Polycyclic aromatic hydrocarbons (PAH) in various types of tea

For the analysis of the 16 PAH (EFSA-PAH), which are classified as priority for different food groups by the Scientific Committee on Food (SCF) and the Joint FAO/WHO Experts Committee on Food Additives (JECFA) in tea, a sensitive analytical Fast-GC/HRMS method was used. The sample preparation included accelerated solvent extraction (ASE) and the highly automated clean up steps, gel permeation chromatography and solid phase extraction. The analytical parameters, limit of detection (0.01–0.02 μg/kg) and limit of quantification (0.03–0.06 μg/kg), were determined. The repeatability (RSD, n = 3) of different PAH in fruit tea ranged from 0.1 to 11%. It was observed that the total contents of the 16 PAH in tea samples ranged from 14 to 2,662 μg/kg. The analysed tea samples showed an increasing presence of PAH in the following order: herbal and fruit tea (n = 7) < black tea (n = 11) < green tea (n = 11) < white tea (n = 3) < mate-tea (n = 8). The correlation coefficient (R) between BaP and the sum of the 16 EFSA-PAH was established considering the contamination amount in all the 40 tea samples analysed.     

Polycyclic aromatic hydrocarbons (PAH) in chocolate on the German market

Benzo[a]anthracene (BaA), chrysene (CHR), cyclopenta[c,d]pyrene (CPP), 5-methylchrysene (5MC), benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF), benzo[j]fluGoranthene (BjF), benzo[a]pyrene (BaP), dibenzo[a,h]anthracene (DhA), indeno[1,2,3-cd]pyrene (IcP), benzo[g,h,i]perylene (BgP), dibenzo[a,l]pyrene (DlP), dibenzo[a,e]pyrene (DeP), dibenzo[a,i]pyrene (DiP) and dibenzo[a,h]pyrene (DhP), the 15 SCFPAH, assessed to be relevant as well as benzo[c]fluorene (BcL) recommended by the European Food Safety Authority (EFSA), were analysed in different types of chocolate. The sample preparation included accelerated solvent extraction (ASE), size exclusion chromatography (SEC) and solid phase chromatography using small silica gel columns. The individual PAH were separated by gas chromatography using a VF-17ms GC column and detected by high resolution mass spectrometry (HRMS). The investigation of 40 samples of various types of chocolate with different cocoa contents resulted in a median benzo[a]pyrene (BaP) content of 0.22 μg/kg. Furthermore, the results showed a linear correlation between the content of BaP and the sum content of the 16 priority PAH. Therefore, the analysis of BaP as a leading substance seems to be suitable to estimate the PAH contamination in chocolate.     

Polycyclic aromatic hydrocarbons (PAHs) in meat products and estimated PAH intake by children and the general population in Estonia

The concentrations of benzo[a]pyrene and 11 other polycyclic aromatic hydrocarbons (PAHs) were analysed from 322 commercial, cured meat products and 14 home-grilled meat samples as part of the Estonian food safety monitoring programme during 2001-2005. The maximum acceptable concentration of 5 µg kg^-1 for benzo[a]pyrene was exceeded in 3.4% of samples. The highest PAH concentrations were detected in home-grilled pork samples. Using of disposable grilling unit resulted in 1.6 times higher PAH concentrations compared to the traditional wood-burning grill. The average intake of benzo[a]pyrene and sum of 12 PAHs from meat products was estimated for children (age 1-16 years) on the basis of an individual food consumption questionnaire and, for the general population, based on national food consumption data. The highest total PAH concentrations detected were 16 µg kg^-1 in smoked meat and ham, 19 µg kg^-1 in smoked sausage and 6.5 µg kg^-1 in smoked chicken samples. Since smoking and grilling are prevalent meat-cooking methods in Estonia, the impact of meat products is assessed to be significant in overall PAH intake.     

