## 5 Environmental Risk Assessment
### Evaluation of Western Corn Rootworm RNAAi Traits on Non-Target Organisms  
Multiple studies have investigated the impact of WCR-active dsRNA constructs on non-target organisms. After the initial report that maize expressing _v-ATPase A_ dsRNA could generate root protection from rootworm feeding damage, researchers started evaluating the potential effects of WCR _v-ATPase A_ dsRNA on non-target arthropods, including a pollinator, _Apis mellifera_ L. [187]; a herbivorous insect, _Danaus plexiprus_ ([PERSON]) larvae [188]; predators including the ladybeetle species, _Coccinella septempunctata L., _Colemegila maculata_ ([PERSON]), _Hippodamia convergens_ [PERSON], _Harmonia axyridis_ ([PERSON]), and _Adalia bipunctata_ ([PERSON]) [189; 190]; and a decomposer, the collembolan _Sinella curviseta_ [PERSON] [191]. Results found that WCR _v-ATPase A_ dsRNA has negligible effects on honey bee larvae and adults, monarch caterpillars, and _S. curviseta_[187; 188; 191]. Interestingly, species-specific _v-ATPase A_ dsRNA experiments found that monarch larvae are not susceptible to dietary RNAi [188], and only a small downregulation from species-specific _v-ATPase A_ dsRNA was observed in honey bees after 24 hours with gene expression recovering after 48 hours [187]. Furthermore, dsRNA was found bound to royal jelly, making it unavailable for honey bee larvae, suggesting environmental factors could render insecticidal dsRNA unavailable for non-target organisms [187]. In contrast, two ladybeetles _H. axyridis_ and _C. septempunctata_, experienced some gene knockdown and mortality when exposed to WCR _v-ATPase A_ dsRNA [190]. In a different study, WCR _v-ATPase A_ significantly reduced the development of _A. bipunctata_ and _C. septempunctata_[189]. In these studies, ladybeetles with the strongest effects had more base pair matches between WCR _v-ATPase A_ and their specific _v-ATPase A_ sequence, highlighting the importance of bioinformatic analyses when designing insecticidal dsRNAs [189; 190]. It is also important to consider that dsRNA concentrations tested in these studies were orders of magnitude higher than what is expected to occur in the field [189]; therefore, potential detrimental effects in a field scenario may be negligible.  
Other studies have examined the impact of _DvSnf7_ on non-target insects [192; 154]. [PERSON] et al. [192] initially evaluated the lethal and sublethal effects of _DvSnf7_ dsRNA on insects representing ten families and four orders. The _DvSnf7_ dsRNA spectrum of activity was narrow, and activity was only observed in beetles within the Galercinae subfamily of Chrysomeliidae, whose homologous _suf7_ sequence shared a 90% identity with WCR _Suff7_[192]. [PERSON] et al. [193] evaluated _DvSnf7_ dsRNA in honey bee larvae and adults and reported no adverse effects at high exposure levels. [PERSON] et al. [154] tested _DvSnf7_ dsRNA activity at field concentrations in seven non-target insects, including honey bee larvae and adults; _C. maculata_; a rove beetle, _Aleochara bilineata_ [PERSON]; a ground beetle, _Poecilus chalcites_ ([PERSON]); the green lacewing, _Chrysoperla carnea_ ([PERSON]); the insidious flower bug, _Orius insidiosus_ [PERSON]; and a parasitic wasp, _Pediobius foveulatus_ ([PERSON]). Results from this study indicated that exposure to _DvSnf7_ dsRNA at field-relevant exposure levels would not cause adverse effects on the non-target insect species described above [154]. These studies demonstrate the impact of _DvSnf7_ dsRNA expressed in maize plants will be negligible on beneficial insects. Recent review papers provide more details regarding the problem formulation for off-target effects of dsRNA products for pest control [194], and sublethal endpoints for non-target organisms for insect-active transgenic crops, including RNAi [172].