Document 8Vw2n8mD7abZ7jJxeZKOg0aQd

PRESENTED AT THE INTERNATIONAL EXPERTS DISCUSSION MEETING ON: LEAD - OCCURRENCE, FATE AND POLLUTION IN THE MARINE ENVIRONMENT ROVINJ, YUGOSLAVIA - OCTOBER 1:8-22, 1977. THE ACUTE TOXICITY AND BIOACCUMULATION OF SOME LEAD ALKYL COMPOUNDS IN MARINE ANIMALS Maddock B G Taylor D Imperial Chemical Industries Ltd Brixham Laboratory Devonshire United Kingdom N 27635 INTRODUCTION Alkyl lead compounds are used as ..additives to petrol as. a convenient and economic method of increasing the octane rating of fuels for use in high com'pression internal combustion engines. The United Kingdom is the largest European producer of these materials, some 11% (40,000 tonnes) of the total amount of lead used in the United Kingdom is converted into these additives, and approximately 80% of the UK production is exported by sea (Anon, 1974). In the last few years, increasing concern has been expressed about the possible harm to aquatic life that might be caused by the accidental release of dangerous materials carried in cargo ships due to shipwreck or collision. An 1MC0 Working Party is preparing regulations that should minimise the risk of environmental damage caused by such accidents, but in order to do this effectively, adequate data is required about the effects of the various compounds on aquatic life. In addition to the possibility of accidental releases of man-made lead alkyls, it has recently been shown (Jarvie et al, 1975; Schmidt and Huber, 1978; Wong et al, 1975) that the possibility exists, in the aquatic environment, for the transformation of inorganic, lead compounds into alkylated forms by chemical or bacterial mechanisms. Furthermore, Sirota and Uthe (1977) have demonstrated that tetra alkyl lead compounds can b.e detected is some Canadian fish products. In terms of Its acute toxicity to aquatic life, lead is one of the least studied of the heavy metals, and most of the data that has been reported concerns the effects of in organic lead compounds. Prior to 1975, When the present study began, the only work reported in the literature, concerning the toxicity of alkyl lead compounds to fish was the paper by Turnbull et al (1954) who reported that the 24 hr LC_Q of Bluegill Sunfish 2 (Lepomis macrpchirus) exposed to tetra ethyl lead Was 2.0 mg/1. He also quoted the figure of 0.2 mg/1 as a "safe level", but without producing evidence to support that Conclusion, This data has been quoted extensively and used as the basis for assessment of the risk caused by spillages of tetra ethyl lead (Anon, 1968b). In addition to this study, there had been two other investigations of the effects of tetra ethyl lead on lower forms of aquatic life. Kozyura et al (1961) had studied its effects on two species of blue-green algae; Cladophera sp. and Scenedesmus sp. and Siegel et al (1971) had compared its toxicity with a range of organo-mercurials to a flat worm. Since 197:5, two further papers have been published. (Krupinska (1976) has studied the effects of tetra ethyl lead on two species of Bryophytes whilst Silverberg et al (1977) have investigated the effect of bacterially produced tetra methyl lead on various species of green algae. The results of all these studies, with the exception of Silverberg et al. must be interpreted with caution since the high volatility and degradability of these materials may have been underestimated. The object of the present study was to measure the acute toxicity of a range of alkyl lead compounds and to gain information on the potential bioaccumulation of these materials in marine animals. .It should be noted that throughout this paper, concentrations of alkyl lead compounds in water are quoted as mg/1 total lead and all tissue concentrations refer to mg/Kg (dry Weight) unless stated otherwise. DUP040008789 3 MATERIALS AMD METHODS Alkyl Lead Compounds The most important organic lead compounds in. terms of scale of manufacture and use, are the lead alkyl derivatives. A wide range of these materials are manufactured, but the most significant are the tetra alkyl lead compounds; Tetra methyl lead (TML) and tetra ethyl lead (TEL). These two materials are unstable in seawater and undergo progressive dealkylation eventually forming inorganic lead. (Grove, 1977). The initial step in this sequence proceeds very rapidly to produce the trialkyl lead chloride, although subsequent steps in the sequence are considerably slower. Thus the acutely toxic effects of a release of a tetra alkyl lead compound into the marine environment will initially be due to the parent compound, but within a few hours the toxic effects of the trialkyl lead compounds will become of considerable significance and in the longer term the effects of the di-substituted, materials may be important. Consequently we have studied both the tetra alkyl lead compounds and the primary and secondary .dealkylation products. In addition, since the mammalian toxicity of TML and TEL are significantly different (Magistre.tti et al, .1963), we have investigated both the methyl and ethyl derivatives of these materials, In the context of accumulation, the tetra alkyl lead compounds are of only minor interest since they have only a transitory existence in seawater. Thus, although some information has been obtained on the accumulation of these materials during short term exposures to high concentrations, the majority of the work has been concerned with the accumulation of the trialkyl lead compounds during long term exposures. The test materials were supplied by the Ellesmere Port Research Laboratories of the Associated Octel Company Limited and with the exception of the tetra methyl lead, all materials had a purity of 98%. Tetra methyl lead was tested as an anti knock y DUP040008790 4 formulation (TML-CB) containing dibromo and dichloroethane in addition to the lead alkyl. Throughout the investigations, the safe handling techniques recommended for these materials (Anon, 196,8 a) were followed. Analytical Techniques Water samples were analysed for lead alkyl compounds using the technique of Noden (1977). The total amount of the tetra, tri .and di-alkyl lead components is obtained by reducing the tetra and tri alkyl lead to dialkyl lead by treatment with .iodine monOchloride, and then measuring the total dialkyl lead content speetrophotometrically as a complex with 4(2-Pyridylazo) resorcinol (TAR) in the presence of 1.2.