Benzo[a]pyrene and total polycyclic aromatic hydrocarbons (PAHs) levels in vegetable oils and fats do not reflect the occurrence of the eight genotoxic PAHs

Concentrations of polycyclic aromatic hydrocarbons (PAHs) were determined in 115 samples of olive oil (extra virgin olive oil, virgin olive oil, olive oil, pomace olive oil and blended olive oil), cooking oil (corn oil, sunflower oil, sesame oil, palm olein oil, soya oil, canola oil, mustard oil, peanut oil and mixed vegetable oil) and fat (butter and table margarine) collected from retail stores in Kuwait. Carcinogenic benzo[a]pyrene (BaP) was detected in 43% of the samples analyzed. Benz[a]anthracene and chrysene were detected in 37 and 45% of the samples, respectively, that did not contain BaP. Of the individual non-carcinogenic PAHs, naphthalene showed the highest mean concentration (14 µg kg^?1), while for the carcinogenic PAHs, BaP (0.92 µg kg^?1) and chrysene (0.87 µg kg^?1) showed the highest mean values. Approximately 20% of the samples within the olive oil and cooking oil sub-categories exceeded the EU maximum tolerable limit for BaP, with the highest level of 6.77 and 11.1 µg kg^?1, respectively. For the fat sub-category, 9% of the samples exceeded the tolerance limit, with the highest level of 3.67 µg kg^?1. The Kuwaiti general population's dietary exposure to the genotoxic PAHs (PAH8: benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, dibenz[a,h]anthracene and benzo[ghi]perylene) was estimated to be 196 ng day^?1 (3.3 ng kg^?1 bw day^?1, assuming an average adult body weight of 60 kg). Results indicated that PAH8 and BaP_eq (total sum benzo[a]pyrene equivalents) are more reliable measures of the concentrations of other carcinogenic PAHs in oil and fat samples, while BaP and PAHs alone are not good indicators of the occurrence or degree of contamination by carcinogenic PAHs in these food products.     

HPLC-fluorescence analysis of polycyclic aromatic hydrocarbons (PAHs) in pork meat and its gravy fried without additives and in the presence of onion and garlic

The effect of onion and garlic on the formation of polycyclic aromatic hydrocarbons (PAHs) was evaluated by comparing the concentrations of PAHs in meat and gravy samples coming from pork dishes prepared in the presence and absence of these spices. PAHs were analysed by high performance liquid chromatography with fluorescence detection. The clean-up procedure included alkaline hydrolysis, solid-phase extraction (on columns with diatomaceous earth and propyl sulphonic acid) and column chromatography on silica gel. Total concentrations of 6 PAHs (benzo[b]fluoranthne, benzo[k]fluoranthne, benzo[a]anthracene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene) were (in ng g⁻¹ of cooked meat) from 2.0 to 7.2 in meat samples and from 0.05 to 0.6 in gravies. Concentration of B[a]P was from 0.38 to 1.61 in meat and from 0.01 to 0.11 in gravy samples. Onion (30/100 g of meat) caused on average decrease of 60% of the total content of PAHs in pan fried meat and of over 90% in gravies. Garlic (15/100 g of meat) lowered the concentration of 54% in meat on average and from 13.5–79% in gravies.     

Animal Welfare Labelling and the Approach of the European Union: An Overview on the Current Situation

There is a growing appreciation of the insistence of consumers that animals used in food production should be well treated. High welfare standards could have both a direct and indirect impact on food safety and quality; regulatory and support systems in agriculture must adapt accordingly. Retailers and producers are increasingly recognising animal welfare as a fundamental aspect of product image and quality which create a need for reliable systems for on farm monitoring of animal welfare status and providing some warranty on appropriate production conditions. The article shows an overview of the European strategies for implementing the animal welfare labelling. Received: July 2, 2008; accepted: July 8, 2008     

Polycyclic aromatic hydrocarbons in cereal products on the Turkish market

The contamination level of four EU marker polycyclic aromatic hydrocarbons (PAHs) in some cereal-derived products was surveyed in this study. Thirty-eight samples, 20 bread and 18 breakfast cereals, were purchased from retail shops and local markets of East Black sea region in Turkey. The samples were analysed for four EU marker PAHs, using ultrasonic extraction, solid-phase extraction (SPE) clean up and stable-isotope dilution gas chromatography with mass-spectrometric (GC/MS) detection. The method was validated with the parameters linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ) and uncertainty. Total content of the four PAHs in bread varied from 0.19 to 0.46 µg kg^?1 and in breakfast cereals from 0.10 to 0.87 µg kg^?1.     

