Environ. Sci. Techno/. 1994, 28, 11 10-1 115
Soil-Plant Transfer of Polychlorinated Dibenzo-p-dioxins and Dibenzofurans to Vegetables of the Cucumber Family (Cucurbitaceae) Anke Hulster,’ Jochen F. Muller,? and Horst Marschner Institute of Plant Nutrition, University of Hohenheim, 70593 Stuttgart, Germany -
In a preliminary study in fruits of zucchini (Cucurbita pepo L. convar. giromontiina) polychlorinated dibenzop-dioxins and dibenzofurans (PCDD/PCDF) concentrations were found which were approximately 2 orders of magnitude higher than in other fruits and vegetables examined. These results formed the basis for field experiments on the uptake of PCDD/PCDF from contaminated soils by zucchini and related plant species (pumpkin and cucumber). The experimental design allowed the discrimination of several uptake pathways and an evaluation of their contribution to the total PCDD/ PCDF contamination of the plants. Using two soils with different organic matter content, additional information was obtained on the influence of soil parameters on the soil-plant transfer of PCDD/PCDF. For zucchini and pumpkin (both belonging to the genus Cucurbita), root uptake of PCDD/PCDF and subsequent translocation to the shoots and into the fruits is the main contamination pathway. Cucumber plants (Cucumis sativus L.), by contrast, are mainly contaminated by deposition of airborne PCDD/PCDF and, consequently, show much lower PCDDiPCDF concentrations. The nature of the mechanism responsible for the unexpectedly high soilplant transfer into plants of the species Cucurbita pepo L. remains to be clarified.
Introduction
It is generally agreed that human diet represents the main route of exposure to PCDDIPCDF. This route is believed to be responsible for over 90% of the total PCDD/ PCDF body burden ( l ) ,with meat, milk, and fish contributing 30 % each, whereas the intake by consumption of vegetables and fruits is considered negligible. However, plants are the first link in the food chain, and therefore it is necessary for full-risk assessment to know by which pathways plants may become contaminated. In principle, PCDD/PCDF may accumulate in plants via different pathways, namely (i) adsorption to the root surface, (ii) root uptake and transport to the shoot, (iii) absorption of volatilized PCDDiPCDF by aerial plant parts, (iv) contamination of the foliage and fruits by soil particles, and (v) deposition of airborne PCDDiPCDF. Although in hydroponic cultures, extremely hydrophobic substances (log KO, > 6) such as PCDD/PCDF may be strongly adsorbed by the roots, the mobility of these substances within plants is thought to be minimal (2-4). Therefore, PCDDiPCDF transport within plants is considered to be unlikely, even over long time intervals (5). In soils, extremely hydrophobic substances, for example, chlorinated hydrocarbons, are very immobile (6) and, thus, scarcely available for root adsorption or subsequent uptake. Plant uptake studies with 2,3,7,8-TCDD in growth cham~
t Present address: Faculty of Environmental Sciences, Griffith
University, Nathan, Queensland 4111, Australia. 1110
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bers (7) and 1,2,3,4-TCDD in greenhouses (8) have demonstrated that volatilization from soil, rather than root uptake and translocation, is the main source of contamination of plant shoots. However, results of field studies reported recently show that under outdoor conditions volatilization is of minor importance; PCDD/PCDF concentrations in plant shoots were not correlated to the level of soil contamination, suggesting that atmospheric deposition is the main entry pathway of PCDD/PCDF in plant shoots (9-12). In field experiments (9, 101, PCDDiPCDF concentrations in shoots of various plant species were consistently below 1ng of I-TEq kg-I dry wt, even in plants grown on soils contaminated with up to 6000 ng of I-TEq kg’. Compared to these results, in a preliminary experiment with zucchini PCDD/PCDF concentrations in fruits grown on a soil contaminated with 148 ng of I-TEq k g ’ were exceptionally high; in both peel and