-Diaminocyclohexane-NNN'N'-tetra acetic acid (CDTA) which masks inorganic lead, calcium, magnesium and other metal salts. The amount of the tri and dialkyl lead compounds is then measured in a similar manner on another portion of the solution which has been extracted with hexane to remove the tetra alkyl lead. Finally the dialkyl lead component is measured ,on a further portion of the extracted solution using the PAR complex. Trialkyl lead does not form a complex with PAR* The precision of this technique is ca. - 5% at a detection limit of 0.05 mg/1. Periodic measurements of total lead were also carried out using atomic absorption techniques. During studies with the tetra alkyl lead compounds a lower detection limit (0,005 mg/1) was required and this was achieved by extracting the tetra alkyl lead from Solution with hexane, converting to inorganic lead with bromine in carbon tetrachloride, followed by determination of the lead using atomic absorption spectrophotometry with deuterium arc background correction. During the investigation of the formulated lead alkyl (TML-CB), measurements were made of the dibromoethane and dichloroethane content of the test solutions. The method adopted was a GLC technique involving direct injection of aqueous samples. DUP040008791 5 Dissected tissue samples (Q.lg - lOg) were ashed at 100C in Aristar grade concentrated nitric acid for several hours prior to measurement of lead by atomic absorption spectrophotometry using background correction.. The precision of the technique was - 10% at a detection limit of 0.5 mg/Kg. The method has been used to analyse two standard biological reference materials, Bowen Standard Kale (Bowen, 1967) and Monoco Oyster (Fukai, 1976). In each case the method produced a value within 25% of the accredited value. Experimental Animals Five species, representing four phyla were selected for investigation (Table 1). With the exception of the algae, the species are of commercial importance and could provide a pathway for the transfer of lead alkyls from seawater to man. In addition, all the species are commonly found in estuarine and coastal areas where there is a greater risk of a serious spillage of lead alkyl compounds than in deeper oceanic waters. All the test animals were obtained from areas free from the effects of industrial and domestic effluent discharges, and, at the commencement of the exposure tests, contained lead concentrations equivalent to those quoted for uncontaminated animals (Bowen, 1966). All animals were maintained in flowing seawater at the laboratory for a minimum of seven days prior to their exposure to test materials. ,s DUP040008792 6 BXOASSAY TECHNIQUES Acute Toxicity Experiments The technique's used are conveniently subdivided into three sections dealing with the tetra alkyl compounds, the degradation products and the algal assay procedure. (a) Tetra Alkyl Lead Compounds The tetra al.ky.1 lead compounds are volatile and relatively insoluble in seawater, hence they are readily lost from solution to the atmosphere. Secondly, they rapidly undergo dealkylation in seawater to produce the trialkyl lead derivatives (Grove, 1977). Consequently it was necessary to design a test System that would permit the exposure of aquatic animals to constant levels of the materials for periods of up to .100 hours. The system used is shown in Figures 1 and 2. In view of the high .mammalian toxicity and .high volatility of the tetra alkyl lead compounds, the test equipment was constructed in a self contained and vented enclosure, and the waste discharged into a sealed drainage system. Seawater (salinity :34,9/oo) was pumped directly from the sea at Brixham and supplied at a constant temperature (15C) to the system. At the design flow rate of 100 ml/min, the 90% replacement time, was 7,5 hours and, with the exception of small pieces of silicone rubber in the peristaltic pumps, those parts of the system which were in contact with the toxicant were made of either glass or PTTE. This system ensured minimum contact between the test solution and the .atmosphere and hence minimal losses due to volatilisation. Stock solutions were prepared by gently agitating 22 litres volumes of water with lOg of the lead alkyl for four hours, allowing the excess to settle for two hours and then pumping the top 20 litres into a sealed stock vessel. This procedure enabled stock solutions of seawater saturated with lead alkyls to be produced without the use of solvents. The stock solutions were used to supply five test vessels simultaneously, DUP040008793 7 and the total rate of utilisation of the stock solution was maintained, with the aid of a bleed line to waste, at a constant rate of 1.2 1/hr. This ensured that the stock solutions were always replaced before decomposition of the lead alkyl became significant. Since the dealkylation of the tetra alkyl lead compounds is promoted by light, the stock vessels were painted black and the complete test system isolated from direct sunlight. Using these procedures it was possible to maintain concentrations of the tetra alkyl lead compounds in the test vessels with a variation of - 10% over the range 0.01 mg/1 to 0,7 mg/1. The halogenated ethanes present in the TML formulation are Very soluble in seawater by comparison to TML, and although the ratio of TML to halogenated ethane in the original formulation was 1:0.4, in a saturated solution this ratio can become 1:1000. In our experiments', if was not possible to maintain the 1:0.4 ratio, but by using peristaltic pump tubing which Was more permeable to the halogenated ethanes than to the lead alkyls, it was possible to achieve ratios of 1:10 for dichloroethane and 1:4 for dibromoethane. The toxicological significance of the presence of these materials, and the dealkylation products in the te:st vessels is discussed in a subsequent section. The test procedure was as follows: Ten individuals of the appropriate species were transferred from the laboratory holding tanks to each of the five test vessels and maintained in flowing seawater for 24 hours. The injector pump was then started, and preselected amounts of the stock solution were fed continuously into four of the vessels. The fifth vessel acting as a control. This procedure results in a gradual increase in the concentration of the toxicant from