Characterisation of white tea – Comparison to green and black tea

An overview is given on the manufacture of the different types of tea along with the most important phenolics present in tea and methods of analysis. Compositional data are presented for green, white and black teas. A differentiation of green and black tea by using the ratio between total phenolics and sum of the major catechins seems to be feasible. For white tea there is no general accepted definition. Possible approaches are geographic origin, the botanical variety and the manufacture or the appearance. The differentiation between green and white teas by the ratio mentioned above is not possible. An erratum to this article is available at .     

Microbiological Attributes of Turkish Butters Sold Under Market Conditions

In this study, microbiological quality of 45 butter samples sold under market conditions at Manisa (Turkey) was investigated. Total coliform, total fecal coliform, Escherichia coli and yeast and mould counts were found between < 1.0 – > 3.15 log₁₀ cfu.g^-1, < 1.0 – > 3.15 log₁₀ cfu.g^-1, < 1.0 – > 3.15 log₁₀ cfu.g^-1 and < 1.0 – > 6.62 log₁₀ cfu.g^-1 respectively. Only in one sample Salmonella was detected. Staphylococcus aureus was not detected in any of the samples. To that extent butters sold under market conditions in Manisa have high coliform, yeast and mould contamination. Received: April 29, 2008; received in revised form: May 28, 2008; accepted: June 3, 2008     

Untersuchungen zur aktuellen Belastung von Tiermehlen mit Tetracyclinen

Zusammenfassung:  Fleischmehle aus Herstellungsbetrieben in Deutschland, in denen die Rohstoffe entsprechend den spezifischen Anforderungen der Verordnung (EG) 1774/2002, Artikel 19 („Inverkehrbringen und Ausfuhr von verarbeitetem tierischem Eiwei? und anderen verarbeiteten Erzeugnissen, die als Futtermittel-Ausgangserzeugnisse verwendet werden k?nnen“) verarbeitet wurden, wurden im Rahmen dieser Studie einer qualitativen und quantitativen Untersuchung auf Tetracycline mittels LC-ESI-MS-MS unterzogen. Insgesamt wurden 108 Proben untersucht, davon 18 Knochenmehle, 16 Griebenmehle, 15 Geflügelmehle, 10 Geflügelfleischmehle und 49 Fleischknochenmehle. In den untersuchten Proben konnten Tetracycline nachgewiesen werden. Im Einzelnen wurden Oxytetracyclin sowie Tetracyclin in allen 108 Proben und Chlortetracyclin in 91 von 108 untersuchten Proben nachgewiesen. Die maximalen Gesamt-Tetracyclin-Gehalte betrugen 828,1 μg/kg in Knochenmehlen, 317,1 μg/kg in Griebenmehlen, 606,9 μg/kg in Geflügelmehlen, 267,1 μg/kg in Geflügelfleischmehlen und 1524,7 μg/kg in Fleischknochenmehlen. Die Ergebnisse deuten darauf hin, dass die rechtlich vorgeschriebene Erhitzung in Verarbeitungsanlagen nicht ausreichend ist, um einen Abbau von Tetracyclin-Rückst?nden sicherzustellen. Es muss angenommen werden, dass es nicht m?glich sein wird, Tetracyclin-freie Tiermehle herzustellen. Die m?glichen Folgen dieser Kontamination mit Tetracyclinen werden diskutiert.

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In this study, meat meals produced in Germany according to article 19 of regulation EC 1774/2002 (“Placing on the market and export of processed animal protein and other processed products that could be used as feed material”) were analysed qualitatively and quantitatively for tetracyclines using LC-ESI-MS-MS. A total of 108 samples was analysed: 18 bone meals, 16 greave meals, 15 poultry meals, 10 poultry meat meals and 49 meat and bone meal samples. Tetracycline derivatives were found in all analysed samples. Oxytetracycline and tetracycline were found in 108 samples, chlortetracycline was detected in 91 of 108 samples. The highest total tetracycline content was 828.1 μg/kg in bone meals, 317.1 μg/kg in greave meals, 606.9 μg/kg in poultry meals, 267.1 μg/kg in poultry meat meals and 1524.7 μg/kg in meat and bone meals, respectively. The results of this study showed that the compulsory heating step actually used in processing plants does not result in a degradation of tetracyclines. From these results it does not seem possible to produce tetracycline free animal meals from contaminated raw materials. Further, the possible implications of the findings are discussed.