pulp, concentrations of about 30 ng of I-TEq kg’dry wt were found. Calculated on a fresh weight basis, the consumption of 100 g of these zucchini fruits represents a PCDD/PCDF intake of more than 100 pg, exceeding the German “value of precaution” of 1 pg of I-TEq kg-l body weight per day (13). In the same contaminated area where these experiments with zucchini were carried out, Wuthe et al. (14) examined PCDD/PCDF levels in human blood. The authors concluded that the consumption of certain vegetables, e.g., zucchini, could be an important source for PCDD/PCDF intake by humans. The main objective of the present study was to identify the principle pathway by which PCDD/ PCDF enter zucchini plants. The PCDDiPCDF contamination of two related plant species, cucumber and pumpkin, were included in the investigations for comparative purposes. Materials and Methods
Experimental Design. The experiments were carried out in Rheinfelden and in Crailsheim, Southern Germany. In Rheinfelden, a highly PCDDiPCDF-contaminated area is located close to the town center. The main contamination, dumping and landfilling of chlorinealkaline-electrolysis residues, occurred between 1900 and 1921. For decades, parts of this land were used for private and commercial fruit and vegetable production. A garden in this area was used for our trials. The experimental setup for zucchini is depicted in Figure 1. The PCDD/ PCDF concentration in the soil was 148 ng of I-TEq k g l (I-TEq according to NATO-CCMS). To evaluate the influence of direct soil contact on contamination levels, zucchini plants (Cucurbitapepo L. convar. giromontiina cv. Diamant F1) were cultivated “conventionally” within the soil. On some of the plants, fruits were fixed to a wire frame to avoid soil contact. The contribution of atmospheric deposition was quantified by growing plants in pots with uncontaminated soil (0.4 ng of I-TEq k g l soil) elevated approximately 1.5 m above the ground level. To 0013-936X/94/0928-1110$04.50/0
0 1994 American Chemical Society
Flgure 1. Schematic representation of experimental setup used to evaluate possible PCDD/PCDf pathways into shoots (leaves and fruits) of zucchini. Arabic numerals indicate PCDD/PCDF concentration in the soils (ng of I-TEq kg-I); roman numerals indicate the treatment.
evaluate the proportion of root uptake and absorption of volatilized PCDD/PCDF, pots containing plants growing in uncontaminated soil were buried into the PCDD/PCDF contaminated soil such that leaves and fruits were in contact with the contaminated soil but the contaminated soil did not have contact with the uncontaminated soil. Cucumber (Cucumis sutiuus L. cv. Delikatess) and pumpkin (Cucurbitu pepo L. cv. Gelber Zentner), both, like zucchini, belonging to the family of Cucurbitaceae, were grown in the same plot. In addition to fruits, leaves from each treatment were harvested and analyzed to determine the PCDD/PCDF distribution within the plants. Soil properties, in particular organic matter content, may affect binding strength and the mobility of PCDD/ PCDF in soils. With an increase in organic matter content, both the availability for root uptake and the extent of volatilization are expected to decrease. Therefore, additional experiments were carried out with zucchini a t another contaminated site (Crailsheim) on a clayey-loam soil with a high organic matter content (8.1%),whereas the organic matter content of the soil in Rheinfelden (loamy-sand/sandy-loam) was rather low (2.5 cm. A sample consisted of all suitable fruits from two plants, which could be harvested in a 6-week period in the summer of 1992. For zucchini and cucumber, a sample consisted of approximately 20 fruits; for the very large pumpkin fruits (>4 kg) a sample consisted of two fruits only. In addition to the fruits, 20 fully developed leaves from each treatment were harvested. All plant samples were freeze-dried, finely ground, and homogenized. Analytical Methods. Extraction, cleanup, and analyses for PCDD/PCDF in the soil and plant material were performed by ERGO-Forschungsgesellschaft mbH, Hamburg, Germany, and