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.     

Seasonal growth and composition of fronds of Macrocystis pyrifera in the Falkland Islands

Frond growth of Macrocystis pyrifera in the Falkland Islands was monitored in shallow coastal water from December 1985 to March 1987, and at a different site in deeper water from December 1985 to June 1986. Growth rates in the deeper bed were generally higher than those recorded in the coastal zone. At both sites, node initiation and elongation rate fluctuated according to the seasonal pattern of light or water temperature. In the shallow coastal area, nitrate was abundant in the winter and below detection levels during late spring and summer. Correlation analysis suggests that the production of the fronds of the giant kelp in this area was probably inhibited during the summer months by extremely low concentrations of nutrients. Internal nitrogen was exhausted approximately one month after a sharp decline in ambient nitrate concentration, and carbon reserves were formed. In the deeper bed of M. pyrifera, nitrogen was abundant all year round and the production of the fronds reflected the seasonal pattern of light or water temperature. The nitrogen content of the tissue probably did not drop below a level that limited production, and no internal carbon reserves were accumulated.     

The effects of mating system on male mate choice in a coral reef fish

Summary We show how mate limitation appears to be critical in determining whether or not males exercise mate choice among available females. Thalassoma bifasciatum is a Caribbean reef fish with two distinct mating patterns: group-spawning and pair-spawning. In both mating systems, female fecundity is variable and size dependent, and female availability is high. However, sperm competition among group-spawning males apparently limits the number of effective matings in which a male may engage, whereas territorial pair-spawning males have little or no such limitation. Group-spawning males should be discriminating in their choice of mates and our data confirm this: there is strong evidence for assortative mating in group-spawns, with more large males joining in mating groups around large females. In contrast, pair-spawning males show no indication of mate preferences, and spawn with all females who arrive at their territories.     

Influence of age,season, body condition and geographical area on concentrations of chlorinated and brominated contaminants in wild mink (Neovison vison) in Sweden

The wild mink has gained acceptance as a sentinel species in environmental monitoring. However, only limited data are available in the literature on factors driving variability in concentrations of organic pollutants in this species. This study characterizes the differences in contaminant concentrations in subcutaneous fat of male mink from four different areas in Sweden and demonstrates how age, season and body condition influence concentrations of polychlorinated biphenyl (PCB) congeners, polybrominated diphenyl ether (PBDE) congeners (including methoxylated forms, MeO-PBDEs), as well as the pesticides dichlorodiphenyldichloroethylene (DDE), chlordane and hexachlorobenzene (HCB). The data were statistically treated using multiple regression and principal component analysis. The ∑PCB concentration and concentrations of PCB congeners 138, 156, 157, 180, 170/190, 189, 194, 206, 209 as well as PBDE 153/154 varied with age. Season had an influence on ∑PCB, PBDE 47 and PBDE 153/154 concentrations, as well as concentrations of most PCB congeners, with the exception of PCB 101, 110, 141 and 182/187. Lean mink had higher concentrations of most PCBs and PBDEs than mink with larger fat depots. The analyzed pesticides (DDE, oxychlordane, HCB) showed no systematic variation with season, age or body condition. The concentrations of MeO-PBDEs were generally low and 6MeO-PBDE 47 was the most commonly detected MeO-PBDE in mink from marine, brackish and freshwater areas. The results indicate that age, season and body condition are factors that may influence the concentrations of PCBs and PBDEs, and it is thus recommended to take these factors into account when analyzing mink exposure data.     

Toxicity of abamectin and doramectin to soil invertebrates

This study aimed at determining the toxicity of avermectins to soil invertebrates in soil and in faeces from recently treated sheep. Abamectin was more toxic than doramectin. In soil, earthworms (Eisenia andrei) were most affected with LC50s of 18 and 228 mg/kg dry soil, respectively, while LC50s were 67-111 and >300 mg/kg for springtails (Folsomia candida), isopods (Porcellio scaber) and enchytraeids (Enchytraeus crypticus). EC50s for the effect on reproduction of springtails and enchytraeids were 13 and 38 mg/kg, respectively for abamectin, and 42 and 170 mg/kg for doramectin. For earthworms, NOEC was 10 and 8.4 mg/kg for abamectin and doramectin effects on body weight. When exposed in faeces, springtails and enchytraeids gave LC50s and EC50s of 1.0-1.4 and 0.94-1.1 mg/kg dry faeces for abamectin and 2.2->2.4 mg/kg for doramectin. Earthworm reproduction was not affected. This study indicates a potential risk of avermectins for soil invertebrates colonizing faeces from recently treated sheep.     

