Measurement of pollutant toxicity to fish I. Bioassay methods for acute toxicity

Wafer Reseurch Pergamon Press 1969. Vol. 3, pp. 793-821. Printed in Great Britain.

REVIEW PAPER MEASUREMENT

OF POLLUTANT

TOXICITY

TO FISH

I.* BIOASSAY METHODS FOR ACUTE TOXICITY J. B. SPRAGUE Fisheries Research Board of Canada, Biological Station, St. Andrews, New Brunswick, Canada (Received 19 May 1969) Abstract-The review describes profitable methods for measuring lethal levels of pollutants for aquatic organisms. Methods for research in the laboratory are emphasized but the same principles could be applied in field work. Greater use of standard toxicological methods and terminology is urged. For 211 out of 375 toxicity tests reviewed, acute lethal action apparently ceased within 4 days, although this tabulation may have been biased towards short times by a large number of static tests. The incipient LCSO (lethal concentration for 50 per cent of individuals on long exposure) is recommended as the most useful single criterion of toxicity. If this cannot be estimated, the Cday LC50 is a useful substitute, and often its equivalent. A desirable first step in toxicity tests is to estimate median lethal time for each of a series of concentrations. A toxicity curve should be drawn by plotting median survival times against concentrations on logarithmic paper. The curve helps to reveal any unusual features of toxicity. Whenever possible, tests should be prolonged until the toxicity curve becomes parallel to the time axis, indicating a lethal threshold concentration. The incipient LC50 is then estimated by selecting an exposure time from the asymptotic part of the toxicity curve; for this exposure time, observed mortality is plotted against concentration on log-probit paper, and the LC50 is read from an eye-fitted line. Confidence limits of the LC50 may also be estimated by simplified methods. These should be given in published work along with a value for slope of the probit line. Alternativeapproaches usereciprocal transformations or estimate LCSO’s for a series of exposure times. A graph is given for estimating partial replacement times of water in tanks of continuousflow tests. Rate of flow should give a short partial replacement time, such as a flow equal to volume in 3-5 hr, and also adequate water for respiration of fish, usually 2 or 3 l/g of fish/day, or more. INTRODUCTION

intended for students or scientists entering the field of toxicity tests with aquatic organisms. It outlines profitable bioassay methods. Later parts (II and III) will cover associated topics, especially those which relate lethal tests to field problems, and to “safe” levels of pollutants. This is intended to be a critical review. Favourable reactions of the reviewer are implied by inclusion of methods and by the amount of space devoted to them. Direct recommendations are sometimes made, and no doubt there is room for healthy disagreement on these. Urgency of water pollution problems is leading us toward official “water quality criteria” for aquatic life, notably in U.S.A. but also in Europe (U.S. National Technical Advisory Committee, 1968 ; European Inland Fisheries Advisory Commission, THIS REVIEW is

* Parts III and II will appear in subsequent issues of voter Research. 793

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J, B SPRAGUE

1965). Information needed for protection of aquatic life has been eloquently described by TARZWELL(1962a, 1962b, 1966), and toxicity tests provide some of the required answers. Because of the urgency, such tests should use efficient methods which yield meaningful results. Effective methods are also appropriate since today's bioasSays may be costly in automatic equipment or in weeks of laboratory time. It seems only sensible to analyze expensive experiments by techniques which gather maximal information. The need for good methods is further documented in every review of toxic limits for aquatic life. The investigator looking up a toxicant in the compendium of MCKEE and WOLFE (1963) should expect a list of "lethal levels" which vary a thousand-fold, and seldom will he be disappointed. It is time to organize the reasons for such variation. This requires perceptive research using the most revealing methods. It is unfortunate that pollution biologists have tended to form a splinter group as far as toxicology is concerned. Standard techniques of analysis, developed by pharmacologists and statisticians for testing drugs, have too often been ignored. Successful adpatations of these methods will be cited frequently. This review deals primarily with methods for research in the laboratory. However the same general principles may also be applied to bioassays for management or control purposes, by industry or government, using aquaria or cages offish in a river. Similarly most examples are for freshwater fish but there is little difference in basic approach with marine organisms. We should avoid erecting unnecessary barriers between the categories above, and between tests with vertebrates and invertebrates, or lethal and sublethal tests. Examples in this review often cross such lines of distinction. The literature contains excellent reviews parallel or complementary to this one. HERBERT(1965) and EDWARDSand BROWN(1967) discuss the methods used so effectively by the British Water Pollution Research Laboratory. Also highly recommended are the reviews by ALDERDICE(1967), BURDICK (1967), WARNER (1967), and BEAK (1958). From Europe, the writing of WtrI-mMANN(1952) remains classic, ROTHSCHEIN (1964) analyzes bioassay method critically, while BREITIG(1966) discusses standardized procedures. Among books, that of MARCHETTI(1962) gives a unique major treatment of aquatic bioassays, JONES (1964) includes much earlier work, while HYNES (1960) evaluates laboratory tests somewhat skeptically from an ecological viewpoint. Dealing with bioassays, but not particularly fisheries, the review by GADDUM(1953) is still a most easily read and informative discussion of methods and is highly recommended. Types of response and analyses are given by BLISS (1957) and BLISS and CATTELL(1943). FINNEY'Sbook (1964) gives considerable detail of statistical methods. The present review has benefited greatly from organization and outlook of these and other works. MECHANICS OF SETTING UP TESTS It is encouraging that most investigators now use very similar methods for acute lethal tests with fish. Furthermore, the methods are advantageous compared to the variety of approaches which could have been taken, and sometimes were taken in the first half of the century. Almost without exception, present-day tests incorporate: (1) a series of test-containers, each with a different but constant concentration of the toxicant;

Measurement of Pollutant Toxicityto Fish. Part I

795

(2) a group of similar fish, usually ten, in each container; (3) observations on fish mortality during exposures which last between 1 day and about 1 week; and (4) final results expressed as concentration tolerated by the median or "average" fish. Given this similar general approach, smaller variations in technique will be compared in this review. Broadly speaking there are two procedures in current use. In the first, approximate mortality-times are recorded for most individual fish. The time taken to obtain 50 per cent mortality is estimated for each test-tank. The series of median lethal times is generally used to estimate an approximate threshold concentration for lethal effect. This has been used with notable success in British studies of pollutants, and in studies of lethal temperatures of fish by F. E. J. Fry and associates in Canada. In the second procedure, customarily used in the U.S.A., mortality is recorded only at 1, 2, and 4 days. The concentration lethal to half the fish in each of these time periods is estimated. The first procedure has more complete observations, and hence will also provide the answers yielded by the second procedure. The first will be the standard technique described below, and the second will be discussed separately. However the two procedures have tended to yield similar estimates of acute toxicity in recent years when exposure-times of 4 days or longer have become usual. Many aspects of methodology which have been well discussed by others will be omitted. These include species, number, size, and condition of fish, kind of dilution water, temperature and the myriad other conditions of testing. Researchers are urged to consider the recommendations of DOUDOROFFand his committee (1951) or APHAet al. (1965), which are clear, sensible, and have stood the test of time. Especially valuable are the sections on care and handling of fish, experimental concentrations based on logarithmic bisection, preparation of test animals, diluent, and "other prescribed conditions". MARCHETTI'S book (1962) is also recommended. ROTHSCHEIN'S(1964) critical review lists ten common deficiencies of bioassays, with remedies for some. A few special aspects of test procedure are mentioned below because they are continuing problems or because of recent progress. Required volume of test-solution One aspect of this is allowing enough space for fish to swim around in a reasonably free manner. This would probably depend on the size and shape of the holding tank to which they were previously accustomed. Some recommendations about minimum depths and volumes are given by DOUDOROFFet al. (1951). However, there does not seem to be any investigation on exactly what sizes and shapes of tanks are necessary to eliminate stressing the fish and affecting test-results. It must be left in large part to the biological judgment of the investigator, to provide enough water for a reasonable amount of free activity by the fish. In the discussion which follows, it will be assumed that this is done. Aside from the above question, there are easily-predictable peculiarities arising from insufficient volume of test-solution. These cause sporadic problems in both static and continuous-flow tests. During the test, fish may deplete the toxicant, the oxygen, foul the water, or all three, with various effects on survival. In the 1920s and 1930s, a great deal of effort was expended in studying changes in results caused by reduced volume of test solution.

796

J . B . SPRAGUE

Today the problems may be avoided for continuous-flow tests, by following good practice such as the suggestions of ALABASTERand ABRA~ (1965). They recommend that the supply of new test solution should be sufficient to maintain dissolved oxygen in the test tank. This also keeps toxicant and waste products within desirable limits. The extreme values which they mention for required amount of replacement solution are 0.5 and 10 1. per gram of fish per day, the lower values sometimes involving artificial aeration. Alabaster and Abram chose 10 1/g/day as satisfactory for harlequin fish, which are small and have relatively high respiratory needs per unit of weight. Calculations from their data suggest that about 2 or 3 l/g/day, preferably the latter, would probably be satisfactory in tests with trout. For 3 1/g/day and an oxygen consumption of 0.16 mg/g/hr, used by Alabaster and Abram, this would mean that trout would use about 1.3 mg of oxygen for each liter of incoming water, so that dissolved oxygen levels would not be greatly depressed. When possible, static tests should aim at the same value of 2 or 3 1. of solution per gram offish, changed daily. This is not too far removed from the preference expressed in the APHA et al. (1965) method for at least 1 1/g fish. For very small tropical fish such as the harlequin fish, higher values equivalent to the 10 I/gm of Alabaster and Abram, might be desirable. Another useful tool in continuous-flow tests is to estimate the amount of time required to replace the body of test water. Sometimes the "replacement time" is published according to the naive calculation: volume of solution divided by flow per unit time. This would be the time required to fill an empty tank or to empty a full one, but it certainly is not a reasonable estimate of displacement time of molecules in the test tank. This may be estimated approximately by using FIG. 1 which is based on calculations by A. A. HEUSNER (personal communication). It may be compared to displacement time in lakes, the mathematics of which are discussed by RAINEY(1967). Many toxicants decrease in concentration with time, apparently by sorption on walls of the test container, etc. Therefore, when deciding flow rates in holding tanks or test tanks, even with artificial aeration, I have considered it a reasonable guide to try to keep the 90 per cent replacement time down to about one-third or one-half of a day, i.e. about 8-12 hr. This means that flow would equal the test-volume in about 3-5 hr (FIG. 1). Such replacement times may be attained by increasing flow or by reducing test volume, within reasonable limits of each. It was mentioned in the first paragraph of this section that the volume of solution should be enough for free movement of fish. The suggested replacement time may be considered to be in general agreement with the recommendations of ALABASTERand ABRAM (1965). For example, 90 per cent replacement in 8 hr of 30 1. test-solution containing 100 g of trout, works out to a replacement of 2 I/g/day. The forthcoming revision of the APHA et aL (1965) method will recommend a minimum flow rate which equals the volume of water in the tank in 6 hr (Q. H. PICKERING,personal communication). This is not much longer than equalling the volume in 3-5 hr as suggested above. The APHA minimum would mean 90 per cent replacement of water in about 15 hr, which seems fairly reasonable. However, the APHA revision will further recommend that flow rates be increased if necessary, until satisfactory levels of dissolved oxygen are maintained. As a basis for calculation, an oxygen uptake by fish of 0.2 mg O2/g fish/hr will be suggested, which is in line with the

Measurement of Pollutant Toxicity to Fish. Part I

797

value of Alabaster and Abram. The recommended increased flow could thus correspond with the suggestions in this section, depending on the weight of fish used. To summarize, continuous-flow tests should allow for both (a) a reasonable rate of flow of test solution per gram of fish, probably 2 or 3 1/g/day or higher for small fish, and (b) fairly rapid replacement-times of water, perhaps 90% replacement in something like 8-12 hr. IO n

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of of

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40

60

IO0

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FIG. 1. Approximate times required for partical replacement of water in tanks, for constantflow situations. Example: for a tank containing 30 l., with a flow of 10 I. per hour, there would be 50 per cent replacement of water in the tank in about 2 hr, 75 per cent in about 4 hr, 90 per cent in 7 hr, and 95 per cent replacement in 9 hr. Another time-period could replace hours, but the same time must be used on each axis, and the same unit of capacity must beused for volume and flow. Based on information supplied by Alfred Heusner.

