Environmental risk assessment of the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins to non-target organisms

ABSTRACT

Lepidopteran pests are a serious threat to soybean production worldwide and have developed resistance to several pest management options, including the use of transgenic soybean expressing crystalline (Cry) proteins derived from the soil bacterium Bacillus thuringiensis Berliner (Bt). For this reason, there is great interest in discovering insecticidal proteins that function via new modes and/or sites of action against lepidopteran pests to support a sustainable and durable management plan. Event COR-23134–4 (hereafter referred to as COR23134 soybean), which expresses Bt-derived insecticidal proteins Cry1B.34.1 and Cry1B.61.1 and a novel plant-derived IPD083Cb insecticidal protein, was developed to provide additional sites of action to confer protection against certain susceptible lepidopteran pests. As part of the environmental safety assessment, the potential risks (exposure and hazard) posed by the cultivation of COR23134 soybean to non-target organisms (NTOs) were assessed. The environmental risk was characterized by comparing the Tier I laboratory hazard study results to worst-case or refined estimated environmental concentrations (EECs) to establish the margin of exposure (MOE) for the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins in COR23134 soybean. Overall, results from the exposure and hazard assessments and the MOE values show that the Cry1B.34.1, Cry1B.61.1, and IPD083Cb insecticidal proteins expressed in COR23134 soybean are not expected to result in unreasonable adverse effects on NTO populations at environmentally realistic concentrations; hence, the risk to NTOs from the cultivation of COR23134 soybean is considered negligible.

1. Introduction

Soybean (Glycine max [L.] Merr.), the world’s leading oilseed crop, is extensively cultivated in several tropical and temperate geographies worldwide. Brazil, the United States, and Argentina are the world’s largest producers of soybean, with a combined total production of 319 million metric tons in 2023/2024, which is equivalent to 80% of global soybean production.^1,2 Lepidopteran insect pests are a serious threat to soybean production worldwide. The feeding damage caused by these insects significantly reduces soybean yield and quality, which results in economic losses worth billions of dollars annually.³ Pest management options for these insects include using crop rotation, broad-spectrum insecticides, and, in certain geographies, transgenic soybean expressing crystalline (Cry) proteins derived from the soil bacterium Bacillus thuringiensis Berliner (Bt). However, the evolution of crop rotation-, insecticide-, and transgenic-resistant lepidopteran insect pest populations in soybean-growing areas of the world has diminished the efficiency of these pest management practices. For instance, several Cry proteins have been effective in controlling lepidopteran pests and have been deployed in multiple crops, including soybean^4,5; however, their benefits are threatened by the rapid evolution of practical insect resistance.⁶ Pyramiding multiple insecticidal proteins with different modes of action and/or target sites offers increased efficacy and target pest spectrum and delays the evolution of resistance in the target pest populations that are susceptible to the proteins.^6,7 Therefore, there is a great interest in discovering insecticidal proteins that function through new modes of action and/or target sites against lepidopteran pests to support a sustainable and durable management plan. Corteva Agriscience LLC has developed an insect-resistant transgenic soybean containing event COR-23134–4 (hereafter referred to as COR23134 soybean), which confers protection against certain susceptible lepidopteran pests via expression of Bt-derived insecticidal proteins Cry1B.34.1 and Cry1B.61.1 and a novel plant-derived IPD083Cb insecticidal protein. These proteins do not bind to the same target sites as Bt-derived insecticidal proteins expressed in current commercial traits. COR23134 soybean also expresses the GM-HRA protein, which was used as a selectable marker during the event development process. By expressing insecticidal proteins that function through distinct target sites, COR23134 soybean is expected to provide growers with an effective, wide-spectrum, sustainable, and durable pest management tool for controlling key susceptible lepidopteran pest populations in soybean production systems.

Before a new genetically modified (GM) crop is registered and/or deregulated for commercial cultivation, a science-based environmental risk assessment (ERA) is performed to quantify the potential risk that the cultivation of the GM crop may pose to beneficial non-target organisms (NTOs) and the ecosystem services that they provide (e.g., pollination, pest regulation, organic matter decomposition, soil structure, nutrient cycling, aesthetic or cultural values, etc.). Such assessment follows an established multi-step iterative process, viz. problem formulation, analysis of exposure and potential adverse effects, and risk characterization.^8–10 The problem formulation stage of the risk assessment is conducted to identify applicable risk hypotheses and an approach to testing them, which includes estimates of relevant exposure and harmful or adverse effects (hazard).¹¹ The potential exposure of NTOs to GM crops is determined using highly conservative assumptions. Potential adverse effects of GM crops on NTOs and the beneficial ecosystem services they provide are evaluated using a tiered testing approach that progresses from highly controlled worst-case or artificially high exposure lower-tier (Tier I) studies in the laboratory to more realistic exposure but less controlled higher-tier studies in the laboratory (Tier II), greenhouse (Tier III) or field (Tier IV).¹² Higher-tier studies are only considered relevant if the results from the lower-tier study detect potential adverse effects or indicate that further hazard assessment is necessary.^12–14 Key indicators of adverse effects may include impacts on survival, growth, development, and reproduction.⁸ Higher-tier studies may also be conducted if unacceptable scientific uncertainty remains following the conduct of a lower-tier study¹⁵ or if some regulatory agencies require in-country field studies to supplement the findings of lower-tier studies.¹⁶ However, a meta-analysis has indicated that results from laboratory studies conducted to quantify the effects of GM crops on NTOs generally predict or overestimate field-level effects.^17,18 In some instances, higher-tier studies may be conducted at an early phase when lower-tier studies are impractical or irrelevant due to a lack of testable surrogate species or validated test protocol.^19,20

An ERA for the cultivation of COR23134 soybean was conducted following the risk assessment framework described by the United States Environmental Protection Agency.¹⁰ This framework provides a robust and suitable science-based tool for assessing potential risks associated with GM crops expressing plant-incorporated protectants (PIPs) derived from Bt and non-Bt sources.²¹ Problem formulation was used to identify potential routes by which adverse environmental effects on functional groups (pollinators and pollen feeders, soil-dwelling organisms, insect predators and parasitoids, aquatic organisms, insectivorous birds, and granivorous mammals) may arise from the cultivation of COR23134 soybean. Key information used to inform the problem formulation step for COR23134 soybean included the characteristics of the receiving environment, the biology of the crop, and the characteristics of the introduced Cry1B.34.1, Cry1B.61.1, and IPD083Cb insecticidal proteins (mode of action, spectrum of insecticidal activity, and similarity with previously assessed proteins). The GM-HRA protein only served as an inert selectable marker during the event development process and was not considered during the problem formulation step. Protection goals identified for the COR23134 soybean risk assessment included protecting NTOs and maintaining the ecosystem and biodiversity services that these organisms provide. Therefore, this ERA tested the hypothesis that the expressed insecticidal proteins (Cry1B.34.1, Cry1B.61.1, and IPD083Cb) in COR23134 soybean are not toxic or harmful to NTOs at the concentration or exposure present in the field. The objective of this ERA was to quantify the potential risk (exposure and hazard) that the cultivation of the COR23134 soybean may pose to NTOs.

2. Materials and Methods

2.1. Exposure Assessment

2.1.1. Concentrations of Cry1B.34.1, Cry1B.61.1, and IPD083Cb in COR23134 Soybean

COR23134 soybean was planted during the 2022 growing season at five locations in the United States (Iowa, Illinois, Indiana, Nebraska, and Pennsylvania) and one location in Canada (Ontario). These locations were selected to represent differences in environmental conditions for commercial soybean production in North America. Agronomic management practices such as fertilization, irrigation, pest management, etc., followed standard local production recommendations. Tissue samples (leaf, flowers, root, forage, and seed) were collected from COR23134 soybean plants at various growth stages (Table 1) at each of the six field locations. The tissue samples were analyzed for Cry1B.34.1, Cry1B.61.1, and IPD083Cb protein concentrations using quantitative enzyme-linked immunosorbent assay (ELISA) methods and adjusted for a dilution factor to provide values (Table 1) for calculations of estimated environmental concentrations.

Table 1.

Dry weight concentrations of the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins in COR23134 soybean tissues.

Tissue Type	Growth Stage	ng Cry1B.34.1/mg Tissue			ng Cry1B.61.1/mg Tissue			ng IPD083Cb/mg Tissue
		Maximuma	Meanb	SD	Maximuma	Meanb	SD	Maximuma	Meanb	SD
Leaf	V5	960	460	220	900	450	150	110	65	17
Leaf	R1	780	310	170	1300	490	220	100	76	14
Leaf	R3	590	210	110	780	480	130	130	83	19
Flower	R1-R2	320	260	28	190	150	19	88	64	13
Root	R3	210	82	40	0.93	0.34c	0.18c	33	21	5.6
Forage	R3	180	150	22	390	200	72	84	59	10
Seed	R8	210	170	22	22	15	2.6	19	15	1.5

^aThe maximum concentrations of the proteins in relevant COR23134 soybean tissues at any single field trial location and any growth stage were used for determining worst-case estimated environmental concentrations (EECs).

^bThe highest mean concentrations of the proteins in applicable COR23134 soybean tissue across field trial locations and at any growth stage were used for determining refined EECs.

^cSome sample results were below the lower limit of quantification (LLOQ). A value equal to the LLOQ value was assigned to those samples to calculate the mean and standard deviation.

SD – Standard deviation.

Maximum and/or mean values determined to be relevant for the exposure assessment are in bold text.

2.1.2. Estimated Environmental Concentrations (EECs) of Cry1B.34.1, Cry1B.61.1, and IPD083Cb from COR23134 Soybean

Estimates of EECs were made from measurements of the concentrations of Cry1B.34.1, Cry1B.61.1, and IPD083Cb in relevant COR23134 soybean tissues to determine potential exposure for various functional groups of NTOs (pollinators and pollen feeders, soil-dwelling organisms, aquatic organisms, predators and parasitoids, insectivorous birds, and granivorous mammals). In each case, worst-case EECs of the proteins (Cry1B.34.1, Cry1B.61.1, and IPD083Cb) in COR23134 soybean were determined using highly conservative assumptions based on the maximum concentrations of the proteins in relevant COR23134 soybean tissues at any single field trial location and any growth stage (Table 1). The worst-case EEC represents exposure via a diet of 100% of the relevant COR23134 soybean tissue. If necessary, refined EECs were calculated based on the highest mean concentrations of the proteins in applicable COR23134 soybean tissue across field trial locations and at any growth stage (Table 1). Refined EECs typically reflect more realistic environmental conditions and ecological processes (e.g., dilution of the proteins through prey, in soil, or by other means) that reduce actual NTO exposure in the field. Dry weight concentrations are considered high estimates since they are unaffected by differences in water content of tissues, as would fresh weight concentrations; therefore, all EECs (worst-case and refined) were estimated using dry weight concentrations of Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins (Table 1). In reality, NTOs would be exposed to levels comparable to fresh weight levels.

