Monitoring of Bt-Maize pollen exposure in the vicinity of the nature reserve Ruhlsdorfer Bruch in northeast Germany 2007 to 2008

Background, aim, and scope Basically, technological innovations are associated with benefits and risks. This is also true for the introduction of genetically modified organisms (GMO) into agriculture. In Germany, precautionary regulations currently demand isolation distances (i.?e. buffer zones) for the cultivation of genetically modified maize (Bt-maize) in the vicinity of conventional (150?m) and organic maize fields (300?m). The Bt-toxin may harm non-target organisms (NTO) such as Lepidoptera. Despite this, corresponding regulations for the protection of nature reserves are lacking to date. Conventional and Bt-maize have been grown in the vicinity of the Flora-Fauna-Habitat (FFH) Ruhlsdorfer Bruch in Brandenburg, Germany. The aim of this study was to investigate whether exposure of maize pollen from surrounding Bt-maize fields to NTOs in the nature protection area could be excluded or not. Two types of exposure were investigated: Firstly, whether maize pollen was dispersed by wind into the nature reserve area exposing resident NTOs. Secondly, foraging NTOs from the nature reserve are exposed by roaming the surrounding fields and collecting maize pollen. In order to fulfil the precautionary principle defined by law, the study should help to determine appropriate isolation distances for the cultivation of Bt-maize with regard to sustainable protection of NTOs in the FFH Ruhlsdorfer Bruch. In 2007, the local authorities issued an isolation distance between Bt-maize fields and nature reserves of 100?m and in 2008, this became 250?m in the northern and 500?m in the westerly direction, respectively. Materials and methods Standardised methods for biological and technical pollen sampling issued by the Association of German Engineers (VDI 4330 Part 3, 2007 and VDI 4330 Part 4, 2006) were applied providing a quality controlled and methodologically harmonised database which does not only serve the needs to be fulfilled by the present case-specific monitoring study but can also be used as a reference database for further investigations. Maize pollen exposure was measured within the FFH Ruhlsdorfer Bruch and its immediate vicinity in July and August 2007 and 2008. In 2007, the sampling was performed at three sites using 12 technical samplers (Sigma-2/PMF) placed at five measuring points at distances from 5?m to 120?m from the maize field edges. Additionally, for biological pollen sampling six bee colonies were situated at these three sites (two colonies at each site). The technical sampler Sigma-2/PMF enables point sampling which is primarily influenced by wind and topography providing information on effective maize pollen input (flow and deposition) at the measuring sites. Honey-bees roaming in the surrounding area with typical foraging distances of several kilometres may act as planar collectors. They may serve as indicators for the exposure to pollen-collecting NTOs. Furthermore, biological sampling is more selective due to the organism’s preferences, whereas the technical sampling is neutral. Hence, both the technical and the biological sampling complement each other in their scope of application. The pollen samples of both matrices were analysed microscopically and the maize pollen loads were quantified. Pollen-DNA was analysed by means of the quantitative PCR-method (qPCR) identifying conventional and Bt-maize pollen by two independent laboratories. In 2008, the monitoring was repeated with additional sites. Eighteen technical samplers were exposed at five sites with eight measuring points at distances from 5?m to 250?m from the maize field edges. Two honey-bee colonies for biological sampling were placed at one site for control purposes. PCR-analyses were performed to measure the amounts of Bt-maize pollen in the samples. Results The results of pollen monitoring at the Ruhlsdorfer Bruch revealed maize pollen exposure for all monitoring sites in both surveys. In 2007, up to 1.75 million maize pollen/m² were deposited at sites closest to the maize field. At 120?m from field edge in the middle of the FFH-reserve, 99,000 maize pollen/m² were detected. In 2008, similar results were found, at distances up to 250?m from the field edges deposition of 164,000 maize pollen/m² was detected. Data on maize pollen deposition show a clear distance relationship and are in accordance with results of further comprehensive surveys based on the same methodology (Hofmann 2007). The results of the microscopic analysis of the pollen pellets demonstrated that bees collected maize pollen at all sites, in 2007 and 2008. Although maize pollen is not the main food source (2007:0.1–0.3?