Studies on the toxicity of cadmium, copper and zinc to the brown shrimp, Crangon crangon L.

Price, Robin Kevin John

March 1979

Thesis or dissertation

© 1979 Robin Kevin John Price. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.

[General introduction:]
During the last two decades, much. public interest and concern has been expressed on the presence of metal ions in the environment and, particularly, in organisms of commercial significance. The concern has arisen because the amounts of such ions has, in local and sometimes general areas , increased to levels which are far higher than 'normal ' background values.

The present thesis is concerned with studies on the effects of 3 metals on the brown shrimp, Crangon crangon - a species which is common in the inshore waters of the Yorkshire coast and which has a commercial significance to a number of local fisheries.

Of the metals selected for study, cadmium is known to be neither biologically essential nor beneficial to living organisms (Eisler, 1971; Thorpe & Lake, 1974; Bryan, 1976; Pascoe & Mattey, 1977). Consequently, this metal is absent, or present only in trace amounts, in organisms from unpolluted waters.

By contrast, copper is an essential element for several living processes, having first been associated with such in studies of blood proteins of Helix pomatia (Harless, 1847 - cited in Severy, 1923). Since then, its functional significance in the oxygen transport of crustacean and molluscan haemocyanins has became well documented (Redmond, 1955). Copper is known also to be an important constituent of certain enzymes such as tyrosinase and cytochrome oxidase (Scott & Major, 1972) and as an activator for certain others (e.g. malate dehydrogenase; Saliba & Krzyz, 1976). Furthermore, copper has been shown to be a necessary component for the successful accomplishment of specific behavioural and physiological phenomena (e.g. the settling and metamorphosis of the oyster, Crassostrea virginica; Prytherch, 1931). Possibly, the ubiquitous distribution of this metal accounts for its wide biological functions in organisms.

Zinc also has been found to occur naturally in the tissues of many marine organisms (Bodansky, 1920) and, subsequently, has been shown to be an essential element in many metal-enzymes (Dixon & Webb, 1964). These enzymes include carbonic anhydrase (Keilin & Mann, 1940; Vallee, 1959; Coombs, 1972), alkaline phosphatase (Vallee, 1962; Wolfe, 1970; Coombs, 1972), carboxypeptidase (Vallee, 1962; Coombs, 1972), glutamate dehydrogenase, lactic dehydrogenase, alcohol dehydrogenase (Vallee, 1959) and -D- mannosidase, (Coombs, 1972). Parker (1962) has suggested that the capacity of organisms to concentrate zinc reflects the biological function of this metal. However, as with copper, zinc is usually found in tissues in quantities far in excess of those, required to satisfy the needs of enzymes. Coombs (1972), for example, used data of Vallee & Wacker (1970) to estimate that the oyster, Ostrea edulis used only 0·1% or less of its total zinc content for enzyme purposes.

Heavy metals are ·normal constituents of estuarine and marine environments and are usually present in trace amounts. Normally, they reach the sea via rivers following the erosion of rocks. However, with the advent of industrialization, man has contributed to the base levels of metals found in coastal waters. Cairns, Dickson, Sparks & Waller (1970) summarised the industrial attitude as "the production of wastes by industry is not related to the capacity of the ecosystem to absorb and transform these wastes, but rather to market demand." Hence, large quantities of cadmium, copper and zinc, among other metals, have found their way in estuarine and coastal waters, mainly from copper and lead mines (McKee & Wolfe, 1963; Mount & Steven, 1967) and zinc smelting and electroplanting plants (Little & Martin, 1972; Jordan, 1975).

By virtue of their physicogeochemical nature, estuaries have the capacity (albeit to a limited extent) to 'detoxify ' heavy metals by altering their biological availability. This is achieved by absorption to particulate material (Krauskopf, 1956) and by precipitation, chelation and sedimentation (Lewis , Whitfield & Ramnarine , 1972; Whitfield & Lewis , 1976; Batley & Gardner, 1978). However, those metal species which remain dissolved in seawater are likely to escape to the open sea (van Bennekom, Gieskes & Tijssen, 1975) and, if present in sufficiently high concentrations, are likely to be directly toxic to the fauna and flora.