Eingegangen: 14. M?rz 2008     

Quantitative Determination of Isoflavones in Soy Based Nutritional Supplements by High-Performance Liquid Chromatography

An increasing number of nutritional supplements contain phytoestrogens, in particular isoflavones, which potentially alleviate climacteric complaints. Intention of the present study was the qualitative and quantitative determination of isoflavones in soy based nutritional supplements in order to compare the actual content with the labeling of these products. For the analysis high-performance liquid chromatography (HPLC) with photo diode array (PDA) detection was used. The amount of isoflavones was determined via external calibration functions. Our analyses revealed certain variations in the isoflavone content of soy based nutritional supplements, with regard to the individual and the total amount. Furthermore, the total amount of isoflavones per serving unit in some products was higher than declared on the labeling, although only the major isoflavones were taken into account. This study shows that there is the necessity to clearly specify the isoflavone composition on the labeling and - due to safety aspects - to standardize and control the isoflavone content.

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Zusammenfassung:  Nahrungserg?nzungsmittel mit Zusatz von Soja werden vermehrt als “Alternative” zur klassischen Hormonersatztherapie für Frauen im Klimakterium angeboten. Diese Pr?parate enthalten Isoflavone, welche zu den Phytoestrogenen z?hlen und eine estrogene Wirkung aufweisen. Die in Sojabohnen enthaltenen Isoflavone sollen unter anderem klimakterische Beschwerden wie z.B. Hitzewallungen oder Tachykardie lindern. In der vorliegenden Studie wird eine Methode beschrieben, mit der aus Nahrungserg?nzungsmitteln mit Zusatz von Soja-Extrakten der Gehalt an Isoflavonen bestimmt werden kann. Dazu wurde eine HPLC-Methode mitDioden-Array-Detektion entwickelt, mit welcher die Quantifizierung der in Soja vorliegenden Isoflavone über externe Kalibrierfunktionen erfolgen kann. Damit war es m?glich, die vom Hersteller gemachten Angaben bezüglich der Isoflavongehalte zu überprüfen. Unsere Untersuchungen wiesen deutliche Unterschiede sowohl im Gesamtgehalt an Isoflavonen als auch in der individuellen Zusammensetzung der einzelnen Produkte auf. In einigen F?llen war der Isoflavongehalt in einigen Produkten pro Verzehrseinheit deutlich h?her als vom Hersteller angegeben. Unsere Untersuchungen belegen die Notwendigkeit im Sinne des Verbraucherschutzes, die Isoflavongehalte in Nahrungserg?nzungsmitteln klar zu spezifizieren und zu deklarieren.

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Received: January 9. 2008; accepted: February 19. 2008     

Polycyclic aromatic hydrocarbons (PAHs) in yerba mate (Ilex paraguariensis) from the Argentinean market

Polycyclic aromatic hydrocarbons (PAHs) occurrence in 50 samples marketed in the main supermarkets from Argentina was surveyed. A high performance liquid chromatography (HPLC) method was applied with fluorescence detection (FLD) and UV—VIS diodes array detector (DAD) for the analysis of 16 PAHs in “yerba mate” (Ilex paraguariensis), with recoveries higher than 89% and limits of detection and quantification lower than that found by other methodologies in previous studies. Contamination expressed as the sum of 16 analysed PAHs ranged between 224.6 and 4449.5 μg kg^?1 on dry mass. The contamination expressed as PAH4 (sum of benzo(a)pyrene, chrysene, benzo(a)anthracene and benzo(b)fluoranthene) varied between 8.3 and 512.4 μg kg^?1. The correlation coefficient for PAH2 (sum of benzo(a)pyrene and chrysene) and PAH4 groups was 0.99, for PAH2 and PAH8 (sum of benzo(a)pyrene, chrysene, benzo(a)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(g,h,i)perylene, dibenzo(a,h)anthracene and indeno(1,2,3cd) pyrene) 0.97 and for PAH4 and PAH8 0.98.     