%; 2008:2–3?%) the collection efficiency of the bee colonies resulted in high amounts of sampled maize pollen with 4 to 11 million per site in 2007 and up to 467 million in 2008. Molecular-biological analysis of maize pollen DNA by qPCR demonstrated that transgenic Bt-MON810 DNA was present in all technical and biological samples, corroborated by two independent laboratories. In 2007, the GMO-content in the samples ranged up to 44?% in the bioaerosols and 49?% in the pollen pellets. In 2008, GMO-proportions of up to 18?% were detected. Discussion The results of this study provide evidence that NTOs in the Ruhlsdorfer Bruch were exposed to Bt-maize pollen under the cultivation conditions in 2007 with a buffer zone of 100?m. The GMO-content reached up to 48?%. The results of the monitoring in 2008 confirmed these findings. Even though the exposure could be reduced by increasing the isolation distances to 250?m and 500?m respectively, the results still show percentages of up to 18?% Bt-MON810 in the pollen samples. The results on maize pollen deposition at the Ruhlsdorfer Bruch in 2007 and 2008 correspond to the results of an investigation which was conducted over several years applying the same standardised method, but covering a wider range of distances. The correlation between maize pollen deposition (n/m²) and distance to the source field (m) fitted best to a power function of the type y = 1.2086 · 10⁶ · x ^–0.548. Despite the same trend, the pollen deposition in the Ruhlsdorfer Bruch revealed above-average findings. Also the analysis of the pollen pellets collected by the bees showed an exposure in 2007 with values for the GMO-content of up to 49?%. For both methods, the exposure decreased in 2008 due to the greater buffer zones up to 500?m. Whereas the GMO-content for the biological sampling were reduced to values below 10?%, the values for the technical sampling were still higher indicating that greater buffer zones would be necessary for safety reasons under the precautionary principle. Conclusions The results of this investigation proved that maize pollen were dispersed by wind to distances farther than 250?m from field edge leading to maize pollen exposure in the centre of the nature reserve. The results also demonstrated that foraging NTOs living in the nature reserve were exposed to maize pollen from surrounding fields. Considering the cultivation of Bt-maize MON810, the assumption of the Environmental Risk Assessment (ERA) that there will be no relevant exposure beyond the Bt-maize fields, cannot be confirmed. Considering the results of this and related studies and with respect to the precautionary principle, one can state that buffer zones between Bt-maize fields and protected areas are an effective measure to minimise the exposure of Bt-maize pollen to NTOs and, thus, to prevent from adverse effects. Recommendations and perspectives Because of still insufficient ecotoxicological data for the risk assessment of Bt-maize MON810 considering butterflies and other protected NTOs, protection standards assuring the precautionary principle have to be implemented to avoid Bt-maize pollen exposure to NTOs. This applies for the case Ruhlsdorfer Bruch and for nature reserve areas in general. In order to exclude risks to protected NTOs occurring in nature reserves, sufficient buffer zones for Bt-maize cultivation should be considered. The statistical analysis revealed that distances of more than 500?m are necessary to decisively reduce exposure to foraging insects. In fact, distances of more than 1,000?m are necessary to prevent maize pollen deposition from values above 100,000 pollen/m² with a certainty of 90?%. An adequate risk assessment can only be attained if based on field measurements accounting for the high variation of pollen deposition due to local environmental site conditions and field management. The monitoring should be based on standardised methods. It should include locations with the highest expected deposition rates, the boundaries of the protected areas and sites of interest within those boundaries, e.?g., specific habitats of sensitive species.     