Life cycle responses of the midge Chironomus riparius to polycyclic aromatic compound exposure

During acute exposure, polycyclic aromatic compounds (PACs) act mainly by narcosis, but during chronic exposure the same compounds may exert sublethal life cycle effects. The aim of this study was therefore to evaluate the chronic effects of sediment spiked PACs on the emergence of the midge Chironomus riparius. Three isomer pairs were selected, and 28-day LC50 values and 50% emergence times (EMt50) were determined. Concentration-response relationships were observed for phenanthrene, acridine, phenanthridine and acridone. Anthracene and phenanthridone had no effect on total emergence, but did cause a delay in emergence. Calculated porewater LC50 values correlated well with logKow values, suggesting narcosis as mode of action. In contrast, effect concentrations for delay in emergence (EMt50) deviated from narcosis, suggesting a specific mode of action during chronic exposure. It is concluded that emergence is a powerful endpoint to detect life cycle effects of PACs on a key sediment inhabiting invertebrate.     

Kinetic extractions to assess mobilization of Zn, Pb, Cu, and Cd in a metal-contaminated soil: EDTA vs. citrate

Kinetic EDTA and citrate extractions were used to mimic metal mobilization in a soil contaminated by metallurgical fallout. Modeling of metal removal rates vs. time distinguished two metal pools: readily labile (QM1) and less labile (QM2). In citrate extractions, total extractability (QM1+QM2) of Zn and Cd was proportionally higher than for Pb and Cu. Proportions of Pb and Cu extracted with EDTA were three times higher than when using citrate. We observed similar QM1/QM2 ratios for Zn and Cu regardless of the extractant, suggesting comparable binding energies to soil constituents. However, for Pb and Cd, more heterogeneous binding energies were hypothesized to explain different kinetic extraction behaviors. Proportions of citrate-labile metals were found consistent with their short-term, in-situ mobility assessed in the studied soil, i.e., metal amount released in the soil solution or extracted by cultivated plants. Kinetic EDTA extractions were hypothesized to be more predictive for long-term metal migration with depth.     

A synchronized amphibian metamorphosis assay as an improved tool to detect thyroid hormone disturbance by endocrine disruptors and apolar sediment extracts

Amphibian metamorphosis assays are used to evaluate potential effects of endocrine disrupting compounds on the thyroid hormone axis. In this study, Xenopus laevis tadpoles are kept in a solution of 0.2% thiourea (TU) to arrest and synchronise them in their development. The advantage of this synchronized amphibian metamorphosis assays is that synchronised tadpoles are available at any time to start metamorphosis experiments, and experimental groups are much more homogenous at the start of experimental exposure compared with groups selected from an untreated pool of animals. The water volume per animal was kept constant throughout the experimental period to overcome the influence of declining numbers of animals per aquarium due to metamorphosis and mortality on the density dependent development of the remaining tadpoles. Clophen A50 (a technical PCB mixture), the single congener 3,3',4,4'-tetrachlorobiphenyl (PCB 77) and apolar sediment extracts that were previously tested positive in the T-Screen, an in vitro proliferation assay for thyroid hormone disruption, were tested in the Synchronized Amphibian Metamorphosis Assay. Endpoints studied were mortality, malformations, body weight, and percentage of metamorphosed froglets at the end of the 60-day experimental period, percentage of tadpoles in different developmental stages, and developmental stage-dependent awarded penalty points. Dietary exposure to Clophen A50 (0.2-50mg/kg food) resulted in a significant increased percentage of tadpoles that did not pass metamorphosis at concentrations higher than 2mg/kg food. Time until metamorphosis in those animals that were able to metamorphose after the 60-days experimental period was significantly decreased. Dietary exposure to PCB 77, a congener that can be readily metabolised, did not result in significant effects in any exposure group (2-500 microg/kg food). Apolar sediment extracts from two of the three sites that are contaminated with a wide variety of chemicals significantly decreased the percentage of metamorphosed animals and significantly increased the number of tadpoles that remained in early and late metamorphic stages. These effects already occurred when the extracts where diluted more than 1000 times (on an organic carbon base) compared to environmental concentrations. The rank of potency was comparable to results obtained with the T-screen. This suggests the presence of thyroid hormone disrupting compounds in the aquatic environment and possible effects of such compounds on animal development in the wild.