Monitoring of polycyclic aromatic hydrocarbons (PAH) in food supplements containing botanicals and other ingredients on the Dutch market

Food supplements can contain polycyclic aromatic hydrocarbons (PAH). The European Food Safety Authority (EFSA) has defined 16 priority PAH that are both genotoxic and carcinogenic and identified eight priority PAH (PAH8) or four of these (PAH4) as good indicators of the toxicity and occurrence of PAH in food. The current study aimed to determine benzo[a]pyrene and other EFSA priority PAH in different categories of food supplements containing botanicals and other ingredients. From 2003 to 2008, benzo[a]pyrene exceeded the limit of quantification (LOQ) in 553 (44%) of 1258 supplements with a lower-bound mean of 3.37?µg?kg^?1. In 2008 and 2009, benzo[a]pyrene and 12 other EFSA priority PAH were determined in 333 food supplements. Benzo[a]pyrene exceeded the LOQ in 210 (63%) food supplements with a lower-bound mean of 5.26?µg?kg^?1. Lower-bound mean levels for PAH4 and PAH8(-indeno[1,2,3-cd]pyrene) were 33.5 and 40.5?µg?kg^?1, respectively. Supplements containing resveratrol, Ginkgo biloba, St. John's wort and propolis showed relatively high PAH4 levels in 2008 and 2009. Before 2008, supplements with these ingredients and also dong quai, green tea or valerian contained relatively high benzo[a]pyrene levels. On average, PAH4 intake resulting from food supplement use will be at the lower end of the range of contributions of main food groups to PAH4 exposure, although individual food supplements can contribute significantly to PAH4 exposure. Regular control of EFSA indicator PAH levels in food supplements may prove a way forward to reduce further the intake of PAH from food.     

Pharmakodynamische und pharmakokinetische Grundlagen der Mastitistherapie

Zusammenfassung:  Bei der Behandlung von Mastitiden steht die Anwendung von lokal oder systemisch verabreichten Antibiotika im Vordergrund. Bei der systemischen Anwendung sind der Bioverfügbarkeit und der Verteilung des Stoffes in die Milchdrüse besonderes Augenmerk zu schenken. Neben der Molekülgrü?e und dem Ausma? der Proteinbindung sind dabei die Lipophilie und der Ionisationsgrad des Arzneistoffs bestimmend. Schwache S?uren, wie beispielsweise β-Lactam-Antibiotika, liegen im Blutplasma überwiegend ionisiert vor; sie k?nnen die Blut-Milch-Schranke daher im Gegensatz zu schwachen Basen nur in ?u?erst geringem Umfang passieren. Bei der intramamm?ren Applikation muss gew?hrleistet werden, dass sich das Antibiotikum in ausreichendem Umfang im Drüsengewebe verteilt. Die Pr?parate sind in den meisten F?llen wie auch Formulierungen mit Langzeitwirkung beim Trockenstellen ?lige Suspensionen.

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The therapy of bovine mastitis usually relies on antibiotics, administered either systemically or by the intramammary route. A key factor in the success of systemic treatment is the bioavailability of the chemotherapeutic agent and the distribution into the mammary gland. Apart from molecular size and protein binding capacity, the lipid solubility of the active principle and its degree of ionisation play a major role. Weak acids (like β-lactams) are present in the ionised form in the blood and their ability to pass through the blood/udder barrier is very limited. The opposite is true for weak bases. In the case of intramammary administration the main consideration is that the substance is distributed sufficiently into the glandular tissue. This is why drugs for topical administration are usually formulated in an oily base, which is also suitable for long-term prophylaxis at drying off.

Eingegangen: 6. M?rz 2008; angenommen: 17. M?rz 2008     

Polycyclic aromatic hydrocarbons (PAHs) levels in propolis and propolis-based dietary supplements from the Italian market

Propolis and propolis-based extracts, attained from beekeepers and the local market, were analysed for the presence of 13 polycyclic aromatic hydrocarbons (PAHs), including eight high molecular weight PAHs (PAH8), recently indicated by the European Food Safety Authority (EFSA) as suitable indicators of the presence of carcinogenic and genotoxic PAH in foods. An analytical procedure based on microwave assisted saponification/extraction, high performance liquid chromatography (HPLC) and spectrofluorometric detection, has been developed. About half of the samples analysed presented benzo[a]pyrene (BaP) concentrations exceeding 2 μg/kg, which is proposed as a regulatory limit for dietary supplements. A product-by-product approach (based on maximum recommended dosage) was used to calculate PAH exposure. Even thought the majority of the samples gave low exposure levels when compared to exposure levels from other diet constituents, PAH intakes deriving from a daily consumption of some of the investigated products provided an important contribution to the total dietary intake and lead to margin of exposure (MOE) values which are of concern for human health.     