Klima-Biomonitoring: Nachweis des Klimawandels und dessen Folgen für die belebte Umwelt

Hintergrund und ZielEs wird vorgeschlagen, Biomonitoring-Verfahren zu verwenden, um Auswirkungen des Klimawandels auf die belebte Umwelt zu erkennen, zu bewerten und zu dokumentieren, weil ? es mit dieser Methode gelingt, klimainduzierte Veränderungen in besonders empfindlichen Gebieten in Deutschland mit ihren Lebensräumen, Lebensgemeinschaften und Arten darzustellen, ? es bezüglich Zuwanderung und Ausbreitung neuer Schädlinge und Krankheitserreger für Mensch, Tier und Pflanze nach bzw. in Deutschland relevante Informationen liefern kann, ? damit der Politik zur Bewertung der Auswirkungen des Klimawandels wichtige Informationen, Handreichungen und Entscheidungsgrundlagen zur Verfügung gestellt werden können und ? auf dieser Grundlage geeignete Anpassungsmaßnahmen eingeleitet und auf ihre Wirksamkeit geprüft werden können, wie beispielsweise in der Deutschen Anpassungsstrategie (BMU 2009) beschrieben. Für Biomonitoring-Verfahren, die geeignet sind, Auswirkungen des Klimawandels anzuzeigen, wird der Begriff Klima-Biomonitoring vorgeschlagen Diese Verfahren sollten aus verschiedenen Gründen (u.?a. abgestimmte Methodik und gleiche Datenbasis, Kostenersparnis) unter Beteiligung aller Bundesländer umgesetzt werden. Bioindikation ist bereits heute ein unverzichtbares Verfahren, frühzeitig Veränderungen in der belebten Umwelt zu erkennen und somit Hinweise auf besondere Gefahren zu liefern (Frühwarnsystem). Für das Klima-Biomonitoring werden vorzugsweise bestehende Monitoringsysteme mit ihren bereits erhobenen Daten ausgewertet und mitgenutzt. Material und Methoden Das Klima-Biomonitoring greift auf bereits eingeführte und bewährte Methoden der Bioindikation von Umweltveränderungen zurück. Bestehende Methoden werden im Hinblick auf die besonderen, durch den Klimawandel bedingten Anforderungen ergänzt, angepasst und weiterentwickelt. Auf der Grundlage einer Auswertung relevanter laufender Bundes- und Länderprogramme werden wesentliche Wirkungen des Klimawandels identifiziert, die datenliefernden Programme zugeordnet und Auswertungen vorgeschlagen. Zusätzlich werden Datenquellen beschrieben, deren Nutzung weitergehende Betrachtungen ermöglichen. ErgebnisseIn einer Übersichtstabelle werden Monitoring-Programme auf Bundes- und Länderebene systematisiert und hinsichtlich ihrer Bedeutung für die Bewertung von Klimafolgen auf die belebte Umwelt dargestellt. Eine Übersicht über Datenquellen sowie eine Darstellung erster Erkenntnisse aus der Auswertung vorliegender Daten belegen die Relevanz des vorgeschlagenen Verfahrens. Der erkennbare Forschungs- und Entwicklungsbedarf wird umrissen und Vorschläge für eine Optimierung des Daten- und Methodenaustausches benannt. Beispiele zur Anwendung und Vertiefung der Thematik sowie Anregungen zur Weiterentwicklung der Methodik und zum Schließen von Kenntnislücken werden in Folgepublikationen aufgezeigt. DiskussionAuf der Basis des Klima-Biomonitorings kann das Ausmaß der durch den Klimawandel bereits eingetretenen Veränderungen beschrieben sowie Szenarien und Prognosen zu den Auswirkungen von Klimaveränderungen erstellt werden. Weiterhin ist es möglich, die sekundären Wirkungen des Klimawandels, insbesondere die Auswirkungen der Maßnahmen zur Anpassung an den Wandel, mithilfe der Bioindikation auf ihre Wirksamkeit zu prüfen. Für einige Klima-Bioindikatoren ist der kausale Zusammenhang zwischen Klimawandel und Reaktion bereits belegt (zum Beispiel die Frühjahrsphasen in der Pflanzenphänologie), in anderen Fällen müssen noch Methoden zur Unterscheidung zwischen Klimawirkungen und anderen Wirkfaktoren entwickelt werden. Die Erkenntnisse aus dem Klima-Biomonitoring sollen Grundlage für entsprechende Handlungen sein, sodass geeignete Anpassungsstrategien und gleichzeitig Maßnahmen zur Vermeidung oder zur Verminderung der Effekte eingeleitet werden können. Zugleich sollen eine angemessene Politikberatung, eine Information der Öffentlichkeit und die Erfüllung entsprechender Berichtspflichten erfolgen. Schlussfolgerungen:Klima-Biomonitoring ist eine geeignete Methode, um klimainduzierte Veränderungen in besonders empfindlichen Gebieten in Deutschland mit ihren Lebensräumen, Lebensgemeinschaften und Arten darzustellen. Erste Auswertungen zeigen, dass bereits Wirkungen des Klimawandels auf die belebte Umwelt nachweisbar sind. Hierbei liefert die Pflanzenphänologie seit vielen Jahren wertvolle Hintergrunddaten. Eine Verschneidung dieser Hintergrunddaten mit Daten aus anderen geeigneten Monitoring-Programmen und weiteren Informationen (zum Beispiel Geobasisdaten) ermöglicht es, diese Erkenntnisse zu ergänzen. Damit ist die Ermittlung und Bewertung von klimainduzierten Wirkungen auch im Bereich der Ausbreitung von Krankheitsüberträgern und -erregern und anderer Phänomene sowie der Bewertung von Maßnahmen möglich. Empfehlungen und PerspektivenEine abgestimmte Anwendung der Methodik in allen Bundesländern und beim Bund, eine Verbesserung des Daten- und Methodenaustausches, die Identifikation, Erschließung und Nutzung weiterer Datenquellen, die Weiterentwicklung der Methodik und eine Publikation weiterer Erkenntnisse werden empfohlen. Es ist davon auszugehen, dass sich das Klima-Biomonitoring wegen seiner Vorteile als Methode zur Erhebung von Klimafolgen für die belebte Umwelt auch international etablieren wird.     