will be described in detail elsewhere (16). Briefly, an aliquot of the soil and plant samples was dosed with 13C-labeled internal standards and soxhlet extracted in acetoneln-hexane (50:50) (plant materials) or toluene (soil) for 24 h. After rotary evaporation, the extracts were passed through a series of four columns: a large H~S0~/silica-silica-KOH/silica combination column; a small CsOH/silica-H~S04/silicacombination column connected to an Alox column; and a Carbopack C column. The samples were then concentrated nearly to dryness and taken up in 10-25 p L of toluene. The analyses were conducted by high-resolution gas chromatography with a DB 5 column for homologue sum measurements and an SP 2330 column for the isomer specific analyses, connected to a VG Autospec mass spectrometer operating on a resolution of 10 000. Results and Discussion
I-TEqand CPCDD + PCDF. PCDD/PCDF concentrations (I-TEq and C (sum of) PCDD + PCDF) of the various plant samples are summarized in Table 1. The PCDD/PCDF concentrations in fruits grown on “conventionally cultivated” plants (treatment Ia), in fruits grown without soil contact (treatment Ib), and in leaves of these Environ. Sci. Technoi., Vol. 28, No. 6, 1994
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Table 1. PCDD/PCDF Concentrations in Various Samples of Zucchini, Pumpkin, and Cucumber PCDDiPCDF concentrations plant species
plant part treatment
I-TEqbiC soil plants
Rheinfelden fruits (Ia)" 148 148 fruits (Ib)" 148 leaves (I)" fruits (11)" 0.4 leaves (11)" 0.4 fruits (111)" 0.4 pumpkin fruits (outer parts) 148 fruits (inner parts) 148 leaves 148 cucumber fruits (outer parts) 148 fruits (inner parts) 148 leaves 148 Crailsheim zucchini fruits 328 leaves 328 fruits 2390 zucchini
21.0115.1 19.4121.6 21.4122.6 0.911.1 4.4110.2 0.510.7 12.0111.6 3.113.4 3.612.4 2.412.3 0.210.2 3.412.0
EPCDD + PCDFblc plants 348.11274.0 368.21380.9 440.61490.9 31.9134.7 196.21348.4 44.2155.8 252.91181.6 68.8159.3 98.0185.5 85.0194.8 13.4110.3 322.71135.2
17.0117.4 647.31651.0 29.2126.7 1322.0/1198.0 54.6155.2 2660.0/3005.0
Treatments are depicted in Figure 1. Concentrations in ng k g l dry wt. Values separated by a slash represent two replicates.
plants are approximately 20 ng of I-TEq kg-1 dry wt, indicating a homogeneous distribution of PCDD/PCDF throughout the aerial parts of zucchini. These results show, furthermore, that the usual soil contact of zucchini fruits during their growth is not responsible for their elevated PCDD/PCDF concentrations. With about 20 ng of I-TEq kg-l dry wt the PCDD/PCDF concentrations in the fruits are in the same order of magnitude as those of the preliminary study with zucchini. When grown in uncontaminated soil 1.5 m above the contaminated soil (treatment 111), PCDD/PCDF concentrations in zucchini fruits are between 0.5 and 0.7 ng of I-TEq kg-l dry wt. Fruits from zucchini grown in pots with uncontaminated soil surrounded by contaminated soil (treatment 11)show similarly low concentrations (0.9/ 1.1 ng of I-TEq kg-l dry wt). The use of toxic equivalents (I-TEq) simplifies data presentation as it reduces the extensive set of analytical data to a single value. Based on the relative toxicities of the individual congeners and homologue groups compared to the most toxic congener 2,3,7,8-TCDD, toxic equivalency factors (TEFs) have been developed. The derived toxic equivalents (I-TEq) may be an appropriate measure for hazard assessment and the estimation of human exposure to PCDDIPCDF. However, due to the different contributions (e.g.,different TEFs) of the individual compounds in the calculation of I-TEq, results can become distorted. For the interpretation of PCDD/PCDF levels in biological samples and of contamination pathways, the sum of PCDD + PCDF therefore is a more appropriate measure. Considering CPCDD + PCDF, with 33 ng kg-' dry wt, zucchini fruits of the plants grown in pots with uncontaminated soil surrounded by contaminated soil are even less contaminated than fruits from the elevated pots (50 ng k g ' dry wt). These results demonstrate that the high PCDD/ PCDF concentrations found in conventionally cultivated zucchini fruits result neither from the atmospheric deposition nor from the absorption of volatilized PCDD/ PCDF. The results suggest rather that zucchini roots take up PCDDiPCDF and that these compounds are translocated to the shoots. The somewhat higher PCDDiPCDF 1112