Many organisms , especially sessile bivalves, can accumulate cadmium, copper and zinc and tolerate high concentrations of these in their tissues without any apparent signs of harm (Brooks & Rumsby, 1965)'. This suggests that such organisms have very efficient methods for preventing these metals from poisoning essential enzyme systems . Other organisms (e .g. Paratya tasmaniensis; Thorpe & Lake, 1974) ,d.o not have the ability to tolerate these metals and are killed by very low concentrations of them. There appear to be few reliable data available on the susceptibility of C. crangon to heavy metals and these studies were undertaken to provide comprehensive data on the toxicity of cadmium, copper and zinc to this species .

One widely used and accepted method for the assessment of the effects of pollutants to organisms, is that of toxicity testing (sometimes referred to as 'bioassays '). When death is used as the criterion of response in such studies, the method suffers serious disadvantages in that accuracy is limited because of the wide disparity of individual susceptibility. However, in toxicity studies, it is accepted universally that the most tolerant and the most susceptible individuals in a test group show greater variability of response than individuals near the median of this group. Consequently, the relevant stdies in this thesis are concerned predominantly with the median responses to the test parameters (i.e. the responses of the average individual ). The median lethal concentration (LC50) is the term used generally to describe the concentration at which 50% of the test population are killed Alderdice, 1967; Brown, Jordan & Tiller, 1967; Sprague, 1969; Eisler, 1971). In situations where time is the effect parameter, the median lethal time (LT50) is used to describe 50% mortality value. Concentration and time, however, are here inextricably linked and to maximise their usefulness in comparative studies, LC50 values need to be qualified by a prefixed time component. APHA (1965) has suggested that the time component may be expressed as hours, days or weeks, whichever is convenient in particular circumstances. Similarly, LC50 values should ~e qualified by the concentration of the toxicant used. Median lethal concentrations and LT50 values are determined by graphical means from plots of concentration or time respectively against the percentage mortality of the test population at specific times (LC50) or concentration (LT50).

Brown (1973) suggested that 'quantal' bioassays (using concentrations and percentage mortalities) are superior to 'quantitive' bioassays involving exposure times and percentage mortalities. His reasoning is based on the fact that quantal bioassays yield mortality curves which are amenable to mathematical definition and thus enable confidence limits to be given in terms of units of concentration. On the other hand, quantitive bioassay mortality curves represent subjective estimates of effective concentrations. However, when experimental methodology imposes limitations to the design of experiments (e.g. in flow systems which permit but one concentration at a time to be tested) then quantitative
methods offer an acceptable alternative.

A very important concept in toxicity studies is that of incipient lethal levels (ILL). Sprague (1969) defined an ILL as "that level of the environmental entity beyond which 50% of the population cannot live for an indefinite time." The same concept has been named the 'lethal threshold concentration' (Lloyd & Jordan, 1963) or the 'asymptotic LC50' (Ball, 1967a). These values or levels, however, should not be construed as safe levels as they represent the concentrations which would kill the average specimen of the test organism, on long exposure. In practice, the value for any particular toxicant is obtained from a composite plot of a series of LC50s at various exposure times - the ILL is the LC50 value at which the resulting plot becomes asymptotic to the exposure-time axis.

The standardisation of toxicity tests has been discussed by several authors. Brown (1973) has stated "as there is nothing 'standard' about any poison, animal or exposure in the environment, no 'standard' test can be advocated. The only standards to be applied in toxicity testing are those of good experimental technique and sound scientific practice. The more standardised the test or the test organism, the less applicable the information is likely to become." For practical and comparative purposes, however, some standardisation of methodology is necessary. Therefore, the initial toxicity tests, described in Chapter 1 of this thesis represent an attempt to assess the effects of certain abiotic variables (e.g. static water, continuously flowing water, presence or absence of a sand substratum) which may affect the toxicity of cadmium, copper and zinc to C. crangon. The aim of these particular aspects of the present investigations was to maximise accuracy and convenience for subsequent toxicity tests.

Until recently, little attention seems to have been paid to the influence of environmental variables in toxicity testing of heavy metals to crustaceans. Although an extensive literature exists on temperature effects on the toxicity of pollutants to fish (for review, see Doudaroff & Katz, 1953) the results are equivocal and emphasise the need for short-term and long-term studies to be carried out on individual species.

