Trace element speciation and aquatic toxicology

162

trends in analytical chemistry, vol. 2, no. 7, 1983 .

14 Nakamura, H., Nishida, H., Takagi, M. and Ueno, K. (1982) Anal. Chim. Acta 139, 2 19 15 Yamashita, T., Nakamura, H., Takagi, M. and Ueno, K. (1980) Bull. Chem. Sot. J/m 53, 1550 16 Kaneda, T., Sugihara, K., Kamiya, H. and Misumi, S. (1981) Tetrahedron

L.ett. 22, 4407

17 Shinkai, S., Nakegi, T., Nishida, Y., Ogawa, T. and Manabe, 0. (1980) J. Am. Chem. Sot. 102, 5860 18 Shinkai, S., Minami, T., Kouno, T., Kusano, Y. and Manabe, 0. (1982) Chem. Lett. 499 19 Sevdic, D., Fekete, L. and Meider, H. (1980) J. Inorg. Nml. Chem. 42, 885 20 Zolotov, Yu. A., Ionov, V. P., Bodnja, V. A., Larikova, G. A., Niz’eva, N. V., Vlasova, G. E. and Rybakova, E. V. (1982) Zh. Anal. Khim. 37, 1543 21 Zolotov, Yu. A., Niz’eva, N. V., Ionov, V. P., Kumina, D. M. and Ivanov, 0. V. (1983) Mikrochim. Acta 1, 381

22 Zolotov,

Yu.

A.,

Bodnja,

V.

Trace element

A.,

La&ova,

G. A. and

Krasnushkina, E. A. (1982) Vestnik Mosk. Univ. Khim. 23, 296 23 Zolotov, Yu. A., Larikova, G. A., Bodnja, V. A., Efremova, 0. A., Davydova, S. L., Yatsimirskii, K. B. and Kol’chinskii, A. G. (1981) Dokl. Acad. Nauk SSSR 258, 889

E. A. Krasnushkina graduated from the Department of Chemistry of the Moscow University in 1981 and is currently Research Associate at the Vernadskii Institute. Her research interests are photometry and macrocyclic compounds. Yu. A. Zolotou is the head of the solvent extraction laboratory of the Vernadrkii Institute of Geochemistry and Analytical Chemistry, USSR Academy of Sciences, 19 Kosigin str., 117975, GSP-1, Moscow, V-334, USSR. In 1978 he also joined the Department of Chemistry of the Moscow University as a Professor. Since 1970 Prof Zolotov has been an Associate Member of the USSR Academy of Sciences. His research interests irulude solvent extraction and trace analysis. Prof. Zolotov is also an advisory editor of TrAC.

weciation

and aauatic .

.

toxicoloav

The toxicity of trace elements towards aquatic organisms is critically de ndent on the chemical form of the element. This expan 8”ing area of research offers many opportunities for collaboration between chemists and biologists. T. M. Florence Sydney, Australia Speciation analysis of an element is the determination of the individual physico-chemical forms of that element, which together make up its total concentration in a sample. As an example, some of the physico+hemical forms of copper that may exist in seawater are shown in Table I. It is now well established that speciation measurements are necessary for the study of aquatic toxicity and for an understanding of trace element transport in rivers and estuaries’s2. Measurement of the total concentration of a trace element provides no information about its bioavailability or toxicity. The concentration of many heavy metals and other elements in natural waters is often below 1 pg l-l, and sometimes below 0.1 pg 1-l. Chemical analysis at these TABLE Physico+hemical

form

Particulate Simple inorganic complexes Organic complexes Adsorbed on inorganic colloids Adsorbed on organic colloids Adsorbed on mixed organic/ inorganic colloids

concentration levels is a specialized science, particularly when the total metal concentration has to be divided into fractions for speciation determination. There are severe problems with contamination from a multitude of sources, and a clean room is essential for reliable results. Even when speciation measurements are carried out on polluted waters, a room with a filtered air supply, reserved for this type of work, is the minimum requirement. Variation in the speciation of trace elements can dramatically change their toxicity. Most studies of the toxicity of heavy metals towards fish and other aquatic life have shown that the free (hydrated) metal ion is the most toxic form. In the case of copper, hydroxy complexes are also believed to have some toxicity. Most stable complexes and species associated with colloidal particles are nontoxic. The most important exceptions to this rule, however, are metal complexes that are lipid