Turner syndrome-omphalocele association: Incidence,karyotype, phenotype and fetal outcome

Objective

Omphalocele is known to be associated with genetic anomalies like trisomy 13, 18 and Beckwith–Wiedemann syndrome, but not with Turner syndrome (TS). Our aim was to assess the incidence of omphalocele in fetuses with TS, the phenotype of this association with other anomalies, their karyotype, and the fetal outcomes.

Method

Retrospective multicenter study of fetuses with confirmed diagnosis of TS. Data were extracted from a detailed questionnaire sent to specialists in prenatal ultrasound.

Results

680 fetuses with TS were included in this analysis. Incidence of small omphalocele in fetuses diagnosed ≥12 weeks was 3.1%. Including fetuses diagnosed before 12 weeks, it was 5.1%. 97.1% (34/35) of the affected fetuses had one or more associated anomalies including increased nuchal translucency (≥3 mm) and/or cystic hygroma (94.3%), hydrops/skin edema (71.1%), and cardiac anomalies (40%). The karyotype was 45,X in all fetuses. Fetal outcomes were poor with only 1 fetus born alive.

Conclusion

TS with 45,X karyotype but not with X chromosome variants is associated with small omphalocele. Most of these fetuses have associated anomalies and a poor prognosis. Our data suggest an association of TS with omphalocele, which is evident from the first trimester.     

Großräumige Regionalisierung der Kohlenstoffbindung in Wäldern Nordrhein-Westfalens

