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Biochem. Physiol. Pflanzen 187,131-138 (1991) Gustav Fischer Verlag lena Measurement of Formaldehyde, Hydrogen Peroxide and Non-protein Thiols in Tobacco Leaves during Ageing GABOR GULLNER and ERNO TYIHAK Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Hungary Key Term Index: ageing, formaldehyde, hydrogen peroxide, thiol; Nicotiana tabacum cv. White Burley Summary Formaldehyde, hydrogen peroxide and non-protein thiols were measured in the leaves of intact tobacco plants at 3 ageing stages as the function of plant age and leaf position. The average formaldehyde level changed only slightly during ageing within the range 1.1 - 1.4 Itmole . (g fresh weight)-l. The highest formaldehyde levels were found in the youngest leaves. The H 2 0 2 level depended strongly on plant age and leaf position. The average H 2 0 2 content of leaves was 3.3 Itmole . (g fresh weight)-l in the oldest and in the middle-aged plants, while this value was 2.3 in the youngest plants. Within one plant gradually increasing H 2 0 2 levels were found from the apex toward the base, but it declined in the oldest, yellowing leaves. The maximum values of H 2 0 2 level were 2.9, 4.4 and 5.2 Itmole . (g fresh weight)-l in the young, middle-aged and old age-groups, respectively. The peroxidase activity increased more significantly with the plant age than the H 2 0 2 level. Reversely, the highest catalase activities were found in the young leaves. Gradually decreasing foliar thiol contents were found from the apex towards the base. The reverse changes of H 2 0 2 and thiol concentration in the leaves refer to the alteration of cellular redox potential during ageing. Introduction According to recent investigations formaldehyde can be detected in both animal (HECK and CASANOVA-SCHMITZ 1984) and plant tissues (TYIHAK et al. 1978). It has been reported that the measurable formaldehyde level increases considerably in virus infected tobacco leaves (SZARVAS et al. 1982). This increase is presumably due to enhanced demethylation processes (BURGYAN et a1.1982). Several enzymes (cytochrome P-450s, peroxidase, catal- ase) can produce formaldehyde in oxidative demethylation reactions. Hydrogen peroxide dependent N-demethylase activity (which produces formaldehyde) has been found in bean leaves (GULLNER and TYIHAK 1987). The temperature dependence of peroxidase and N- demethylase activities in bean leaf extracts can explain the recently observed increased formaldehyde levels in heat-shocked bean leaves (TYIHAK et al. 1989). These observations and the data showing the increased H 2 0 2 production in plant leaves or cell suspensions during drought stress (MUKHERJEE and CHOUDHURI 1983) and fungal elicitor stress (LINDNER 1988) suggest that the metabolism of formaldehyde and H 2 0 2 may be connected. Hydrogen peroxide is a normal plant metabolite (HALLIWELL 1974) but its overproduc- tion (oxidative stress) is deleterious to plant tissues. Chloroplasts contain an efficient ascorbate-glutathione system to scavenge H 2 0 2 (GILLHAM and DODGE 1986). The relation- 9* BPP 187 (1991) 2 131

Measurement of Formaldehyde, Hydrogen Peroxide and Non-protein Thiols in Tobacco Leaves during Ageing

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Biochem. Physiol. Pflanzen 187,131-138 (1991) Gustav Fischer Verlag lena

Measurement of Formaldehyde, Hydrogen Peroxide and Non-protein Thiols in Tobacco Leaves during Ageing

GABOR GULLNER and ERNO TYIHAK

Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Hungary

Key Term Index: ageing, formaldehyde, hydrogen peroxide, thiol; Nicotiana tabacum cv. White Burley

Summary

Formaldehyde, hydrogen peroxide and non-protein thiols were measured in the leaves of intact tobacco plants at 3 ageing stages as the function of plant age and leaf position. The average formaldehyde level changed only slightly during ageing within the range 1.1 - 1.4 Itmole . (g fresh weight)-l. The highest formaldehyde levels were found in the youngest leaves. The H20 2 level depended strongly on plant age and leaf position. The average H20 2 content of leaves was 3.3 Itmole . (g fresh weight)-l in the oldest and in the middle-aged plants, while this value was 2.3 in the youngest plants. Within one plant gradually increasing H20 2 levels were found from the apex toward the base, but it declined in the oldest, yellowing leaves. The maximum values of H20 2 level were 2.9, 4.4 and 5.2 Itmole . (g fresh weight)-l in the young, middle-aged and old age-groups, respectively. The peroxidase activity increased more significantly with the plant age than the H20 2 level. Reversely, the highest catalase activities were found in the young leaves. Gradually decreasing foliar thiol contents were found from the apex towards the base. The reverse changes of H20 2 and thiol concentration in the leaves refer to the alteration of cellular redox potential during ageing.