2.1.2.1. Pollinators and Pollen Feeders

Pollinators and pollen feeders (honey bee larvae, honey bee adults, and non-target Lepidoptera) are primarily exposed to pollen, either directly or indirectly, from a variety of different plant species in the agroecosystem and are likely to be present and actively foraging during soybean flowering. However, it was not possible to conduct protein expression analysis for pollen due to the small quantity produced and the difficulty in collecting pollen from the soybean flowers. Thus, the concentration of the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins expressed in flowers were used as a surrogate tissue for pollen. To this end, a worst-case scenario was assumed based on the consumption of only COR23134 soybean pollen and maximum observed concentrations of Cry1B.34.1 (320 ng/mg flower dry weight), Cry1B.61.1 (190 ng/mg flower dry weight), and IPD083Cb (88 ng/mg flower dry weight) proteins in COR23134 soybean flower (Table 1). Honey bee larvae were presumed to ingest 2.0 mg of soybean pollen throughout larval development.²² Honey bee adults were presumed to ingest 4.3 mg of soybean pollen daily.²³

2.1.2.2. Soil-Dwelling Organisms

Soil-dwelling organisms (decomposers and detritivores) are most likely to consume senescent soybean tissues incorporated into the soil post-harvest.⁸ Therefore, the exposure route of soil-dwelling organisms to the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins in COR23134 soybean is via ingestion of senescent COR23134 soybean tissues. Since the protein concentrations in senescing tissues are expected to decline as they degrade, the protein concentrations in R3 forage tissue were used as worst-case and refined exposure scenarios for these taxa to achieve extremely conservative EECs. The worst-case EEC for soil-dwelling organisms was calculated based on the maximum concentration of the Cry1B.34.1 (180 ng/mg forage dry weight), Cry1B.61.1 (390 ng/mg forage dry weight), and IPD083Cb (84 ng/mg forage dry weight) proteins in R3 forage tissue (Table 1). A refined EEC for soil-dwelling organisms was also calculated based on the highest mean concentration of the Cry1B.34.1 (150 ng/mg) and Cry1B.61.1 (200 ng/mg) proteins in R3 forage tissue (Table 1).

2.1.2.3. Aquatic Organisms

The most likely route of exposure of aquatic organisms to Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins were considered to be from COR23134 soybean tissues deposited in water bodies (e.g., via runoff, wind, etc.) adjacent to soybean fields. The worst-case and refined exposure scenarios for these taxa were estimated following the Environmental Protection Agency (EPA) standard agricultural field-farm pond model (also called the US EPA standard pond model) previously described in Jones et al.²⁴ For these scenarios, the assumptions made were 1. All of the aboveground soybean tissue from a 10-hectare (ha) field was deposited via runoff in a 1-ha pond that is 2 m deep and contains 20,000,000 L of water, 2. Soybean planting density was 346,000 plants/ha, 3. One soybean plant weighs 0.021 kg dry weight²⁵ (Bhanwar et al., 2020), which is equivalent to 7266 kg plant tissue/ha, and 4. The insecticidal proteins (Cry1B.34.1, Cry1B.61.1, and IPD083Cb) in COR23134 soybean tissue are freely soluble and instantaneously bioavailable, and no degradation occurs in the field or the pond. The maximum forage concentration of the Cry1B.34.1 (180 ng/mg forage dry weight), Cry1B.61.1 (390 ng/mg forage dry weight), and IPD083Cb (84 ng/mg forage dry weight) proteins at the R3 growth stage (Table 1) were used to calculate the worst-case EEC for aquatic organisms. A refined EEC for aquatic organisms was also calculated based on the highest mean forage concentration of the Cry1B.61.1 (200 ng/mg forage dry weight) and IPD083Cb (59 ng/mg forage dry weight) proteins at the R3 growth stage (Table 1).

2.1.2.4. Predators, Parasitoids, and Insectivorous Birds

Predators, parasitoids, or insectivorous birds may be exposed to the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins via consumption of insect herbivores that have previously consumed tissue from a COR23134 soybean plant. The assumptions made for estimating the worst-case exposure scenario for these organisms were 1. 100% of the protein (Cry1B.34.1, Cry1B.61.1, or IPD083Cb) in the COR23134 soybean tissue transfers to the insect herbivore and then subsequently is transferred to the predator, parasitoid, or insectivorous bird (there is no degradation or loss of the protein during tri-trophic transfer) and 2. Predators, parasitoids, and insectivorous birds were exposed to the maximum concentration of Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins expressed in any aboveground COR23134 soybean tissue at any growth stage (960 ng Cry1B.34.1/mg V5 leaf dry weight, 1300 ng Cry1B.61.1/mg R1 leaf dry weight, and 130 ng IPD083Cb/mg R3 leaf dry weight; Table 1). The same assumptions were also made for estimating the refined EEC for these organisms, the lone exception being that the predators, parasitoids, and insectivorous birds were assumed to be exposed to the highest mean concentration of the proteins expressed in any aboveground COR23134 soybean tissue at any growth stage (460 ng Cry1B.34.1/mg V5 leaf dry weight, 490 ng Cry1B.61.1/mg R1 leaf dry weight, and 83 ng IPD083Cb/mg R3 leaf dry weight; Table 1).

2.1.2.5. Granivorous Mammals

The main route of exposure of granivorous mammals to Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins is via consumption of COR23134 soybean seed. For these organisms (e.g., wild rodents), the worst-case exposure scenario was based on the maximum concentration of the Cry1B.34.1 (210 ng/mg seed dry weight), Cry1B.61.1 (22 ng/mg seed dry weight), and IPD083Cb (19 ng/mg seed dry weight) proteins in COR23134 soybean seed (Table 1) and assumed that 100% of the diet contained COR23134 soybean seed. A daily dietary dose (DDD) for wild rodents was estimated following the procedure previously described by Crocker et al.²⁶:

$$\mathrm{DDD}\mathit{\hspace{0.17em}}=\mathit{\hspace{0.17em}}\left(\mathrm{FIR}/\mathrm{bw}\right)\mathit{\hspace{0.17em}}\times \mathrm{C}$$

where FIR is the food intake rate, bw is the body weight, and C is the maximum concentration of Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein in soybean seed. The worst-case FIR/bw ratio for seed-eating rodents previously determined for the harvest mouse (Micromys minutus) is 0.33²⁶ and was used to calculate the worst-case EECs.

2.2. Hazard Assessment

2.2.1. Test Substances

For a more rigorous test of risk hypothesis and to expose the test species to higher protein test concentrations than are possible with COR23134 plant-derived test substances, all laboratory effects tests were conducted using microbially (Escherichia coli) or plant (Nicotiana benthamiana)-produced test substances containing Cry1B.34.1 (or Cry1B.34 for some bioassays), Cry1B.61.1, or IPD083Cb protein. Each protein was expressed in Escherichia coli (for Cry1B.34.1/Cry1B.34 or Cry1B.61.1) or Nicotiana benthamiana (for IPD083Cb), lyophilized, and characterized.^27–30 The biochemical and functional equivalence between the COR23134 plant-expressed and Escherichia coli-produced Cry1B.34.1 (or Cry1B.34 for some bioassays) or Cry1B.61.1 protein was confirmed. Likewise, the biochemical and functional equivalence between the COR23134 plant-expressed and Nicotiana benthamiana-produced IPD083Cb protein was also confirmed: for each protein, both COR23134 plant-expressed and Escherichia coli or Nicotiana benthamiana-produced test substances had the expected molecular weight, immunoreactivity, and amino acid sequence; demonstrated bioactivity against a sensitive insect species; and lacked glycosylation.^27–36 Both the Cry1B.34 and Cry1B.34.1 proteins, when expressed in planta, provide protection from certain susceptible Lepidopteran pests of maize and soybean, respectively. The Cry1B.34 is a full-length protein consisting of 1149 amino acids, with a molecular weight (MW) equal to 128.987 kDa, and is expressed in an insect-resistant transgenic maize event DP-910521-2 (referred to as DP910521) developed by Corteva Agriscience LLC. The Cry1B.34.1 protein is a truncated version of the Cry1B.34 and consists of 665 amino acids (formed by the removal of approximately 484 amino acids from the C-terminal end of the Cry1B.34 protein), with a MW of 75.294 kDa. This alteration does not affect the core toxin of the protein, which remains identical in both Cry1B.34 and Cry1B.34.1 proteins. The similarity in the potency of full-length and truncated versions of Cry proteins has been well documented.^37–40 Therefore, when compared on an equimolar basis, the full-length Cry1B.34 protein used in the safety studies for DP910521 maize and the truncated Cry1B.34.1 protein in COR23134 soybean should exhibit similar potency against a sensitive insect. To this end, for every bioassay where Cry1B.34 protein was used, the MW was adjusted by dividing the MW of Cry1B.34.1 (75.294 kDa) by that of Cry1B.34 (128.987 kDa):

Cry1B.34.1 MW/Cry1B.34 MW = 75.294 kDa/128.987 kDa = 0.584

2.2.2. Selection of Surrogate Non-Target Test Species

Tier I laboratory studies were conducted using representative surrogate test species (Table 2) at concentrations exceeding the worst-case EECs to assess for potential adverse effects of the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins on NTOs. Whenever possible, the surrogate test species were exposed to protein concentrations that targeted a maximum hazard dose of at least 10 times the worst-case EECs. The surrogate test species were selected based on the exposure assessment, understanding of the spectrum of activity of the Cry1B.34.1 (due to an identical toxin domain, the spectrum of activity data generated using the Cry1B.34 protein was used for Cry1B.34.1 protein), Cry1B.61.1, and IPD083Cb proteins,^7,41–44 and practical considerations (e.g., availability of laboratory-reared insects and other test species, amenability to testing, and availability of established, reproducible, and robust methods). The selected surrogate test species also fulfill other criteria for choosing a suitable representative indicator species for hazard assessments of insecticidal proteins: phylogenetic relation to the target insects, ecological function, and presence in the agroecosystem.^9,20,45–48 Surrogate test species assessed included honey bee (Apis mellifera) larvae and adults, springtail (Folsomia candida), daphnia (Daphnia magna), green lacewing (Chrysoperla rufilabris), pink spotted lady beetle (Coleomegilla maculata), parasitic hymenoptera (Pediobius foveolatus), Northern bobwhite quail (Colinus virginianus), and mouse (Mus musculus), which represent functional groups of pollinators and pollen feeders, soil-dwelling organisms, aquatic organisms, predators and parasitoids, insectivorous birds, and granivorous mammals (Table 2).