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concentrations (CPCDD + PCDF) of fruits of the elevated pots (44.2/55.8) compared to those of plants grown in uncontaminated soil on ground level (31.9/34.7) can be attributed to a higher exposure of these plants to atmospheric deposition. This peculiarity is also observed in experiments with standardized grass cultures used for biomonitoring of atmospheric PCDD/PCDF ( I 7). Generally, the exponated standardized grass cultures, which are elevated to 1.5 m, show higher levels of contamination than grass samples from the natural environment. In our preliminary experiment with zucchini, similar PCDDiPCDF concentrations were found in the peel and pulp of the fruits. In contrast to these results, the outer parts of pumpkin and cucumber fruits show distinctly higher PCDDiPCDF concentrations than the inner parts. Compared to the results of our studies with other plant species (9, I O ) , the PCDD/PCDF concentrations of whole pumpkin fruits (7.5 ng of I-TEq k g l dry wt) are relatively high (data not shown in Table 1). In contrast, the PCDD/ PCDF concentrations of whole cucumber fruits (0.6 ng of I-TEq k g l dry wt) are in a range normally found in other plant species (9, I O ) . Zucchini and pumpkin both belong to the genus Cucurbita, whereas cucumber belongs to the genus Cucumis. Therefore, it appears that plants of the genus Cucurbita show some propensity to take up and translocate PCDD/PCDF. The higher PCDD/PCDF contamination of the soils at Crailsheim are reflected in elevated CPCDD PCDF concentrations in fruit and leaf samples from zucchini (Table 1). Calculated on CPCDD PCDF, the increase in soil contamination from 13 832 ng kg1(=328ng of I-TEq k g l ) to 169393 ng k g l (=2390 ng of I-TEq kg'), corresponding to a factor of 12,was associated with a 4-fold increase in fruit contamination (from 649 to 2833 ng kg-l). Homologue Profiles and Bioaccumulation Factors. As discussed previously, I-TEq values are not entirely appropriate for the interpretation of PCDD/PCDF levels in plant materials. For an identification of the principle pathway by which PCDD/PCDF enter plants, the PCDD/ PCDF homologue profiles are a better measure. Conventionally grown fruits and fruits grown without soil contact not only show similar I-TEq concentrations (Table 1)but also show corresponding homologue profiles (Figure 2a). The profiles are characterized by a domination of the furans, in particular the tetra- to hexachlorinated compounds (with increasing concentrations from TCDF to HxCDF). In the soil, OCDD is the main compound (Figure 2e). Homologue profiles of samples from zucchini grown in uncontaminated soil (Figure 2b) are clearly different to those of zucchini grown in contaminated soil. As in contaminated soil, furans are the predominating compounds, but in contrast, concentrations decrease from the lower to the higher chlorinated furans. Zucchini plants cultivated in pots with uncontaminated soil buried in the contaminated ground (treatment 11) show similar homologue profiles as samples from the elevated plants (treatment 111). This suggests that the plants of both treatments were contaminated from the same PCDD/PCDF source, namely, atmospheric deposition. In contrast, uptake of volatilized PCDD/PCDF (from the contaminated soil) seems to be of little relevance in accounting for the contamination of zucchini plants. These results represent indirect evidence of the ability of zucchini to take up PCDD/PCDF via roots. Calculation of the bioaccumu-
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Figure 2. Homologue profiles of the various plant samples (a-d) compared to the soil homologue profile (e); location Rheinfelden. For details of treatments described in the legends see Figure 1.