Introduction

According to recent investigations formaldehyde can be detected in both animal (HECK and CASANOVA-SCHMITZ 1984) and plant tissues (TYIHAK et al. 1978). It has been reported that the measurable formaldehyde level increases considerably in virus infected tobacco leaves (SZARVAS et al. 1982). This increase is presumably due to enhanced demethylation processes (BURGYAN et a1.1982). Several enzymes (cytochrome P-450s, peroxidase, catal­ase) can produce formaldehyde in oxidative demethylation reactions. Hydrogen peroxide dependent N-demethylase activity (which produces formaldehyde) has been found in bean leaves (GULLNER and TYIHAK 1987). The temperature dependence of peroxidase and N­demethylase activities in bean leaf extracts can explain the recently observed increased formaldehyde levels in heat-shocked bean leaves (TYIHAK et al. 1989).

These observations and the data showing the increased H20 2 production in plant leaves or cell suspensions during drought stress (MUKHERJEE and CHOUDHURI 1983) and fungal elicitor stress (LINDNER 1988) suggest that the metabolism of formaldehyde and H20 2 may be connected.

Hydrogen peroxide is a normal plant metabolite (HALLIWELL 1974) but its overproduc­tion (oxidative stress) is deleterious to plant tissues. Chloroplasts contain an efficient ascorbate-glutathione system to scavenge H20 2 (GILLHAM and DODGE 1986). The relation-

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ship between plant senescence and hydrogen peroxide has been already investigated using excised leaves (PARIDA et al. 1978; MONDAL and CHOUDHURI 1981; MONDAL and CHOUDHURI 1982). It is suggested that the transition from juvenility into senescence is accompanied by a progressive shift towards an oxidative state (BRENNAN and FRENKEL 1977; MONDAL and CHOUDHURI 1982). This shift may result in the breakdown of autooxidizable metabolites, e.g. sulfhydryl groups (STONIER and YANG 1973).

The increase of peroxidase activity has been observed in many species in the course of physiological or ethylene-induced senescence (KAR and MISHRA 1976; BIRECKA et al. 1979). Catalase activity declines during the senescence of both attached and detached leaves of several plants (KAR and MISHRA 1976; PARIDA et al. 1978).

To obtain more information on formaldehyde and on its connection to H20 2 metabolism we have investigated the level of endogenous formaldehyde and H20 2 in the leaves of intact tobacco as function of plant age and leaf position. In spite of many studies on plant thiols, relatively little is known on the localisation of thiols throughout a given plant (BIELAWSKI and JOY 1986). In order to gain a deeper insight in the the redox state of tobacco leaves we have determined also the non-protein thiol content in the leaves. Peroxidase and catalase activities were measured, too.

Materials and Methods

Plant material Tobacco plants (Nicotiana tabacum cv. White Burley) were grown from seeds in soil under normal

greenhouse conditions (temperature: 18-25°C, supplemental light: 160!-tE· m-2 • S-1 for 8 h per

day). The plants were watered daily with tap water. Three plant groups with different ages were investigated: I. 55-60 d old plants (after sowing) with 5 expanded leaves, II. 80-85 d old plants with 10 expanded leaves, III. 105-110 d old plants with 15 expanded leaves (still in the pre-flowering status). Leaf samples were taken at each leaf positions from intact plants.

Assays

Formaldehyde was measured by thin-layer chromatography (TLC) in formaldemethone adduct form after derivatization with dimedone according to TYIHAK et al. (1989) with modifications. Leaves (1 g) were frozen in liquid nitrogen then 10 ml of 25 mM dimedone solution in acetone was added. The suspension was filtered and completed to 10 ml. Aliquots (25 !-tl) of these solutions were spotted on Kieselgel 60 F 254 plates (Merck) and the chromatograms were developed in chloroform-dich­loromethane mixture (35: 65, v/v). Spots were evaluated by a Shimadzu CS-930 scanner. Formal­dehyde concentrations were calculated by using a formaldemethone calibration curve.