Dosing apparatus Happily, recent years have seen the advent of a number of mechanical devices for regulating flow of test water and/or toxicant. Although electric pumps, especially persistaltic tubing pumps, would seem suitable, most investigators have apparently concluded that they are too undependable or expensive. Most new devices are homemade. MARCHETTI (1962) covers the earlier systems and illustrates the best ones so these will not be repeated. Since then, STARK(1967) has described a desirable-looking dosing apparatus which operated for 24 weeks within 5 per cent error. It is based on earlier ones of ABRAM (1960) and GP,ENIZR (1960). All three have the advantage of "failing safe" because the flow of dilution water controls input of toxicant; if water slows down, fish are not killed by overdose of toxicant. MOUNT and BRUlqGS (1967) describe a "proportional diluter" simplified from an earlier serial diluter (Moutqr and

798

J. ]3. SPRAGUE

WARNER, 1965). Both have long-term dependability and fail-safe characteristics, but

successive test concentrations are somewhat dependent on each other in magnitude.* Other designs are simpler but do not fail safely (BURKEand FERGUSON, 1968), or offer little advantage over a simple Mariotte bottle (SoLoN et aL, 1968). Certain specialized pieces of apparatus have been described. CHADWICK and KnGEMAGI(1968) succeeded in dosing relatively insoluble insecticides by first sorbing them on a sand column. MOUNT and BRUNGS(1967) inject them as a slurry. A closed continuous-flow apparatus for small fish is shown by ALABASTERand ABRAM(1965). Recirculation of test water makes fish swim and would be suitable for static or continuous-flow bioassays (BETrS et al., 1967). ALOEROICEet aL (1966) describe a simple tank, suitable for holding or testing, which is self-cleaning and makes fish swim continuously. Acclimation and holding Acclimation of fish to water and general test conditions remains poorly known. "At least a week" of acclimation is sometimes suggested on little factual basis. The topic may often be of direct importance for bioassay results. LLOYD(1965) states that trout transferred from hard to soft water required at least 5 days of acclimation, before their response to a toxic metal became the same as the reponse of fish held permanently in soft water. Similarly, Motr~T (1966) mentions that complete acclimation to water hardness may not have been achieved in his tests with metal at various levels. Probably more is known about acclimation by fish to temperature than to any other entity. Even here the knowledge is very incomplete, and has been gained mostly from responses of fish to lethal temperatures. Judging by this response, change in acclimation state is particularly slow for downward acclimation to low temperatures (BRETT, 1956). However, the rule of thumb sometimes used in such lethal temperature work, of "at least 1 day per Centigrade degree of change" has no particular scientific basis and may sometimes contradict the facts (e.g. DOUDOROFF, 1942). Of much greater interest in toxicity work is metabolic acclimation which could be different from rate of acclimation as judged by lethal temperatures. The state of acclimation of metabolism could determine rates of detoxification and other processes governing survival in a pollutant. On this topic, PEa'ERSOr~and ANDERSON(1969) have recently concluded that complete acclimation of metabolism of a fish to a temperature change requires about 2 weeks, regardless of direction of change. Much more information of this kind is needed on acclimation before tests. Meanwhile the investigator should allow generous time for this process, and state clearly what regime was followed. A long-needed set of blood tests, for routinely measuring physiological condition of fish being held for bioassays, has been provided by HtrNN et al. (1968). They also suggest desirable holding conditions. Randomization Randomization of fish among test tanks is probably neglected in many bioassays. A formal process of randomization should be used according to FINNEY (1964) and * N o t e added to proof: This difficulty may be circumvented by using the dosing apparatus for individual tanks which is described by BRUNGS and MOUNT (1967) A device for continuous treatmerit of fish in holding chambers. Trans. A m . Fish Soc., 96, 55-57. That device seems equally as good as the one described by Stark.

Measurement of Pollutant Toxicity to Fish, Part I

799

GADDUM (1953). T h e y cite a p r o v e n e x a m p l e o f c o r r e l a t i o n between weight o f experim e n t a l animals (mice) a n d o r d e r in which they were caught. W i t h fish, it w o u l d be easy to catch the w e a k e s t swimmers in a h o l d i n g t a n k first. W e a k s w i m m i n g m i g h t c o n c e i v a b l y be related to w e a k resistance to a pollutant. A serious systematic e r r o r c o u l d result f r o m p l a c i n g each successive b a t c h o f ten captives in a test t a n k in o r d e r o f c o n c e n t r a t i o n . D i s t r i b u t i o n o f a n i m a l s by a process like dealing o u t a p a c k o f c a r d s still has a slight t e n d e n c y to p u t m o r e easily c a u g h t animals into certain c o n c e n t r a t i o n s a c c o r d i n g to G a d d u m ; to a v o i d this, F i n n e y suggests using r a n d o m numbers. D i s t r i b u t i o n o f fish as in d e a l i n g a p a c k o f cards w o u l d m e a n t h a t if, for example, six t a n k s were to receive fish, the first fish which was c a u g h t w o u l d be p l a c e d in the first t a n k , the s e c o n d into the s e c o n d t a n k , etc., the seventh into the first t a n k , the eighth into the second t a n k , etc. A n i m p r o v e m e n t o f this has been used in research by the U.S. F e d e r a l W a t e r P o l l u t i o n C o n t r o l A d m i n i s t r a t i o n (Dr. C. E. STEPHAN, p e r s o n a l c o m m u n i c a t i o n ) , a n d is h e r e b y r e c o m m e n d e d : for six tanks, the first six fish to be c a u g h t f r o m the h o l d i n g t a n k are d i s t r i b u t e d one to each o f the test tanks, in r a n d o m o r d e r a c c o r d i n g to occurrence o f the n u m e r a l s one to six in a table o f r a n d o m n u m b e r s o r b y d r a w i n g n u m b e r e d slips o f p a p e r ; the seventh to twelfth fish are d i s t r i b u t e d one to each o f the six t a n k s b y the same p r o c e s s ; this is c o n t i n u e d until the t a n k s are filled. In a d d i t i o n , o f course, t e s t - c o n c e n t r a t i o n s s h o u l d also be assigned t o the t a n k s b y f o r m a l r a n d o m i z a t i o n to g u a r d a g a i n s t a n y effect o f position. TERMINOLOGY A variety of terminology has been used in work covered here. I have generally attempted to use one set of terms and symbols, chosen because of wide usage, early publication, or lack of ambiguity. Median Effective Concentration (EC50) and Median Lethal Concentration (LC50) will be primary terms in this review. EC50 may refer to lethal or sublethal responses. The terms correspond to ED50 and LD50, universally used in pharmacology and toxicology, for example insect toxicology (HosKINS, 1960). D represents dose, the amount of drug inside the animal, by injection or ingestion. Fish toxicology may sometimes be concerned with internal dose, (e.g. pesticide residues), but usually deals with concentrations in the surrounding water, for which EC50 and LC50 are appropriate. Use of LC50 (and EC50) in fish toxicology should make the notation more immediately comprehensible to workers in other fields of science. LC50 is used in German pollution literature according to BREmO (1966), is used in Canada ALDERDICE,1967), and generally in Britain where explicit preference is sometimes stated (BROWNet al., 1967a). In the United States, LC50and EC50 are used to some extent (U.S. Dept. Interior, 1963; EiSLER,1965; JOHNSON, 1968; STEWARTet aL, 1967b) but the term median tolerance limit (TLm) has been customary with fish. U.S. readers may wish to substitute TLm for LC50 throughout this review. Unfortunately we cannot speak with grammatical accuracy of lethal "concentrations" of water temperature and pH, but the abbreviation will perhaps still be understood. The wording "median lethal limit" or "median lethal level" could be used if necessary. The symbol LD50 was first proposed by TREVAN(I 927). He wrote the symbols all on the same line (not LDso), and major writers in pharmacology follow him (BLISS, 1952; FINNEY, 1964; GADDUM, 1953). Making the 50, or 10, 90, etc., into a subscript is historically incorrect and seems to serve little purpose beyond complicating the lives of typists and proofreaders. Time and concentration are inseparably linked in tests with aquatic organisms. Hence LC50's should be qualified by an indication of exposure-time, such as 24-hour LC50, 4-day LC50, etc. Another important parameter is the concentration at which acute toxicity ceases, given several suitable names: incipient lethal level (FRY, 1947); ulitimate median tolerance limit (DouDoROFF, 1945; DotrDOgOrF et al., 1951); lethal threshold concentration (LLOYDand JORDAN,1963); asymptotic LC50 (BALL, 1967a); kritische schwelle (DENZER, 1959); Schwellenwerte (WUHRMANNand WOKER, 1950; NEHRING, 1962); Todlichkeitsgrenze (LmPOLT and WEBER, 1958). For the English language, incipient lethal level has the advantage of being part of a general scheme of classifying environmental factors