I. Some possible physico-chemical forms of copper in seawater Possible examples

Approximate diameter (nm)

Retained by 0.45 pm filter Cu(OH)z, CuCOs Cu-fulvate, C&porphyrins Cu*+-FezOs, Cu*+-MnOz

>450 1 2.4 1O-500

Cu’+-humic acid, Cu’+detritus

1O-500

Cu*+-humic acid/Fez03

1O-500

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twds in y.a&thal chemishy, vol. 2, M. 7, 1983

TABLE II. The relationship ASV-labile Chemical species

(%)

between speciation and toxicity to algae

Chelex-100 labile (96)

Solvent extractable (%)

Cu-fulvic acid Cu-tannic acid Cu-oxine Cu-oxine-5-sulphonate Cu-ethylxanthogenate CHsHgCl n-CsH7HgCl n-CsHllHeCl

1.5 5.5 64 100 11 -

66 100 98 77 63


Toxicity Iactor (compound/ metal chloride) 0.36 0.60 >lO 1.7 >lO 7 20 300

ASV = anodic stripping voltammetry.

soluble. Lipid-soluble complexes can rapidly penetrate a biomembrane and cause highly damaging reactions in the cell. Nature has provided fish and other aquatic animals with effective defences against ingested heavy metals, which are eliminated via the gut and detoxified in the liver, kidneys and spleen by a group of proteins called metallothioneins. These defences allow the animals to cope with quite high levels of heavy metals in the food chain and sediment. Evolution has not, however, equipped them to tolerate free metal ions or toxic lipid-soluble complexes in the water pumped through their gills. Unpolluted seawater or river water contains very low concentrations of these toxic metal forms, most of the dissolved metal being adsorbed on colloidal particles or combined in non-toxic complexes. The situation with dissolved pollutants which interact directly with the gill of an aquatic animal is analogous to the danger posed to terrestrial animals by volatile heavy metal compounds such as lead aerosols from automobile exhausts, which can enter the bloodstream by direct absorption through the lungs. Natural waters have powerful detoxification mechanisms which convert free ionic metal species into non-toxic forms, but considerable damage can be caused close to the source of pollution.

most commonly determined metals. The ASV procedure can be adjusted to measure either total or labile metal. Labile metal represents the fraction of the total dissolved metal that can be deposited at the electrode under carefully defined conditions, and has often been assumed to be similar to the toxic fraction of the dissolved metal (Table II). Distinct advantages of the ASV-labile measurements are that no preliminary separations are needed, no blanks are involved, and the opportunity for contamination is minimal. Ion exchange Ion exchange is an attractive technique for trace metal speciation because separations can be achieved with little manipulation of the sample. The iminodiacetate chelating resin, Chelex-100 (Bio-Bad), has found wide application for the measurement of labile metal in water. Metal combined in inert complexes will not be retained by a column of Chelex-100 resin, and metal associated with colloidal particles will be only partly removed. The resin may therefore be used to separate inert metal forms from dissociable complexes. However studies with algae indicate that Chelex-loo-labile metal (i.e. the fraction of total metal removed by the resin) is a serious overestimate of the toxic metal fraction (Table II)‘.

At present it is not possible to measure the concentrations of all the individual chemical species that make up the total concentration of an element in a natural water (Table I). Ion selective electrodes can determine the activity of free metal ions, such as Cu2+, Cd*+ and Pb*+, but are usually insufficiently sensitive for use in natural waters and, in addition, interference effects are often severe. Currently available speciation techniques allow the dissolved elements to be divided only into groups of species based on behavioural differences’. Some of these techniques are described below.

Solvent extraction Solvent extraction of water samples with solvents such as noctanol, chloroform, or hexane-butanol, which simulate the solvent properties of lipid material, is used to estimate the metal fraction that is lipid soluble. Synthetic copolymers, such as Bio-Bad SM2 resin, which has a high affinity for large molecules that have both hydrophobic and hydrophilic moieties, may also be useful models for lipid solubility*. In seawater and some fresh waters, organically-associated metal can be determined by ASV-labile measurements before and after UV irradiation of the sample to destroy organic matter4.