Hydrogen peroxide was determined spectrophotometrically by titanium-tetrachloride reagent (PILZ and JOHANN 1965), according to FERGUSON et al. (1983) with modifications. All steps were carried out as described by them, but after the final absorbance measurement the samples were mixed with equal volumes of 1 M mercaptoacetic acid solution (dissolved in 1 M H2S04) in order to remove the color of the Ti-H20 2 complex. The absorbance of these mixtures were read again at 415 nm. The residual absorbance is caused by green pigments remained in the samples. PATTERSON et al. (1984) pointed out that these pigments interfere in this assay. H20 2 concentrations were calculated by using a calibration curve.

The concentration of water-soluble, non-protein thiols were determined spectrophotometrically with 5.5'-dithiobis-(2-nitrobenzoic acid) reagent according to DE KOK and GRAHAM (1989).

Enzyme assays

Leaf tissue (2 g) was homogenized in a mortar with 40 ml of 0.1 M sodium phosphate buffer (PH 7.5) containing 1 % insoluble polyvinyl-pyrrolidone and 0.1 mM EDTA-Na2' The homogenate was

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stained through muslin and centrifuged at 8000 g for 20 min. The supernatants were used as enzyme sources. All steps were carried out at 0-4 0c.

Peroxidases were assayed according to CHANCE and MAEHLY (1955). The catalase activity was determined by following the decomposition of H20 2 spectrophotometrically at 240 nm according to AEBI (1970). The extinction coefficient of H20 2 is 0.040 mM- 1 • cm- 1 at 240 nm (PATTERSON et al. 1984).

All experiments were performed with 3 independent parallel determinations. Mean values and standard deviations were calculated.

Results

At the beginning of our experiments we investigated the effect of dirnedone concentration on the amount of formaldehyde detected in plant extracts. The formaldehyde levels found in the acetone extract of tobacco foliar tissues depended strongly on the concentration of the formaldehyde trapping agent dirnedone (Fig. 1). In each further experiments 25 rnM dirnedone concentration was used. The formaldehyde levels determined in tobacco leaves are shown in Fig. 2. The highest average formaldehyde concentration was observed in the age­group II (l.42 [tmole . (g fresh weight) -I). In the age-groups I and III these values were

1.5

1.0

0.5

,umole formaldehyde

9 fresh leaf

10 20 30 mM dimedone

}.lmole formaldehyde

9 fresh leaf I

5 4 3 2 1 IT

10 9 B 7 6 5 4 3 2 1 ]I

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 leaf position

Fig. 1. The dependence offormaldehyde concentration detected in tobacco leaf extracts on dimedone concentration. Leaf samples were taken from 85-d-old plants (middle leaf position). Formaldehyde was collected by dimedone in acetone solution and the product formaldemethone was determined by TLC.

Fig. 2. Formaldehyde levels in the leaves of tobacco plants. Groups I, II and III represent plants with 5, 10 and 15 expanded leaves (see in the Materials and Methods section). Arabic numbers show the leaf positions, No.1 denotes the oldest leaf and the increasing numbers refer to gradually younger leaves. Formaldehyde was determined by TLC after derivatization with dimedone reagent.

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,0 mole H202 9 fresh leaf

15 14 13 12 11 10 9 8 7 6 5

I

][

][

leaf position

,umole guaiacol 50 min· 9 fresh lea!

40

30

20

10

50

40

30

20

10

50

40

30

20

10

II

![

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 leaf posit.ion

Fig. 3. Hydrogen peroxide levels in the leaves of intact tobacco plants. H20 2 was detennined spectrophotometrically by using TiC14 reagent. For symbols see Fig. 2.

Fig. 4. Peroxidase activities in the leaf cell-free extract of tobacco plants. Peroxidase activities were detennined spectrophotometrically by using guaiacol. For symbols see Fig. 2.

slightly lower (1.30 and 1.18llmole· (g fresh weight) - l, respectively). The highest formaldehyde concentrations were detected in the youngest leaves (l.5-2.0 Ilmole . (g fresh weight)-l) in each age-group. The lowest formaldehyde level was found at middle leaf positions and in the oldest leaves it increased slightly again.