800

J . B . SPRAGU~

and may be defined as "that level of the environmental entity beyond which 50 per cent of the population cannot live for an indefinite time" (modified slightly from FRY, 1947). The word "ultimate" in the second term was used earlier (FRY, BRETT and CLAWSON, 1942) not to describe the length of exposure-time, but the highest degree of resistance attainable by acclimation to the lethal condition. Perhaps it might be reserved for acclimation effects, which hopefully will become more widely studied in toxicity tests. Lethal threshold concentration seems a good term although some investigators still feel that threshold effects should not deal with 50 per cent mortality but with the most sensitive individual in a population. That concept has no precise mathematical meaning as discussed further on in the review. According to BUERKLE(1967), most modern investigators would agree that a threshold is "that stimulus intensity at which a positive response can be expected in 50 per cent of the presentations." Nor should the threshold for acute toxicity be misinterpreted as a "safe" concentration. It is merely a convenient and reproducible reference point: that concentration which would kill the average fish on long exposure. To avoid such misunderstandings, the term incipient LC50 is proposed to describe threshold values as defined in the previous paragraph. The term will be used in this review, and also lethal threshold

concentration. Another set of terms is used for response time in fixed concentrations, usually Median Lethal Time (LT50), and Median Effective Time (ET50). Other words such as median survival time, and resistance time are also used. The symbols follow the established pattern, are useful with fish (ALDERDIeE,1967), and are standard for time-series in pharmacology (LITCHFIELD, 1949). Unfortunately confusion is likely between this notation and the abbreviation TL50 which is to replace TLm (P. Doudoroff, personal communication) in the 13th edition of"Standard Methods" (APHA et al., 1965,12th edition). To avoid misinterpretation, LC50 will be used in preference to TL50, and LT50 will be written in words when required, as in the section immediately following. MEASURING

RESPONSE

AT EACH

CONCENTRATION

T h e r e a s o n f o r u s i n g a g r o u p o f fish in e a c h t e s t - t a n k , i n s t e a d o f o n e fish, is t h a t i n d i v i d u a l s v a r y in resistance. I n this section, v a r i o u s m e t h o d s o f e s t i m a t i n g s u r v i v a l t i m e o f t h e " a v e r a g e " o r " t y p i c a l " fish will be d e s c r i b e d . O b t a i n i n g t h e s e m e d i a n l e t h a l t i m e s is t h e first step in a n a l y z i n g results o f t o x i c i t y tests. T h e y c a n be u s e d to construct median survival curves and estimate incipient LC50's.

M o r t a l i t y within each test-tank Ever since TREVAN (1927) it has been generally recognized that in bioassays, the least and most resistant individuals in a group show much greater variability in response than individuals near the median for the group. A good deal of accuracy may therefore be gained by measuring some average response rather than a minimum or maximum response, which might represent one fish in ten or might happen to represent only one fish in a thousand. A special analytical procedure must be used for toxicity tests in which time plays a double role, as in tests with aquatic organisms. Both concentration and exposure-time are part of the experimental stimulus, yet time is also part of the response (GADDtrM, 1953). Since concentration is fixed within a given test-tank, exposure-time becomes the stimulus controlled by the investigator according to the times which he choses for inspection. Percentage mortality becomes the response. The effect on each animal is quantal (all-or-none), and the test in each container is essentially a quantal bioassay. If cumulated percentage mortality from such a test is plotted against exposure-time, a skewed sigmoid curve usually results. The curve tails off more gradually toward longer survival-times. This skew apparently results from the logarithmic nature of biological time (GADDUM,1953; LECOM'fEDO NO0X', 1937). It is usually eliminated by using a logarithmic scale on the time-axis. The sigmoid nature results from variable individual response causing the curve to resemble a cumulated normal curve, or rather a cumulated lognormal curve (BLiss, 1937; FINNEy, 1964). It is usually straightened by plotting percentage response on a probability or probit scale instead of an arithmetic scale. Probits merely express mortality in terms of standard deviations above and below the mean response, with the value 5.0 added for the convenience of eliminating negative numbers (BLISSand CAa~rELL, 1943). The suitability of a log-probit transformation for time-mortality experiments was shown thirty

Measurement of Pollutant Toxicity to Fish. Part I

801

years ago by BLISS(1937). It seems to have been first used for fish by FRY, HART,and WALKER(1946) for testing lethal temperatures, and had been extensively used for that purpose since. The same transformation is applicable to tests of pollutants against fish, as documented for cyanide by HERBERT and MERKENS(1952) and demonstrated for many toxicants such as pulp mill wastes (ALDERDICEand BRE'rr, 1957), fluoride (HERBERTand SHURBEN,1964), phenol (BROWNet al., 1967a; 1967b), and zinc (SPRAGLIEand RAMSAY,1965).

The first step in analysis of aquatic toxicity tests uses standard pharmacological procedures for experiments involving time and percentage effect. These are described by BLISS (1937). Usually the equivalent but faster methods of LITCHFmLD (1949) are used; they provide nomograms so that numbers can be used in their original form without transforming to probits and logarithms. The essential feature of analyses is simply to plot the data on log-probit paper and fit straight lines by eye. A good example is shown by the results of SHEPARD (1955) in FIG. 2. Seven o f the probit lines are complete and straight. At the three least toxic concentrations, there were incomplete kills of 73 (8 of 11 fish?), 40, and 10 per cent. The important point here is that Shepard continued his tests until the breaks in the probit lines were made obvious, i.e. it was apparent that acute mortality had ceased. ! 90/-

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FIG. 2. An example of mortality in individual test-tanks plotted against time on logarithmicprobability paper. Results are for trout tested at 10 levels of oxygenindicated in the body of the graph. Similar results could be obtained with a toxicant. The lines on the left are for more severely lethal conditions. Exposure was for 5000 rain, equal to 3.5 days. Modified from SHEPPARD

(1955).

Plotting results on log-probit paper also makes it apparent whether there are two or more different modes o f toxic action. Changes in slope or grouping of lines provide the clues. In FIG. 2, the slopes change for the less toxic concentrations at the right, but Shepard interpreted this merely as increasing variability. Much more complicated and unusual "split probit" lines are demonstrated in FIG. 3. From these and similar results, TYLER (1965) concluded that high temperature had three separate mechanisms of death which operated sequentially, the fastest apparently being heat shock. Split probit lines of the same pattern are used by entomologists as the classical indication that insecticide-resistance is developing in a population. The

802

J.B. SPRAGUE

two components of the log-probit line represent normal and resistant individuals (HosKINS, 1960). HARRIS (1968) has shown how two such super-imposed normal distributions may be separated into individual components. Application of this technique could be very useful in evaluating modes of toxicity.

Estimating median response-time The commonest method is to plot the results on logarithmic-probability paper and fit straight lines by eye as described above and shown in FIG. 2. Median survival times may then be read directly from the graph, where the fitted lines cut 50 per cent mortality (LITCHFIELD,1949). The 95 per cent confidence limits may also be estimated easily by Litchfield's method, and this should be done especially if the median lethal times are to be published without supporting data. This is further discussed in the section on estimating error. Again, more exact mathematical methods could be used if desired (BLISS, 1937). Non-lethal effects may also be analyzed by the very same methods; BRETT (1967) used log-probit methods to estimate median fatigue time of fish swimming at various speeds and temperatures. Median survival times have occasionally been estimated from arithmetic plots (HERBERT and DOWNING, 1955). Smooth sigmoid curves are drawn and medians read

90:

0

o//

70

0

50t

./.

27. 5

30

]

f

/ 26.5

J I

J

i t t zI I0

t

,

l L2_a~_,[ 50 I00 Time

of

~

J ~2_~t_L~l_ -_ 500 ~000

Observat[o n,

5000

rain

Fio. 3. Split probit response of a fish to certain lethal temperatures. This unusual response indicates two modes of lethal action at 27.5° and 29.5°C, with a less distinct effect at 30.5°C. The format of the graph is the same as Fie. 2, Modified from TYLER(1965). from the graph. Use o f log-probit paper seems preferable: firstly, differences in slope of lines are more easily detected; and secondly, confidence limits may be placed on the median lethal time. Median survival time m a y also be calculated by various arithmetic procedures.

Measurement of Pollutant Toxicity to Fish. Part I

803

The simplest is to adopt the response-time of the median fish and this is often adequate (DouDOROFF et al., 1966). However, this does not fully use information provided by response times of the other fish and provides no estimate of error. Geometric mean survival time has often been used. It is calculated as the antilog of the mean of logarithms of individual survival times (e.g. BROWN et al., 1967a). This would be the same value as the median for regular lognormal data. Such purely arithmetic procedures should be confirmed by simple graphs (FINNEY, 1964; GADDUM, 1953). For irregular or truncated data, "graphical analysis is essential for the derivation of unbiased statistical constants" (Buss, 1937). The harmonic mean survival times has been effectively used by ABRAM (1964, 1967). It was about equivalent to the median for some toxicants, and ALDERDICEand BRETT (1957) found the same thing for pulp mill waste. However, SSEPARD (1955) found that harmonic means were consistently and significantly shorter than observed times to 50 per cent mortality. The harmonic mean may be calculated as the reciprocal of the arithmetic mean of reciprocals of individual response times. Or it may be estimated by plotting survival time on a reciprocal scale against per cent mortality on an arithmetic scale. Such a graph fitted toxicity data for Malachite green as well as a log-probit plot or better (ABRAM, 1967). Unfortunately, reciprocal of exposure times does not always yield a linear plot with percentage response. For example, logarithm of time is more suitable for cyanide poisoning (HERBERTand MERI
804

J.B. SPRAOUE

Logarithmic toxicity curves Most investigators use logarithmic scales for both time and concentration. An arithmetic plot of concentration against survival-time is unsatisfactory for interpretation for several reasons. The apparent logarithmic nature of time has already been commented upon. Concentrations are also conveniently handled by a logarithmic scale since a wide range may be plotted in reasonable detail. Most importantly, increasing concentration by a given multiple, say doubling, is represented by the same linear distance at any place along the scale. Accordingly the shapes of curves, indicating responses of animals, may be compared for two or more toxicants which are effective at different concentrations. The suitability of a logarithmic transformation of dose is generally accepted in pharmacology (BLISS, 1957) for these and other reasons (GADDUM, 1953). In fish toxicology, DOUDOROFF et al. (1951) simply state that a logarithmic series of concentrations is "always advantageous". For many results presented logarithmically, curvilinear relations have been shown, as in FIG. 4. WUHRMANN(1952) demonstrates such curves for phenol, cyanide, and ammonia. HERaERT (1965) reviews similar curves for metals, sewage, and complex industrial wastes. 4000

End

of Experiment

48 hei

NH 3 =

c

I000

g I00

o ZnSO 4 as p.p.m. Z n l • NH4 Cl asp.p.m. N l

I

i

I

[

i

i

i

ii

i

i

i

lO

Concentration

of

~

1

[

I00

Poison , p.p.m.