Anodic stripping voltammetry (ASP’) This electroanalytical technique can be used for the determination of about 15 elements, although, in natural waters, copper, lead, cadmium and zinc are the

Dia&sis, ultrafiltration, and gel permeation chromatography These techniques separate molecules on the basis of molecular size. No clear relationship has been established between molecular size and toxicity,

Measurement of trace element speciation

164

trenh in analytical chemistry, vol. 2, no. 7, 1983 *

although, in natural waters, metals associated with most large molecules (e.g. copper-humic acid) are relatively non-toxic. There are significant problems with contamination using all 3 techniques, blank values are high in gel permeation chromatography and, as in all speciation techniques, the ionic equilibrium in the sample is disturbed.

The relationship between speciation and toxicity Heavy metal ions, such as Cu2+ and Cd2+, are readily adsorbed on the cell surface of algae, or by the gill mucous of fish. The adsorption probably involves metal-protein interactions. The adsorbed metal slowly diffuses through the cell membrane to the interior ofthe cell, where it may participate in injurious reactions. The toxic effect appears to be related to the rate of diffusion through the membrane5. Chromate ion, for example, is much more toxic than chromium(III), because the small, non-hydrated chromium (VI) anion diffuses rapidly through biomembranes. Lipid-soluble metal complexes diffuse very rapidly and, for several metals, toxicity increases with increasing lipophilicity (Table II). Likewise, the copper complex of 8-hydroxyquinoline (oxine), which is soluble in organic solvents, is remarkably toxic towards algae, while its water-soluble sulphonate derivative has a much lower toxicity (Table II)3. The metallothioneins are a class of low molecular weight, sulphur-rich proteins which occur widely in vertebrate and invertebrate animals, including humans, rats, fish, oysters and mussels. These proteins have a high affinity (via sulfbydryl bonding) for such metals as cadmium, copper, zinc and mercury, and their biological function is to bind, detoxify and store the heavy metals in the liver, kidney and spleer?. The synthesis of metallothioneins in these organs is induced when the animal is exposed to heavy metals. Even when fish are raised in water which is highly polluted with copper or cadmium, the fish muscle (flesh) contains extremely low concentrations of these metals because they are bound as metallothionein complexes in the liver (Cd, Cu, Zn and Ag), kidney (Cd, Cu, Hg, Ag) and spleen (Cd). It is believed that heavy metal toxicity in animals manifests itselfonly when ‘spillover’ from the metallothionein proteins occurs, i.e. when the amount of metal ingested exceeds the animal’s ability to synthesize the detoxifying metallothionein6. The complexing capacity of a water system can be likened to the metallothionein-synthesizing ability of an animal. Complexing capacity is an important water quality parameter’T2 because it measures the ability of

Computer modeling

By using sophisticated ionic equilibria computer programs and known data for pH, total ion concentrations and equilibrium constants, trace element speciation can be calculated. This approach is adequate for synthetic solutions and for major ions in natural waters, but cannot be used for the speciation of trace heavy metals in natural waters because neither the nature, nor the concentrations, of all the complexing agents present are known1p2.

Trace element speciation in natural and polluted waters Results for the speciation of Cu, Pb, Cd and Zn in some seawater, river water, and polluted water samples are shown in Table III. Copper tends to associate with organic ligands, especially those having sulfhydryl or other sulphur donor groups, whereas lead has an affinity for inorganic adsorbents such as colloidal ferric hydroxide and silica4. Most of the lead in surface seawater, even at a great distance from land, is believed to originate from lead alkyl compounds added to petrol. Similarly, sewage outfalls are the source of most zinc in seawater samples taken up to 100 km seaward of some large coastal cities2. A high percentage of mercury in fresh waters is associated with organic matter and, both in seawater and fresh waters, some of the mercury may be present as the extremely toxic alkylmercury compounds. In mercury-contaminated fish, 900/b of the mercury content may be as methylmercury5. The disaster at Minamata Bay, Japan, during 1953-1960, when over 100 people died after eating mercury-contaminated fish and shellfish, revealed to the world the dangers of environmental pollution with heavy metals. TABLE

III.

Speciation

of copper

in some natural

and polluted waters

Labile copper, Total Sample Seawater, Sydney Seawater

1.40

from a marina

3.21

River water after sewage treatment plant Tap water

ASV

(CLg 1-l)

1 km east of

River water before sewage treatment plant

= anodic stripping

voltammetry.