The hydrogen peroxide levels of tobacco leaves are compiled in Fig. 3. The average hydrogen peroxide level of each leaves was the same in the age-groups II and III (3.3 Ilmole . (g fresh weight)-l), but it was significantly lower in the youngest plants (2.3 Ilmole . (g fresh weight)-l). The highest values in the age groups were 2.9, 4.4 and 5.2 I-lmole . (g fresh weight)-l (groups I, II and III, respectively). The H20 2 levels depended strongly on the leaf position. Gradually increasing H20 2 levels were found from the apex (youngest leaves) toward the base, but it declined in the old, yellowing leaves.

In accordance with previous papers, the peroxidase activity of the cell-free extract considerably increased during plant ageing (Fig. 4). The older was the leaf, the higher activity was found, except the oldest, yellow leaves . Catalase activities were heavily dependent on leaf position in the age-groups II and III (Fig . 5), but to a lesser extent in the youngest plants (group I). The youngest leaves showed high activity while gradually declining activities were detected in the older leaves. However, the average catalase activity

134 BPP 187 (1991) 2

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40

20

100

80

60

40

20

100

80

60

40

20

,umole H202 min· 9 fresh leaf

~

TI

10 9 8 7 6 5 4 3 2 1

ill

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 leaf position

0.8

0.6

0.4

0.2

0.8

0.6

0.4

0.2

0.8

0.6

0.4

0.2

)Jmole thiol 9 fresh leaf

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 leaf position

Fig. 5. Catalase actlvltles in the leaf cell-free extract of tobacco plants. Catalase activities were determined by following the degradation of H20 2 spectrophotometrically at 240 nm. For symbols see Fig. 2.

Fig. 6. The concentration of non-protein thiols in tobacco as the function of plant age and leaf position. Thiol concentrations were determined spectrophotometrically by using 5.5'­dithiobis-(2-nitrobenzoic acid) reagent. For symbols see Fig. 2.

changed in a different manner, its values were 30, 53 and 42 f.!mole H20 2 (g fresh weight)-l . min- 1 in the age-groups I, II and III, respectively.

In Fig. 6 the distribution of water-soluble non-protein thiols in tobacco leaves is shown. A gradual decrease of foliar thiol content was found from the apex towards the base in the case of each age-group. The observed thiol concentrations (0.09-0.84 f.!mole . (g fresh weight)-l are similar to those observed in.Pisum sativum (BIELAWSKI and JOY

1986).

Discussion

The endogenous formaldehyde is reversibly bound to various cellular nuc1eophiles (among others free and bound amino acids, glutathione) in the living tissue (HECK and CASANOVA-SCHMITZ 1984). To collect formaldehyde in tobacco leaf extracts 20-25 mM dimedone had to be applied (Fig. 1). This methodological result shows that at relatively high dimedone concentrations formaldehyde can be freed from the various reversible adducts e.g. from different methylol groups. The origin of the high formaldehyde level

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found in the youngest leaves is unknown. A possible explanation is the enhanced rate of methylation reactions in juvenile tissues. HUSZTI and TYIHAK (1986) found that the formation of formaldehyde from the methyl group of S-adenosyl-L-methionine (SAM) as general methyl donor is probably linked to enzymatic methylation reactions. During this and other methylation processes the formaldehyde formed from SAM is bound (in methylol form) to guanidine groups of L-arginine residues in transmethylases. Dimedone can react with the formaldehyde formed during the enzyme reaction.

Our results show that the hydrogen peroxide level of leaves increases with ageing, but it declines in the oldest, yellowing leaves. Peroxide accumulation may cause changes in plant metabolism in several ways. Peroxides participate in free radical reactions which play an important role in senescence (LESHEM 1988). It is possible that the primary function of peroxides is to bring about a shift in the redox potential of tissues.

No obvious correlation was observed between formaldehyde levels and peroxidase or catalase activity. The reverse changes of peroxidase and catalase activities during ageing suggest that while in the initial phase of growing the destruction of H20 2 is predominant (maintenance of reductive state in the cell), the utilization of H20 2 in various oxidative processes becomes more important during senescence.