F=G.4. Examplesof toxicitycurves for trout. The lethal thresholdconcentrationof ammonia has obviouslybeen defined.This is not clear for zinc; longer experimentsmight have shown additional mortality.From HERBERT(1961). Sometimes the relation appears to be straight instead of curved. Such a situation is shown in FIG. 5 for metal toxicity. Linear logarithmic plots have been shown for cyanide toxicity (DOUDOROFF et al., 1966; HERBERT and MERKENS, 1952), fluoride (HERBERT and SHURBEN, 1964), and hydrochloric acid (LLOYD and JORDAN, 1964). NE(-mING'S (1962) data for thallium plot linearly for times from half a day to at least 14 days. In his review article, ROTHSCHEIN(1964) assumes such a linear relationship as typical for intermediate ranges of toxicity with fish. A linear relation has also been

Measurement of Pollutant Toxicity to Fish. Part I

805

shown for toxicity of certain drugs to mammals, plotting log dose against log survival time (SCHOLZ, 1965), and GADDUM(1953) considers this usual. Thresholds. An approximate and subjective lethal threshold concentration may be estimated by visual inspection of toxicity curves. Straight or curved lines are usually fitted to the points by eye. These are made asymptotic to the time axis either gradually (Fro. 4) or abruptly (FIG. 5) depending on the data. The asymptote marks the value of the incipient LC50 approximately. From FIG. 4, the incipient LC50 of ammonia is about 25 mg/1, and from FIG. 5, the values for copper and zinc are in the vicinities of 50 and 600/tg/l. Since these are subjective, no mathematical confidence limits may be placed on them. Usually an asymptote on the concentration axis may also be seen, representing a minimum time for lethal action. This may be from 10 min (HERBERTand MERKENS, 1952, for cyanide) to about 3 hr (LLOYD, 1960, for zinc). More detailed mathematical consideration has been given to the logarithmic toxicity curve by a few investigators. Because it becomes asymptotic with both axes, it resembles a rectangular hyperbola (ALDERDICE, 1967). It may be mathematically Conceatratlon

of

Metal

moll

,

0.1 J

O.Oa

1.0 ~

I0.

=

,

,

,

200

= o =

I00

:'z o

50

Copper Zinc

ti ° o

o

.i I

i=

20

o

i.=

2

, 2O

i

[

50

r

illl

k

I00

I

t ~ l l t

300

Concentration

f

I000 of

Metal

t

i

i__.i

t

II

5000

.~.O / I

FIG. 5. Examples of straight-line toxicity curves for salmon. The lethal thresholds are indicated by sharp breaks in the lines. F o r each median survival time, 95Yo confidence limits are indicated

From SPP.AGtm(1964). described using Ostwald's formula of concentration, time, their threshold values, and two constants (WUHRMA~% 1952; GADDtrM, 1953; MATIDA, 1960). If the lethal threshold concentration and minimum survival time are estimated and included in the formula, the hyperbolic function is expressed linearly (WUrmMA~,q,~and WAKER, 1950). A simple trial-and-error method of doing this is described by BtmDICK (1957). He shows that transformation of the toxicity curve to a straight line indicates that the lethal threshold concentration has been estimated correctly. Burdick's method of

806

J.B. SPRAGUE

estimating the threshold seems to have been little used, since proposed, but could be a useful tool and is worthy of further trials. Further mathematical consideration of toxicity curves is given by MATIDA (1960) with applications to thresholds, body size, and modifying factors. HEY and HE',"(1960) show efficient calculation of thresholds using the same formula for a rectangular hyperbola mentioned in the previous paragraph. Irregular curves appear for some toxicants. Tests with suspensions of inert solids failed to show any well-defined relation between concentration and survival time (HERBERT and MERKENS, 1961). BALL(1967C) reports an anomalous S-shaped toxicity curve for cadmium, in which all concentrations from 0.01 to 1.0 rag/1 killed fish in about 6 days, although a threshold appeared at a somewhat lower concentration. ALDEgDICE and WORTmNOTON (1959) show a "bizarre distribution of resistance times" for DDT-in-oil and emulsifier. Maximum toxicity was at intermediate concentration, and fish survived longer in higher concentrations! (The authors credited this to unstable emulsions.) One such peculiar case has been given an adequate chemical explanation. DOOOOROFF(1956) found distinct "primary" and "secondary" mortality in tests with mixtures of cyanide and nickelous sulfate. He proved that this resulted from a great change in cyanide toxicity with a small change in pH from respiratory carbon dioxide. Sometimes the relation is regular but no threshold is apparent. The toxicity curve continued with no threshold during 15 days for high pH (JORDAN and LLOYD, 1964) and during 12 weeks for detergent (HERBERTet al., 1957). These examples of unusual toxicity curves by no means invalidate the usefulness of logarithmic plotting. On the contrary, they show the value of constructing such a survival curve, so that unusual toxicity relationships will be uncovered. Such relations could easily be obscured by incomplete toxicity curves obtained by arbitrarily restricting length of exposure or frequency of inspection times. Weak or erroneous interpretations could result. The value of obtaining complete toxicity curves is particularly well illustrated in the work of BROWNet al. (1967a); complete curves allowed them to interpret and demonstrate a complicated reversal of temperature effects on toxicity of phenol. DOUDOROFFet al. (1966) used the toxicity curve to demonstrate that molecular hydrocyanic acid is the cause of acute toxicity, not total cyanide. BALL(1967a) showed that full curves were necessary to evaluate ammonia. One-day tests indicated trout were more sensitive than coarse fish, but actually the coarse fish were merely slow to react. Completion of toxicity curves, requiring 3- to 5-day tests, showed that all fish were equally sensitive in terms of incipient LC50. An additional minor irregularity has been pointed out by ROTHSCnEtN(1964). Some pollutants which occur naturally in trace amounts (e.g. copper) are necessary for life. Below an optimum concentration, the toxicity curve would double back to shorter times. However, this would only occur if the test-water were deficient in the necessary element. Furthermore the irregularity would not exist for most man-made pollutants. Two approaches f o r construction of toxicity curves such as those in FIGS. 4 and 5 may be used: firstly, median lethal times may be estimated for a series of concentrations, as described above; secondly, median lethal concentrations may be estimated for a series of inspection-times (see section on U.S. standard method). If the response is reasonably regular and inspections of mortality are equally frequent, either method should give essentially the same curve.

Measurement of Pollutant Toxicityto Fish. Part I

807

Semi-log toxicity curves These are relatively uncommon in fish toxicity work. However, the logarithm of survival time plotted against an arithmetic scale of temperature is customary in lethal temperature work with fish, and yields linear or nearly linear relations (FRY, 1947). Curves with reciprocal survival time The reciprocal of time was used by early investigators of fish toxicology (see JONES, 1964). Recently, the usefulness of this transformation has been demonstrated for several toxicants by ABRAM (1964, 1967). Since infinity appears as one extreme value on reciprocal graph paper, suitable choice of time units can cause infinite time to be physically very close to useful finite times--Abram shows times from 1 week to 10 weeks in such proximity. Extrapolations can be made to infinite exposure-time. Abram extrapolates to (a) ~ kill at infinite time for various constant concentrations, and (b) concentration lethal to half the fish at infinite time. The extrapolated values from (a) could also be used to estimate (b). The technique would seem very useful. Obviously no fish would live for an infinite time. Also, the linear relation would often be disturbed by secondary or tertiary toxic effects at long exposures. However, this is essentially a technique for extrapolating to the threshold of a given kind of toxic action, say primary. Unfortunately, this reviewer's experience has been that .not all data are amenable to analysis on reciprocal paper. However, Abram showed relatively straight plots of log concentration vs. reciprocal time for data on phenol, sodium sulphide, potassium cyanide, and 3 pesticides. Undoubtedly the technique would be applicable to other pollutants. Results could easily be plotted on reciprocal paper on a trial basis according to Abram's methods. Such trials would seem desirable as an additional way of estimating reasonable threshold concentrations. ALDERDICE and BRETT (1957) concluded that reciprocals were the best transformation for both survival time and concentration in tests with salmon and pulp mill effluent. Their procedure for mathematically estimating the threshold concentration would no doubt frighten most biologists, but a line fitted to their data by eye would almost certainly give much the same threshold for infinite survival. They obtained confidence limits from their caclulations. Toxicity curves using the U.S. standard method The "routine bioassay method" given in "Standard Methods" (APHA et al., 1965) is more or less standardin the U.S.A. and will be called hereafter the "U.S. standard method". It is discussed separately since it has a somewhat different approach. It is described by DOUDOROFFand his committee (1951) and almost identically by APHA et al. (1965). These descriptions are based on a more extensive outline, now out of print, by HART, DOUDOROFF,and GREENBANK(1945), in turn derived from research methods of DOUDOROFF(1942, 1945). The method is well-known, and yields median tolerance limits (TLM's or LC50's), customarily for 1, 2, and 4 days. For each fixed time, percentage mortalities are plotted against test concentrations. Concentration lethal to half the fish is estimated by interpolation. This is very much the same as standard quantal bioassays in pharmacology, which plot percentage response against dose. -Hence, standard techniques of analysis may be easily adapted.

808

J.B. SPRAGU~

It is clear that the U.S. standard method was intended to be a valid tool for pollution control, yet simple enough for industrial laboratories unfamiliar with bioassays and fish. The committee succeeded admirably in its purpose, judging by the wide use of the method. However it is unfortunate that many biologists, particularly in the U.S.A., have been content to use for research, the method described by DOUDOROFFet al., as "the simplest routine procedure". Only in recent years have researchers started to follow some of the low-key suggestions for obtaining increased information. For example, following the description of the routine method for calculating LCS0, the A P H A et al. 0965) suggest: "Other widely accepted and often more satisfactory procedures include graphical methods which involve fitting a smooth, sigmoid c u r v e . . . or fitting a straight line to data plotted on logarithmic probability paper, as well as the more refined methods o f probits, logits, or angles." Some recent research papers have followed suggestions of the committee on three topics which will be described below: (1) placing confidence limits on the LCS0; (2) longer exposure times: and (3) more frequent inspection of mortality. Criticism as a research method eentres mainly on using data in its quantal form (reacted or not reacted at an arbitrary time) instead of utilizing the graded time-response which is available. Each animal supplies more information if its survival time is measured, rather than being classified as positive or negative (BLISSand CATTEL,1943). If most of the animals die during the test, "neglect of the information conveyed by the detailed time measurements may seriously reduce the precision. . . . " (FINNEY, 1964). GADDUM(1953) estimates that "theoretically it may be expected that about half the information will be lost, so that twice as many observations will be needed for any given degree of accuracy." An even higher estimate is "ten times the number of replicates to obtain the same confidence limits that would be reached through the use of the logarithmic mean time of the death time of all fish" (BURDICK,1960). This criticism would seem to be negated if estimates of LC50, by the regular U.S. standard method, were made more frequently during a test. The resulting toxicity curve should look the same as one constructed from median lethal times. They should be equally valid if based on the same observations of mortality at successive times. Since the same data on fish mortality could be used either way, my own preference is to estimate time to 50 per cent mortality in each concentration; this shows in a simple way what happens in each test tank (FIG. 2) and is easy to understand if complications arise (FIG. 3). LC50'S based on short exposure times, often 24 hr, were frequently published in research articles some years ago, although the U.S. standard method recommends continuation for at least 48 hr. DOUDOROFFet aL (1951) seem to have had research work in mind when they stated: "Median tolerance limits for longer exposure periods (for example 8 days, etc.) thus can be determined. The concentration at which half of the animals are killed eventually, while the rest . . . survive indefinitely ( . . . the ultimate median tolerance limit) sometimes can be established." Obviously this describes a lethal threshold. Happily, most researchers in recent years have carried on their tests for at least 48 hr and usually 96 hr, and for certain purposes to 7-12 days (PICKERINGand VIGOR, 1965), or even 30 days (PICKERINGe t al., 1962; JENSEN and GAUFIN, 1966). Confidence limits have customarily been omitted for LC50's estimated by this method. However this has been done recently, by American workers CAmNS and SCHEIER (1963) and PICKERINGand VIGOR (1965). The topic is an important one, and is discussed in a separate section. Differences between 1-, 2-, and 4-day LC50's, or lack of difference, can indicate whether or not a threshold has been reached. This may be formalized by statistical