lation factors (BAF) of the particular homologue groups supports this conclusion. The pattern of the bioaccumulation factors (Figure 3a) reflects a soil-plant transfer, namely, a decrease of the BAF from the penta- to the octachlorinated compounds (both dioxins and furans), which is in agreement with the expected decrease in bioavailability due to decreasing solubility in water and increasing sorption to soil particles. As already indicated by the I-TEq concentrations, pumpkin plants show asimilar behavior as zucchini, which
is reflected in corresponding homologue profiles (Figure 2c). This supports the assumption that pumpkin plants, like zucchini plants, were mainly contaminated by root UptakeofPCDDiPCDF. ThereasonforthehigherPCDD/ PCDF concentrations of outer parts compared to inner parts of pumpkin fruits are not known. In contrast to pumpkin, the homologue profiles of cucumber (Figure 2d) resemble the profiles of zucchini grown in uncontaminated soil (Figure 2b). As airborne PCDD/PCDF is the only possible source of contamination Emriron. Sei. Technd.. Vol. 28. NO. 6. 1994 i i 1 3
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Bioaccumulation factors (BAF) of the particular homologue groups in zucchini. Calculation: ng kg-‘ plant dry wtlng kg-’ soil dry wt. Numbers in parentheses in the legend of panel b indicate PCDDIPCDF concentrations (ng of I-TEq kg-’) in the respective soil. Flgure 3.
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Figure 4. Zucchini homologue profiles compared to the soil homologue profile; location Crailsheim. Numbers in parentheses in the legend of panel a indicate PCDDIPCDF concentrations (ng of I-TEq kg-’) in the respective soil.
for zucchini plants grown in uncontaminated soil, the corresponding homologue profiles are probably an indication that atmospheric deposition of PCDDiPCDF is the main contamination pathway of cucumber plants. Asarule,leaves have higher OCDD concentrationsthan fruits. The main component in wet and dry depositions is OCDD (18-20). This could be confirmed by measurements of wet and dry deposition in Rheinfelden and Crailsheim (data not presented). All three plant species used in the trials have large hairy leaves and, hence, can be considered as a “sink for atmospheric PCDDIPCDF. Moreover, a substantial proportion of the fruits was covered by the leaves and, thus, not readily accessible to atmospheric depositions. Both facts are likely to contribute to the elevated OCDD levels of leaf samples compared to fruit samples. Yet, the exceptionally high OCDD concentrations of cucumber leaves cannot be explained satisfactorily. In Figure 4, the PCDD/PCDF homologue profiles of zucchini plants and soils for Crailsheim are presented. In all zucchini plant parts sampled, the furans are the dominating compounds (Figure 4a). In contrast to the zucchini profiles of Rheinfelden (Figure Za),concentrations decrease with increasing degree of chlorination. The homologue distributions in the soil samples of the two locations (Figures 2e and 4b) are also distinctly different. Thus, for a comparison of soil-plant transfer at the two 1114
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locations, the hioaccumulation factors of the particular PCDD/PCDF homologue groups need to he considered (Figure 3). The patterns of the BAF profiles of zucchini grown in Rheinfelden and those grown in Crailsheim are quite similar: with increasing degree of chlorination, soil-plant transfer decreases (decreasing BAF). However, on the soils of Crailsheim, the BAF (and thus soil-plant transfer) are distinctly lower than on the soil of Rheinfelden. As the organic matter content of soils may affect binding strength and thus the mobility of PCDD/PCDF in soils, the lower soil-plant transfer on the soil of Crailsheim can probably a t least in part be attributed to the very high soil organic matter content of that soil.
Conc’usions The presented study clearly demonstrates that the relatively high PCDD/PCDF concentrations in the aboveground parts of zucchini and pumpkin can be attributed to the uptake of these substances via the roots and subsequent translocation to the shoots. Taking into account the low mobility of PCDD/PCDF in soils (6)and within plants (2-5),this result was unexpected. All other plant species tested to date, including cucumber, like zucchini and pumpkin belonging to the family Cucurbitaceae, are mainly contaminated uia atmospheric deposition of PCDD/PCDF (S12).