zero to its set value over the first 3-4 hours of exposure, but allows the animals to acclimatise to the actual test conditions. s DUP040008794 8 The test vessels were regularly monitored for the deaths of the individual animals> and the time of death recorded.. Water samples were taken from the stock solutions and the test vessel overflow lines twice daily and analysed for the tetra alkyl component and its degradation products and where appropriate for the halogenated ethanes. In addition the temperature, pH and dissolved oxygen content of the water in each vessel was measured daily. The test exposure terminated after 96 hours. The test animals were fed prior to but not during the exposures. (b) Tri and Pi Alkyl Lead Compounds The methyl and ethyl derivatives of the tri and di alkyl lead chlorides are all soluble in water at>2g/100 ml. (Calingaert et al> 1948).. They are not readily lost from solution by volatilisation, adsorption or dealkylation, the latter process proceeding only Slowly in seawater. Consequently, a much simpler bipassay procedure was used for these materials. A series of 15 litre volumes of seawater containing a range of concentrations of the appropriate material were prepared in 30 litre glass tanks. Each tank was fitted with an aeration system supplying a flow of 300 ml/min of air, covered with a loose fitting lid and maintained at 15C in a constant temperature room. Experimental animals, in groups of ten, were transferred to the test vessels from the laboratory holding tanks and observed at regular intervals over a period of 96 hours. Dead animals were removed, deep frozen for subsequent analysis, and the time of death recorded. The levels of the toxicant, its dealkylation product and the pH, temperature and dissolved oxygen levels were measured daily and the test solutions changed at 24 hour intervals. This procedure permitted concentrations of tri and di alkyl lead chlorides to be DUP040008795 9 maintained to an accuracy of 5% oyer the range 0,1 - 420 mg/1. The level of the dealkylation products ie. dialkyl lead dichloride was always less chan the detection limit of the method used ie. 0.05 mg/1, (c) Algal Assay The procedure used to -study the effects of these materials on algal productivity was based on the technique developed by Johnstone (1974) from the organic pro ductivity assessment technique of Steeman-Nielsen (1952). A standardised quantity of algae, in this case Phaeodactylum tricornutum was incubated for 6 hours., under controlled conditions, in the presence of a range of concentrations of the toxicant and a standard amount of labelled carbonate. The accumulation of by the con trol cultures, after correction for adsorption indicates the ''normal" activity of the algae which can be compared to the activity of the algae exposed to the toxicant. Accumulation Experiments Two accumulation experiments have been undertaken involving the exposure of mussels (Mytilus edutis) and Dabs (Limanda limaada) to .seawater (salinity 34.9/oo) containing either trimethyl or triethyl lead chloride. Since these materials are soluble and relatively stable in seawater, the relatively simple experimental system, shown dlagrammatically in Figure 3, was used. In the experiment with mussels, constant temperature seawater (14C) was supplied at a rate of 200 ml/min. to exposure tanks consisting of 35 litre all glass aquaria, fitted with glass lids. This produced a 95% replacement time of 6 hours. The dabs were exposed in substantially larger (ie. 300 litre) glass fibre tanks and these received a flow of 900 ml/min. of constant temperature seawater (12C) producing DUP040008796 10 a .95% replacement time of 13 hours. In order to maintain a homogenous toxicant distribution, it was necessary to equip each of these large tanks with a low volume aeration system. All the apparatus which was in contact with the toxicant was made of glass, fibre glass or PTFE with the exception of peristaltic pump tubing. A standard procedure was used. An .appropriate number of animals (60) mussels, 50 dabs) were transferred from the laboratory holding tanks to each of four test tanks (two for tr.iethyl'.and two for tr.imethyl exposure) and a control tank. These animals were then maintained in flowing seawater for at least 48 hours prior to the commence ment of the experiment. At the start of the exposure period, the peristaltic pumps were started and then a suitable volume of stock solution was .added to each tank, with gentle agitation, to produce the appropriate test concentration. During the first few days of the exposure period, the trialkyl and dialkyl lead content of the test solutions was monitored at three hourly intervals, subsequently the frequency was reduced to daily measurements of trialkyl lead with occasional checks on the dialkyl lead content. The level of this latter material in the test vessels was always <0.05 mg/1. The temperature of the test tank water was recorded continuously and the pH, dissolved oxygen content and the flow rates of seawater and stock solution were measured daily. At the end of the exposure period, the peristaltic pumps supplying the toxicant were stopped and 95% of the test tank water replaced immediately with clean .seawater at the same temperature. The mussels were able to obtain sufficient food from the seawater supply used. However, the fish were fed three times per week on a diet of ox liver.. At each feed, the total amount of food given was equivalent to 1% of the total weight of the experimental animals. DUP040008797 1.x Animals were removed from the tanks at regular intervals. On each occasion three individuals were selected at random, .dissected and the tissues analysed immediately for total lead content. Four tissues from the mussel (gill, digestive gland, gonad and foot) and two tissues from dabs (liver and flesh) were examined. In any long term experimental work with animals, aimed at detecting chronic or sub" lethal effects of pollutants, it is necessary to know if the experimental animals are under excessive stress. Bayne (1975) has defined stress in this context as "Ameasurable alteration of a physiological (or behavioural, biochemical Or psychological) steady state which is induced by an environmental change and which renders the individual (or the population or the community) more Vulnerable to further environmental