% of total

Chelex-100

Solvent extractable

co.5

<5

copper

4.45

ASV <

1.5 16.2

<

0.5

46 <2

53 5.8

9.9

< 0.5

48

57

15.6

79

75

40

165

trerds in analytical chemistry,vol. 2, no. 7,1983

t . TABLE IV. Effect of complexing

Complexing None

agent

agents on the toxicity of aluminium

Concentration (mg 1-l)

Labile aluminium (pug 1-l)

towards Chlorella pyrenoidosa Algal growth rate (% of aluminium-free blank)

-

20.5

Fulvic acid

10.0

17.0

85

Tannic acid

4.0

<2

113

10.0 1.0

10.1 8.5

125 62

6.0

<5

106

Phosphate Fluoride Silicate

Synthetic hard water of pH5.2, total aluminium

48

content = 3Opg 1-l.

the water to complex and detoxify added heavy metals. Copper (II) is usually chosen as the heavy metal titrant because it is a common ion, highly toxic to aquatic life. Complexing capacity is defined as the concentration of cupric ion (moles 1-l) that must be added to a water sample before free Cu’+ appears. It reflects the concentration of organic and inorganic substances in the water sample, both molecular and colloidal, that bind copper ions (Table I)‘. Near-shore surface seawater has a copper complexing capacity of about 2 X lo-* M, while river waters range f&m 1 to 50 X lo-’ M.

The toxicity of aluminium in acidified waters Acidification of fresh water lakes in Scandinavia, Canada, and the northeastern USA has led to a serious decline in the fish populations’. Most of this increase in acidity originates from sulphur and nitrogen oxide emissions from coal-fired power stations and ore smelters. The SO2 and NOX eventually find their way into the waterways as H&O4 and HNOs. Lakes most at risk are those which have granite basins, with little limestone in the surrounding soil, and hence are poorly buffered. The lowered pH results in important changes in trace element speciation. Copper may be dissolved from sediment and suspended particles to release toxic cupric ion, and the concentrations of labile cadmium, lead and zinc will also increase’. However, perhaps the most important consequence ofthe higher acidity is the change in aluminium speciation. Aluminium, mobilized from the sediment by the increased acidity, is now believed to be the main cause of fish deaths in waters of pH 4.5-6.0. The toxic aluminium species is the dihydrolysed species, Al(OH):; other hydrolysed aluminium species, including Al(OH):, Al (OH)3 and Al(O are nontoxic’. The selective toxicity of Al(OH): is probably due to the fact that it is the kinetically-favoured form in the reaction between aluminium and the target compounds, most probably proteins in the gill membrane. Several naturallyoccurring complexing agents, such as fulvic acid, phosphate, and silicic acid, complex aluminium and reduce its toxicity (Table IV)“. Maximum toxicity towards the fresh water green alga Chlorella pyrenoidosa occurred between pH 5.8 and 6.2, where as little as 5Fg 1-l of labile aluminium significantly inhibited growth’.

It is likely that dissolved aluminium would be highly toxic to aquatic organisms in fresh waters which-ark slightly acidic and low in organic matter and nutrients. The specific toxicity of the relatively small Al( ion could also explain the phenomenon of dialysis dementia, a disease which occurs in patients undergoing haemodialysis when the dialysis fluid is prepared from water containing high concentrations of aluminium (e.g. a town water supply treated with alum to flocculate organic matter). Diffusion of the small Al( ion through the dialysis membrane could occur rapidly, whereas polymerized aluminium species are unlikely to cross the membrane into the bloodstream’.

Conclusions and prospects By the turn of the century, the supply of clean water for domestic, agricultural and industrial use may well be the factor limiting the growth of population and industry in many countries. Water recycling and purification will assume increasing. importance, and water supplies will need to be analysed in much greater detail, and for a wider range of impurities. Procedures for the speciation of trace elements are likely to be included in future water quality legislation. It is oflittle use to impose a legal limit of, for example, 100 pg 1-l of copper on an industrial discharge since this limit gives no information about its likely toxicity. If the copper is associated with suspended hydrated ferric oxide it may pose no danger but, if present as e.g. the ethyl xanthogenate complex, a common mineral flotation agent (Table II), poisoning of aquatic life would probably occur. The development of alternative liquid fuels from oil shale and coal conversion will eventually become economic as the world’s deposits of petroleum are depleted. These new industries will produce large amounts of organometallic compounds in their waste streams, whose toxic effects on human and aquatic life are only beginning to be studied. The speciation of trace elements critically affects their accumulation in, and toxicity towards, aquatic organisms. More work is required to understand the relationships between element speciation and toxicity; this area of research will provide many opportunities for important collaborative work by analytical chemists and biologists.