Measurement of Pollutant Toxicity to Fish. Part I

809

tests of difference. THATCHER (1966) compared successive LCS0's of detergent by means of t-tests, since he had several separate estimates for each time-interval. PICKER1NG and HENDERSON(1966a, 1966b, 1966c) used computer programs to test significant differences. Such procedures are useful for publication. Many biologists would be assisted in their interpretation by also plotting a simple toxicity curve as the tests proceeded. Constructing toxicity curves requires more frequent estimates of LC50. It is surprising that more researchers using the U.S. standard method have not followed the lead given in research papers by DOUDOROFF (1942, 1945); he used a logarithmic series of 0.5, 1, 3, 6, 12, and 24 hr followed by daily inspections for as long as 11 days. HART et al. (1945) in an appendix intended as instructions for research laboratories, outlined a similar series up to 10 days. They constructed and named a logarithmic time-concentration curve of tolerance, and estimated the ultimate median tolerance limit (DouDOROFF, 1945). These are exactly equivalent to the toxicity curves and thresholds described in sections above. A logarithmic curve of tolerance has been used more recently by Doudoroff and colleagues (1966). Research workers using the U.S. standard method should probably make more frequent observations of mortality and construct a time-concentration toxicity curve. Effort required for making extra observations within the 96-hr period, and for plotting a few graphs, would be negligible compared with total effort in preparing fish, apparatus, and toxicant. It would produce results which could be interpreted by any of several approaches. Furthermore, the vast amount of past information on 48- and 96-hr LC50's could still be compared directly, conforming with the objective of Doudoroff and colleagues " . . . the accumulation of comparable data". Acute vs. chronic toxicity Harmful effects of pollutants may be divided into a number of categories but they overlap somewhat. Of most interest here are primary lethal effects, those which are seen in short-term tests. Acutely lethal toxicity would be considered that which causes severe and rapid damage to the organism by the fastest acting mechanism of poisoning, fatal unless the organism escapes the toxic environment at an early stage. Neither this nor any other definition has been found by this reviewer to separate acutely lethal action from other kinds of effect. Most effects could probably be described accurately by using one of the following five terms, or by using two of them together. All of them have good dictionary meanings. In the present context of toxicity to aquatic life, the terms might be briefly defined as follows: acute--coming speedily to a crisis; chronic--continuing for a long time, lingering; lethal--causing death, or sufficient to cause it, by direct action; sublethal--below the level which directly causes death; and cumulative brought about, or increased in strength, by successive additions. These and other terms have sometimes been used in careless or conflicting ways, to the extent that they have lost a good deal of their precision. In general we may distinguish two broad categories of effect (ALDEPaglCE, 1967), acute toxicity which is usually lethal, and chronic toxicity which may be lethal or sublethal. Concerning acute toxicity to fish, there seems to be a working consensus that it B

810

J . B . SPRAGUE

TABLE I. ESTIMATESOF TIME REQUIRED FOR CESSATIONOF ACUTE LETHAL ACTION, IN VARIOUSBIOASSAYS REPORTED IN THE LITERATURE Toxicant

Species*

Apparent time of lethal threshold hr = hour, d = day, wk = week

Cyanide Cyanide Ammonia Ammonia Ammonia Fluoride Chlorine High pH Zinc Copper/zinc Zinc Copper Copper/zinc Heavy metals

Phoxinus trout trout 4 fw. fishes Phoxinus trout trout trout minnow fry salmon zebrafish trout trout fw. fishes

Zinc Zinc Zinc Cadmium Eighteen metals

minnow eggs 4 fw. fishes bream trout stickleback

Copper Thallium Various (6)

crayfish Perca tubificid worms Gambusia

10-15d (delayed mortality) more than 14d 2d or less

Pickering, Vigor 1965 Ball 1967b Ball 1967b Ball 1967c Doudoroff and Katz 1953 from data of Jones 1938, 1939 Hubschmann 1967 Nehring 1962 Marvan 1963

4d

Hart et aL, 1945

trout

14d or more

Herbert 1965

bluegill l l fw. fishes trout 5 fw. fishes minnow eggs

1d or less (static tests) 2d or less (continuous-flow) acute l d ? subacute continued 12 wk more than 4d? (continuous-flow) 9d or more (continuous-flow)

Lemke, Mount 1963 Thatcher 1966 Herbert et al., 1957 Thatcher, Santner 1966 Pickering 1966

trout 4 fw. fishes trout

l d or less (saline water) 5 hr-ld 1d

Brown et al., 1967b W u h r m a n n 1952 Brown et aL, 1967a

fw. fishes

62 of 75 cases, l d or less; remainder 4d or more (static tests) 1.Sd acute 1.5d ? subacute 2wk ? 30d or more (several modes of action .9) 14 cases, 2d or less; 8 cases, 4d or more (static tests). Cont. flow tests, 20d or more

Pickering, Henderson 1966b

Oil-field brine Corrosion inhibitor ABS detergent ABS detergent Detergents LAS detergent ABS, LAS detergents Phenol. Phenol. Various phenolics Various petrochem, D D T (acetone) DDT Five insecticides Chlor. Hyd. insecticides

salmon trout 2 stoneflies 4 fw. fishes

about 2d 4d or more 5 hr < ld--4d about 2d about 7d more than 7d more than 15d l d or less l-3d 1-6d, various young stages 2-4d 4d or less 2d or less for about half of 59 cases; 4d or longer for other half (static tests) 7d or less 4-5d 7d or more 7d 7d or more in each case

Authors

Wuhrmann 1952 Herbert, Merkens 1952 Lloyd 1961b Ball 1967a W u h r m a n n 1952 Herbert, Shurben 1964 Merkens 1958 Jordan, Lloyd 1964 Pickering, Vigor 1965 Sprague, Ramsay 1965 Skidmore 1965 Liepolt, Weber 1958 Lloyd 1960, 1961a Pickering, Henderson 1966a

Alderdice, Worthington 1959 A b r a m 1967 Jensen, Gaufin 1966 Henderson et aL, 1959

Measurement of Pollutant Toxicity to Fish. Part I

811

TABLE 1 (continued)

Toxicant Organophosphate insecticides Various pesticides Sewage effluents Pulp mill effluent Pulp mill effluent Many pollutants

Species*

Apparant time of lethal threshold hr = hour, d = day wk -- week

Authors

6 fw. fishes

41 cases, 2d or less; 27 cases, 4d or Picketing et al., 1962 more (static tests)

various fw. fishes trout

25 cases, 2d or less; 13 cases, 4d or Pickering, Henderson 1966c more (static tests) one, 8hr; three, about 3d Lloyd, Jordan 1963

salmon

about 12d

Alderdice, Brett 1957

Sprague, McLeese 1968 salmon about 7d lobster larvae various invert, of 82 cases: ld or less, 26 cases; Dowden, Bennet 1965 esp. Daphnia 1-3d, 14 cases; 2d or more, 13 cases; 4d or more, 29 cases (static tests)

* fw. = freshwater. Trout are rainbow trout. generally occurs within the first I00 hr of exposure (WARNER,1967) or about 4 days (CAIRNS, 1966). The 2-day and 4-day exposures of the U.S. standard method have generally been accepted as covering the period of acute lethal action. Some assessment of the usual time limits for acute lethality may be made from TABLE 1. It is compiled from selected published material which gave sufficient detail that a threshold could be estimated. This was done by inspection of toxicity curves or from lack of change between LCS0's at successive time intervals. The review in TABLE 1 is not complete and involves many subjective interpretations of the reviewer. Tabulation by cases is heavily weighted by the multiple tests of Picketing, Henderson, and colleagues and Dowden and Bennet, which use m a n y toxicants and several species. These are are mostly static tests which may tend to show lethal thresholds in relatively short times. This effect of static tests is suggested by the incipient LC50's shown in TABLE 1 for detergents. That from a static test is 1 day or less while those from continuous-flow tests apparently range from less than 2 days to 4 or 9 days, possibly up to 12 weeks, admittedly, the comparison is not exact. Many of the pesticides appear to be cumulative poisons and are not strictly comparable with other pollutants. Despite these and other deficiencies, the information in Table 1 is instructive and is tabulated in the text table. 1 day or less 2 days or less 4 days or less 4 days or more 7 days or more 14 days or more N o t fitting above classification 13

98] 83) 30

211 cases 122 cases

26]? 16.)