change". Several indices have been proposed for describing this physiological condition and an estimation of one of these indices gives an easily det ermined general guide to the metabolic state of the experimental animals and allows an estimate to he made of the stress induced in the control population by the test system itself in the absence of any toxic materials. In this work we have used the following condition factors Condition Factor Mussel = Flesh Weight lnn Total Body Weight31 Condition Factor Dab ,= Liver Weight mo Total Body Weight* The factor for mussels has been used previously by several workers e.g. Bayne and Thompson, 1970; Roberts, 1972, The factor for dabs was suggested by Thompson, 1976, DUP040008798 12 RESULTS AND DISCUSSION Acute Toxicity The acute toxicity of four alkyl lead compounds (tetra methyl lead., tetra ethyl lead, trimethyl lead chloride and triethyl lead chloride) have been measured to three marine species (mussel, shrimp and plaice) and the effect of these materials oh the photosynthetic activity of Phaeodactylum tricornutum has been established. In addition the toxicity of dimethyl and diethyl lead dichlorides have been measured to plaice. Results The results, as 96 hour LC^g values are presented in Table 2 and diagrammatieally in Figure 4, All .the 96 hour LC,.q values have been calculated after the method proposed by Doudoroff et al, (1951) and in addition., where appropriate data was available, by the method of Litchfield and Wilcoxon (194.9). Since the values calculated using the Doudoroff technique fell within the 95% confidence limits of the other method, only the Doudoroff results are presented. It can be clearly seen from the results that in general the ethyl derivatives are more toxic than the corresponding methyl derivatives and that the toxicity decreases with decreasing degree of alkylation; the dialkyl lead bichlorides having a similar order of toxicity to inorganic lead. There are two exceptions to this Order, mussels are more sensitive to trimethyl than triethyl lead chloride, although the difference in toxicity is so slight that it is probably justifiable to consider them of similar order of toxicity. The other exception is that plaice Seem to be rather resistant to the effects of tetra ethyl lead. DUP040008799 13 The results of the algal exposures are shown in greater detail in Figure 5, from Which it can be seen that the nature ,of the alkyl group has much more effect than the degree of alkylation. Several other materials were present in the test.vessels during part of the Study, In the investigations of the toxicity of the tetra alkyl compounds, trialkyl lead chlorides were inevitably present as breakdown products. However, in all but one instance (where mussels were exposed to tetra ethyl lead), the concentration of 'trialkyl lead chloride present in the test solution was always well below the level that had been shown to cause no deaths in that species over 96 hours. In the study of the t.etramethyl lead formulation, dibromoethane (EDB) and dichloroethane (EDC) were also present in the test solutions. The concentration of EDC in the test solutions was always t 5 mg/1 and Taylor (1974) has shown that levels of 60 mg/1 and 25 mg/l had no apparent effect on dabs (Limanda limanda) and prawns (Palaenon serratus) respectively in 96 hour exposures. The only data in the literature on EDB is the report by .Davis and Hardcastle (1959) that the 48 hour LCj q to Bluegill sunfish (Lepomis macro.chirus) in freshwater was 18 mg/l. Compared to our test concentrations in seawater of i2 mg/l. We conclude that, although synergistic and antagonistic effects cannot be ruled Out, the contaminants measured in the test vessels did not materially affect the recorded toxicity of the lead alkyl compounds with the possible exception of mussels exposed to tetra ethyl lead, where the presence of triethyl lead chloride may have had a minor effect. DUP040008800 Discussion the toxic effects of inorganic lead compounds on aquatic animals are largely governed by the chemical form in which the lead is present. In freshwater the dominant control ling factor has been shown to be the hardness ,o.f the water, (Pickering and Henderson, 1966). Lead has a low solubility of 0.5 mg/1 in soft water and an even lower sol ubility ..(0.(303 mg/1) in hard water, (Anon, 1972a)-although considerably higher concentrations of suspended .and colloidal lead may remain in the water. In hard water, the lead will be mainly present as carbonate salts and the amount of "free ionic" lead ie. uncomplexed, which is thought to be the most toxic species, will be relatively small compared to the situation is soft water, this has been confirmed by Davies et al (1976), who were able to monitor electrochemically the levels of "free ionic" lead in their test solutions in addition to the total lead concentration. They reported that on the basis of the "free ionic" lead concentration there was no difference in the toxicity in hard and soft waters, whereas in terms of total lead concentration the toxicity differed by a factor of .100. As in the case with many materials, juveniles are more sensitive than adult animals. MCKim et al (1975) state that the 96 hour LC,-0 for juvenile rainbow trout (Salmo gairdneri) is 0.14 mg/1, ie. 10% of the value recorded for adults by Brown (1.968). There have been few studies of the acute toxicity of lead to marine animals, primarily because of the difficulty of maintaining solutions of lead in seawater at concentrations large enough to cause toxic effects. The most reliable data available at the present time is that of Po.rtmann and Wilson (1971) who report 48 hour LC^q values for the Pink Shrimp (Pandalus montagui) and the cockle (Cardium edule) of 375 and 500 mg/1 respectively. 