[1] Site preference (SP), the difference in d15 N between the central and outer nitrogen atoms in N 2 O, is a powerful approach for apportioning fluxes of N 2 O from soils to nitrification and denitrification (Sutka et al., 2006). A critical aspect of the use of SP data to apportion sources of N 2 O to nitrification and denitrification is the need to evaluate data for isotope shifts that may have occurred during N 2 O reduction in soils prior to its escape to the atmosphere. We present data on the isotopologue effects during reduction of N 2 O during anaerobic incubation of soils and pure cultures of denitrifying bacteria. N a with increased reduction. Consequently, a deviation from the linear mixing relationship between soil-derived and atmospheric N 2 O is an indication of extensive reduction. On the basis of our characterization of isotopic fractionation during N 2 O reduction, we show that the rate of reduction would have to be substantially greater than 10% of that of production to impact SP estimates of N 2 O from denitrification by more than a few percent. Nonetheless, reduction results in a small, but potentially important, increase in SP away from values proposed for bacterial denitrification (0%) toward those associated with production from nitrification (33%) (Sutka et al., 2006). On this basis, estimates of the proportion of N 2 O derived from denitrification obtained from SP values are underestimates and therefore conservative.Citation:

A laboratory soil degradation study was conducted to determine the biodegradation potential of a DvSnf7 dsRNA transcript derived from a Monsanto genetically modified (GM) maize product that confers resistance to corn rootworm (CRW; Diabrotica spp.). This study provides new information to improve the environmental assessment of dsRNAs that become pesticidal through an RNAi process. Three agricultural soils differing in their physicochemical characteristics were obtained from the U.S., Illinois (IL; silt loam), Missouri (MO; loamy sand) and North Dakota (ND; clay loam), and exposed to the target dsRNA by incorporating insect-protected maize biomass and purified (in vitro-transcribed) DvSnf7 RNA into soil. The GM and control (non-GM maize) materials were added to each soil and incubated at ca. 22°C for 48 hours (h). Samples were collected at 12 time intervals during the incubation period, extracted, and analyzed using QuantiGene molecular analysis and insect bioassay methods. The DT50 (half-life) values for DvSnf7 RNA in IL, MO, and ND soils were 19, 28, and 15 h based on QuantiGene, and 18, 29, and 14 h based on insect bioassay, respectively. Furthermore, the DT90 (time to 90% degradation) values for DvSnf7 RNA in all three soils were <35 h. These results indicate that DvSnf7 RNA was degraded and biological activity was undetectable within approximately 2 days after application to soil, regardless of texture, pH, clay content and other soil differences. Furthermore, soil-incorporated DvSnf7 RNA was non-detectable in soil after 48 h, as measured by QuantiGene, at levels ranging more than two orders of magnitude (0.3, 1.5, 7.5 and 37.5 µg RNA/g soil). Results from this study indicate that the DvSnf7 dsRNA is unlikely to persist or accumulate in the environment. Furthermore, the rapid degradation of DvSnf7 dsRNA provides a basis to define relevant exposure scenarios for future RNA-based agricultural products.

MON 87411 maize, which expresses DvSnf7 RNA, was developed to provide an additional mode of action to confer protection against corn rootworm (Diabrotica spp.). A critical step in the registration of a genetically engineered crop with an insecticidal trait is performing an ecological risk assessment to evaluate the potential for adverse ecological effects. For MON 87411, an assessment plan was developed that met specific protection goals by characterizing the routes and levels of exposure, and testing representative functional taxa that would be directly or indirectly exposed in the environment. The potential for toxicity of DvSnf7 RNA was evaluated with a harmonized battery of non-target organisms (NTOs) that included invertebrate predators, parasitoids, pollinators, soil biota as well as aquatic and terrestrial vertebrate species. Laboratory tests evaluated ecologically relevant endpoints such as survival, growth, development, and reproduction and were of sufficient duration to assess the potential for adverse effects. No adverse effects were observed with any species tested at, or above, the maximum expected environmental concentration (MEEC). All margins of exposure for NTOs were >10-fold the MEEC. Therefore, it is reasonable to conclude that exposure to DvSnf7 RNA, both directly and indirectly, is safe for NTOs at the expected field exposure levels.

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