Xylem Structure in Pine Trees Grown Near the Chernobyl

IAWA Journal, Vol. 21 (4), 2000: 379–387

XYLEM STRUCTURE IN PINE TREES GROWN NEAR THE 1 CHERNOBYL NUCLEAR POWER PLANT / UKRAINE by Uwe Schmitt, Claudia Grünwald & Dieter Eckstein Federal Research Centre for Forestry and Forest Products and University of Hamburg, Leuschnerstrasse 91, 21031 Hamburg, Germany

SUMMARY

Pine trees around the Chernobyl Nuclear Power Plant, Ukraine, were investigated by light- and electron-microscopy as well as UV-microspectrophotometry in order to find out whether the accident on April 26 in 1986 affected the structure of xylem formed. The analysis of increment cores did not reveal any influence on the amount of xylem formed in 1986, but there was a distinct reduction during the subsequent three years. The xylem formation had recovered in 1990. ʻForkingʼ in the rows of tracheids was frequently seen at the cellular level in the 1986 tree ring. This phenomenon also occurred in the 1987 tree ring, but was less pronounced. It appears that the radioactive irradiation had no direct effect on the cambium in 1986, but affected the differentiation of xylem mother cells. The reduced wood formation between 1987 and 1989 appeared to be a consequence of the massive losses in needles in the year of the accident rather than of the uptake of radiobiologically active elements. The layering and lignification of the tracheid walls were not affected. Key words: Pinus sylvestris, Chernobyl Nuclear Power Plant, 1986 accident, tree-ring widths, xylem structure, light- and electron-microscopy.

INTRODUCTION

On April 26 in 1986 in one of the reactors of the Chernobyl Nuclear Power Plant (CNPP), Ukraine, an accident occurred, which was the most serious in the history of the nuclear industry. This resulted in the release of an extremely high amount of radioactive substances into the environment until May 6. Agachanjanz and Breckle (1994) estimated that the emission of radioactivity exceeded that from the atomic bomb explosions in Hiroshima or Nagasaki by 100-fold. The cloud of radioactivity moved mainly in the northwestern and northern directions (Ipatjew & Ignattschik

1) We are deeply indebted to our friend Erjigit K. Mousaev, Russian Academy of Science, Svertsov Institute of Evolution, Morphology, and Ecology of Animals, Leninsky Prospect 33, 117071 Moscow, Russia, who passed away unexpectedly at the age of 41. He did the sampling and spent three months at our laboratory; further collaboration had been antici- pated.

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1994). The surroundings of the CNPP were heavily contaminated for several kilometers in all directions (Fig. 1). The effect of this could be visually recognized by the dieback of trees and other plants within a few weeks after the accident, and pine trees, the dominant tree species in some forests, developed reddish-brown needles. Character- istically, these needles remained on the branches, and the forests were thus named ʻred forestsʼ (Matsko et al. 1996). Some pine trees survived in certain other areas close to the reactor and were investigated in the following years (Fig. 2).

Fig. 1. Map of central and eastern Europe and enlarged section showing the location of Cher- nobyl and the radioactive load of the area in the year 1989 (dark gray = more than 40 Ci/km2; medium gray = 15–40 Ci/km2; light gray = 1–15 Ci/km2). The broad line in the middle indicates the border between Belarus and the Ukraine.

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To reveal possible short-term effects of the nuclear accident on tree-ring formation, increment cores as well as stem discs from pine trees around the CNPP were collected and investigated by means of light- and electron-microscopy within a pro- gramme of collaboration between the Russian Academy of Science, Moscow, and the University/Federal Research Centre of Hamburg. The study focuses on the tree ring formed in the disaster year of 1986 as well as on rings formed afterwards.

Fig. 2. Pine trees 1 km from the CNPP, eight years after the accident; for study tree see arrow.