In studies on the soil-plant transfer of hexachlorobenzene (HCB) into various plants, pumpkin showed as well a particularly high transfer (21, 221, and uptake uia the roots was discussed as a possible uptake pathway (22). Moreover, the authors of both studies concluded that HCB is translocated within pumpkin plants. The results of the present study are in agreement with those of the cited studies (21,22). The nature of the mechanism responsible for the unexpectedly high transfer of PCDD/PCDF, as well as that of HCB, into Cucurbita pepo L. remains to be clarified. The release of root exudates with PCDD/ PCDF “mobilizing”properties is one possible explanation. Certain compounds in these exudates may desorb PCDD/ PCDF from soil particles (in particular organic matter) in the rhizosphere of Cucurbita pepo L. and thus possibly enhance their availability for root uptake. It well might be that the same compounds also facilitate the radical transport of PCDD/PCDF in the roots and the long distance transport in the vascular system of Cucurbita pepo L. The hypothesis that zucchini root exudates mobilized PCDD/PCDF is currently being tested. Acknowledgments The authors wish to thank Dr. M. T. Treeby, Merbein, Australia, and Dr. M. Warne, Brisbane, Australia, for reading the manuscript critically and for offering valuable comments and amendments. The financial support of the Baden- Wurttemberg Ministry for Environment is gratefully acknowledged. Literature Cited (1) Beck, H.; Eckhart, K.; Mathar, W.; Wittkowski, R. Chemosphere 1989,18, 417-24. (2) Briggs, G. G.; Bromilow, R. H.; Evans, A. A. Pestic. Sei. 1982, 13,495-504. (3) Bacci, E.; Gaggi, C. Bull. Environ. Contam. Toxicol. 1985, 35, 673-81.
(4) McCrady, J.; McFarlane, C.; Lindstrom, F. T. J.Exp. Bot. 1987, 38, 1875-90.
( 5 ) Reischl, A.; Reissinger, M.; Thoma, M.; Hutzinger, 0. Chemosphere 1989,19,467-74. (6) Karickoff, S. W.; Brown, D. S.; Scott, T. A. Water Res. 1987, 13, 241-8. (7) McCrady, J.; McFarlane, C.; Gander, L. K. Chemosphere 1990,21, 359-76.
(8) Bacci,E.; Cerejeira, M. J.;Gaggi,C.; Chemello, G.;Calamari,
D.; Vighii, M. Bull. Environ. Contam. Toxicol. 1992, 48, 401-8. (9) Hulster, A.; Marschner, H. Chemosphere 1993,27,439-46. (10) Muller, J. F.; Hulster, A,; Papke, 0.;Ball, M.; Marschner, H. Chemosphere 1993,27, 195-201. (11) Prinz, B.; Krause, G. H. M.; Radermacher, L. Chemosphere 1991,23, 1743-61. (12) Kuhn, T.; Steeg, E. Organohalogen Compd. 1993,12,1836. (13) Schulz, D. Chemosphere 1993,27, 501-7. (14) Wuthe, J. Current Views on the Impact of Dioxins and
Furans on Human Health and on the Environment; The Toxicology Forum: Berlin, Germany, 1992; pp 260-2. (15) Umweltbundesamt. Texte 21/92; Umweltbundesamt: Berlin, Germany, 1992; p 36. (16) Hulster, A. Doctoral Thesis, University of Hohenheim, 1994, submitted. (17) Nobel, W.; Maier-Reiter, W.; Sommer, B.; Finkenbeiner, M. VDI-Ber. 1991, 901, 813-26. (18) Czuczwa, J. M.; Hites, R. A. Environ. Sci. Technol. 1986, 20, 195-200. (19) Eitzer, B. D.; Hites, R. A. Environ. Sci. Technol. 1989,23, 1389-95. (20) Koester, C. J.; Hites, R. A. Environ. Sei. Technol. 1992,26, 1375-82. (21) Hafner, M. Mitteilungsbl. Dtsch. Phytomed. Ges. 1982,12, 49. (22) Ecker, S.; Horak, 0. VDI-Ber. 1991,901, 851-62.
Received for review August 23, 1993. Revised manuscript received February 8, 1994. Accepted February 10, 1994.” ~
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Abstract published in Advance ACS Abstracts, March 15,1994.
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