Thiol gradients were found in tobacco plants with decreasing foliar thiol concentrations from the apex towards the base. Similar gradient was found in Pisum sativum (BIELAWSKI and JOY 1986) and in other plants (STONIER and YANG 1973). It is interesting to note that in our case the slope of thiol gradient diminished during plant ageing. The concentrations of H20 2 and thiols change in reverse directions in the plants of the age groups II and III (except the oldest leaves). This phenomenon refers to the alteration of cellular redox potential during senescence. Glutathione is the major cellular low molecular weight thiol component (RENNENBERG 1982). PAULS and THOMPSON (1984) observed a loss of both reduced and oxidized glutathione in cotyledons of Phaseolus vulgaris during senescence. DE KOK and GRAHAM (1989) suggested that the oxidation of glutathione is not a primary event of senescence and that it occurs during the last stages of senescence when cellular integrity is lost.

Finally, it is supposed that endogenous H20 2 can oxidize formaldehyde and the singlet oxygen formed in this reaction (TREZL and PIPEK 1988) can participate in biotic stress reactions.

Further experiments are necessary to elucidate the metabolic role of formaldehyde and the connections between formaldehyde and hydrogen peroxide, especially in various stress reactions.

References

AEBI, H. : KataIase. In: Methoden der Enzymatischen Analyse. Vol. I (Ed. BERGMEYER, H. U .) pp. 636-647 . Verlag Chemie, Weinheim 1970.

BIELAWSKI, W., and JOY, K. W.: Reduced and oxidised glutathione and glutathione-reductase activity in tissues of Pisum sativum. Planta 169, 267-272 (1986).

BRENNAN, T., and FRENKEL, c.: Involvement of hydrogen peroxide in the regulation of senescence in pear. Plant Physiol. 59,411-416 (1977) .

BURGYAN, 1., SZARVAS, T., and TYIHAK, E. : Increased formaldehyde production from L-methionine­(S-14CH3) by crude enzyme of TMV infected tobacco leaves . Acta Phytopathol. Acad. Sci. Hung. 17,11-15 (1982).

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CHANCE, B., and MAEHLY, A. C.: Assay of catalases and peroxidases. In: Methods in Enzymology, Vol. II (Eds. COLOWICK, S. P., and KAPLAN, N. 0.) pp. 764-775. Academic Press. New York 1955.

DE KOK, L. J., and GRAHAM, M.: Levels of pigments, soluble proteins, amino acids and sulfhydryl compounds in foliar tissue of Arabidopsis thaliana during dark-induced and natural senescence. Plant Physiol. Biochem. 27, 203-209 (1989).

FERGUSON, I. B., WATKINS, C. B., and HARMAN, J. E.: Inhibition by calcium of senescence of detached cucumber cotyledons. Plant Physiol. 71, 182-186 (1983).

GILLHAM, D. 1., and DODGE, A. D.: Hydrogen-peroxide-scavenging systems within pea chloroplasts. Planta 167, 246-251 (1986).

GULLNER, G., and TYIHAK, E.: Hydrogen peroxide dependent N-demethylase activity in the leaves of normal and heat shocked bean plants. Plant Sci. 52, 21-27 (1987).

HALLIWELL, B.: Superoxide dismutase, catalase and glutathione peroxidase: solutions to the problems of living with 02. New Phytol. 73, 1075-1086 (1974).

HECK, H. D' A., and CASANOVA-SCHMITZ, M.: Biochemical toxicology of formaldehyde. Rev. Biochem. Toxicol. 6, 155-189 (1984).

HUSZTI, Z., and TYIHAK, E.:Formation of formaldehyde from S-adenosyl-L-[methyPHlmethionine during enzymic transmethylation of histamine. FEBS Lett. 209, 362-366 (1986).

KAR, M., and MISHRA, D.: Catalase, peroxidase and polyphenol oxidase activities during rice leaf senescence. Plant Physiol. 57, 315-319 (1976).

LESHEM, Y. Y.: Plant senescence processes and free radicals. Free Rad. BioI. & Med. 5, 39-49 (1988).

LINDNER, W. A., HOFFMANN, C., and GRISEBACH, H.: Rapid elicitor-induced chemiluminescence in soybean cell suspension cultures. Phytochemistry 27, 2501-2503 (1988).

MONDAL, R., and CHOUDHURI, M. A.: Role of hydrogen peroxide in senescence of excised leaves of rice and maize. Biochem. Physiol. Pflanzen 176, 700-709 (1981).

MONDAL, R., and CHOUDHURI, M. A.: Regulation of senescence of excised leaves of some C-3 and C-4 species by endogenous H20 Z ' Biochem. Physiol. Pflanzen 177, 403-417 (1982).