42 cases

Of 375 cases, 211 or 56 per cent showed a lethal threshold in 4 days or less. Only 42 cases are clearly longer than 4 days. The remaining 122 cases may be about 4 days or may be longer. The overall distribution tends to substantiate that 4 days is a

812

J.B. SPRA~UE

reasonable limit for occurrence of acutely lethal toxicity of most pollutants. However this must not be taken as an arbitrary time limit, since acute effects of some toxicants obviously continue beyond 4 days. Caution in generalizing too much from these results is particularly necessary since such a tabulation may apparently be easily biased. The possible influence of static bioassays has already been mentioned. As another example, TABLE 1 includes results for 18 metals studied by JONES(1938, 1939). The summary of his results by DOUDOROFF and KATZ (1953) shows that mortality continued after 4 days, in every one of the 18 cases. Although these results are difficult to interpret because of mortality in controls, nevertheless they tend to run directly contrary to the general trend in Table 1 toward short incipient LC50's. Indeed, there is considerable contradiction evident if we consider only the threshold times for metals taken from the 15 publications included in Table 1. How long should acute toxicity tests be continued, in view of this information ? Apparently no single time can be stated. It would seem prudent to continue tests for 4 days as a rule. Tests could then be stopped if mortality had ceased and the toxicity curve showed a threshold. I f this were not the case, research tests should be continued until the shape of the toxicity curve is clearly established. Usually this would be a week or 10 days (TABLE 1). However, it could be longer, in which case tests might be stopped for practical or economic reasons. If no threshold were apparent, this should be clearly reported since it is of considerable interest. Quantitative estimates of thresholds for acute toxicity An extremely felicitous combination of approaches has recently come into general use by British workers (HERBERT and SrIURBEN, 1964; BROWN et al., 1967a, 1967b; BALL, 1967a, 1967b). In brief, they drew toxicity curves to show the general pattern of response. However they did not attempt to read an incipient LC50 from a given toxicity curve. Instead, they re-used part of the same data to make an accurate estimate of the incipient LC50 by log-probit methods. The whole approach is highly recommended and is described more fully below. Complete toxicity curves of time vs. concentration are first constructed (similar to FIGS. 4 and 5). These are based either on median mortality times (BROWN et al.) or on frequent median lethal concentrations (Ball). By inspection of each curve, a time is chosen which is obviously equal to or longer than the time when acute lethality has ceased. These times turned out to be from 1 to 5 days for the various species and toxicants in the papers listed above. For the selected time in each case, the median lethal concentration was estimated by standard log-probit methods (FIG. 6 and described below under Procedure). This is termed the incipient LC50 here and was termed by the British authors asymptotic LC50 or simply 48-hr LC50 since 48 hours included the lethal threshold in some cases. Nothing in the method is particularly new, but the application of the entire technique to fish toxicity work is novel. SHEPARD(1955) seems to have been the first to use this log-probit technique to estimate thresholds with fish. He calculated incipient lethal levels of oxygen for trout. He did not provide confidence limits, but gave a "slope function" for each probit line as an indication of variability. Sublethal responses may also be analyzed by exactly the same log-probit technique; for example the median avoidance concentration has been estimated (SPRAGLr~, 1968).

Measurement of Pollutant Toxicity to Fish. Part I

813

As a standard method, this way of reporting toxicity data seems well worth emulating. It has the advantages of" (1) complete toxicity curves for easy interpretation of results; (2) an incipient LC50 instead of one for an arbitrary time; (3) a mathematical instead of a subjective estimate of incipient LC50; and (4) confidence limits on that LC50. The advantages of an incipient LC50 are succinctly stated by Wuhrmann at the head of this section. It allows the toxicities of different pollutants to be compared easily and meaningfully. In addition, this threshold value serves advantageously as a basic measuring unit for predicting joint toxicity of two or more pollutants (see Part II), and and for describing sublethal effects (Part III). Procedure for estimating the incipient LC50 is shown in FIG. 6. This is similar to the 0,2

o u

5

o

o- 2 0

vo

x

5O

80

(_)

a.

95

99

99.8

I 2

I

t

I

4

Concentrotion

6 of

I Itll 8

I I0

Fluoride,

20

t

I 40

p.p.m. F

FIG. 6. Estimating the median lethal concentration. In this case the incipient LC50 is estimated since the exposure time was long. Percentage response of trout is plotted on the vertical probit scale. The median lethal concentration is 8.5 mg/l, and its confidence limits could be estimated as described in the text. The 5 per cent response is also shown. F r o m HERBERT and

SmmBEN (1964). routine U.S. standard method except that it uses probits, following standard pharmacological techniques. Analysis of results by the rapid graphic methods of LITCHFIELDand WILCOXON(1949) is recommended. (These improvements are also suggested by A P H A et al. (1965) as steps beyond the routine method.)To carry out the LitchfieldWilcoxon procedure, actual percentage mortality in each test tank at the selected time beyond the lethal threshold, is plotted on log-probit paper as in FIG. 6. A line is fitted to the points by eye. Its goodness of fit is estimated by a rapid chi-square test. In

814

J.B. SPRAGU~

doubtful cases, several lines may be drawn and the best chosen by minimum chisquare value. The incipient LC50 is then read from the graph. Its confidence limits may be estimated as described in the section on errors. If desired, the more formal and time-consuming mathematical procedures of FINNEY (1962) may be used to estimate the incipient LC50. The line is fitted by successive approximations based on maximum likelihood methods, since the well-known method of least squares is unfortunately not valid for these quantal log-probit relations. The extra computations would seldom seem warranted, since it has been shown (LITCHFIELDand WILCOXON,1953) that log-probit lines fitted by eye are often highly accurate. EISENBERG(1952) finds graphical estimates of ED50 are "in very good agreement" with the formal mathematical estimates. Even FINNEY (1964) gives limited approval to eye-fitted lines for moderately good data. GADDUM(1953) states bluntly that "there is no evidence against the view that the calculation of successive approximations is a pure waste of time." As mentioned in previous sections, sometimes a lethal threshold may not be found, even after weeks of testing. If not, some arbitrary time period could be substituted, and a 4-day LC50, one-week LC50, etc., could be estimated in the same way as described above. The incipient LC50 must be estimated by using the actual observed percentage mortalities from each test tank. Derived values for a selected exposure time should not be taken from fitted lines such as those in FIG. 2. Only by using original observations to construct graphs such as FIO. 6 can confidence limits be calculated and comparisons of statistically significant differences be made.

Alternative methods of estimating thresholds Other techniques have been developed and may sometimes be preferable or could be used for confirmation. As mentioned before, the logarithmic toxicity curve may be treated as a hyperbola, and its asymptote with the time axis estimated mathematically (WUHRMANN,1952; MATIDA,1960; HEY and HEY, 1960) or graphically (BuRDICK, 1957). In the section on curves with reciprocal survival time various techniques of ABRAM (1964, 1967) and ALDERDICEand BRETT(1957) were described, for estimating the infinite LC50. Another method used to calculate LC50's is that of moving averages, in conjunction with angular transformations. Because of special interest in the error calculation, all aspects of this method are discussed together for convenience in the section on confidence limits. Mortality in controls Substantial mortality in control tests, merging with the mortality at some concentrations of toxicant, sometimes interferes with interpretation of experiments, especially long ones. For example, this occurred in tests lasting a few weeks against lobster larvae, which are difficult to rear in the laboratory without some mortality (SPRAGUE and MCLEESE, 1968). It is possible to consider the threshold concentration as that which allows survival time equal to the control. However this is not deemed entirely satisfactory although it would be similar to medical studies, in which equal survival in control and experimental groups is taken to indicate absence of chronic illness. A correction can be made by the simple method of TATTERSFIELDand MORRIS (1924). The observed percentage mortality at a given test concentration is corrected by

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815

deducting the percentage dead in the control; the result is multiplied by 100, then divided by the quantity (100 minus the percentage dead in the control). This yields a corrected percentage mortality at the given concentration. However, even that procedure does not solve the main problem, which is the possibility of interaction of stress from the experimental condition with whatever stress causes mortality in the controls. In view of the additive effects now shown for a number of diverse toxicants, such interaction of stresses should be considered a probability rather than merely a possibility. (Joint toxicity will be discussed in Part II). Working with fish, it would seem such preferable, and usually quite possible, to create laboratory conditions which permit indefinitely long survival of the animals. ESTIMATING ERROR For the sake of completeness, pertinent references and procedures have been gathered in this section, although this involves some repetition. Confidence limits of median response For median lethal times, confidence limits are often omitted. However, they can be very useful to the investigator when fitting a line by eye to a set of median survival times (e.g. FIo. 5). Also the reader could more easily evaluate results to his own satisfaction. The rapid graphic methods of LITCHFIELD(1949) are suitable for estimating confidence limits of median survival times and have been used (SHEPARD,1955; SPRAGUE and RAMSAY, 1965) as have the more formal methods of BLISS (1937) by BROWN et al. (1967a). Litchfield also provides simple methods for testing whether two median survival times are significantly different, and whether slopes of two probit lines are different. These could sometimes be useful in evaluating results. For median lethal concentrations, confidence limits are even more valuable (BALL, 1967a). The standard and preferred (BLISS, 1956; FINNEY, 1964; GADDUM, 1953) methods are based on logarithmic-probability transformation. The easiest and most rapid of these is the graphical procedure of LITCHFIELDand WILCOXON(1949). It has been used for 96-hr LC50's (CAIRNSand SCHEIER,1963), and incipient LC50's (BALL, 1967a; BROWN et al., 1967a). The more time-consuming formal method of probit analyses (FINNEY, 1952) has also been used (BROWN et al., 1967b). However, the Litchfield-Wilcoxon method compares well with the formal mathematical procedure, and if anything is somewhat more conservative in that it tends to give larger values for confidence limits (EISENBERG,1952). A case has been made (BALL, 1967a) for publishing the parameter termed "slope function" or " S " by Litchfield and Wilcoxon. It is the factor by which a dose must be multiplied or divided to produce a standard deviation change in response. Knowing LC50 and S, a probit line may be reconstructed. The point seems a good one, and publication not only of incipient LCS0 and its confidence limits, but also S, should be done inasmuch as it may have value for future investigators. Confidence limits based on reciprocal transformations have been calculated by ALDERDICEand BRETT(1957) for an infinite LC50. The only other method which seems to have been used in fish toxicity studies is based on the angle transformation instead of probits, and a moving average to estimate the LC50 instead of a graph (HARRIS, 1959; THATCHERand SANTNER,1966). It was conclusively shown that LC50's for zinc, petrochemicals and detergent were

816

J.B. SPRAGUE

almost identical for this moving average method and the U.S. standard method (PICKEmNG and Vie,OR, 1965; PICKERII,rG and HENDERSON, 1966b; PICKERING, 1966). Nevertheless it would seem prudent to plot the original unsmoothed data on a graph and examine them for any interesting irregularities, as a supplement to the arithmetic estimate. Estimating confidence limits by the angle transformation instead o f probits could have some disadvantages. Equal numbers of animals must be used at each concentration, and concentrations must be separated by a constant interval, usually logarithmic (HARRIS, 1959). Malfunction of apparatus, which breaks the series of concentrations, prevents use of the method, as encountered by PICKEmNG (1966). The angular transformation is considered reasonable for bioassays only if extreme responses of zero and one hundred per cent do not occur (BLISS, 1956; FINNEY, 1964), but these are frequent when using the U.S. standard method, as pointed out by PICKERING and HENDERSON (1966b). Probably they would also be frequent in calculations of incipient LC50's. GADDUM (1953) recommends that the angular transformation should only be used when responses are within the limits of 20 to 8 0 ~ . Serious misinterpretations of estimates of error are possible by any of the above methods. The confidence limits are based only on internal evidence from one experiment. They are merely indicators of what might be expected if the very same stock of animals were immediately retested under identical conditions! They do not show the differences that might be expected in any repetition of an experiment (FINNEY, 1952); those would be considerably larger (LITCHFIELDand WmCOXON, 1953). For example, at different times of year, the same stock of fish in the same laboratory may give LC50's which differ by a factor of 2.5 (BRowN, 1968). Variation with time, place, and species is well known to most biologists. DOUDOROFF et al. (1951) make it clear that such variation played an important part in arriving at a suitable compromise between precision and simplicity of methods for their routine bioassay procedure.