15 The acute toxicity of inorganic lead to aquatic animals can be summarised as follows: In soft freshwater, the acute toxicity, as indicated by the 96 hour LG^ value fall between 1-10 mg/1. In hard freshwater or in saline waters the acute toxicity falls between 300-500 mg/1. No data has been published relating to the aquatic toxicity of the trialkyl or dialkyl lead compounds and in general, the data published on the toxicity of the tetra alkyl lead compounds is not directly comparable with the present study. The .24 hour LC50 value of 2.0 mg/1 quoted by Turnbull et al (1954) for the Bluegi.ll sunfish (Lepomis macrochirus) in freshwater may be an underestimate when compared to data reported .in the present study for marine fish (ie, 0.2 mg/1) which would be expected to be less sensitive. This may be due to losses of the toxin from the test environment, by volatilisation and degradation, in previous studies. The reduction in algal photosynthesis was studied by Silverberg et al (1977) with a method similar to that used in the present Study. They report a 4 hour fCjg value of <0.3 mg/1 for the freshwater alga Ankis.trodesmus fplcatus compared to the .6 hour EC,.q of 1.3 mg/1 for the marine alga Phaeodactylum tricornutum determined in the present study. The toxicity of inorganic lead compounds to aquatic animals was originally attributed to coagulation film anoxia (Westfall, 1945), This phenomenon, which was first report ed for ..lead by Carpenter in 1949, involves the formation, by the action of a toxicant, of a veil like film of coagulated mucus on the body surface of the fish. If this film affects the gill tissue, the fish suffers acute respiratory distress and dies from lack of oxygen. This type of mucus development was not detected in the present study. More recently, other workers (Jackim, 1970) have indicated that direct action of the metal on enzyme systems may be responsible for their acute effects, and such responses may be involved in the case of the alkyl lead compounds. DUP040008802 16 In mammals, the toxicity of tetraethyl lead is several times greater than the toxicity of tetramethyl lead. This has been demonstrated in studies with rats (Magistretti et al 1963; Schepers, 1964) and in man (De Treville et al, 1962). However, the toxicity is not simply dependant on the size of. the alkyl group since Cremer and Callaway (1961) have shown that tetraproply lead is less toxic than tetraethyl lead. In the present study a Similar order has been established for marine animals ie.' terra ethyl lead is more toxic than tetramethyl lead. However, in mammals, the trialkyl lead compounds are more toxic than the tetra alkyl lead compounds and the presently accepted view (Cremer, 1959) is that the tetra alkyl lead compounds are rapidly broken down in the liver of mammals to produce trialkyl lead derivatives which are then responsible for the toxic effects, primarily to the central nervous system. This study, however, appears to show the reverse situation in marine animals since the trialkyl compounds are shown to be .considerably less toxic than the tetra alkyl compounds. This is probably due to the fact that the hydrophobic tetra alkyl lead compounds can be readily absorbed into the aquatic animal where they may exert their toxic action after dealkylation as suggested by Cremer (1959), whereas the more hydro philic and ionic trialkyl lead chlorides have more difficulty in penetrating the gill membranes. It is desirable, for monitoring and control purposes, to obtain an estimate of the "safe" concentration of any contaminant. "Safe" is defined here to mean that concentration of the contaminant which is not expected to cause ..any acutely toxic response in the animal. The "safe" concentration can be obtained from the 96 hour LCj0 value using an appropriate "application factor". This factor, which may range from 0.1-0.001, is intended to allow for the existence of more sensitive species than those studied and to compensate for the difference between the 96 hour DUP040008803 17 1C50 value and the incipient LC^0 dr lethal threshold value which is the -concentration just lethal to 50% of the animals after indefinite exposure, A more satisfactory procedure is to obtain the incipient value from a plot of the concentration against the median time of death of the animal (Median .Survival Period - MSP curve) and apply an appropriate safety factor to this value. This procedure, which is applied below, eliminates errors caused by the variable slope of the MSP curve-. Insufficient data is available from this study to allow the complete median survival period curve to be plotted, since in some of the experiments, the maximum exposure level that could satisfactorily be obtained, did not cause sufficient mortality within 9.6 hours. However, sufficient data :is available to produce points on the curve representing 24, 48 and 96 hour LCg0 values. (Figure 6) . This data shows that in most cases, a threshold value had not become evident after 96 hours. This is in agreement with the work of Cairns (1957) who reported similar findings with tetra ethyl lead. However, the data can be used to predict threshold Values for plaice of 0.04 .mg/1 for TML-CB and 1.0 mg/1 for triethyl lead chloride. This information, coupled with a reasonable application factor of 0,1 (Sprague, 1971) produces estimated safe levels of 0.004 mg/1 for TML-CB and 0.1 mg/1 for triethyl lead chloride. This latter figure will also apply to trimethyl lead chloride, since although no threshold value can be predicted from the data, its toxicity is far less than the triethyl derivative. Similarly since the toxicity of TEL is similar to that of TML- CB and ca. 70-80x that of the trialkyl compounds a ''safe" level in the region of 0.001 mg/1 for both the tetra alkyl compounds seems reasonable. Support for these levels is provided by the data obtained during the accumulation experiments, which are discussed in the next section. In these studies, marine animals were maintained for periods in excess of 30 days without apparent ill effect, in concentrations of t.ri- alkyl lead chlorides ranging from 0.1-2.0 mg/1. DUP040008804 Thus in the light of existing evidence we can propose tentative "safe" levels for the avoidance of acutely toxic effects in the marine environment of 0.001 mg/.l for te.tra alkyl lead compounds and 0.1 mg/1 for trialkyl lead compounds. Since the tetra alkyl lead compounds are rapidly degraded to the trialkyl lead derivatives it is the latter figure Which is of most importance. Bioaccumulation > There are two principal routes by which aquatic animals may accumulate a contaminant, via food or via water.. Exposure of animals to contaminants in food is usually used to assess the significance of food chain transfer and is usually considered to be secondary to accumulation direct from the water. At this stage in the investigation, the water route was considered to be of primary importance, since