Editorial
Chronic Toxicity
Editorial
Chronic Toxicity: How Can We Measure It? Peter Wangersky Centre for Earth and Ocean Research, University of Victoria, P.O. Box 1700, Victoria, BC VdW 2Y2, Canada
Acute T o x i c i t y While journalists make much of the "green revolution", and politicians hasten to demonstrate that they are on the side of truth, light, and ecology, we are still far from putting into place truly effective regulations for the preservation of natural ecosystems. Our very measures of pollution are largely based on the concentration of pollutant necessary to kill half of a test population in some specified time period. We are concerned only with acute toxicity; the residual, longterm effects of lower concentrations are seldom considered for any species other than Homo sapiens. Even this concern has come rather late and is expressed only weakly. Perhaps it is difficult to interest the general public in the fate of zooplankton or baby trout, but one wonders how far up the phylogenetic tree one must go before accepting that acute to toxicity is too severe a test for the preservation of any environment. When we are concerned with the health of an ecosystem, it is certainly not sufficient to conclude that anything which doesn't kill half of the test species almost immediately is probably all right. We have no adequate tests for effects which result in lowered resistance to disease or in generally lowered capabilities. For these we depend upon purely statistical reasoning, the correlations which are a commonplace of the medical columns of our daily newspaper, and which have been known to link the prevalence and intensity of the common cold to the strength of the ozone layer or this year's E1 Nifio.
The Rate of Increase of the Test Population Some measure of the ability of the test organisms to continue to function more or less normally under a continuing administration of a given pollutant is clearly needed. I would suggest that this Measure should be the rate of increase of the test population. Continuance of population growth is a much more sensitive test of the condition of the environment than is simple survival. Obviously, this measure is not feasible for a population breeding only once a year, and perhaps not even for one breeding once a month. Populations with a shorter life span and a quicker reaction to adversity must be used. Since I am an oceanographer, I will concern myself here only with aquatic ecosystems. The populations of choice in this case would be bacteria and
ESPR-Environ. Sci. & Pollut. Res. 2 (1) 3 - 4 (1995) 9 ecomed publishers, D-86899 Landsberg, Germany
phytoplankton, and perhaps some species of zooplankton. The choice of larger, longer-lived organisms would entail the long-term maintenance of colonies, a labour-intensive and therefore expensive procedure. As well, the use of any organism large enough to be recognized as such without the employment of a microscope could bring down the wrath of the various animal rights groups, with all the attendant disruption and publicity, and the necessity for filling out and filing endless official forms, a major growth industry in North America. How should we go about measuring changes in growth rates? Since organisms even as small as bacteria are notorious for refusing to behave like chemical reagents, we cannot simply add medium, pollutant, organism inoculum, and mix; we must first establish the growth rate for this clone of this organism in this medium at this time of year. Once the rate has been established, we are free to add pollutant and observe the consequences. However, (and the ecological literature is rife with "however's), we must understand the sometimes hidden assumptions in our culture methods in order to understand our results.
Chronic T o x i c i t y While batch culture is the simplest method of testing for acute toxicity, and is the recommended method for LDs0 and EDs0 testing, it is not suitable for studies of chronic toxicity. The organisms at risk are exposed to the maximum concentration of the pollutant at the very start of the experiment. As the pollutant is metabolized, incorporated into the organisms, or adsorbed on particulate matteer, its effective concentration decreases. In such systems, we are never quite sure how much of the pollutant is actually biologically available. At the same time, the available nutrients are being depleted; if the cultures are truly axenic, prolonged periods of even no population growth will result in nutrient stress, thus complicating the interpretation of the experimental results. Some form of continuous culture, where both pollutant and nutrients are kept constant, is clearly indicated. Unfortunately, both of the most common forms of continuous caflture have serious drawbacks for toxicity testing. The chemostat controls population size by a balance between nutrient addition and population growth, the excess population be-
3