Table 2.

Representative surrogate test species used to assess the toxicity of the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins on non-target organisms.

Surrogate Test Species	Common Name	Order	Family	Functional Group
Apis mellifera	Honey bee (larvae and adult)	Hymenoptera	Apidae	Pollinators and pollen feeders
Folsomia candida	Springtail	Collembola	Isotomidae	Soil-dwelling organisms
Daphnia magna	Daphnia	Diplostraca	Daphniidae	Aquatic organisms
Chrysoperla rufilabris	Green lacewing	Neuroptera	Chrysopidae	Predators and parasitoids
Coleomegilla maculata	Pink spotted lady beetle	Coleoptera	Coccinellidae	Predators and parasitoids
Pediobius foveolatus	Parasitic hymenoptera	Hymenoptera	Eulophidae	Predators and parasitoids
Colinus virginianus	Northern bobwhite quail	Galliformes	Odontophoridae	Insectivorous birds
Mus musculus	Mouse	Rodentia	Muridae	Granivorous mammals

2.2.3. Test for Synergism Among Cry1B.34.1, Cry1B.61.1, and IPD083Cb Proteins on a Sensitive Organism to Inform Tier I Laboratory Hazard Study Design

The combined potency of Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins were evaluated by assessing the survival of an organism (soybean looper, Chrysodeixis includens) sensitive to each of the proteins alone. To assess for synergism among the three protein actives, the median lethal concentrations (LC₅₀) of each active alone were first established using multiple independent 7-d bioassays,^42,49,50 (Table 3). Subsequently, three additional 7-d bioassays were conducted using mixtures of the three actives at one-third of their respective LC₅₀ concentrations. Additional treatments included a negative control, and 5-fold and 10-fold dilutions of the three protein mixtures at one-third of their respective LC₅₀ concentrations⁵² (Table 3) to evaluate the degree of possible synergism if it were to be observed. Predictions derived from the dose addition model⁵¹ were used to estimate the relative combined potency of the mixtures given the similar modes of action of the proteins (all three proteins are receptor-mediated, gut-active membrane disrupters). For each treatment, the mortality rate of soybean looper across bioassays was estimated with 95% confidence intervals (Table 3). Across the three bioassays, the observed mortality of soybean looper fed a diet containing Cry1B.61.1 protein at the median lethal concentration (LC₅₀) was as expected, indicating the response of soybean looper to Cry1B.61.1 protein used to assess synergism among the three protein actives was consistent with the previous bioassays used to generate the LC₅₀ values (Table 3). The observed mortality of soybean looper fed a diet containing Cry1B.34.1 (LC₅₀), IPD083Cb (LC₅₀), or mixtures of the three protein actives at one-third of their respective LC₅₀ concentrations exceeded 50% (Table 3). Due to the additive action of the proteins and considering that two of the three proteins individually resulted in soybean looper mortality exceeding 50% (86.4% and 93.3% for IPD083Cb and Cry1B.34.1, respectively, Table 3), the mortality observed with the combination of the three proteins at one-third of their respective LC₅₀ concentrations was not surprising. The observed mortality for the 5-fold and 10-fold dilutions of the three protein mixtures at one-third of their respective LC₅₀ concentrations was well below 50% and comparable to the buffer control mortality (Table 3), signifying no indication that Cry1B.34.1, Cry1B.61.1, and IPD083Cb result in synergistic effects. A five-fold increase in potency is considered a threshold beyond which additional work would be necessary further to characterize synergism.⁵³ The observed discrepancy between the expected and observed mortality for each treatment is presented in Table 3. None of the treatments (including those where observed mortality exceeds expectation) met this threshold, further establishing that Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins do not result in biologically relevant synergistic effects. Therefore, hazard assessments (bioassays) were conducted with individual test substances and were considered informative for this ERA.

Table 3.

Combined potency of the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins on soybean looper (chrysodeixis includens).

	Expected Mortality	Observed Mortality	Clopper-Pearson 95% Confidence Interval
Treatment	%			Differenced
Buffer Control Diet	≤ 20a	11.7	4.8 - 22.6	NA
LC50 of Cry1B.34.1	50b	93.3	83.8 - 98.2	1.9×
LC50 of Cry1B.61.1	50b	43.1	30.2 - 56.8	0.86×
LC50 of IPD083Cb	50b	86.4	75.0 - 94.0	1.7×
1/3 the LC50 of each protein active	50c	76.3	63.4 - 86.4	1.5×
5× dilution of 1/3 the LC50 of each protein active	< 50	13.3	5.9 - 24.6	> 0.27×
10× dilution of 1/3 the LC50 of each protein active	< 50	13.3	5.9 - 24.6	> 0.27×

^aThe bioassay acceptability criteria are dead and missing organism count ≤ 20%.

^bMedian lethal concentration (LC₅₀) values for Cry1B.61.1, Cry1B.34.1, and IPD083Cb proteins were derived from^42,49 and⁵⁰ respectively.

^cMortality estimated using the dose addition model.⁵¹

^dMagnitude of difference between the expected and observed mortality.

NA – Not applicable.

2.2.4. Effects of Cry1B.34.1, Cry1B.61.1, and IPD083Cb Proteins on Surrogate NTOs

All laboratory effects studies (bioassays) followed Good Laboratory Practice regulations provided in EPA 40 CFR part 160⁵⁴ and were conducted with diet-incorporation methodology unless stated otherwise. Details of each bioassay are summarized in Tables 4–6. Insect bioassays were conducted in small environmental chambers, with temperatures ranging from 20–35°C (depending on the surrogate non-target insect of interest), light maintained at either continuously dark or with a 16-hour light:8-hour dark photoperiod, and relative humidity maintained at 65% (except for honey bees which varied between 50–100% depending on life stage). Quail and mouse bioassays were conducted in test rooms, with temperatures ranging from 20–25.3°C, light maintained at either 8-hour light:16-hour dark, 10-hour light:14-hour dark, or 12-hour light:12-hour dark, and relative humidity ranging from 18–82%. Most insect bioassays were initiated with neonates under 30-h old unless stated otherwise, while the quail and mouse bioassays were initiated with young adults (Tables 4–6). In all bioassays, the Cry1B.34.1 (or Cry1B.34 for some bioassays), Cry1B.61.1, or IPD083Cb protein concentrations in diets (vehicle or solution in some bioassays) were characterized to confirm stability, homogeneity in diet, and concentration. In all bioassays, the maximum Cry1B.34.1 (or Cry1B.34 for some bioassays), Cry1B.61.1, or IPD083Cb protein concentration or dose in diets, vehicle, or solution exceeded the relevant worst-case EECs by at least 10 whenever possible. Insect bioassays were based on wet weight or dry weight concentrations, depending on the nature of the test diet or solution used during the bioassays. Depending on the bioassay, a fresh diet was supplied daily, every other day, every three to four days, or as needed to maximize exposure of the surrogate NTOs to bioactive Cry1B.34.1 (or Cry1B.34 for some bioassays), Cry1B.61.1, or IPD083Cb protein. The duration of each bioassay exceeded the time (within three to four days; data not shown) known for each protein to cause mortality to a sensitive organism. In each study, appropriate positive controls (toxic reference substance) were included to verify exposure of each surrogate NTO (apart from northern bobwhite quail and mouse) to the Cry1B.34.1 (or Cry1B.34 for some bioassays), Cry1B.61.1, or IPD083Cb protein in the test diet or solution (Tables 4–6). Depending on the surrogate NTO, a negative control, consisting of diet, vehicle, solution, or comparative protein control (e.g., bovine serum albumin) without either the Cry1B.34.1 (or Cry1B.34 for some bioassays), Cry1B.61.1, or IPD083Cb protein, was included in each study to assess bioassay performance. For the study to be valid and acceptable, mortality had to be either ≤ 10%, ≤ 15%, or ≤ 20% (depending on the bioassay) in the negative control over a six-day duration or at study termination (depending on the bioassay) and > 80% in the positive control (except for honey bee bioassays where mortality acceptability criteria were set at ≥ 50%) for Cry1B.34.1 (or Cry1B.34 for some bioassays), Cry1B.61.1, or IPD083Cb (Tables 4–6). To demonstrate the bioactivity of the Cry1B.34.1 (or Cry1B.34 for some bioassays), Cry1B.61.1, or IPD083Cb protein in test diet or solution, a portion of the test diet or solution that each surrogate NTO was fed or immersed in was used to prepare a diet fed to a sensitive organism (soybean looper, fall armyworm - Spodoptera frugiperda, or codling moth - Cydea pomonella, depending on the bioassay). Depending on the surrogate NTO of interest, relevant endpoints, viz. survival, pupation, days to adult emergence, weight, reproduction, food consumption, behavioral changes, and evidence of acute oral toxicity, were assessed (Tables 7–9). For each surrogate NTO, the no-observed-effect-concentration (NOEC) or the no-observed-effect-dose (NOED) was determined for relevant endpoints for each protein (Cry1B.34.1, Cry1B.61.1, or IPD083Cb). The NOEC or NOED for every bioassay where Cry1B.34 was used was adjusted for molecular weight by multiplying the NOEC or NOED value by 0.584 to determine the NOEC or NOED value for Cry1B.34.1.

Table 4.

Summary of tier I laboratory study design used to assess the toxicity of Cry1B.34.1 (or Cry1B.34) on non-target organisms.