if no significant accumulation occurred by this route, the possibility of food chain transfer and magnification would be relatively small. The tetr.a alkyl lead compounds have only a short lifetime in seawater and therefore they are unlikely to be accumulated over long periods by marine life. Thus, although some information has been obtained on the accumulation of lead from these materials during short-term exposures to high concentrations;, most of the experimental work discussed is concerned with the accumulation of lead from trialkyl lead compounds during long term exposures. DUP040008805 19 Results (a) Short. Term Exposures The animals, which had been exposed for 96 hours to a wide range of concentrations of four alkyl lead compounds during the acute toxicity investigations, were subsequently analysed for their lead content. The data obtained fro the experiment with shrimps (Crangon erangon) is shown in Figure 7, The results obtained must however, be treated with caution since at exposure concentrations in excess of the 96 hour LCj q value a significant proportion of the animals would have diedbefore the end of the test period. The behaviour of these animals under high stress, Such as immediately prior to death, may have caused anomalies in the rate of accumulation of the material being investigated, and, in addition, since it was not practicable to remove dead animals from- the test system the analytical data obtained for these species may he unrepresentative due to decomposition. The data obtained is of little use for predicting the results of long term exposures, but can be used to indicate the likely concentrations of lead in the tissues of animals exposed to high levels of lead alkyls in the immediate vicinity of a spillage. The results of these short term exposures are summarised in Table .3, The relevant tissue levels, as far as hazards to fish consumers are Concerned is that produced by exposure to the 96 hour LC^ value since higher exposure levels will lead to' substantial mortalities in the exposed population. As can he seen the concentration factors (see Table 3) measured were all relatively small with the tetra alkyl compounds showing a greater tendency to accumulate. (b) Long Term Exposures Two species, the common mussel (Mytilus edulis) and the dab (Ljmanda limanca) both of DUP040008806 20 which are of conmerical importance and provide a pathway for the transfer of lead alkyls to man were chosen for investigation. The test conditions were selected to represent typical seawater and the exposure levels set at approximately 10% of the 96 hour LCj0 values. 1. Mussel Experiment The results of this experiment are summarised in Table 4. The shape of the accumulation curves was similar for both compounds and both concentrations tested and a typical example is shown in Figure 8. After an initial rapid accumulation of lead, during which intake was greater than loss, the mussels reached an equilibrium. The time taken to reach this plateau varied from 5-9 days but was apparently independent of the concentration of alkyl lead. No further accumulation took place although the exposures were continued for up to 35 days. After termination of the exposure the accumulated lead was rapidly lost with a half life, of ea. 3 days. At equilibrium, the average tissue level of lead was directly proportional to the exposure concentration with a concentration factor of ca. 70X. The maximum lead concentration, 68 mg/Kg, was measured in the mussels that had been exposed to 0.1 mg/1 of trimethyl lead chloride (20% of the 96 hour LC^q value) and represented an order of magnitude increase in lead concentration compared to the control population. The distribution of lead within the mussels could be represented as follows: GILL > DIGESTIVE GLAND > FOOT > GONAD The condition factor of the control population indicated no significant signs of stress and, this is confirmed by the low cumulative mortality of * 5% observed over the 60 day experimental period. There was some evidence of a decline in.. DUP040008807 21 condition in the exposed animals for the first ten days of the experiment but this was within the natural variation.. 2. Dab Experiment A summary of the data is shown in table 5 and the .accumulation curves are shown in Figures 9 and 10. In this animal, two tissues were analysed, muscle since it con stitutes the major part of the animal and is eaten by human beings and the liver since this is both a storage organ and a major part of the animals detoxification system. In contrast to the study with mussels, the increase in tissue concentration of lead, with one exception, was linear with respect to time, and there was no evidence for an equilibrium being reached even though the exposure was continued for 41 days. The exception is illustrated in Figure 9 where, in the liver of fish exposed to trimethyl lead chlpride, an equilibrium was reached after ca> 20 days. The reason for this is not known. The rate of accumulation of lead into the fish tissues was very low. After 41 days, the concentration factors were 2x for trimethyl and 12x for triethyl lead chloride. The liver and muscle tissues accumulated similar levels of lead with the average concentration reaching a maximum 30 mg/Kg, In contrast to the mussel experiment, the rate of loss of lead from the fish, after termination of the exposure, was very slow with half lives in excess of 41 days. This low rate of loss was expected, since the linear accumulation curves had implied that the rate of loss was lower than the rate of accumulation. Since the accumulated lead levels in liver and muscle were very similar it seems unlikely that the liver is involved in any major detoxification mechanism in the animal, although the observation that the liver concentrations tended to increase after the exposure had terminated DUP040008808 22 (Fig. 10) implies that some trails location of lead to the liver may take place. During the .course of the experiment, there was a gradual but minor decline in condition of all the experimental animals;. It is interesting to note that the mean deterioration in condition of the fish exposed to triethyl lead chloride was identical to that of the control population (0.004%/day) whereas the corresponding figure for fish exposed to trimethyl lead chloride was 0.008%/day. Several workers have studied