Testing differences between median lethal concentrations Any of the methods in the previous section, which estimate the LC50 and its error, also make it possible to test for significant difference between two LC50's. In procedures originally developed for pharmacology (e.g. L~TCHEIELDand WILCOXON, 1949), the appropriate instructions are those for testing whether two drugs differ in potency. Testing significant differences between LC50's has been done very little in fish toxicology but seems to be coming into use. Particularly good examples have been provided recently by PICKERING and HENDERSON (1966a, 1966b) who compared toxicity o f many petrochemicals and several metals to four species of fish in two types of water. They tested by a computer program based on the moving average-angle transformation. Analyses of variance and multiple range tests have also been used to test for different responses among species or replicated experiments (THATCHERand SANTNER, 1966). Acknowledgements--Discussions with Dr. D. F. ALDERDICE,Dr. PETERDOUDOROFF,and Mr. IAN R. BALLhave been of great profit to me in preparing this review, as have several unpublished writings of Dr. ALDEROICE.Dr. JOHN M. ANDERSON,Dr. CHARLESE. WARREN,Mr. RtcHARo LLOYDand Mr. THOMASO. THATCHERcommented on the manuscript. I thank these gentlemen and acknowledge that they would probably not agree with all that is written here. Dr. ALFREDA. HEUSN~R,now of the University of California, Los Angeles, kindly supplied information on replacement times of flowing

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water systems. QUENTIN H. PICKEg~qG gave me details of the forthcoming revision of the A P H A bioassay method. I appreciate facilities, especially those of the library, extended by Oregon State University, during my stay on sabbatical leave, when this review was written. I am grateful to the following publishers, journals, and their editors for permission to reproduce figures: Blackwell Scientific Publications, Fisheries Research Board of Canada; Marine Biological Laboratory; National Research Council; Pergamon Press; Society for Water Treatment and Examination; United States Department of the Interior; Water and Waste Treatment Journal. REFERENCES ABRAMF. S. H. (1960) An automatic dosage apparatus. Lab. Pract. 9, 796-797. ABRAM F. S. H. (1964) An application of harmonics to fish toxicology. Int. J. Air Wat. Pollut. 8, 325-338. ABRAMF. S. H. (1967) The definition and measurement of fish toxicity thresholds. Advances in Water Pollution Research, Proc. 3rd Int. Conf. Miinich 1966. Vol. 1, pp. 75-95. Pergamon Press, Oxford. ALABASTER,J. S. and ABRAMF. S. H. (1965) Development and use of a direct method of evaluating toxicity to fish. Advances in Water Pollution Research Proc. 2nd Int. Conf., Tokyo 1964. Vol. 1, pp. 41-54. Pergamon Press, Oxford. ALOEROICE D. F. (1967) The detection and measurement of water pollution biological assays. Canada Dept. Fisheries: Can. Fish. Rept. No. 9, 33-39. ALDERICED. F. and BRETT J. R. (1957) Some effects of kraft mill effluent on young Pacific salmon. J. Fish. Res. Bd Can. 14, 783-795. ALOERDICED. F., BRETT, J. R. and StrrHERLANDD. B. (1966) Design of small holding tank for fish. J. Fish. Res. Bd Can. 23, 1447-1450. ALD~RDICED. F. and WORTHINGTONM. E. (1959) Toxicity of a DDT forest spray to young salmon. Can. Fish Cult. 24, 41--48. A P H A et aL (1965) Standard Methods for the Examination of Water and Waste Water Including Bottom Sediments and Sludges. Am. Pub. Health Assoc., New York, 12th edition, 769 pp. BALL I. R. (1967a) The relative susceptibilities of some species of freshwater fish to poisons--I. Ammonia. Water Research 1,767-775. BALL I. R. (1967b) The relative susceptibilities of some species of freshwater fish to poisons--II. Zinc. Water Research 1, 777-783. BALL I. R. (1967c) The toxicity of cadmium to rainbow trout (Salmo gairdnerii Richardson). Water Research 1, 805-806. BEAKT. W. (1958) Toleration offish to toxic pollution. J. Fish. Res. Bd Can. 15, 559-572. BETTSJ. L., BEAKT. W. and WILSONG. G. (1967) A procedure for small-scale laboratory bioassays. J. Wat. Pollut. Control Fed. 39, 89-96. BLISS C. I. (1937) The calculation of the time-mortality curve. Ann. appL BioL 24, 815-852. BLiss C. I. (1952) The Statistics of Bioassay, with Special Reference to the Vitamins. pp. 445-628. Academic Press, N.Y. BLISS C. I. (1956) Confidence limits for measuring the precision of bioassays. Biometrics 12, 491-526. Buss C. I. (1957) Some principles of bioassay. Am. ScL 45, 449--466. BLIss C. I. and CATTELLMcK. (1943) Biological assay. Ann. Rev. Physiology 5, 479-513. BREITIG G. (1966) Zur Notwendigkeit der Standardisierung von Toxizitgttstesten. Verh. int. Verein. theor, angew. LimnoL 16, 979-984. BRETTJ. R. (1956) Some principles in the thermal requirements of fishes. Q. Rev. Biol. 31, 75-87. BRETT J. R. (1967) Swimming performance of sockeye salmon (Oncorhynchus nerka) in relation to fatigue time and temperature. J. Fish. Res. Bd Can. 24, 1731-1741. BROWN V. M. (1968) The calculation of the acute toxicity of mixtures of poisons to rainbow trout. Water Research 2, 723-733. BROWN V. M., JOROAN D. H. M. and TILLER B. A. (1967a) The effect of temperature on the acute toxicity of phenol to rainbow trout in hard water. Water Research 1,587-594. BROWN V. M., SntlRBEN D. G. and FAWELLJ. K. (1967b) The acute toxicity of phenol to rainbow trout in saline waters. Water Research 1, 683-685. BtmRKLE U. (1967) An audiogram of the Atlantic cod, Gadas morhua L. J. Fish. Res. Bd Can. 24, 2309-2319.

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BURDICK G. E. (1957) A graphical method for deriving threshold values of toxicity and the equation of the toxicity curve. N. Y. Fish & Game 4, 102-108. BURDICK G. E. (1960) The use of bioassays by the water pollution control agency. Biol. Problems Wat. Pollut. Trans. of Second Seminar, 1959, U.S. Public Health Service, R.A. Taft Sanit. Engng Center, Tech. Rept. W 60-3, 145-148. BURDICK G. E. (1967) Use of bioassays in determining levels of toxic wastes to aquatic organisms. Trans. Am. Fish. Soc. 96(1) Suppl., Spec. Publ No. 4, 7-12. BURKE W. D. and FERGUSOND. E. (1968) A simplified flow-through apparatus for maintaining fixed concentrations of toxicants in water. Trans. Am. Fish. Soc. 97, 498-500. CAIRNS, J. and SCHEIER m. (1963) Environmental effects upon cyanide toxicity to fish. Notulae Naturae, Philad. Aead. Nat. Sci. 361, 11 pp. CHADWICK G. G. and KUGEMAGI U. (1968) Toxicity evaluation of a technique for introducing dieldrin into water. J. Wat. Pollut. Control Fed. 40, 76-82. DENZER H. W. (1959) Merkblatt iiber die Sch~digung der Fischerei durch Abw~'sser. L Schwellenwerte flit Fisehe und Fischn~hrtiere. Landesanstalt ffir Fishcherei, Nordrhein-Westfalen, 78 pp. DOUDOROFEP. (1942) The resistance and acclimatization of marine fishes to temperature changes. L Experiments with Girella nigricans (Ayres). Biol. Bull. 83, 219-244. DOUDOROEFP. (1945) The resistance and acclimatization of marine fishes to temperature changes. IL Experiments with Fundulus and Atherinops. Biol. Bull. 88, 194-206. DOUDOROFF P. (1956) Some experiments on the toxicity of complex cyanides to fish. Sewage ind. Wastes 28, 1020-1040. DOUDOROFFP., ANDERSONB. G., BURDICKG. E., GALTSOFFP. S., HART W. B., PATRICKR., STRONG E. R., SURBER E. W. and VAN HORN W. M. (1951) BiD-assay methods for the evaluation of acute toxicity of industrial wastes to fish. Sewage ind. Wastes 23, 1380-1397. DOUDOROFF P. and KATZ M. (1953) Critical review of literature on the toxicity of industrial wastes and their components to fish. IL The metals, as salts. Sewage ind. Wastes 25, 802-839. DOUDOROFFP., LEDUCG. and SCHNEIDERC. R. (1966) Acute toxicity of solutions containing complex metal cyanides, in relation to concentrations of molecular hydrocyanic acid. Trans. Am. fish. Soc. 95, 6-22. DOWDEN B. F. and BENNETTH. J. (1965) Toxicity of selected chemicals to certain animals. J. Wat. Pollut. Control Fed. 37, 1308-1316. EDWARDS R. W. and BROWN V. M. (1967) Pollution and fisheries: a progress report. Wat. Pollut. Control, Lond., J. Proc. Inst. Sewage Purif 66, 63-78. EISENaERG FREIDA F. (1952) A comparison of Litchfield-Wilcoxon and Bliss estimates. Biometrics 8, 120-121. EISLER R. (1965) Some effects of a synthetic detergent on estuarine fishes. Trans. Am. Fish. Soc. 94, 26-31. EUROPEAN INLAND FISHERIES ADVISORY COMMISSION(1965) Water quality criteria for European freshwater fish. Report on finely divided solids and inland fisheries. Int. J. Air Water Pollut. 9, 151-168. FINNEY D. J. (1952) Probit Analysis. A statistical Treatment of the Sigmoid Response Curve 318 pp. 2nd ed. Cambridge Univ. Press, Lond. FINNEV D. J. (1964) Statistical Methodin BiologicalAssay 2nd ed. 668 pp. Hafner, N.Y. FRY F. E. J. (1947) Effects of the environment on animal activity. Univ. Toronto Stud., Biol. Ser. No. 55, Pub. Ontario Fish. Res. Lab., No. 68, 62 pp. FRY F. E. J., BRETTJ. R. and CLAWSONG. M. (1942) Lethal limits of temperature for young goldfish. Rev. Canad. BioL 1, 50-56. FRY F. E. J., HART J. S. and WALKERK. F. (1946) Lethal temperature relations for a sample of young speckled trout, Salvelinusfontinalis. Univ. Toronto Stud., Biol. Ser. No. 54, Pub. Ontario Fish. Res. Lab. 66, 9-35. GADDUMJ. H. (1953) Bioassays and mathematics. Pharmacol. Rev. 5, 87-134. GRENIER F. (1960) A constant flow apparatus for toxicity experiments on fish. J. Wat. Pollut. Control Fed. 32, 1117-1119. HARRIS D. (1968) A method of separating two superimposed nornal distributions using arithmetic probability paper. J. animal Ecol. 37, 315-319. HARRIS E. K. (1959) Confidence limits for the LDso using the moving average-angle method. Biometrics 15, 424-432.