Surrogate Test Species	Guideline	Concentration or Dosea	Positive Control (Mortality Acceptability Criteria)	Negative Control (Mortality Acceptability Criteria)	Exposure Route	Life Stage at Initiation	Sample Size	Environmental Conditionsb	Test Duration (Days)
Honey bee larvae (A. mellifera)	OECD Document No. 23955	3800 and 7500 ng Cry1B.34.1/larva/day	Yes1 (≥ 50%)	Yes (≤ 15%)	Artificial diet	Neonates (≤ 30 hours old)	36 replicates per treatment	33 ± 2°C; 50–100% RH; 0hL:24hDc	22
Honey bee adult (A. mellifera)	OECD Document No. 24556	6500 and 14,000 ng Cry1B.34.1/bee/day	Yes1 (≥ 50%)	Yes ( < 15%)	Artificial diet	Adults (≤ 2 days post-emergence)	30 replicates per treatment	33 ± 2°C; 50–70% RH; 0hL:24hD	10
Springtail (F. candida)	None	125, 250, 500, and 1000 ng Cry1B.34/mg diet	Yes2 ( > 80%)	Yes (≤ 20%)	Artificial diet	Adults (10 days old)	80 replicates per treatment	20°C; 65% RH; 0hL:24hD	28
Daphnia (D. magna)	None	3, 15, and 30 mg Cry1B.34/L water	Yes3 ( > 80%)	Yes (≤ 20%)	Hard reconstituted water	Neonates (≤ 24 hours old)	20 replicates per treatment	20°C; 65% RH; 16hL:8hD	21
Green lacewing (C. rufilabris)	None	2000 and 8000 ng Cry1B.34/mg diet	Yes4 ( > 80%)	Yes (≤ 20%)	Artificial diet	Neonates (≤ 24 hours old)	40 replicates per treatment	25°C; 65% RH; 16hL:8hD	19
Pink spotted lady beetle (C. maculata)	None	2000 and 8000 ng Cry1B.34/mg diet	Yes4 ( > 80%)	Yes (≤ 20%)	Artificial diet	Neonates (≤ 24 hours old)	30 replicates per treatment	27°C; 65% RH; 16hL:8hD	19
Parasitic hymenoptera (P. foveolatus)	None	2000 and 8000 µg Cry1B.34/ml diet	Yes5 ( > 80%)	Yes (≤ 20%)	Sucrose diet	Adults (≤ 48 hours post-emergence)	30 replicates per treatment	25°C; 65% RH; 16hL:8hD	7
Northern bobwhite quail (C. virginianus)	OCSPP 850.210057	1700 mg Cry1B.34/kg body weight	No	Yes6 (≤ 10%)	Oral gavage using gelatin capsule	Young adults (27 weeks and 6 days)	10 replicates (5 males and 5 females) per treatment	20.1–24.4°C; 64.3–79.9% RH; 10hL:14hD	14
Mouse (M. musculus)	OECD Section 4 Part 42358d	5000 mg Cry1B.34/kg body weight	No	Yes7	Oral gavage	Young adults (7 weeks old)	12 replicates (6 males and 6 females) per treatmente	20–25°C; 30–70% RH; 12hL:12hD	15

¹ Dimethoate; ² Teflubenzuron; ³ Potassium Chloride; ⁴ Cryolite; ⁵ Boric acid; ⁶ Bovine serum albumin (BSA; administered at an equivalent target dose to that of the test substance); ⁷ Vehicle control (deionized water) and BSA control (administered at an equivalent target dose to that of the test substance).

^aValues are reported in active ingredient of the test substance.

^bRH, Relative humidity; hL:HD, hour light:hour dark.

^cLarval phase, 33 ± 2°C and ≥90% RH; Pupal phase, 33 ± 2°C and 50–85% RH.

^dThe mouse acute oral toxicity test followed OECD guidelines for testing chemicals section 4 (part 423) with the following exceptions: 6 males and 6 females were used per treatment; in addition to the nominal dose level tested, treatments also included a vehicle control (deionized water) and BSA control (administered at an equivalent target dose to that of the test substance).

^eMale and female (nulliparous and non-pregnant) Crl:CD1 (ICR) mice were used in the study.

Table 5.

Summary of tier I laboratory study design used to assess the toxicity of Cry1B.61.1 on non-target organisms.

Surrogate Test Species	Guideline	Concentration or Dosea	Positive Control (Mortality Acceptability Criteria)	Negative Control (Mortality Acceptability Criteria)	Exposure Route	Life Stage at Initiation	Sample Size	Environmental Conditionsb	Test Duration (Days)
Honey bee larvae (A. mellifera)	OECD Document No. 23955	3000 and 6000 ng Cry1B.61.1/larva/day	Yes1 (≥ 50%)	Yes (≤ 15%)	Artificial diet	Neonates (≤ 30 hours old)	36 replicates per treatment	33 ± 2°C; 50–100% RH; 0hL:24hDc	22
Honey bee adult (A. mellifera)	OECD Document No. 24556	6100 and 12,000 ng Cry1B.61.1/bee/day	Yes1 (≥ 50%)	Yes ( < 15%)	Artificial diet	Adults (≤ 2 days post-emergence)	30 replicates per treatment	33 ± 2°C; 50–70% RH; 0hL:24hD	10
Springtail (F. candida)	None	1500 and 3000 ng Cry1B.61.1/mg diet	Yes2 ( > 80%)	Yes (≤ 20%)	Artificial diet	Adults (10 days old)	80 replicates per treatment	20°C; 65% RH; 0hL:24hD	28
Daphnia (D. magna)	None	1 and 3 mg Cry1B.61.1/L water	Yes3 ( > 80%)	Yes (≤ 20%)	Moderately Hard reconstituted water	Neonates (≤ 24 hours old)	20 replicates per treatment	20°C; 65% RH; 16hL:8hD	21
Green lacewing (C. rufilabris)	None	8000 and 16,000 ng Cry1B.61.1/mg diet	Yes4 ( > 80%)	Yes (≤ 20%)	Artificial diet	Neonates (≤ 24 hours old)	30 replicates per treatment	25°C; 65% RH; 16hL:8hD	21
Pink spotted lady beetle (C. maculata)	None	8000 and 16,000 ng Cry1B.61.1/mg diet	Yes4 ( > 80%)	Yes (≤ 20%)	Artificial diet	Neonates (≤ 24 hours old)	30 replicates per treatment	27°C; 65% RH; 16hL:8hD	17
Parasitic hymenoptera (P. foveolatus)	None	8000 and 16,000 µg Cry1B.61.1/ml diet	Yes5 ( > 80%)	Yes (≤ 20%)	Sucrose diet	Adults (≤ 48 hours post-emergence)	30 replicates per treatment	25°C; 65% RH; 16hL:8hD	7
Northern bobwhite quail (C. virginianus)	OECD Document No. 22359	2000 mg Cry1B.61.1/kg body weight	No	Yes6 (≤ 10%)d	Oral gavage using gelatin capsule	Young adults (27 weeks old)	5 replicates (3 males and 2 females) per treatment	21.5–25.3°C; 18–69% RH; 8hL:16hD	14
Mouse (M. musculus)	OECD Section 4 Part 42358e	5000 mg Cry1B.61.1/kg body weight	No	Yes7	Oral gavage	Young adults (7 weeks old)	12 replicates (6 males and 6 females) per treatmentf	20–25°C; 30–70% RH; 12hL:12hD	15

¹ Dimethoate; ² Teflubenzuron; ³ Potassium Chloride; ⁴ Cryolite; ⁵ Boric acid; ⁶ Bovine serum albumin (BSA; administered at an equivalent target dose to that of the test substance); ⁷ Vehicle control (deionized water) and BSA control (administered at an equivalent target dose to that of the test substance).

^aValues are reported in active ingredient of the test substance.

^bRH, Relative humidity; hL:HD, hour light:hour dark.

^cLarval phase, 33 ± 2°C and ≥90% RH; Pupal phase, 33 ± 2°C and 50–85% RH.

^dThe method was designed to minimize the number of birds tested. Thus, an initial limit test with five birds was conducted with a dose level of 2000 mg a.i./kg body weight. If one death is observed and no signs of toxicity are observed in other birds, five more birds may be dosed at the limit. If there is still only one death in the total of 10 birds after this, then the mortality acceptability criteria are met. However, no further testing was required based on the initial limit test results.

^eThe mouse acute oral toxicity test followed OECD guidelines for testing chemicals section 4 (part 423) with the following exceptions: 6 males and 6 females were used per treatment; in addition to the nominal dose level tested, treatments also included a vehicle control (deionized water) and BSA control (administered at an equivalent target dose to that of the test substance).

^fMale and female (nulliparous and non-pregnant) Crl:CD1 (ICR) mice were used in the study.

Table 6.

Summary of tier I laboratory study design used to assess the toxicity of IPD083Cb on non-target organisms.

Surrogate Test Species	Guideline	Concentration or Dosea	Positive Control (Mortality Acceptability Criteria)	Negative Control (Mortality Acceptability Criteria)	Exposure Route	Life Stage at Initiation	Sample Size	Environmental Conditionsb	Test Duration (Days)
Honey bee larvae (A. mellifera)	OECD Document No. 23955	2500, 5000, and 7500 ng IPD083Cb/larva/day	Yes1 (≥ 50%)	Yes (≤ 15%)	Artificial diet	Neonates (≤ 30 hours old)	36 replicates per treatment	33 ± 2°C; 50–100% RH; 0hL:24hDc	22
Honey bee adult (A. mellifera)	OECD Document No. 24556	13,000, 13,000, and 19,000 ng IPD083Cb/bee/day	Yes1 (≥ 50%)	Yes ( < 15%)	Artificial diet	Adults (≤ 1 day post-emergence)	30 replicates per treatment	33 ± 2°C; 50–70% RH; 0hL:24hD	10
Springtail (F. candida)	None	5000, 10,000, and 20,000 ng IPD083Cb/mg diet	Yes2 ( > 80%)	Yes (≤ 20%)	Artificial diet	Adults (10–12 days old)	80 replicates per treatment	20°C; 65% RH; 0hL:24hD	28
Daphnia (D. magna)	None	1 and 3 mg IPD083Cb/L water	Yes3 ( > 80%)	Yes (≤ 20%)	Hard reconstituted water	Neonates (≤ 24 hours old)	20 replicates per treatment	20°C; 65% RH; 16hL:8hD	21
Green lacewing (C. rufilabris)	None	5000, 10,000, and 20,000 ng IPD083Cb/mg diet	Yes4 ( > 80%)	Yes (≤ 20%)	Artificial diet	Neonates (≤ 24 hours old)	30 replicates per treatment	25°C; 65% RH; 16hL:8hD	21
Pink spotted lady beetle (C. maculata)	None	5000, 10,000, and 20,000 ng IPD083Cb/mg diet	Yes4 ( > 80%)	Yes (≤ 20%)	Artificial diet	Neonates (≤ 24 hours old)	30 replicates per treatment	27°C; 65% RH; 16hL:8hD	20
Parasitic hymenoptera (P. foveolatus)	None	5000, 10,000, and 20,000 µg IPD083Cb/ml diet	Yes5 ( > 80%)	Yes (≤ 20%)	Sucrose diet	Adults (≤ 48 hours post-emergence)	30 replicates per treatment	25°C; 65% RH; 16hL:8hD	7
Northern bobwhite quail (C. virginianus)	OECD Document No. 22359	2000 mg IPD083Cb/kg body weight	No	Yes6 (≤ 10%)d	Oral gavage using gelatin capsule	Adults (49 weeks old)	5 replicates (3 males and 2 females) per treatment	20.4–21.8°C; 61–82% RH; 8hL:16hD	14
Mouse (M. musculus)	OECD Section 4 Part 42358	5000 mg IPD083Cb/kg body weight	No	Yes7	Oral gavage	Young adults (7 weeks old)	12 replicates (6 males and 6 females) per treatmentf	20–25°C; 30–70% RH; 12hL:12hD	15

