the water, and the free carbon dioxide excreted by the gills of the fish. An estimate of the concentration of excreted carbon dioxide in the respiratory water (as mg. carbon dioxide/1.) is given by the following relation mol wt. COj, P D.O. X R.Q. X mol wt. O, 100' (0 where D.O. is the dissolved oxygen concentration of the water in mg./l., R.Q. the respiratory quotient of the fish (assumed to be o-8), and P the percentage of oxygen removed from the respiratory water by the fish; the values of P used are given later in the discussion. As the oxygen concentration of the water is reduced, the concen- tration of excreted carbon dioxide at the gill surface is also reduced and the pH value of the water at this surface rises, resulting in an apparent increase in the toxicity of ammonia. This increase in toxicity will become greater as the concentration of free carbon dioxide in the bulk of the solution is reduced. Thus, theoretical values of X8jX for ammonia can be calculated on the assumption that the relation between dissolved oxygen concentration and the toxicity of this poison is essentially similar to the relation for the other four poisons (Fig. 1), but that 450 R. LLOYD in water of lowered oxygen content the toxicity is further increased, because of the reduction in the concentration of excreted carbon dioxide at the gill surface. An estimate of this additional increase in toxicity can be derived from the theory given by Lloyd & Herbert (i960). Theoretical curves for the factor XsjX for ammonia under the conditions of the experiments described here are shown in Fig. 2 where they are in good agreement with the experimental points. From data in a paper by Merkens & Downing (1957) on the effect of a reduction of the dissolved oxygen concentration to 47 % of the air-saturation value on the toxicity of ammonia, it can be calculated that the experimental factor for X8jX for a 500 min. median period of survival was 3*64; the factor expected for the experimental conditions, in which the concentration of free carbon dioxide was between 0-75 and i-o mg./l., is between 3-35 and 4-17. The good agreement between predicted and experimental results strengthens the view that the effect of low dissolved oxygen concentrations on the toxicity of this poison is basically similar to that for the other four poisons, but that its toxicity is increased still further by the rise in pH value of the water at the gill surface. DISCUSSION Although the toxic actions of heavy metals, ammonia, and monohydric phenols are probably dissimilar, the common effect on their toxicity resulting from a reduction in the concentration of dissolved oxygen suggests that this is a result of a physiological reaction by the fish to such a change of the environment, and is independent of the nature of the poison. The most obvious reaction of fish to a lowered oxygen content of the water is to increase the volume of water passed over the gills, and this may increase the amount of poison reaching the surface of the gill epithelium, the site at which most poisons are absorbed. Weiss & Botts (1957) have shown that an increase in the oxygen uptake of several species of fish results in a decrease of their survival times in toxic solutions; they found, however, that a reduction in the dissolved oxygen concentration of the water reduced the oxygen uptake of the fish, yet increased the toxicity of the solution, and thought that this reduction in uptake was insufficient to compensate for the reduced oxygen content of the solution and that it was the increased rate of respiratory flow through the gills which led to an increased toxicity of the poison. However, the design of their experiments does not allow the results to be compared in detail with those from the experiments described here. Therefore, although there is some evidence that an increase in respiratory flow increases the toxicity of poisons, there is no evidence to show that this accounts for the whole of the increase in the toxicity of poisons in water of low dissolved oxygen concentration.