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HART W. B., DOUDOROFFP, and GREI~NBANKJ. (1945) The Evaluation o f the Toxicity o f Industrial Wastes, Chemicals, and Other Substances to Freshwater Fishes 317. pp. Atlantic Refining Co., Philadelphia, Pa. HENDERSON C., PICKERrNG Q. H. and TARZWELL C. M. (1959) Relative toxicity of ten chlorinated hydrocarbon insecticides to four species of fish. Trans. Am. Fish. Soc. 88, 23-32. HERBERT D. W. M. (1961) Freshwater fisheries and pollution control. Proc. Soc. Wat. Treat, J. 10, 135-156. HERBERT D. W. M. (1965) Pollution and Fisheries. Ecol. ind. Soc., 5th Symp. British Ecol. Soc. 173-195. Blackwell Scientific, Oxford. HERBERTD. W. M. and DOWNING K. i . (1955) A further study of toxicity of potassium cyanide to rainbow trout (Salmo gairdnerii Richardson). Ann. appl. Biol. 43, 237-242. HERBERT D. W. i . , ELKINS G. H. J., MANN H. T. and HEMENSJ. (1957) Toxicity of synthetic detergents to rainbow trout. Water Waste Treat. J. 6, 394-397. HERBERT D. W. i . and MERKENSJ. C. (1952) Toxicity of potassium cyanide to trout. J. exp. Biol. 29, 632-649. HERBERT D. W. M. and MERKINSJ. C. (1961) The effect of suspended mineral solids on the survival of trout. Int. J..4Mr Wat. Pollut. 5, 46-55. HERBERTn . W. M. and SHURBEND. S. (1964) The toxicity of fluoride to rainbow trout. Water Waste Treat. J. 10, 141-142. HEY E. N. and HEY M. H. (1960) The statistical estimation of a rectangular hyperbola. Biometrics 16, 606-617. HOSKINS W. M. (1960) Use of the dosage-mortality curve in quantitative estimation of insecticide resistance. Entomol. Soc. Am., Misc. Pub. 2(1), 85-91. HUBSCHMAN J. H. (1967) Effects of copper on the crayfish Orconectes rusticus (Girard). I. Acute toxicity. Crustaceans 12, 33-42. HUNN J. B., SCHOETTGERR. A. and WHEALDON E. W. (1968) Observations on the handling and maintenance of bioassay fish. Progr. Fish.-Cult. 30, 164-167. HYNES H. B. N. (1960) The Biology o f Polluted Waters 202 pp. Liverpool Univ. Press, Liverpool. JENSEN L. n . and GAUFIN A. R. (1966) Acute and long-term effects of organic insecticides on two species of stonefly naiads. J. War. Pollut. Control Fed. 38, 1273-1286. JOHNSON n . W. (1968) Pesticides and fishes--a review of selected literature. Trans. Am. Fish. Soc. 97, 398-424. JONES J. R. E. (1938) The relative toxicity of salts of lead, zinc, and copper to the stickleback (Gasteroswus aculeatus L. ) and the effect of calcium on the toxicity of lead and zinc salts. J. exp. Biol. 15, 394-407. JONES J. R. E. (1939) The relation between the electrolytic solution pressures of the metals and their toxicity to the stickleback (Gasterosteus aeuleatus L.) J. exp. Biol. 16, 425-437. JoNEs J. R. E. (1964) Fish and River Pollution 203 pp. Butterworth, London. JORDAN DOROTHY H. M. and LLOYD R. (1964) The resistance of rainbow trout (Salmo gairdnerii Richardson) and roach (Rutilus rutilus (L)) to alkaline solutions. Int. J. Air Water. Pollut. 8, 405-409. LECOMTEDO NOOY D. (1937) Biological Time 180 pp. Macmillan, New York. LEMKEA. E. and MOUNT D. I. (1963) Some effects of alkyl benzene sulfonate on the blue-gill, Lepomis macrochirus. Trans. Am. Fish. Soc. 92, 372-378. LEPOLT R. and WEBER E. (1958) Die giftwirkung yon Kupfersulfat auf Wasserorganismen. Wass. u. Abwass. 355-353. LITCHFmLDJ. T. (1949) A method for rapid graphic solution of time-per cent effect curves. J. Pharmac. exp. Ther. 97, 399--408. LITCHFIELDJ. T. and WlLCOXONF. (1949) A simplified method of evaluating dose-effect experiments. J. Pharmac. exp. Ther. 96, 99-113. LITCI-IrIELDJ. T. and WILCOXONF. (1953) The reliability of graphic estimates of relative potency from dose-per cent effect curves, d. Pharmac. exp. Ther. 108, 18-25. LLOYD R. (1960) The toxicity of zinc sulphate to rainbow trout. Ann. appL BioL 48, 84-94. LLOYD R. (1961a) The toxicity of mixtures of zinc and copper sulphates to rainbow trout (Salmo gairdnerii Richardson). Ann. appL BioL 49, 535-538. LLOYD R. (1961b) The toxicity of ammonia to rainbow trout (Salmo gairdnerii Richardson). Water Waste Treat. 3". 8, 278-279.

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LLOYD R. (1965) Factors that affect the tolerance of fish to heavy metal poisoning. Biological Problems in Water Pollution, Third Seminar, 1962. U.S. Public Health Service Publ. No. 999-WP-25, pp. 181-187. LLOYD R. and JORDAN DOROTHY H. M. (1963) Predicted and observed toxicities of several sewage effluents to rainbow trout. 3". Inst. Sewage Purif., Pt. 2, 167-173. MARCHETTI R. (1962) Biologia e tossicologia delle acque usate 386 pp. Editrice Tecnnica Artistica Scientifica, Milano, Italy. MARV^N P. (1963) Zur Methodik der Auswertung yon Toxizit?itstesten. Sbornlk Vysok6 ~koly chemickotechnologick~ v Praze, Technologic vody 7(1), 271-313. MATIDA Y. (1960) Study on the toxicity of agricultural control chemicals in relation to fisheries management No. 3. A method for estimating threshold value and a kinetic analysis of the toxicity curve. Bull. Freshwat. Fish. Res. Lab., Tokyo 9(2), 1-12. McKEE J. E. and WOLF H. W. (1963) Water quality criteria. Calif. State Water Quality Control Bd., Sacramento Pub. No, 3A, 548 pp. MERKENSJ. C. (1958) Studies on the toxicity of chlorine and chloramines to the rainbow trout. Water Waste Treat. J. 7, 150-151. MOUNT D. I. (1966) The effect of total hardness and pH on acute toxicity of zinc to fish, Air Wat. Pollut. lnt. J. 10, 49-56. MOUNT D. I. and BRtrNGS W. A. (1967) A simplified dosing apparatus for fish toxicology studies. Water Research 1, 21-29. MOUNT D. I. and WARNERR. E. (1965) A serial-dilution apparatus for continuous delivery of various concentrations of materials in water. U.S. Publ. Health Service, Pub. No. 999-WP-23, 16 pp. NEHRrNG D. (1962) Untersuchungen uber die toxikologische Wirkung von Thallium-Ionen auf Fische und Fischntihrtiere. Z. Fisch. 11, 557-561. PETERSONR. H. and ANDERSONJ. M. (1969) Influence of temperature change on spontaneous locomotor activity and oxygen consumption of Atlantic salmon acclimated to two temperatures. J. Fish. Res. Bd Can. 26, 93-109. PICKERrNO Q. H. (1966) Acute toxicity of alkyl benzene sulfonate and linear alkylate sulfonate to the eggs of the fathead minnow, Pimephalespromelas. Int. d. Air Wat. Pollut. 10, 385-391. PICKERING Q. H. and HENDERSONC. (1966a) The acute toxicity of some heavy metals to different species of warm water fishes. Int. J. Air Wat. Pollut. 10, 453-463. PICKERING Q. H. and HENDERSONC. (1966b) Acute toxicity of some important petrochemicals to fish. J. Wat. Pollut. Control Fed. 38, 1419-1429. PICI~RING Q. H. and HENDERSONC. (1966c) The acute toxicity of some pesticides to fish. Ohio J. ScL 66, 508-513. PICKERING Q. H., HENDERSONC. and LEMKE A. E. (1962) The toxicity of organic phosphorus insecticides to different species of warm water fishes. Trans. Am. Fish. Soc. 19, 175-184. PICKERING Q. H. and VIGOR W. N. (1965) The acute toxicity of zinc to eggs and fry of the fathead minnow. Progr. Fish.-Cult. 27, 153-157. RAINEy R. H. (1967) Natural displacement of pollution from the Great Lakes. Science, N. Y. 155,1242. ROTrtSCHEINJ. (1964) Einige kritische Betrachtungen zum Thema "toxikologische Experimente und hrchstzultissige Konzentrationen von Giftstoffen im Wasser." Sbornlk Vysok6 ~koly chemickotechnologiske v Praze, Technologic vody 8(2), 303-325. SCHOLZ, J. (1965) Chronic toxicity testing. Nature, Lond. 207, 870--871. SI-IEPARD M. P. (1955) Resistance and tolerance of young speckled trout (Salvelinus fontinalis) to oxygen lack, with special reference to low oxygen acclimation. J. Fish. Res. Bd Can. 12, 387-446. SKIDMORE J. F. (1965) Resistance to zinc sulphate of the zebrafish (Brachydanio rerio HamiltonBuchanan) at different phases of its life history. Ann. appL BioL 56, 47-53. SOLONJ. M., LINCERJ. L. and NAIR J. H. (1968) A continuous-flow, automatic device for short-term toxicity experiments. Trans. Am. Fish. Soc. 97, 501-502. SPRAGUE J. B. (1964) Lethal concentrations of copper and zinc for young Atlantic salmon. J. Fish. Res. B d Can. 21, 17-26. SPRAGUEJ. B. (1968) Avoidance reactions of rainbow trout to zinc sulphate solutions. Water Research 2, 367-372. SPRAGtm J. B. and McLEESE D. W. (1968) Toxicity of kraft pulp mill effluent for larval and adult lobsters, and juvenile salmon. Water Research 2, 753-760.

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