¹ Dimethoate; ² Teflubenzuron; ³ Potassium Chloride; ⁴ Cryolite; ⁵ Boric acid; ⁶ Bovine serum albumin (BSA; administered at an equivalent target dose to that of the test substance); ⁷ Vehicle control (deionized water) and BSA control (administered at an equivalent target dose to that of the test substance).

^aValues are reported in active ingredient of the test substance.

^bRH, Relative humidity; hL:HD, hour light:hour dark.

^cLarval phase, 33 ± 2°C and ≥90% RH; Pupal phase, 33 ± 2°C and 50–85% RH.

^dThe method was designed to minimize the number of birds tested. Thus, an initial limit test with five birds was conducted with a dose level of 2000 mg a.i./kg body weight. If one death is observed and no signs of toxicity are observed in other birds, five more birds may be dosed at the limit. If there is still only one death in the total of 10 birds after this, then the mortality acceptability criteria are met. However, no further testing was required based on the initial limit test results.

^eThe mouse acute oral toxicity test followed OECD guidelines for testing chemicals section 4 (part 423) with the following exceptions: 6 males and 6 females were used per treatment; in addition to the nominal dose level tested, treatments also included a vehicle control (deionized water) and BSA control (administered at an equivalent target dose to that of the test substance).

^fMale and female (nulliparous and non-pregnant) Crl:CD1 (ICR) mice were used in the study.

Table 7.

Summary of exposure, hazard, and risk assessments of Cry1B.34.1 to non-target organisms.

	Exposure Assessment (EEC)a		Hazard Assessment			Risk Assessment (MOE)c
Surrogate Test Species	Worst-case EEC	Refined EEC	Endpoints Assessed	Results	NOEC or NOEDb	MOE Based on Worst-case EEC	MOE Based on Refined EEC
Honey bee larvae (A. mellifera)	640 ng/larvad	NA	larval survival, pupal survival, adult emergence, adult weight at emergence	No effects on larval survival, pupal survival, adult emergence, or adult weight at emergence were observed	7500 ng Cry1B.34.1/larva/day	11.7×	NA
Honey bee adult (A. mellifera)	1376 ng/adult/dayd	NA	survival, adult body weight	No effects on adult body weight or survival were observed	14,000 ng Cry1B.34.1/bee/day	10.2×	NA
Springtail (F. candida)	180 ng/mg	150 ng/mg	survival, reproduction	No adverse effects on F. candida reproduction and survival were observed	584 ng Cry1B.34.1/mg diete	3.2×	3.9×
Daphnia (D. magna)	0.654 mg/L	NA	survival, reproduction	D. magna exposed to 15 mg Cry1B.34 protein/L showed no statistically significant adverse effects in mortality or mean number of offspring produced, as compared with the negative control	8.8 mg Cry1B.34.1/L watere	13.5×	NA
Green lacewing (C. rufilabris)	960 ng/mg	460 ng/mg	survival, pupation rate, number of days to pupation	No adverse effects on survival or pupation of C. rufilabris were observed	4672 ng Cry1B.34.1/mg diete	4.9×	10.2×
Pink spotted lady beetle (C. maculata)	960 ng/mg	460 ng/mg	Survival, weight, number of days to adult emergence	No adverse effects on survival, weight, or number of days to adult emergence of C. maculata were observed	4672 ng Cry1B.34.1/mg diete	4.9×	10.2×
Parasitic hymenoptera (P. foveolatus)	960 ng/mg	460 ng/mg	Survival	No adverse effects on the survival of P. foveolatus were observed	4672 µg Cry1B.34.1/ml diete	4.9×	10.2×
Northern bobwhite quail (C. virginianus)	960 ng/mg	460 ng/mg	Survival, body weight, food consumption, behavioral changes	No mortality, abnormal behavior, or signs of toxicity were observed	992.8 mg Cry1B.34.1/kg body weighte	1.0×	2.2×
Mouse (M. musculus)	69.3 mg/kgf	NA	Survival, evidence of acute oral toxicity (based on evaluation of body weight, clinical signs, and gross pathology)	No mortality or other evidence of acute oral toxicity (based on evaluation of body weight, clinical signs, and gross pathology) was observed	2920 mg Cry1B.34.1/kg body weighte	42.1×	NA

^aEEC – Estimated environmental concentration.

^bNOEC or NOED – No observed effect concentration (for all other surrogate test species) or no observed effect dose (for A. mellifera larvae).

^cMOE – Margin of exposure.

^dHoney bee larvae were presumed to ingest 2.0 mg of soybean pollen throughout larval development.²² Honey bee adults were presumed to ingest 4.3 mg of soybean pollen daily.²³

^eCry1B.34 was used for this bioassay, and subsequent NOEC or NOED was determined; however, it was adjusted for molecular weight by multiplying the NOEC or NOED value by 0.584 to determine the NOEC or NOED for Cry1B.34.1.

^fThe worst-case FIR/bw ratio for seed-eating rodents previously determined for the harvest mouse (Micromys minutus) is 0.33²⁶ and was used to calculate the worst-case EEC.

NA, Not applicable – No refinement to worst-case EEC was considered, and MOE based on refined EEC was not determined as a result.

Table 8.

Summary of exposure, hazard, and risk assessments of Cry1B.61.1 to non-target organisms.

	Exposure Assessment (EEC)a		Hazard Assessment			Risk Assessment (MOE)c
Surrogate Test Species	Worst-case EEC	Refined EEC	Endpoints Assessed	Results	NOEC or NOEDb	MOE Based on Worst-case EEC	MOE Based on Refined EEC
Honey bee larvae (A. mellifera)	380 ng/larvad	NA	larval survival, pupal survival, adult emergence, adult weight at emergence	No effects on larval survival, pupal survival, adult emergence, or adult weight at emergence were observed	6000 ng Cry1B.61.1/larva/day	15.8×	NA
Honey bee adult (A. mellifera)	817 ng/adult/dayd	NA	survival, adult body weight	No effects on adult body weight or survival were observed	12,000 ng Cry1B.61.1/bee/day	14.7×	NA
Springtail (F. candida)	390 ng/mg	200 ng/mg	survival, reproduction	No adverse effects on F. candida reproduction and survival were observed	3000 ng Cry1B.61.1/mg diet	7.7×	15.0×
Daphnia (D. magna)	1.42 mg/L	0.727 mg/L	survival, reproduction	No adverse effects on D. magna reproduction or survival were observed	3 mg Cry1B.61.1/L water	2.1×	4.1×
Green lacewing (C. rufilabris)	1300 ng/mg	NA	survival, pupation rate, number of days to pupation	No biologically relevant adverse effects on survival or pupation of C. rufilabris were observed at 16,000 ng Cry1B.61.1/mg diet	16,000 ng Cry1B.61.1/mg diet	12.3×	NA
Pink spotted lady beetle (C. maculata)	1300 ng/mg	NA	Survival, weight, number of days to adult emergence	No adverse effects on survival, weight, or number of days to adult emergence of C. maculata were observed	16,000 ng Cry1B.61.1/mg diet	12.3×	NA
Parasitic hymenoptera (P. foveolatus)	1300 ng/mg	NA	Survival	No adverse effects on the survival of P. foveolatus were observed	16,000 µg Cry1B.61.1/ml diet	12.3×	NA
Northern bobwhite quail (C. virginianus)	1300 ng/mg	490 ng/mg	Survival, body weight, food consumption, behavioral changes	No mortality, abnormal behavior, or signs of toxicity were observed	2000 mg Cry1B.61.1/kg body weight	1.5×	4.1×
Mouse (M. musculus)	7.3 mg/kge	NA	Survival, evidence of acute oral toxicity (based on evaluation of body weight, clinical signs, and gross pathology)	No mortality or other evidence of acute oral toxicity (based on evaluation of body weight, clinical signs, and gross pathology) was observed	5000 mg Cry1B.61.1/kg body weight	684.9×	NA

^aEEC – Estimated environmental concentration.

^bNOEC or NOED – No observed effect concentration (for all other surrogate test species) or no observed effect dose (for A. mellifera larvae).

^cMOE – Margin of exposure.

^dHoney bee larvae were presumed to ingest 2.0 mg of soybean pollen throughout larval development.²² Honey bee adults were presumed to ingest 4.3 mg of soybean pollen daily.²³

^eThe worst-case FIR/bw ratio for seed-eating rodents previously determined for the harvest mouse (Micromys minutus) is 0.33²⁶ and was used to calculate the worst-case EEC.

NA, Not applicable – No refinement to worst-case EEC was considered, and MOE based on refined EEC was not determined as a result.

Table 9.

Summary of exposure, hazard, and risk assessments of IPD083Cb to non-target organisms.