Much attention has centred on the effects of salinity on various aspects of crustacean activities (e.g . Prosser, Green & Chow, 1955; Dehnel, 1960; Hagerman, 1970; Spaargaren, 1973). These reports, however, include little on the effects of salinity on the toxicity of pollutants and the few such reports that exist include those of Jones (1974, 1975) and Vernberg, Decoursey & O'Hara (1974). The effects of temperature and salinity on the toxicity of cadmium, copper and zinc to C. crangon have been investigated here and the results are described in Chapter 1 of this thesis .

The uptake, subsequent translocation and removal of pollutants in marine organisms will be allied closely to the metabolic disposition of the test organisms. Consequently, the rates of these activities will be affected not only by the environmental variables mentioned above, but also by the physiological status of the test organisms.

The moult cycle of crustaceans is known to be accompanied by profound changes to several aspects of the physiology of these organisms. Moulting can be taken to include not only the shedding of the old exoskeleton rut also the physiological and behavioural changes that precede and succeed it. A convenient method for the determination of stage in the moult cycle of decapod crustaceans has been to follow the growth of new setae in the uropods. Several workers (e.g. Drach, 1944; Passano, 1960; Scheer, 1960; Drach & Tchernigovtzeff, 1967) have proposed certain criteria which distinguished the various moult stages in particular species of decapod. Although certain generalizations are common to this large taxonomic group, many inter specific differences exist. Furthermore, a full description of the various moult stages of C. crangon does not appear to exist and the aim of the work described in Chapter 2 of this thesis was to provide a clear description of the criteria which characterise the various moult stages in this species. To support this work, the opportunity was taken to assay the concentration of haemolymph total protein, and chloride ion and whole body water content of animals sampled at the various moult stages and data has been discussed in relation to similar, published information.

As Crangon crangon is a species which moults frequently throughout the year and .as the rate of the uptake of ions may vary according to moult stage, moult stage as a biotic variable which may affect the toxicity of the test metals to this species, has been studied. This aspect of these studies, and the effects of other biotic variables (sex, size and reproductive condition) on the toxicity of the metals. are included in Chapter 3 of this thesis.

The literature includes reports which show that, within marine organisms, there are a number of modes of uptake and loss of heavy metals and that these vary even within taxonomic groupings. Certain species are known to rely on absorption from the external milieu for the uptake of ions (see Bryan, 1971; Penreath, 1973a; Bryan & Hummerstone, 1973) whilst other species ingest the metals with their food (see Bryan & Ward, 1965; Bryan, 1967; Young, 1974).

In decapod crustaceans, some regulation of essential metals such as copper (Zuckerkandl, 1960; Bryan, 1968; Djangmah, 1970) and zinc (Bryan, 1964, 1967) does occur. Regulation of metals requires both input and output elements and, as with metal uptake, ways that metals are lost varies between species - some species excreting excess ions via the gills (Wright, 1977a), and others voiding them via the urine or faeces (Bryan, 1967). Between the time of uptake and loss, excess metals are sometimes stored - usually in the form of granules in hepatopancreas tissue (Djangmah, 1968, 1970; Djangmah & Grove, 1970).

The aim of the work described in Chapter 4 of this thesis was to elucidate possible paths of uptake and internal translocation of copper, cadmium and zinc in C. crangon. The studies also aimed to determine whether the biologically non-essential metal, cadmium, is voided in this species. Within these studies, the opportunity was taken to examine the particular tissues associated with uptake and storage (gills and hepatopancreas respectively) to see whether the metals caused any gross morphological or ultrastructural damage to them.

Oxygen consumption of crustacean gill tissue has been found to be depressed on treatment with some heavy metals (e.g. cadmium, Thurberg, Dawson & Collier, 1973; Collier, Miller, Dawson & Thurberg, 1973). However , no such depression has been found for whole animal oxygen consumption (Vernberg, Decoursey, Kelly & Johns, 1977; Collier et al , 1973). Recently, Cumberlidge (1977) and Dyer (1978) have modified impedance techniques so that they are suitable for continuous and simultaneous monitoring of cardiac and ventilatory activities in species like C. crangon.. Chapter 5 of this thesis, describes the effects of acute exposures to high concentrations and chronic exposures to low-concentrations of the 3 test metals to the qualitative and quantitive beating behaviour of the heart and scaphognathites of C. crangon. The use of cardiac and ventilatory responses as sensitive indicators of pollution stress was assessed and discussed in Chapter 5.

Department of Zoology, The University of Hull
Uglow, Roger F.
Sponsor (Organisation)
Science Research Council (Great Britain)
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