the accumulation of inorganic lead compounds by members of the Mytilus family. (Coombs., 1975; Majori and Petronio, 1973; Schulz-Baldes 1972, 1974). Majori and Petronio, using a Mediterranean Species Mytilus galloprovincialis reported concentration factors of 200-300x at exposure levels of 0.05 - 0.10 mg/1. However, most other workers have found the accumulation of inorganic lead to be substantially greater in .mussels than these authors .have reported, the most extensive study has been made by Schulz-Baldes (197.2, 1974) with Mytilus ednljs and a direct comparison of his results with the present study is presented in Table 6. the major difference between inorganic lead, as reported by Schulz-Baldes, and trialkyl lead, as reported here, is that for inorganic lead there is no evidence of a steady state being achieved. Thus, although tbe tissue levels recorded by both groups after 7 ..days exposure are similar, after 35 days exposure the mussels exposed to trialkyl lead chloride have not accumulated any more lead (Concentration Factor (Dry wt)= 400)whereas the mussels exposed to inorganic lead have maintained their initial high rate of accumulation to reach a substantially higher level (Concentration Factor (Dry wt) = 6000). These results are Shown diagrammatically in Fig. 11. These high concentration factors for inorganic lead have been confirmed by Coombs (1975) who reported values of 3,000 in tissues of Mytilus edulis exposed to 0.1 'Hg/1 inorganic lead in seawater. .a DUP040008809 23 This difference between inorganic and trialkyl lead compounds appears to be due to the existence of a rapid excretion process for trialkyl lead in this animal, in the present study the half life of the lead accumulated from trialkyl lead solutions was ca. 3 days. However, Schulz-Baldes quotes a half life for inorganic lead of > 35 days and it has been reported (Kauranen and Jarvenpaa, 1972) that the half life of lead in mussels could be as long as 300 days. At the present time the form in which lead is present in those animals that have been exposed to trialkyl lead chlorides is not known, but it would appear from these results that it is not present simply as inorganic lead. Some similarities do exist between the accumulation of lead from inorganic and trialkyl lead compounds. In both cases the tissue'residue levels at a fixed time are directly proportional to the exposure concentration (Fig. 11). Also, the tissue distribution of lead within the mussel appears to be unaffected by the type of lead compound to which the animal is exposed. Coombs and Schulz-Baldes both found the same ranking order for the tissue distribution as was found in this study. No data has been found on the accumulation of inorganic lead in marine fish that can be contrasted with the present study. Some work has been reported with freshwater fish (Berg, 1973; Merlini and Pozzi, 1977a, b) but comparisons must be made with care. Merlini and Pozzi (1977) showed that the accumulation of inorganic lead by the sunfish (Lepomis gibbosus) was directly related to the species of lead present in the water and they quote concentration factors of 400-900x after 27 day exposures (compared to 2-lOx in the present study). They also report in contrast to this study that the liver tissue contained from 20-30x more lead than the muscle tissue. It is interesting to compare the data obtained in the present study with that produced in the case of mercury accumulation. Several workers have studied the accumulation of inorganic mercury into fish (Macleod and Pessah., 1973; McKone, 1971) and shellfish / DUP040008810 (Kopfler, 1974; tJnlu et al, 1970) and the generally agreed concentration factor is ca. 20-60x. However, experiments by Burki.tt (1974) and Kopfler (1974) indicate that the concentration factor for alkyl mercury compounds in fish and shellfish is approximately an order of magnitude greater at ca. 500-8QQX, Thus alkyl mercury compounds have higher concentration factors in fish and shellfish than inorganic mercury compounds whereas the reverse situation applies in the case of lead. DUP040008811 25 COSCLDSIOKS The aim of the present study was to provide data which could be used to assess the risks caused by an accidental release of lead alkyl compounds in the marine environment;. It has been demonstrated that the toxicity of the alkyl lead compounds is directly related to the degree of alkylation and to a lesser extent to the nature of the alkyl group. The tetra alkyl lead compounds are the most toxic with 96 hour LC^ values of approximately 0.1 mg/1, but these rapidly degrade in seawater to the trialkyl lead chlorides which have 96 hour LC<.q values in the range of 1-10 mg/1, Tentative "safe" levels for these two groups of compounds in the marine environment have been calculated as 0.001 mg/1 and 0.1 mg/1, and these levels would not be expected to cause any acutely toxic effects. The accumulation of lead by -marine animals is important both because of the possible effect on the animal itself and due to the possible effect that the accumulated levels might have on predatory organisms, one of which is man. There is, at the present time, some considerable dispute with regard to the toxicological significance of lead residues in food and in man (Anon, 1972b), however the Joint FAO/WHO Expert Committee on Food Additives (1972) have recommended a provisional tolerable weekly intake of 3 mg lead which is equivalent to 430 ug/day. In the United Kingdom, the lead content of food for human consumption is regulated by either the Food and Drugs Act or the Lead in Food Regulations, These regulations have recently been reviewed fay the Food Additives and Contaminants Committee of the Ministry of Agriculture Fisheries and Food (Anon, 1975) and they have recommended in the light of all the available evidence that in future there should be a statutory lead limit of 2,0 mg/Kg for fish and 5.0 mg/Kg for shellfish (equivalent to 14.0 and 35.0 mg/Kg on a dry weight basis). DUP040008812 26 It has long been known that lead is present in marine animals (Vinogradov, 1953), although due to the inaccuracies of early analytical methods, data prior to 1970 must be treated with caution. The distribution of lead ,in molluscs and particularly mussels obtained from the environment has recently been reviewed by a number of authors (Alexander and Young, 