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This paper provides recommendations on experimental design for early-tier laboratory studies used in risk assessments to evaluate potential adverse impacts of arthropod-resistant genetically engineered (GE) plants on non-target arthropods (NTAs). While we rely heavily on the currently used proteins from Bacillus thuringiensis (Bt) in this discussion, the concepts apply to other arthropod-active proteins. A risk may exist if the newly acquired trait of the GE plant has adverse effects on NTAs when they are exposed to the arthropod-active protein. Typically, the risk assessment follows a tiered approach that starts with laboratory studies under worst-case exposure conditions; such studies have a high ability to detect adverse effects on non-target species. Clear guidance on how such data are produced in laboratory studies assists the product developers and risk assessors. The studies should be reproducible and test clearly defined risk hypotheses. These properties contribute to the robustness of, and confidence in, environmental risk assessments for GE plants. Data from NTA studies, collected during the analysis phase of an environmental risk assessment, are critical to the outcome of the assessment and ultimately the decision taken by regulatory authorities on the release of a GE plant. Confidence in the results of early-tier laboratory studies is a precondition for the acceptance of data across regulatory jurisdictions and should encourage agencies to share useful information and thus avoid redundant testing.

species should be representative of valued taxa or functional groups that are most likely to be exposed to the arthropod-active compound in the field; and (iii) Availability and reliability: suitable life-stages of the test species must be obtainable in sufficient quantity and quality, and validated test protocols must be available that allow consistent detection of adverse effects on ecologically relevant parameters. Our proposed approach ensures that the most suitable species are selected for testing and that the resulting data provide the most rigorous test of the risk hypothesis of no adverse effect in order to increase the quality and efficiency of ERAs for cultivation of GE crops.

Abstract.A technique for reliable labeling of the carbon reserves of the trunk and roots without labeling the current year's growth of grapevines was developed in order to study retranslocation of carbon from the perennial storage tissues into the fruit in response to defoliation stress during the ripening period. A special training system with two shoots was used: the lower one (feeding shoot) was cut back and defoliated to one single leaf (14CO2-feeding leaf)while the other (main shoot) was topped to 12 leaves. The potted plants were placed in a water bath at 30~ to increase root temperature and therefore their sink activity. Additionally, a cold barrier (2-4~ was installed at the base of the main shoot to inhibit acropetal 14C translocation. Using this method, we were able to direct labeled assimilates to trunk and roots in preference to the current year's growth. On vines with root and shoot at ambient temperature, 44% of the 14C activity was found in the main shoot 16 h after feeding whereas only 2% was found in the temperature-treated vines. At the onset of fruit ripening, and at three-week intervals thereafter until harvest, potted grapevines were fed with 14CO2 using the temperature treatment described above. Sixteen hours after feeding, half of the vines of each group were defoliated by removing all except the two uppermost main leaves. Three weeks after each treatment, vines were destructively harvested and the dry weight and 14C incorporation determined for all plant parts. Under non-stressing conditions, there was no retranslocation of carbon reserves to support fruit maturation. Vines responded to defoliation stress by altering the natural translocation pattern and directing carbon stored in the lower parts to the fruit. In the three weeks following veraison (the inception of ripening in the grape berry), 12% of the labeled carbon reserves was translo- cated to the fruit of defoliated plants compared to 1.6% found in the clusters of control vines. Retranslocation from trunk and roots was highest during the middle of the ripening period, when 32% of the labeled carbon was found in the fruit compared to 0.7% in control plants. Defoliation during this period also caused major changes in dry-matter partitioning: the fruit represented 31% of total plant biomass compared to 21% measured in the control vines. Root growth was reduced by defoliation at veraison and during the ripening period. Defoliation three weeks before harvest did not affect dry matter or 14C partitioning.