Chronic Toxicity ing removed by simple washout. If population growth slows or ceases, population washout continues, thus ending the experiment. The turbidostat monitors population size continuously; when the population reaches a predetermined upper limit, new medium (and presumably new pollutant) is added, washing out some of the population. If population growth slows or ceases, addition of new medium also slows or ceases, and we have what is effectively a batch culture, with the drawbacks already mentioned. A better alternative might be the cage culture turbidostat. In this technique, first described by SKIPNES,et al. (1980), the test population is confined within a cage through which fresh medium flows continuously, thus keeping nutrient and pollutant levels constant. The size of the test population is monitored by measuring the solution turbidity; population size is kept between narrow limits by 'harvesting' excess population, pumping out both medium and organisms until a selected lower limit has been reached. Since the harvest rate is a simple function of the population growth rate, changes in growth rate induced by the addition of pollutants are easily detected. As normally used for pollution work, populations are maintained in culture until relatively constant growth rates have been recorded, after which the concentration of pollutant to be tested is incorporated into the medium supply. Addition of pollutant to the culture thus takes place gradually, the rate of increase depending upon the rate of medium replacement selected. In this manner the organisms are not subjected to the shock of instantaneous imposition of the full concentration of the pollutant. This technique has been used with some success in studies on the effect of Ekofisk crude oil on phytoplankton growth (OSTGaaRD, et al., 1984a, b) and on the effects of extracts of polluted sediment on a diatom and a green flagellate (WANGERSKYand MAASS, 1991). The results have sometimes been surprising. As we would have expected, the toxicity of the Ekofisk crude depended on the test species chosen. While all of the species succumbed to the test solutions as prepared, the oceanic species Skeletonema costatum was the most sensitive and the coastal species Pheodactylum tricornutum the most resistant. The latter species is commonly used in teaching culture techniques, since it, like the zucchini, will usually grow in spite of the worst efforts of a ham-handed gardener.
T h e Choice of a Test Species This result points out one of the pitfalls of all toxicity testing, the choice of a test species. The severity of the test will be
set by the sensitivity of the organism used. Furthermore, differences in culture technique sometimes so slight as to go unrecorded, or indeed unnoticed, can result in major differences in apparent sensitivity. Bioassay methods in general,
4
Editorial and culture techniques in particular, are not something to be applied directly from a book of recipes. Anyone working in this field must understand the use of controls, and most particularly must understand that before an effect can be assigned to an apparent cause, the normal variability of the system under investigation must be known. Our own work has pointed up the difference between concentration and bioavailability of toxic materials. The sediment extract with the highest concentration of toxic materials actually had less effect on the test organism than did the extract from a somewhat less polluted sediment. This difference between bioavailability and concentration can become very important when the question of ocean dumping arises, but that could be the subject of another sermon. Another advantage of the cage culture technique is that it allows cultures to be maintained for considerable periods. Some diatoms and flagellates have been kept in log phase growth for months at a time. This longevity becomes important in testing for the effects of low-level chronic pollution. In some as yet unpublished experiments on the effects of cadmium, we found that growth rates-dropped to zero within hours of the addition of pollutant, although population numbers decreased only slightly. Within two days, population growth had restarted, and within a week the populations were growing at the same rate as the controls. If the experiment had ended after 24 or 48 hours, we would have missed the recovery entirely. Outlook There is much to be done before this methodology can be suggested as a standard method. Perhaps the most important step must be to simplify the apparatus and control system, to take them from the bits of plastic, lengths of tubing, and hand-wired boxes beloved of the experimentalist to the plug-and-play systems necessary for commercial test units. Some unanimity on test organisms must also come about, or be imposed, since the severity of the test is so dependent on the sensitivity of the species used. At the present time, however, I feel this to be the most promising approach to testing for low-level chronic toxicity.