Green Tea Polyphenolic Compounds and Human Health

Polyphenols are a structurally diverse group of compounds that occur widely throughout the plant kingdom. Polyphenolic compounds are ubiquitous in all plant organs and are, therefore, an integral part of the human diet. Recent interest in food phenolics has increased greatly because of the antioxidant and free radical-scavenging abilities associated with some phenolics and their potential effects on human health. Most of the polyphenols in green tea are commonly known as catechins. The regular consumption of green tea is related to benefits in some diseases as atherosclerosis and cancer. Although consumption of dietary polyphenols such as flavonoids has been suggested to have beneficial biological effects, there are considerable evidences to suggest that such compounds are not without risk of adverse effects. Received: July 23, 2007; accepted August 8, 2007     

Cross border Classical Swine Fever control: Improving Dutch and German crisis management systems by an integrated public-private approach

The objective of this research approach is to analyse in which ways crisis management measures against Classical Swine Fever (CSF) can be improved by a public private cross border model. A core activity contains the analysis of information and communication systems: In a case study it has been empirically analysed if a sufficient supply of public and private information enables crisis managers at both sides of the Dutch-German border area to take decisions about CSF control more efficient. At the end of this approach a new crisis management model had been developed. One of the most important aspects thereby is the assessment of data: (1) within private quality management systems in normal times according to the benefit for public management tasks in times of crisis and (2) within public crisis management systems according to the benefit for cross-border CSF-control activities. To this effect two different methodological approaches have been combined within the model: (1) a method to identify and illustrate public actors and their options in crisis management decision making and (2) a system of communication and information exchange between public and private as well as Dutch and German actors (engage& exchange model) which permit to collect and to evaluate data in addition for a predefined time period are activated. Received: September 30, 2008; accepted: October 1, 2008     

Die wirtschaftliche Bedeutung von Kaffee, Tee und Kakao – eine Übersicht für Deutschland

Zusammenfassung:  Um Auskunft über die wirtschaftliche Bedeutung von Kaffee, Tee und Kakao zu geben, werden in diesem Mini-Review u.a. aktuelle Angaben zur weltweiten Produktion dieser drei „Exoten“ gemacht (Menge und Herkunftsl?nder), die jeweiligen Import-Anteile der Produktionsl?nder nach Deutschland (bzw. in die EU) benannt, der Pro-Kopf-Verbrauch an Kaffee, Tee oder Kakao in Deutschland mit dem anderer Staaten verglichen und – sofern m?glich – Einblick gegeben in die Entwicklung von weltweitem Angebot und Nachfrage und die damit verbundene Preisentwicklung in den vergangenen Jahren. Eingegangen: 2. Juli 2007     

Bewertung von Grund- und Trinkwassermetaboliten von Pflanzenschutzmittelwirkstoffen in verschiedenen regulatorischen Verfahren

Ohne Zusammenfassung

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Eingegangen: 21. September 2007     

Acute View Transport biofuels: Can they help limiting climate change without an upward impact on food prices?

Comprehensive life cycle assessments show that current transport biofuels often do worse than conventional fossil transport fuels as to the emission of greenhouse gases. Biofuels from microalgae grown with present technology and lignocellulosic biofuels from current arable land or land that is to be deforested are unlikely to do better regarding the emission of greenhouse gases than fossil transport fuels. When crops characterized by relatively low fossil fuel inputs and relatively high biomass yields are grown on abandoned agricultural and marginal soils which currently sequester little carbon, cropping for transport biofuels may help in limiting climate change without an impact on food prices. For such cropping one probably has to go beyond the market mechanism. Worldwide, there is some scope for the use of harvest residues in biofuel production. However, European arable soils show on average large losses of soil carbon and this rather favors increased addition of such residues to soils. Received: November 17, 2008; accepted: December 3, 2008