MUKHERJEE, S. P., and CHOUDHURI, M. A.: Implications of water stress-induced changes in the level of endogenous ascorbic acid and hydrogen peroxide in vigna seedlings. Physiol. Plant. 58, 166-170 (1983).

PARIDA, R. K., KAR, M., and MISHRA, D.: Enhancement of senescence in excised rice leaves by hydrogen peroxide. Can. 1. Bot. 56, 2937-2941 (1978).

PATTERSON, B. D., MACRAE, E. A., and FERGUSON, I. B.: Estimation of hydrogen peroxide in plant extracts using titanium (IV). Anal. Biochem. 139, 487-492 (1984).

PAULS, K. P., and THOMPSON, 1. E.: Evidence for the accumulation of peroxidized lipids in membranes of senescing cotyledons. Plant Physiol. 75, 1152-1157 (1984).

PILZ, W., and JOHANN, I.: Spezielle analytische Methoden fUr die Biochemie und physiologische Chemie. II. Mitteilung. Die photometrische Bestimmung von Wasserstoffperoxid und Natriumper­borat. Z. Anal. Chern. 210, 358-364 (1965).

RENNENBERG, H.: Glutathione metabolism and possible biological roles in higher plants. Phytochemis­try 21, 2771-2781 (1982).

STONIER, T., and YANG, H.-M.: Studies on auxin protectors XI. Plant Physiol. 51, 391-395 (1973).

SZARVAS, T., JANOS, E., GABORJANYI, R., and TYIHAK, E.: Increased formaldehyde formation: an early event ofTMV infection in hypersensitive host. Acta Phytopathol. Acad. Sci. Hung. 17,7-10 (1982).

TREZL, L., and PiPEK, J.: Formation of excited formaldehyde in model reactions simulating real biological systems. J. Mol. Struct. (Theochem) 170, 213-223 (1988).

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TYIHAK, E., BALLA, J., GABORJANYI, R., and BALAZS, E.: Increased free formaldehyde level in crude extract of virus infected hypersensitive tobaccos. Acta Phytopathol. Acad. Sci. Hung. 13, 29-31 (1978).

TYIHAK, E., KIRALY, Z., GULLNER, G., and SZARVAS, T.: Temperature-dependent formaldehyde metabolism in bean plants. The heat shock response. Plant Sci. 59, 133-139 (1989).

Received April 23,1990; revised form accepted July 27,1990.

Authors' address: G. GULLNER and E. TYIHAK, Plant Protection Institute, Hungarian Academy of Sciences, H - 1525 Budapest, P.O.B. 102, Hungary.

Biochem. Physiol. Pflanzen 187,138 (1991) Gustav Fischer Verlag Jena

B uchbesprechung

MERBACH, W., and MULLER-URI, Ch: Lead in the Environment. Part 1. Bibliography. Serie: Terrestrische Okologie. Sonderheft 9. VII, 192 S. Univ.-u. Landesbibliothek Sachsen-Anhalt, Halle 1990.

Bibliographien haben ihren festen Platz im modemen Forschungsgetriebe. Sie erleichtem dem Anfanger die rasche Orientierung tiber bereits vorliegende Ergebnisse, ermoglichen es ihm, Schwer­punkte des Gebietes rasch zu orten und nicht zuletzt zu finden, wo die Zentren der Forschung auf dem betreffenden Feld in der zuruckliegenden Zeit waren. Dem Insider ermoglichen sie es, eine halbverges­sene, aber fUr wichtig erachtete Literaturstelle rasch wiederzufinden und zu tiberprufen. Die Basis fUr ein solches Arbeiten mit Bibliographien sind die Indexe. Ohne solche sind sie wertlos! Hier nun wird eine Bibliographie vorgelegt tiber "Leads in the Environment - Part 1", ein Thema also, das aile "okologiebewuBten" Wissenschaftler alarmieren sollte. Es enthiilt eine Literaturauflistung von 1374 Zitaten in alphabetischer Ordnung von Arbeiten zur Bleivergiftung unserer Umwelt sowie ein Register "Analytik". Moglicherweise bringt der angektindigte Bd. 2 die Indexe, die man von solchen Bibliographien erwarten darf. Vor dessen Erscheinen sollte potentiellen Kiiufem von Bd. 1 abgeraten werden. P. APEL, Gatersleben

138 BPP 187 (1991) 2