References OSTGAARD,K.; EIDE, I.; JENSEN, A.: Exposure of phytoplankton to Ekofisk crude oil. Marine Environmental Research 1 1 , 1 8 3 - 200 (1984) OSTGAARD,K.; HEGSETH,E. N.; JENSEN, A.: Species-dependent sensitivity of marine planktonic algae to Ekofisk crude oil under different light conditions. Botanica Marina 27, 3 0 9 - 318 (1984) SKIPNES,O.; EIDE,I.; JENSEN,A.: Cage culture turbidostat: a device for rapid determination of algal growth. Applied Environmental Microbiology 40, 318 - 325 (1980) WANGERSKY,P. J.; MAASS,R. L.: Bioavailability: the organism as sensor. Marine Chemistry 36, 199-213 (1991)
ESPR-Environ. Sci. & Pollut. Res. 2 (1) 1995
Chronic Toxicity
Editorial
Chronic Toxicity: How Can We Measure It? Peter Wangersky Centre for Earth and Ocean Research, University of Victoria, P.O. Box 1700, Victoria, BC VdW 2Y2, Canada
Acute T o x i c i t y While journalists make much of the "green revolution", and politicians hasten to demonstrate that they are on the side of truth, light, and ecology, we are still far from putting into place truly effective regulations for the preservation of natural ecosystems. Our very measures of pollution are largely based on the concentration of pollutant necessary to kill half of a test population in some specified time period. We are concerned only with acute toxicity; the residual, longterm effects of lower concentrations are seldom considered for any species other than Homo sapiens. Even this concern has come rather late and is expressed only weakly. Perhaps it is difficult to interest the general public in the fate of zooplankton or baby trout, but one wonders how far up the phylogenetic tree one must go before accepting that acute to toxicity is too severe a test for the preservation of any environment. When we are concerned with the health of an ecosystem, it is certainly not sufficient to conclude that anything which doesn't kill half of the test species almost immediately is probably all right. We have no adequate tests for effects which result in lowered resistance to disease or in generally lowered capabilities. For these we depend upon purely statistical reasoning, the correlations which are a commonplace of the medical columns of our daily newspaper, and which have been known to link the prevalence and intensity of the common cold to the strength of the ozone layer or this year's E1 Nifio.
The Rate of Increase of the Test Population Some measure of the ability of the test organisms to continue to function more or less normally under a continuing administration of a given pollutant is clearly needed. I would suggest that this Measure should be the rate of increase of the test population. Continuance of population growth is a much more sensitive test of the condition of the environment than is simple survival. Obviously, this measure is not feasible for a population breeding only once a year, and perhaps not even for one breeding once a month. Populations with a shorter life span and a quicker reaction to adversity must be used. Since I am an oceanographer, I will concern myself here only with aquatic ecosystems. The populations of choice in this case would be bacteria and
ESPR-Environ. Sci. & Pollut. Res. 2 (1) 3 - 4 (1995) 9 ecomed publishers, D-86899 Landsberg, Germany
phytoplankton, and perhaps some species of zooplankton. The choice of larger, longer-lived organisms would entail the long-term maintenance of colonies, a labour-intensive and therefore expensive procedure. As well, the use of any organism large enough to be recognized as such without the employment of a microscope could bring down the wrath of the various animal rights groups, with all the attendant disruption and publicity, and the necessity for filling out and filing endless official forms, a major growth industry in North America. How should we go about measuring changes in growth rates? Since organisms even as small as bacteria are notorious for refusing to behave like chemical reagents, we cannot simply add medium, pollutant, organism inoculum, and mix; we must first establish the growth rate for this clone of this organism in this medium at this time of year. Once the rate has been established, we are free to add pollutant and observe the consequences. However, (and the ecological literature is rife with "however's), we must understand the sometimes hidden assumptions in our culture methods in order to understand our results.
Chronic T o x i c i t y While batch culture is the simplest method of testing for acute toxicity, and is the recommended method for LDs0 and EDs0 testing, it is not suitable for studies of chronic toxicity. The organisms at risk are exposed to the maximum concentration of the pollutant at the very start of the experiment. As the pollutant is metabolized, incorporated into the organisms, or adsorbed on particulate matteer, its effective concentration decreases. In such systems, we are never quite sure how much of the pollutant is actually biologically available. At the same time, the available nutrients are being depleted; if the cultures are truly axenic, prolonged periods of even no population growth will result in nutrient stress, thus complicating the interpretation of the experimental results. Some form of continuous culture, where both pollutant and nutrients are kept constant, is clearly indicated. Unfortunately, both of the most common forms of continuous caflture have serious drawbacks for toxicity testing. The chemostat controls population size by a balance between nutrient addition and population growth, the excess population be-
3