	Exposure Assessment (EEC)a		Hazard Assessment			Risk Assessment (MOE)c
Surrogate Test Species	Worst-case EEC	Refined EEC	Endpoints Assessed	Results	NOEC or NOEDb	MOE Based on Worst-case EEC	MOE Based on Refined EEC
Honey bee larvae (A. mellifera)	176 ng/larvad	NA	larval survival, pupal survival, adult emergence, adult weight at emergence	Exposure to 5000 ng IPD083Cb/larva/day had no effects on larval survival, pupal survival, adult emergence, or adult weight at emergence	5000 ng IPD083Cb/larva/day	28.4×	NA
Honey bee adult (A. mellifera)	378.4 ng/adult/dayd	NA	survival, adult body weight	No effects on adult body weight or survival were observed	19,000 ng IPD083Cb/bee/day	50.2×	NA
Springtail (F. candida)	84 ng/mg	NA	survival, reproduction	No adverse effects on F. candida reproduction and survival were observed	20,000 ng IPD083Cb/mg diet	238.1×	NA
Daphnia (D. magna)	0.305 mg/L	0.214 mg/L	survival, reproduction	No adverse effects on D. magna reproduction or survival were observed	3 mg IPD083Cb/L water	9.8×	14.0×
Green lacewing (C. rufilabris)	130 ng/mg	NA	survival, pupation rate, number of days to pupation	No adverse effects on survival or pupation of C. rufilabris were observed	20,000 ng IPD083Cb/mg diet	153.8×	NA
Pink spotted lady beetle (C. maculata)	130 ng/mg	NA	Survival, weight, number of days to adult emergence	No adverse effects on survival, weight, or number of days to adult emergence of C. maculata were observed	20,000 ng IPD083Cb/mg diet	153.8×	NA
Parasitic hymenoptera (P. foveolatus)	130 ng/mg	NA	Survival	No adverse effects on the survival of P. foveolatus were observed	20,000 µg IPD083Cb/ml diet	153.8×	NA
Northern bobwhite quail (C. virginianus)	130 ng/mg	NA	Survival, body weight, food consumption, behavioral changes	No mortality, abnormal behavior, or signs of toxicity were observed	2000 mg IPD083Cb/kg body weight	15.4×	NA
Mouse (M. musculus)	6.3 mg/kge	NA	Survival, evidence of acute oral toxicity (based on evaluation of body weight, clinical signs, and gross pathology)	No mortality or other evidence of acute oral toxicity (based on evaluation of body weight, clinical signs, and gross pathology) was observed	5000 mg IPD083Cb/kg body weight	793.6×	NA

^aEEC – Estimated environmental concentration.

^bNOEC or NOED – No observed effect concentration (for all other surrogate test species) or no observed effect dose (for A. mellifera larvae).

^cMOE – Margin of exposure.

^dHoney bee larvae were presumed to ingest 2.0 mg of soybean pollen throughout larval development.²² Honey bee adults were presumed to ingest 4.3 mg of soybean pollen daily.²³

^eThe worst-case FIR/bw ratio for seed-eating rodents previously determined for the harvest mouse (Micromys minutus) is 0.33²⁶ and was used to calculate the worst-case EEC.

NA, Not applicable – No refinement to worst-case EEC was considered, and MOE based on refined EEC was not determined as a result.

2.3. Data Analyses

Data collected during each bioassay (excluding honey bee, Northern bobwhite quail, and mouse bioassays) were statistically analyzed using SAS version 9.4.⁶⁰ Statistical analysis of survival (or mortality) and/or pupation rate data was conducted using Fisher’s Exact Test (SAS PROC MULTTEST) to determine if the survival and/or pupation rate of insects fed diets (or immersed in a test solution) containing Cry1B.34.1 (or Cry1B.34 for some bioassays), Cry1B.61.1, or IPD083Cb protein treatments were less than the survival and/or pupation rate of those fed (or immersed in) the negative control treatment included in each study. The statistical analysis procedures used to assess the number of days to pupation (or adult emergence) and/or weight of surviving insects were dependent upon the validity of statistical assumptions for each data set. For some bioassays, the normality assumption was satisfied by the number of days to pupation (or adult emergence) data, and an analysis of variance (SAS PROC GLIMMIX) was conducted as a result to assess if the test diet caused a developmental delay. In instances where the normality assumption was not satisfied by the number of days to pupation (or adult emergence) data, SAS PROC NPAR1WAY was used to conduct the Wilcoxon rank-sum test to determine if exposure to the test diet caused a developmental delay compared to exposure to the negative control diet included in each study. For each applicable bioassay, the normality assumption was satisfied by the weight data; thus, an analysis of variance was conducted to assess if the test diet caused growth inhibition. For reproduction data collected during springtail and daphnia bioassays, a generalized linear mixed model (SAS PROC GLIMMIX) was fit to the reproduction data assuming a Poisson distribution of the number of offspring, a log link function, and the Laplace method of integral approximation. Treatment was considered fixed effect, while jar (for springtail) or cup number (for daphnia) nested within each treatment and block was considered random effect. The estimated model was used to test if the reproduction from the adults fed the artificial insect diet (or immersed in a test solution) containing Cry1B.34.1 (or Cry1B.34 for some bioassays), Cry1B.61.1, or IPD083Cb protein was less than the reproduction from the adults fed the negative control diet (or immersed in negative control test solution). For each insect bioassay, treatment differences for all data were considered significant at the 5% probability level (p < .05).

All data collected during the honey bee (larvae and adults) bioassay were subjected to statistical analysis using CETIS version 1.9.7⁶¹ to compare Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein treatments with the negative control treatment included in each study. For Cry1B.34.1 and Cry1B.61.1 survival (larval, pupal, and adult) and adult emergence data, Fisher’s Exact Test with Bonferroni-Holm’s Adjustment was used for comparison. The statistical analysis of IPD083Cb survival and adult emergence data followed the same approach as Cry1B.34.1 and Cry1B.61.1, the lone exception being that Cochran-Armitage’s Step-Down Test was used for comparison of larval survival data. Weight data (pupal (adult weight at emergence) and adult) were first evaluated by conducting Shapiro-Wilk’s Test to assess normality of the distribution and Bartlett’s Test to assess homogeneity of variance, and then the results of these tests were used to select the statistical method used in comparisons and determination of NOEC, NOED, lowest-observed-effect-concentration (LOEC), or lowest-observed-effect-dose (LOED) values. For Cry1B.34.1 and Cry1B.61.1 pupal and adult weights, Dunnett’s multiple comparison test was used for comparisons. For IPD083Cb, Wilcoxon’s Test with Bonferroni-Holm’s Adjustment was used for the comparison of pupal weight data, while Jonckheere-Terpstra’s Step-Down Test was used for the comparison of adult weight data. All comparisons for determination of a NOEC, NOED, LOEC, or LOED were made at ≥ 95% level of certainty (p < .05) and compared on a per replicate (individual well or bee) basis.

Data collected during the Northern bobwhite quail bioassays were analyzed using SEDEC version 1.3⁶² or SAS version 9.4.⁶⁰ For Cry1B.34.1 (or Cry1B.34), data were checked to determine if statistical assumptions were met. After confirming that data met statistical assumptions (Shapiro-Wilk’s Test was used to assess normality of the distribution while the F-test was used to assess homogeneity of variance), appropriate t-tests were conducted to determine whether there were differences in pre-treatment body weight between sexes (male vs. female) and between treatments (negative control vs. test). Mean measured body weights, calculated body weight change, and weekly feed consumption per bird per day were similarly analyzed at the end of the study. For Cry1B.61.1 and IPD083Cb, data were subjected to descriptive statistics (mean, standard deviation, and coefficient of variation). For all Northern bobwhite quail bioassays, the nominal oral limit dose levels tested and corresponding mortality data derived from the toxicity test were used to empirically assess whether the LD₅₀ and the NOEC were greater or less than the limit dose level tested. All data collected during the mouse bioassays were subjected to descriptive statistics, and the nominal oral limit dose levels tested and corresponding mortality data were used to inform whether the LD₅₀ and the NOEC were greater or less than the limit dose level tested.

2.4. Risk Assessment

The risk assessment for NTOs exposed to COR23134 soybean was based on the hypothesis that Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins are not toxic or harmful at the EEC or exposure present in the field. This hypothesis was tested by comparing the NOEC or NOED obtained from the Tier 1 laboratory effects studies with the worst-case or refined EECs to determine the margin of exposure (MOE). If the ratio of NOEC or NOED to EEC (NOEC or NOED/EEC) is greater than or equal to 10 (the value that indicates concern in environmental risk assessment and routinely used to gauge the need for either additional hazard or exposure assessment⁶³) for all functional groups of NTOs, the risk posed by exposure of the NTOs to Cry1B.34.1, Cry1B.61.1, and IPD083Cb from cultivation of COR23134 soybean was considered negligible. In cases where calculated MOEs based on worst-case EECs were less than 10, refined EECs were used instead to reflect more realistic environmental conditions and ecological processes that reduce actual NTO exposure in the field. All MOE calculations were conducted using 1. Dry weight-based EECs of the Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein from COR23134 soybean tissues. The dry weight concentrations are considered high estimates of the expressed protein since, in reality, NTOs would be exposed to levels comparable to fresh weight levels; and 2. Either wet weight- (as used in the honey bee, green lacewing, and parasitic hymenoptera bioassays) or dry weight-based (as used in other insect bioassays) NOEC or NOED for Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein, depending on the nature of the test diet or solution used during the bioassays.

3. Results and Discussion

All bioassay studies for each protein (Cry1B.34/Cry1B.34.1, Cry1B.61.1, or IPD083Cb) met the prescribed mortality acceptability criteria for positive and negative controls.