1976; Chow et al, 1976; MAFF 1971, 1975; Nickless etal, 1972; Topping, 1973A) and their results are shown in Table 7(a) alongside data from the present study. The corresponding data for fish is presented in Table 7(b), although in this case the data is not as extensive and does not always refer to the dab, but to other similar Species such as the plaice (Fleuronectes platessa) and flounder (Pleuronectes flesus). The results of the present study indicate that the long term exposure of mussels and dabs to concentrations of trialkyl lead chlorides equivalent to 10% of the 96 hour LCj0 value produce lead residues equivalent to twice that permitted for human Consumption, although the form of the accumulated lead is not known. The production of significantly higher residue levels would require exposure to higher concentrations of the lead alkyl which would cause significant mortality. For example. Table 7 indicates that residue levels of 7x the permitted maximum could be achieved by exposure of mussels to tetra methyl lead for 96 hours but under these conditions 50% of the exposed population would die. Thus, the major environmental impact of a release of tetra alkyl lead compounds into the marine environment is more likely to be due to acutely toxic effects than those of bioaccumulation. DUP040008813 27 ACKKOWI.EDGEMENT The authors would like to express their thanks to the Associated Octel Company for the provision of materials and considerable technical assistance, Mr J E Gaunter, and Mr E Gillings of the Brixham Laboratory for assistance with the experimental work and to Dr 1 Carter of the same establishment for his helpful and constructive criticism. DUP040008814 DUP040008815 Bioassay System Bioassay System Fig 3. TEST APPARATUS FOR MUSSEL ACCUMULATION Constant temperature seawater To waste DUP040008818 Fig 4. TOXICITY OF LEAD COMPOUNDS TO MARINE ANIMALS 96 hr LC50 (m g /I) R4 R3 R2 Ri Ro DEGREE OF ALKYLATION DUP040008819 % ACTIVITY DUP040008820 Fig6 (A) MEDIAN SURVIVAL PERIOD PLOT - T.M.LrC.B. Fig 6(B) MEDIAN SURVIVAL PERIOD PLOT 10,000 j TEL. 1,000----0-01 T 1 I------ 1----- 1 1 T~T- 0-1 ------|----r--i--i--i--m 1-0 CONCENTRATION (mg/I) DUP040008821 Fig 6(C) MEDIAN SURVIVAL PERIOD PLOT -- TRIMETHYL LEAD CHLORIDE Fig 6(D) MEDIAN SURVIVAL PERIOD PLOT - TRIETHYL LEAD CHLORIDE DUP040008822 EXPOSURE CONCENTRATION (m g /I) EXPOSURE DAYS DUP040008824 03 SI / DUP040008825 DAYS DUP040008826 FIG 11 COMPARISON OF ACCUMULATION OF INORGANIC AND DUP040008827 im tono>t 3sc ii-xo g tm is IS a: r : ro fD d o&O>. o CL o tn ro 3D && a & 3--S .3 P oo .e OV to cp ,!p* .--* JNP l>A CD OP j -j trt ro* ro* "S ""S <5! 4 9 S?- S d fp fp as 8 tfi 2<PTf'P3 -toiwn go.wc o S?aT cr.3n>*3 -n o. <? ' s DUP040008828 Parameter measured is a reduction in photosynthetic a c tiv ity (6 hr ECg0) Figures in brackets re fe r to related species - 1. Portman. and Wilson (1971) 2. Jackim e t al (1970) * to ro to a o (-Co5l .ftl s 3. ct- 3. m cf rs t r2+T IB S' fp EU SC. S'. 2 <fup CL Q> & o 3. S' O a 2 9. o 3o S'' CL CL 8 ICO I *.* 1 r- m ro tIO j-n i ir* i< I? i I1 to tcno. co co o' ofsS o -*--mm<l '<6 to .sr 2 3 "P tVn oo to coo ro CVJ1 00 wJ DUP040008829 oe 96 h r - LC50 Concn- at ____ exP0SUre After t.) (mg/Kg. Wet W a & P TmgTT)------ eAnntirmaatilonTiTssnuwe ninc -C--o--n- c--e-n--t-r-a--ti-o-ng = Concentration Factor % :g rm* :i s u> *o 0> C Q. faDt ;cx I *i|3no> O 9Mi 2. o 2. 8- l*--i i= I t 3 P ITJ :!8 -Q"')OcWSC?-CSO w. </j -i. S 2,3 ro "--CPU ct3iO.s--5r --i Vi --> 8 !Lg ro --fiy cw foDrvo-->/ Jar --2Q5 ro j-y*'V/>> --sJ. O ro 2 ro --"o TS n ro *im iIX tuo/> --o-- b^o --,o--i.roo O' O O o' 11C50 iXo iit t cn i i i (i O O tn * o O CO CoO o ooo fo `ro ro oo *>JNSI -- _j cn Vjt-. bo 6vtn Op OoO TcoO--I roo Ooo S" *f*oo -o nX pr* o ro o ro XXX i -p* ^ XXX co ro 51 c d >o ro X XX oXoXXo n-rrso*sr DUP040008830 Omitted from Mean Value C? q<j rz<y** S' TXJ Hto* rl* afu IgS O *< --O m to m TxO is1 o- a 22 .8 5. .o t3o a o ngnoD rf QJ O --> Cl --tu' o> o j vo cn c t i i cn o 12 il5(/mt tr im tx fimro !S !S iZ 5*H jl :g J5 to to .c3s Ii.rC-S a.I*-H o l.o co 4s* 4^ ro ro co a> ro --* o cnwoo i vo o nsoj -u-pj r--o croo ctoo up 5cSo o 'a &, co UP ovo "si SO ssod S4 ro ^o 3. co co rc cn i l\5 o ^ cr CO CO o --t -- --> ro CO cri O--- I CD o 4> co -c cri cn ro oo cn ro cr> o cn -- 0 2. DUP040008831 Could not be determined S o X . </ O VCi Vi fl> >b*2 O ft* s *: <S! Sfg s m o 3 IQ C 5 oo :=? f </> .< n fl> e cS: r-- v < n ffi Tf1 sc -r* ot < o n> TS? --* -- -- II -- | O 4S. Xo ro ro. ui ro o ro ro ->jj ro so .t* ro o ro CO 00 troo _S_O^ -J 1ir0o 1 iimx-o rn *23 iS i(/> !i S SI 3 IO ilO> in iicsr IP j? s I5> :zs 00 ro 00 O to 22 O'O ro DUP040008832 H a lf L ife (d a ys) In o rg a n io D ata from Sohulz-Badea (1974) >35 \H O VJi o p> o p a> ss -5 -3 ft FF C6o> 3O 1X . ct .<+ O CD *1 M S-f 04 > a 'JlSF ct CD Or CD CO rs J? *^ 3 O B .O 3 W CO O wl? 0* * CD .*c1t $ Stf 3 , . v-* Oj ct CD JD <3 C*1D CO c _ : KJ <N.X O i'g i C*1t DP -s =Si ct V ON Vw 8 vn O ON o l O w o w Fs Op 3 F O * t-d H Fg o| 04 o lo VO f F (D vo 1 O h In o rg a n ic * T rim e th y l ON o 04 8 o o Ul I O CO e *i CD O o S o3 PO o vn s :S i? DO ro F CD v^i o VO vo ft S' .3 ft s V cr\ o Vj vn o o ON 8 F f In o rg a n ic * ON ro r o ON CD 6 v o i? F S tfON F CD t13 F . 1 IS*IO |F3 IO 13 w t*1 tF ICO IO s 3 o \*t it* Vo 3 IF* IO l ! ftf IF w IO* r* IS IS ICO I CO DUF040008833 Table 7 Distribution of Dead_in_Marine_Animals (A) Mussels Author Concn. Range (mg/Kg. Dry Wt.) Alexander and Young, 1976 Chow et. al. 197$ MAFF. 1971, 1975 Nickless et. al 1972 Topping. 1973 A. This Study MAFF. Recommended Limit (Anon, 1975) 2.4 - 38 0.2 - 42, <3,5 - 35* 1 - 30 <1.4 - 38* 70 - 250 9-68 35 Source California England and Wales Bristol Channel Scotland Short Term Exposure Long Term Exposure * Converted from Wet Wt. Data (B) Flatfish Author Species Concn. Range Source (mg/Kg. Dry Wt.) Hardisty et al. 1974 MAFF 1971, 1975 Topping, 1973 Flounder 14 - 28 All Flat Fish <1,2 - 29* All Plat Fish 2.4 - 7.2* Plaice <1.4 - 5,6* Bristol Channel England and Wales Greenland Scotland 1 1 m 04 This Study MAFF Recommended Limit (Anon, 1975) Dabs Dabs 10 - 32 14 Short Term Exposure Long Term Exposure * Converted from Wet Wt. Data DUP040008834 REFERENCES Alexander G.V., Young D.R. Trace Metals in South Californian Mussels Mar. Poll. 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