Chronic Toxicity ing removed by simple washout. If population growth slows or ceases, population washout continues, thus ending the experiment. The turbidostat monitors population size continuously; when the population reaches a predetermined upper limit, new medium (and presumably new pollutant) is added, washing out some of the population. If population growth slows or ceases, addition of new medium also slows or ceases, and we have what is effectively a batch culture, with the drawbacks already mentioned. A better alternative might be the cage culture turbidostat. In this technique, first described by SKIPNES,et al. (1980), the test population is confined within a cage through which fresh medium flows continuously, thus keeping nutrient and pollutant levels constant. The size of the test population is monitored by measuring the solution turbidity; population size is kept between narrow limits by 'harvesting' excess population, pumping out both medium and organisms until a selected lower limit has been reached. Since the harvest rate is a simple function of the population growth rate, changes in growth rate induced by the addition of pollutants are easily detected. As normally used for pollution work, populations are maintained in culture until relatively constant growth rates have been recorded, after which the concentration of pollutant to be tested is incorporated into the medium supply. Addition of pollutant to the culture thus takes place gradually, the rate of increase depending upon the rate of medium replacement selected. In this manner the organisms are not subjected to the shock of instantaneous imposition of the full concentration of the pollutant. This technique has been used with some success in studies on the effect of Ekofisk crude oil on phytoplankton growth (OSTGaaRD, et al., 1984a, b) and on the effects of extracts of polluted sediment on a diatom and a green flagellate (WANGERSKYand MAASS, 1991). The results have sometimes been surprising. As we would have expected, the toxicity of the Ekofisk crude depended on the test species chosen. While all of the species succumbed to the test solutions as prepared, the oceanic species Skeletonema costatum was the most sensitive and the coastal species Pheodactylum tricornutum the most resistant. The latter species is commonly used in teaching culture techniques, since it, like the zucchini, will usually grow in spite of the worst efforts of a ham-handed gardener.
T h e Choice of a Test Species This result points out one of the pitfalls of all toxicity testing, the choice of a test species. The severity of the test will be
set by the sensitivity of the organism used. Furthermore, differences in culture technique sometimes so slight as to go unrecorded, or indeed unnoticed, can result in major differences in apparent sensitivity. Bioassay methods in general,
4
Editorial and culture techniques in particular, are not something to be applied directly from a book of recipes. Anyone working in this field must understand the use of controls, and most particularly must understand that before an effect can be assigned to an apparent cause, the normal variability of the system under investigation must be known. Our own work has pointed up the difference between concentration and bioavailability of toxic materials. The sediment extract with the highest concentration of toxic materials actually had less effect on the test organism than did the extract from a somewhat less polluted sediment. This difference between bioavailability and concentration can become very important when the question of ocean dumping arises, but that could be the subject of another sermon. Another advantage of the cage culture technique is that it allows cultures to be maintained for considerable periods. Some diatoms and flagellates have been kept in log phase growth for months at a time. This longevity becomes important in testing for the effects of low-level chronic pollution. In some as yet unpublished experiments on the effects of cadmium, we found that growth rates-dropped to zero within hours of the addition of pollutant, although population numbers decreased only slightly. Within two days, population growth had restarted, and within a week the populations were growing at the same rate as the controls. If the experiment had ended after 24 or 48 hours, we would have missed the recovery entirely. Outlook There is much to be done before this methodology can be suggested as a standard method. Perhaps the most important step must be to simplify the apparatus and control system, to take them from the bits of plastic, lengths of tubing, and hand-wired boxes beloved of the experimentalist to the plug-and-play systems necessary for commercial test units. Some unanimity on test organisms must also come about, or be imposed, since the severity of the test is so dependent on the sensitivity of the species used. At the present time, however, I feel this to be the most promising approach to testing for low-level chronic toxicity.
References OSTGAARD,K.; EIDE, I.; JENSEN, A.: Exposure of phytoplankton to Ekofisk crude oil. Marine Environmental Research 1 1 , 1 8 3 - 200 (1984) OSTGAARD,K.; HEGSETH,E. N.; JENSEN, A.: Species-dependent sensitivity of marine planktonic algae to Ekofisk crude oil under different light conditions. Botanica Marina 27, 3 0 9 - 318 (1984) SKIPNES,O.; EIDE,I.; JENSEN,A.: Cage culture turbidostat: a device for rapid determination of algal growth. Applied Environmental Microbiology 40, 318 - 325 (1980) WANGERSKY,P. J.; MAASS,R. L.: Bioavailability: the organism as sensor. Marine Chemistry 36, 199-213 (1991)
ESPR-Environ. Sci. & Pollut. Res. 2 (1) 1995