3.1. Pollinators and Pollen Feeders

The amount of pollen consumed by one honey bee larva has previously been estimated to be 1.5–2.0 mg over the course of larval development.²² Based on the maximum concentrations of Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins in COR23134 soybean flower (representing pollen) and the assumption that one honey bee larva consumes the maximum amount of soybean pollen (2.0 mg) throughout larval development, the worst-case EECs for honey bee larvae were determined to be 640 ng Cry1B.34.1/larva, 380 ng Cry1B.61.1/larva, and 176 ng IPD083Cb/larva (Tables 7–9). For all endpoints assessed (larval or pupal survival, adult emergence, or adult weight at emergence), no adverse effects were observed on honey bees fed diets containing Cry1B.34.1 or Cry1B.61.1 protein at any of the dose levels tested compared to the negative control (Tables 7, 8; Supplemental Tables S1, S2). While the consumption of diets containing IPD083Cb protein did not affect honey bee pupal survival at any of the dose levels tested when compared to the negative control, a significant reduction in larval survival was observed in honey bees fed a diet containing 7500 ng IPD083Cb/larva/day (the highest dose level tested) compared with the negative control (Table 9; Supplemental Table S3). However, no effect on honey bee larval survival was observed at the 2500 ng IPD083Cb/larva/day or 5000 ng IPD083Cb/larva/day dose level when compared to the negative control (Table 9; Supplemental Table S3). Due to the significant reduction in larval survival at the 7500 ng IPD083Cb/larva/day, this dose level was excluded from the remaining statistical comparison (adult emergence and adult weight at emergence). Neither adult emergence nor adult weight at emergence was affected when honey bee larvae were fed diets containing 2500 ng IPD083Cb/larva/day or 5000 ng IPD083Cb/larva/day compared to the negative control (Table 9; Supplemental Table S3). The NOEDs for honey bee larvae were determined to be 7500 ng Cry1B.34.1/larva/day, 6000 ng Cry1B.61.1/larva/day, and 5000 ng IPD083Cb/larva/day, which is equivalent to cumulative doses of 30,000 ng Cry1B.34.1/larva, 24,000 ng Cry1B.61.1/larva, and 20,000 ng IPD083Cb/larva (Tables 7–9) since larvae were fed for four days. Based on the daily NOEDs, the MOE for honey bee larvae exposed to Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein in COR23134 soybean flower (representing pollen) was 11.7×, 15.8×, or 28.4× the worst-case EEC, respectively (Tables 7–9). Daily NOEDs were preferred over cumulative NOEDs, in this case, to achieve an extremely conservative MOE; despite this, the MOEs for honey bee larvae exposed to the insecticidal proteins in COR23134 soybean flower (representing pollen) were still above 10× the worst-case exposure.

Similarly, honey bee adults have previously been estimated to consume 3.4–4.3 mg pollen per day.²³ Based on the maximum concentrations of Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins in COR23134 soybean flower (representing pollen) and the assumption that honey bee adults consume the maximum amount of soybean pollen (4.3 mg) per day, the worst-case EECs for honey bee adults were determined to be 1376 ng Cry1B.34.1/adult/day, 817 ng Cry1B.61.1/adult/day, and 378.4 ng IPD083Cb/adult/day (Tables 7–9). No adverse effects were detected on honey bee adults fed diets containing Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein at any of the concentration levels tested compared to the negative control for any of the measured endpoints (survival or adult body weight; Tables 7–9; Supplemental Tables S1–S3). The NOECs for honey bee adults were determined to be 14,000 ng Cry1B.34.1/bee/day, 12,000 ng Cry1B.61.1/bee/day, and 19,000 ng IPD083Cb/bee/day (Tables 7–9). Based on these NOECs, the MOE for honey bee adults exposed to Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein in COR23134 soybean flower (representing pollen) was 10.2×, 14.7×, or 50.2× the worst-case EEC, respectively (Tables 7–9).

Refined EECs were not estimated for honey bees (larvae and adults) due to the high MOEs based on worst-case assumptions (Tables 7–9). Notwithstanding, several factors would further reduce the exposure of honey bees (larvae and adults) to the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins in COR23124 soybean pollen under a more realistic environmental scenario. Because pollen is the primary food source, honey bees are found in habitats where abundant supplies of flowering plants are present, including agricultural fields during flowering, and may forage over long distances (up to 6–8 miles) in search of pollen and nectar when resources are scarce.^64,65 However, population-level exposure of honey bees to COR23134 soybean pollen is expected to be low because honey bees feed on pollen from a variety of different plant species,^66,67 and soybean pollen, particularly COR23134 soybean pollen, is unlikely to be the only dietary component or food consumed. The aforementioned factors, the worst-case assumptions, and the overall MOE values determined in this ERA for honey bees indicate that the expressed proteins (Cry1B.34.1, Cry1B.61.1, and IPD083Cb) in COR23124 soybean are not expected to result in adverse effects on pollinators and pollen feeders at environmentally realistic concentrations; hence the potential risk of cultivating COR23134 soybean on pollinators and pollen feeders is considered negligible.

Most non-target Lepidoptera do not feed on pollen directly but are indirectly exposed to pollen as they feed on host plants. For instance, Monarch butterfly larvae (Danaus plexippus) use milkweed (Asclepias syriaca L.) as their host plant.⁶⁸ Thus, the degree of potential exposure of non-target Lepidoptera to COR23134 soybean pollen will depend on the presence of host plants in and adjacent to COR23134 soybean fields and the rate of soybean pollen deposition. Typical agronomic weed management practices decrease host plant density within the confines of the soybean field and field margins⁶⁹ and limit the potential exposure of non-target Lepidoptera to COR23134 soybean pollen. Additionally, soybean flower anatomy allows for a high percentage of self-fertilization and limits pollen dispersal,⁷⁰ further reducing the chances of pollen deposition on non-target Lepidoptera host plants. Environmental conditions, such as heat, relative humidity, and ultra-violet radiation, may compromise the integrity of the pollen capsule and impact protein stability in pollen as well.^71,72 The aforementioned factors will drastically reduce the potential exposure of non-target Lepidoptera to the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins via ingestion of COR23134 soybean pollen. As a result, the potential exposure of non-target Lepidoptera to the expressed proteins (Cry1B.34.1, Cry1B.61.1, and IPD083Cb) in COR23134 soybean pollen is considered negligible; hence, minimal risk to non-target Lepidoptera is concluded in this ERA without Tier I laboratory effect or hazard testing of these organisms.

3.2. Soil-Dwelling Organisms

Based on the maximum concentrations of Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins in COR23134 soybean R3 forage tissues, the worst-case EECs for springtails, which represent the soil-dwelling organism functional group in this ERA, were determined to be 180 ng Cry1B.34.1/mg, 390 ng Cry1B.61.1/mg, and 84 ng IPD083Cb/mg (Tables 7–9). Similarly, refined EECs, based on the highest mean concentrations of Cry1B.34.1 and Cry1B.61.1 proteins in COR23134 soybean R3 forage tissues, were determined to be 150 ng Cry1B.34.1/mg and 200 ng Cry1B.61.1/mg (Tables 7 and 8). No refinement to worst-case EEC was considered for IPD083Cb protein for these taxa. For any of the measured endpoints (survival or reproduction), no adverse effects were observed on springtails fed diets containing Cry1B.34 (representing Cry1B.34.1), Cry1B.61.1, or IPD083Cb protein at any of the concentration levels tested compared to the negative control (Tables 7–9; Supplemental Tables S1–S3). The NOECs for springtails were determined to be 584 ng Cry1B.34.1/mg (after adjusting Cry1B.34 for the molecular weight), 3000 ng Cry1B.61.1/mg, and 20,000 ng IPD083Cb/mg (Tables 7–9). Based on these NOECs, the MOE for springtails exposed to Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein in COR23134 soybean forage (representing senescent tissue) was 3.2×, 7.7×, or 238.1× the worst-case EEC, respectively (Tables 7–9). Refinement to worst-case MOE was determined to be 3.9× or 15.0× the exposure for Cry1B.34.1 or Cry1B.61.1, respectively (Tables 7 and 8). Although the estimated MOE was still less than 10× the EEC for Cry1B.34.1 even after refinement, it is important to note that the worst-case and refined EECs for springtails were estimated using the maximum and highest mean dry weight concentrations of Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins in COR23134 soybean R3 forage tissues instead of senescent COR23134 soybean tissues. In reality, senescent COR23134 soybean tissues would likely contain substantially reduced protein concentrations due to tissue degradation, as noted previously. Even at concentration levels that exceed worst-case EECs, Cry1B.34 (representing Cry1B.34.1), Cry1B.61.1, and IPD083Cb proteins have been shown to dissipate rapidly and lose bioactivity against a sensitive organism in diverse soil types^73–75; thus, these proteins are unlikely to persist or accumulate in the field where COR23134 soybean is cultivated. Additionally, Tier I laboratory effect testing of springtails was conducted using concentrations of Cry1B.34 (representing Cry1B.34.1), Cry1B.61.1, and IPD083Cb proteins that exceed anticipated field exposure (worst-case and refined EECs) to maximize the potential for observing and documenting off-target effects. A lack of adverse effects at high-concentration level testing provides sufficient confidence to address uncertainties such that it is reasonable to conclude an overall lack of environmental risk.⁸ None of the measured endpoints were affected when springtails were exposed to diets containing Cry1B.34 (representing Cry1B.34.1), Cry1B.61.1, or IPD083Cb protein at the highest concentration levels tested (1000 ng Cry1B.34/mg diet, 3000 ng Cry1B.61.1/mg diet, and 20,000 ng IPD083Cb/mg diet) when compared to the negative control (Supplemental Tables S1–S3). Furthermore, given the empirically observed spectrum of activity for Cry1B.34 (representing Cry1B.34.1), Cry1B.61.1, and IPD083Cb proteins being within the order Lepidoptera,^41–44 the likelihood for adverse effects on soil-dwelling organisms, resulting from exposure to COR23134 soybean, is very low. Therefore, based on the aforementioned mitigating factors, the worst-case assumptions, the absence of adverse effects at high-concentration level testing, the extremely conservative MOE values, and the spectrum of activity of the insecticidal proteins (Cry1B.34/Cry1B.34.1, Cry1B.61.1, and IPD083Cb) and their stability in soil, the expressed proteins (Cry1B.34.1, Cry1B.61.1, and IPD083Cb) in COR23124 soybean are unlikely to cause adverse effects on soil-dwelling organisms at environmentally realistic concentrations; hence the potential risk of cultivating COR23134 soybean to soil-dwelling organism is considered negligible.