166

trena!s in analyticalckemistry,vol. 2, no. 7, 1983 .

References 1 Florence,

T. M. and Batley, G. E. (1980) CRC Critical Rev. Anal.

Ckem. 9,219

2 Florence, T. M. (1982) Talanta 29, 345 3 Florence, T. M. and Lumsden, B. G. Anal. Chim. Acta (in press) 4 Florence, T. M. (1982) Anal. Chim. Acta 141, 73 5 Bryan, G. W. (1976) in Marine Pollution (Johnston, R., ed.), p. 185, Academic Press, London 6 Roesijadi, G. (1980) Mar. Environ. Res. 4, 167 7 Hart, B. T. (1981) Environ. Technol. Lett. 2, 95

Electrophoric molecular

8 Babich, H., Davis, D. L. and Stotzky, G. (1980) Environment 22,6 9 Helliwell, S., Batley, G. E., Florence, T. M. and Lumsden, B. G. Environ. Tech& Lett. (in press)

Mark Florence received his D.Sc. in analytical chemis@ from the University of New South Wales in 1974, and is now Leader of Analytical Chemistry Section, CSIRO Division of Energy Chemistry, Sutherland, New South Wales, Australia. His current scientific interests include ecotoxicology, trace analysis, electso-analytical chemistry and free radical chemistry.

release tags: labels providing

ultrasensitive multiplicity

Electrophoric release tags are a new class of analytical reagents which function as molecular labels. These tags can be more sensitlve than radioisotopes and their potential for multiplicity allows release-tagged reagents to be used as ‘teams’ in chemical analysis. Monoclonal antibodies, nucleic acid probes, receptor ligands and enzyme substrates may all benefit from this approach. Roger W. Giese Northeastern University, Boston, MA, USA We recently introduced a new class of analytical reagents termed ‘release-tags”. The purpose of these reagents is to function as molecular labels analogous to the use of radioisotopes or fluorophores. Release tags comprise 3 molecular groups, known as ‘signal’, ‘release’ and ‘reactivity’ groups. The signal group is for detection purposes, the release group provides a site for specific covalent cleavage, and the reactivity group attaches the release-tag to a substance of interest. The key property of a release-tag is that detection of the signal group occurs after this group has been detached from a tagged substance. This information is summarized in Fig. 1.

Gas phase electrophores We developed release-tags primarily to broaden the application of gas phase electrophores as signal groups in chemical analysis. These are compounds that are detected with high sensitivity by gas chromatography with electron capture detection (GC-ECD), since they readily capture the thermal electrons placed in their path in the ECD. The high sensitivity of this detection process is also helped by the near-perfect electrical insulator properties of the gas phase under normal conditions. Polyhalogenated aliphatics and aromatics are common examples of gas phase electrophores.

interference with the biological, chemical, or physical properties of the substances which they tag. Secondly, electrophores avoid the decay problem of radioisotopes which, for the latter, complicates sampling handling including subsequent problems of and purification, contamination, cost, and disposal. (Some radiolabels, such as 1251, are also chemically labile.) Thirdly, electrophores offer relatively unique structural and detection properties. This contrasts with the use of fluorophore and lumiphore signal groups, for example, which tend to encounter contamination signals or quenching in some samples2. A contrasting advantage for these latter signal groups is the opportunity for visual detection.

Ultrasensitivity and multiplicity of electrophores The most sensitive electrophores can be measured with greater sensitivity than is available for measuring Release Tag

reactivity

I,

0165-9936/83/$01.00

REACTION

I

/

reactivity

signal

2.

Size, stability, and uniqueness of electrophores Gas phase electrophores offer several useful properties as signal groups in chemical analysis. Firstly, they are small in molecular size, relative to such labels as enzymes or microspheres (although radioisotope labels are smaller). This minimizes their

I

RELEASE

pi$q~+&q+’ ‘_7 reactwlty

substance of interest

Fig. I. Structure, attachment and release of a release-tag. Reprinted

permission

with

from Ref. 1. 0

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