Background, aim and scope The United Nations Framework Convention on Climate Change understands carbon fixation in forests as an important contribution for the reduction of atmospheric pollution in terms of greenhouse gases. According to the German forest inventory on carbon in biomass an amount of 191?t C/ha was roughly estimated, without any spatial differentiation. Therefore, the aim of this investigation was to statistically identify factors that are significant for the carbon fixation and to map the spatial patterns of C sequestration in the federal state North Rhine-Westphalia. Materials and methods Together with information on climate, elevation, vegetation, and deposition, data from two forest monitoring networks were analysed statistically. Geostatistics and the decision tree algorithms Classification and Regression Trees (CART) and Chi-Square Automatic Interaction Detection (CHAID) were applied to calculate surface maps from punctual data on C in vegetation, in dead wood and in soil. Whereas spatial autocorrelation could be detected for the C loads in the humus layers, no surface maps could be calculated for the C contents of the mineral soils and for the forest trees/dead wood. Here, CART and CHAID were used to derive decision trees that were applied on available surface data to predict C loads for the entire study area. Results About 19?t C/ha could be predicted for the humus layer, 67?t C/ha for forest trees/dead wood and 90.7?t C/ha for the soil. An overall mean of 177?t C/ha was calculated for North Rhine-Westphalia lying 14?t C/ha below the German wide mean. Discussion Compared to the calculated results in another investigation a total of 68?mio. t C for the above ground dendromass was estimated. This is 11?mio. t C/ha higher than the amount calculated in this study and may be due to the fact that this value includes the C-pools in both, the brushwood and herbaceous layer in their estimations. The average C concentration in the humus layer all over Germany was found to amount for 20.7?t C/ha which is slightly above the C storage calculated for North Rhine-Westphalia. In the same study a Germany wide C average of 87.9?t C/ha was calculated which is very close to the 90.7?t C/ha calculated in this study. Conclusions The surface estimations of the C-pools in the above-ground biomass, the humus layer and the mineral soil enable to map the efficiency of the C-bounding capacity regarding the fixation of the greenhouse gas CO₂. The mean values derived in this study are in good accordance with estimations based on other techniques. Recommendations and perspectives The approach presented should be verified by application to Germany wide inventory data and by means of Regression Kriging. Furthermore, the C-fixation under climate change should be calculated by combining statistical methods and the dynamic modelling tool WASMOD.     

Effect of herbicides on glutathione S-transferases in the earthworm, <Emphasis Type="Italic">Eisenia fetida</Emphasis>

AIM AND BACKGROUND: Earthworms have been studied as a readily available, easily maintainable and cheap test species for assessing chemical pollution, and may be an alternative to in vivo rodent bioassays. The current investigation aims to characterize detoxification enzymes in Eisenia fetida and stress response against two herbicides with different modes of action, namely, fenoxaprop and metolachlor. METHODS: Herbicides were applied to soil containing earthworms. Animals were then collected, sacrificed and shock-frozen. Extracted protein was analyzed for glutathione S-transferase (GST) activity using CDNB (1-chloro-2,4-dinitrobenzene), DCNB (1,2-dichloro-4-nitrobenzene), pNBC (p-nitrobenzylchloride), PNOBC (p-nitrobenz-o-ylchloride) and selected herbicides. GST isoenzymes were partially purified by affinity chromatography and molecular weights were estimated by SDS-PAGE. RESULTS: In E. fetida protein extracts, GST activity towards model compounds ranked as CDNB>DCNB>PNBOC>PNBC. Fluorodifen was not conjugated at all, but fenoxaprop and metolachlor were conjugated at low rates. Furthermore, the GST isoenzyme pattern changed during the incubation with herbicides, either due to stress or as a defense reaction. After incubation with monochlorobimane, a strong fluorescence of the intestinal tract and the intersegments was observed, indicating organ-specific GST induction. DISCUSSION: According to the author's knowledge, here, for the first time, evidence is presented that E. fetida GST are also capable of conjugating a wider range of xenobiotic substrates. Different forms of GST were observed and changes in GST isoforms due to the herbicide treatment were also noticed. GST conjugation rates varied between different herbicides used in this experiment. It might be assumed that herbicides may well be detoxified by earthworms, to a certain extent, but that they are also potent stress factors influencing the detoxification system of the animal. High doses or long exposure might lead to deleterious effects on earthworms and limit their survival rate. The use of the animals as bioindicators for herbicides and herbicide residues seems very promising, but is surely influenced by the lack of detoxification for some compounds. CONCLUSIONS: Conjugation of several xenobiotics with model substances and herbicides is proven in the earthworm E. fetida. However, E. fetida has only limited capabilities of detoxifying herbicidal compounds. Different isoforms of GST were involved and altered in their activity after treatment. RECOMMENDATIONS AND PERSPECTIVES: The accumulation of GS-conjugates and their determination via fluorescence microscopy is a quick and secure, additional marker for exposure that should be further developed to complement existing biotests. The described methods and endpoints might help to understand the complex reaction of earthworms towards herbicides and lead to an adapted test methodology.