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MATERIALS AND METHODS

The measurement of tree-ring widths was carried out with one core per tree, taken between 1990 and 1993 from 285, 30–200-year-old pine trees (Pinus sylvestris L.) from various sites around the CNPP (Mousaev 1994, unpublished). The dendrochronological data processing, including standardisation and cross-dating, was done according to Shiyatov (1973). Microscopy work was limited to only a few samples. They were taken in January 1995 from pine trees at sites 1, 8, and 20 km from the CNPP. One tree per site was felled and discs from various stem heights were cut off and frozen for later inves- tigation. After thawing, xylem portions of 1 ×1×1 cm3 containing the tree rings formed between 1984 and 1990 were taken out with a chisel and processed for light- microscopy. After boiling for two hours, sections of 15–20 μm thickness were cut with a sliding microtome and mounted on glass slides. For electron-microscopic studies samples came only from the xylem at breast height. The samples for scanning electron-microscopy (SEM) were 5 ×5×5 mm3 and for transmission electron-microscopy (TEM) 2 ×2×5 mm3 in size. SEM samples were boiled for two hours and were subsequently cut with a razor blade to obtain a smooth transverse surface, air-dried, mounted onto aluminium stubs, and sputter-coated with gold before viewing with a Hitachi S-520 SEM at an accelerating voltage of 15 kV. For TEM, samples were treated with 2% aqueous OsO4 (osmium tetroxide), dehydrated with acetone, and embedded in Spurrʼs epoxy resin. Lignin determination was done with samples taken from the same breast height discs as used for conventional TEM. Those samples were also embedded in Spurrʼs epoxy resin but without boiling, osmication, and dehydration. Semithin sections of 1 μm in thickness were mounted on quarz slides and were ana- lysed by means of UV-microspectrophotometry with a UMSP 80 UV-microscope (Zeiss). Ultrathin sections taken from osmicated samples were doublestained with uranyl acetate and lead citrate, those taken from unosmicated samples were stained with KMnO4 (potassium permanganate) according to Donaldson (1992). Potassium permanganate-stained samples were examined with a Philips CM 12 TEM at an accelerating voltage of 40 or 60 kV and osmicated samples at a voltage of 80 kV.

RESULTS AND DISCUSSION

The dendrochronological analysis of the pine trees from various sites around the CNPP revealed a variety of responses in the tree ring formed in 1986 and in those formed subsequently (Mousaev 1994, unpublished). As an example, the tree-ring pattern of one pine tree grown 1 km southwest of the CNPP and which was alive in 1994 is shown in Figure 3. It reflects the typical vertical distribution of growth throughout a trunk from the crown to the roots showing the influences of the accident on the formation of wood in 1986 and during the following years until recovery. The tree-ring width of around 2 mm in 1986 was in the same range as in the previous years. How- ever, in 1987 the increment was reduced to less than half. The most severe effect occurred only in 1988, when no wood at all was formed. In 1989 wood formation

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1 1

2 0

1 1

2 0 [mm] 1 1 Size 2 0

1 1

2 0

1 1

1 0 Fig. 3. Tree-ring widths from 1984–1993 at different heights throughout the trunk. 0 A = 14 m (crown region), B = 10 m (just be- 1982 1986 1990 1994 low crown), C = 6 m, D = 1.3 m, E = 0.55 m, Time [years] F = root.

recovered, but the activity was distinctly reduced as compared to the tree rings formed before the accident. Beginning 1990, wood formation recovered progressively. According to these observations, the amount of wood formed in 1986 did not indicate any direct effect of radioactivity on the cambium, although meristematic tissue in general displayed higher radiobiological sensitivity than other plant tissues (Grodzinsky 1989). The meristems were highly sensitive to irradiation particularly during reactivation after dormancy; Mousaev (1993) assumed a 1.5-fold increased sensitivity of the meristems at the beginning of the growing season. Under central European temperate climatic conditions both shoot elongation and wood formation in softwoods start around the end of April and beginning of May (Ladefoged 1952). The Chernobyl accident happened just around this time. Damage was dramatic for the apical meristems of many trees, and numerous shoots did not elongate at all in spring 1986 (Mousaev 1993). Obviously, the radiobiologically highly active iodine (131I) was emitted in high dosages until the second week of May 1986 (Kozoubov & Taskaev

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—

— 90 — 89 —→88 87 —

—

— Fig. 4 & 5. SEM micrographs of the xylem. – 4: Tree rings 1985 until 1991, note the numerous structural anomalies in the 1986 84 tree ring and the missing 1988 tree ring. Scale bar = 0.5 mm. – 5: Detail of the 1986 tree ring with ʻforkingʼ of tracheidal rows. Scale bar = 0.1 mm.

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1990) which played an important role causing short-term effects directly on contaminated tissues, such as the apical meristems and the parenchyma in needles. The influence of iodine, however, decreased within a few weeks because of its short half- life of only eight days. Therefore, the apical meristems, which are more exposed, were more affected by the irradiation than the cambium because of protection by the surrounding bark. The contamination of inner tissues was generally less than of outer ones; as observed by Matsko et al. (1996) radioactivity decreases in the following order: needles>bark>xylem of the branches>xylem of the stem. Other short-term effects of the accident became obvious within three years after the event as deduced from the distinctly reduced widths of the tree rings formed between 1987 and 1989. These effects are probably to a large extent the result of massive needle losses in the year of the catastrophy. There is no information on whether the uptake of 90Sr and 137Cs through the roots has an effect on pine trees (Matsko et al. 1996). At the cellular level, however, the tree ring formed in 1986 showed anomalies regarding the size and arrangement of the tracheids, the so-called ʻforkingʼ of tracheid rows (Fig. 4 & 5), as indicated by the occurrence of small-diameter tracheids radially adjacent to normal sized tracheids. The tangential diameter of tracheids was distinctly reduced, sometimes nearly to half, with the consequence that two small-diameter tracheids abaxially followed a normal sized tracheid. Tracheids in the neighbouring rows were also slightly reduced in size. Such ʻforkingʼ was frequently observed in the transition zone between earlywood and latewood and less frequently in the latewood, but was absent in the earlywood. The tree ring formed in 1987 also showed this phenomenon, but it was less pronounced. ʻForkingʼ was not observed in any tree rings formed in subsequent years. The reason for this anomaly may be an additional anticlinal division of a differentiating xylem mother cell. Transmission electron-microscopy of tracheids of the tree ring formed in 1986 confirmed that they had a normal wall structure, being differentiated into middle lamella/primary wall, S1, S2 and S3 layers of the secondary wall, and a warty layer covering the S3 on the lumen side. The middle lamella stained most intensely with KMnO4, indicating a high lignin content. S1 and S2 appeared distinctly less stained, which suggested that these layers were less lignified (Fig. 6). There was a similar lignification level of the wall

Fig. 6. TEM micrograph of cell walls from earlywood tracheids. Wall layering and staining with KMnO4 is without anomalies. Scale bar = 2.5 μm.

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Chernobyl 1985 tree ring 1.4 [1] 1.3 [1] Cell corner 1.2 [2] CML-tang. 1.1 [3] S2-tang. 1.0 [4] S2-rad. 0.9 [2] 0.8

absorbance 0.7

0.6 [3] 0.5 [4]

Relative 0.4 0.3 0.2 0.1 0.0 a 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 Wavelength (nm)

Chernobyl 1986 tree ring 1.4 [1] 1.3 [1] Cell corner 1.2 [2] CML-tang. 1.1 [3] S2-tang. 1.0 [4] S2-rad. 0.9 [2] 0.8 0.7 absorbance [3] 0.6 [4] 0.5 0.4 Relative 0.3 0.2 0.1 0.0 b 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 Wavelength (nm)

Fig. 7. UV-spectra of wall layers and cell corner regions of tracheids in the 1985 (a) and the 1986 (b) tree ring. UV-absorption at 280 nm indicates the lignification, which is around the same levels in the 1985 and the 1986 tracheids. layers and also of the cell corner regions as compared with tracheids from 1985. Lignin determination by UV-microspectrophotometry confirmed these results without significant differences between the tracheids from 1985 and 1986 (Fig. 7). Both earlywood- and transition zone-tracheids showed lignin contents to be the same as commonly known for Scots pine and spruce in central Europe, and radiata pine in New Zealand (Kleist et al. 1999; Schmitt et al. 2000).

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In conclusion, in the secondary xylem of pine trees grown nearby the CNPP and surviving the accident in 1986 there were no direct effects of the radiation regarding the amount of wood formation in the 1986 tree ring. However, ring widths became reduced in subsequent years until 1989, with a recovery after 1990. On the cellular level, the only effect within the 1986 tree ring, which was also present in the tree ring formed in 1987 but was less pronounced there, was the so-called ʻforkingʼ of tracheidal rows. Wall layering as well as lignification were without anomalies.

ACKNOWLEDGEMENTS

We thank Dr. Eugene Vaganov, Director of the Institute of Forest, Krasnoyarsk, Russia, for organi- sational support during the sampling, as well as the two reviewers of this manuscript for helpful comments. The study was made possible financially by support from the Volkswagenstiftung.

REFERENCES

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