3.3. Aquatic Organisms

The possible exposure of non-target aquatic organisms to PIPs in transgenic crops has been examined previously, with the movement of plant tissue identified as the most likely route of exposure.⁷⁶ The expressed proteins (Cry1B.34.1, Cry1B.61.1, and IPD083Cb) in COR23134 soybean are no different and may also present a potential route of exposure to non-target aquatic organisms via the movement of COR23134 soybean tissues into water bodies close to fields where COR23134 soybean is cultivated. Notwithstanding, the exposure of aquatic organisms to transgenic crops has been shown to be temporally and spatially limited and of negligible concern.⁶³ Following the US EPA standard pond model previously described by Jones et al.,²⁴ the worst-case EECs for aquatic organisms were determined to be 0.654 mg Cry1B.34.1/L, 1.42 mg Cry1B.61.1/L, and 0.305 mg IPD083Cb/L (Tables 7–9). Following a similar approach,²⁴ refined EECs were determined to be 0.727 mg Cry1B.61.1/L and 0.214 mg IPD083Cb/L, respectively (Tables 8 and 9). Refinement to worst-case EEC was not considered for Cry1B.34.1 protein for these taxa. No adverse effects were detected on daphnia (a representative indicator for the aquatic organism functional group) immersed in hard reconstituted water containing Cry1B.61.1 or IPD083Cb protein at any of the concentration levels tested compared to the negative control for survival or reproduction (Tables 8 and 9; Supplemental Tables S2, S3). Although immersion in hard reconstituted water containing 3 mg Cry1B.34/L or 15 mg Cry1B.34/L did not affect daphnia survival or reproduction when compared to the negative control (Table 7; Supplemental Table S1), significant reductions in survival and reproduction were observed in daphnia immersed in 30 mg Cry1B.34/L (the highest concentration level tested) compared with the negative control (Table 7; Supplemental Table S1). The decreased daphnia survival and reproduction observed at 30 mg Cry1B.34/L were not related to the Cry1B.34 protein itself but were likely due to artificial effects arising from the solubility limits of the Cry1B.34 protein. The NOECs for daphnia were determined to be 8.8 mg Cry1B.34.1/L (after adjusting Cry1B.34 for the molecular weight), 3 mg Cry1B.61.1/L, and 3 mg IPD083Cb/L (Tables 7–9). Based on these NOECs, the MOE for daphnia exposed to Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein in COR23134 soybean forage was 13.5×, 2.1×, or 9.8× the worst-case EEC, respectively (Tables 7–9). Refinement to worst-case MOE was determined to be 4.1× or 14.0× the exposure for Cry1B.61.1 or IPD083Cb protein, respectively (Tables 8 and 9). Even though the estimated MOEs were less than 10× the worst-case and refined exposure for Cry1B.61.1, the general lack of adverse effects observed after exposing daphnia, to concentrations of Cry1B.61.1 in excess of very conservative anticipated field exposure, combined with the highly conservative assumptions used in the US EPA standard pond model, indicates that exposure to Cry1B.61.1 via the cultivation of COR23134 soybean poses negligible risks to aquatic organisms. The assumptions used to estimate the worst-case and refined EEC for all the proteins (Cry1B.34.1, Cry1B.61.1, and IPD083Cb) were extremely conservative because it is very unlikely that all aboveground COR23134 soybean tissue from a 10-ha field will enter a 1-ha pond. It is also very unlikely that all the insecticidal proteins (Cry1B.34.1, Cry1B.61.1, and IPD083Cb) in COR23134 soybean will be freely soluble and instantaneously bioavailable with no degradation in the field or pond, given that these proteins have been shown to degrade rapidly and lose bioactivity in soils,^73–75 and similar degradation would be expected in aquatic systems. Due to the aforementioned extremely conservative assumptions used to estimate aquatic exposure and MOE and the general lack of adverse effects at high-concentration levels, the actual exposure of aquatic organisms to Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein will be lower than the exposure modeled in this ERA. Therefore, the risk to non-target aquatic organisms from the cultivation of COR23134 soybean is considered negligible.

3.4. Predators and Parasitoids

Predators and parasitoids are an important group of NTOs that may be found within the agroecosystem, including fields where COR23134 soybean may be cultivated; hence, they may prey or feed on insects (herbivores) that have previously consumed tissues from a COR23134 soybean plant. Since predators and parasitoids are not expected to feed directly on the COR23134 soybean plant, the amount of Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein that transfers and accumulates in the insect herbivore is an important factor that may influence the degree of predators and parasitoids’ exposure to the insecticidal proteins. Other factors include 1. The rates of ingestion, digestion, and excretion of the COR23134 plant material by the insect herbivore, 2. The concentration of the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins within the insect herbivore, which will vary depending on the species, its developmental stage, and the concentration of the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins in the COR23134 soybean plant parts they are feeding, and 3. The stability of the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins within the insect herbivore. Based on the maximum concentrations of Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins expressed in any aboveground COR23134 soybean tissue at any growth stage and the assumption that 100% of the protein (Cry1B.34.1, Cry1B.61.1, or IPD083Cb) transfers to the insect herbivore and then subsequently to the predator (no protein degradation or loss during tri-trophic transfer), the worst-case EECs for predators and parasitoids were determined to be 960 ng Cry1B.34.1/mg, 1300 ng Cry1B.61.1/mg, and 130 ng IPD083Cb/mg (Tables 7–9). Following a similar assumption (100% tri-tropic transfer of the insecticidal proteins without degradation or loss) and based on the highest mean concentrations of Cry1B.34.1 protein expressed in any aboveground COR23134 soybean tissue at any growth stage, refined EEC was determined to be 460 ng Cry1B.34.1/mg (Table 7). Refinement to worst-case EECs was not considered for Cry1B.61.1 and IPD083Cb proteins for these taxa. Three surrogate species representing the predator and parasitoid functional group were subjected to Tier I laboratory effect or hazard testing: one Neuroptera (green lacewing), one Coleoptera (pink spotted lady beetle), and one Hymenoptera (parasitic hymenoptera). For any of the measured endpoints, there were no adverse effects detected on any of the representative surrogate predators and parasitoids fed diets containing Cry1B.34 (representing Cry1B.34.1), Cry1B.61.1, or IPD083Cb protein at any of the concentration levels tested compared to the negative control (Tables 7–9; Supplemental Tables S1–S3), except for green lacewing fed diets containing Cry1B.61.1. Although exposure to diet containing 16,000 ng Cry1B.61.1/mg did not affect green lacewing survival or pupation rate when compared to the negative control (Table 8; Supplemental Table S2), significant reductions in survival and pupation rate were observed in green lacewing fed 8000 ng Cry1B.61.1/mg diet compared with the negative control (Table 8; Supplemental Table S2). Similarly, while exposure to diets containing 8000 ng Cry1B.61.1/mg did not delay the time of green lacewing pupation when compared to the negative control (Table 8; Supplemental Table S2), pupation was prolonged for green lacewing fed diets containing 16,000 ng Cry1B.61.1/mg compared with the negative control (Table 8; Supplemental Table S2). The observed reduction in green lacewing survival and pupation rate at the 8000 ng Cry1B.61.1/mg diet was considered biologically irrelevant given the lack of a dose-dependent response detected. Additionally, the observed differences (82.8% survival at 8000 ng Cry1B.61.1/mg diet vs. 100% survival at negative control; 79.2% pupation rate at 8000 ng Cry1B.61.1/mg diet vs. 100% pupation rate at negative control; and 12 days to pupation at 16,000 ng Cry1B.61.1/mg diet vs. 11 days to pupation at negative control) were well below the 50% effect threshold recommended for triggering additional hazard testing.¹⁴ The NOECs for each of the representative surrogate predator and parasitoid species (green lacewing, pink spotted lady beetle, and parasitic hymenoptera) were determined to be 4672 ng Cry1B.34.1/mg (after adjusting Cry1B.34 for the molecular weight), 16,000 ng Cry1B.61.1/mg, and 20,000 ng IPD083Cb/mg (Tables 7–9). Based on these NOECs, the MOE for predators and parasitoids exposed to Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein in COR23134 soybean tissue was 4.9×, 12.3×, or 153.8× the worst-case EEC, respectively (Tables 7–9). Refinement to worst-case MOE was determined to be 10.2× the exposure for Cry1B.34.1 protein (Table 7). Although the estimated MOE values were greater than 10× the worst-case (Cry1B.34.1 and Cry1B.61.1) and refined (Cry1B.34.1) exposures for all three representative surrogate predators and parasitoids, several factors would further reduce the actual exposure of these non-target organisms to the Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein (via preying or feeding on insect herbivores that may have consumed COR23134 soybean tissues) below the worst-case and refined EECs determined in this ERA. For instance, the assumption that 100% of the protein (Cry1B.34.1, Cry1B.61.1, or IPD083Cb) transfers from the COR23134 soybean tissue to the insect herbivore and subsequently to the predator without degradation or loss is extremely conservative and unlikely. In reality, some protein degradation or loss within the insect herbivore (via digestion and excretion) is expected, reducing the amount transferred to the predator. Results from previous studies examining the concentration of Cry proteins in herbivores relative to that in the plants they were feeding have shown lower concentrations of the proteins in the herbivores.^77–82 Additionally, not all insect herbivores consumed by these predators and parasitoids would feed exclusively on COR23134 soybean tissue since herbivores consume a variety of plants other than COR23134 soybean. Likewise, most predators and parasitoids in crop fields are generalist feeders that do not rely on a sole insect herbivore species for food,⁸³ and some may require a mixed diet to complete metamorphosis⁸⁴; hence, non-target predators and parasitoids are highly unlikely to exclusively feed on only insect herbivores that may have consumed COR23134 soybean tissue. Therefore, based on the aforementioned mitigating factors, the worst-case assumptions, and the established MOE values, the chances of detecting any adverse effects on populations of non-target predators and parasitoids at environmentally realistic concentrations are extremely low and unlikely; hence, the potential risk of cultivating COR23134 soybean on non-target predators and parasitoids is considered negligible.

3.5. Insectivorous Birds

Similar to predators and parasitoids, some wild birds are insectivorous and could be exposed to the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins via the consumption of insect herbivores that may have previously consumed tissues from a COR23134 soybean plant. The EECs (worst-case and refined) for insectivorous birds were also based on the extremely conservative assumption that 100% of the protein (Cry1B.34.1, Cry1B.61.1, or IPD083Cb) transfers from the COR23134 soybean tissue to the insect herbivore and subsequently to the bird with no degradation or loss. Therefore, the worst-case EECs for insectivorous birds are the same as the worst-case EECs for predators and parasitoids (Tables 7–9). Refined EECs, based on the highest mean concentrations of Cry1B.34.1 and Cry1B.61.1 proteins expressed in any aboveground COR23134 soybean tissue at any growth stage, were determined to be 460 ng Cry1B.34.1/mg and 490 ng Cry1B.61.1/mg (Tables 7, 8). Refinement to worst-case EEC was not considered for IPD083Cb protein for these taxa. No mortality, abnormal behavior, or overt signs of toxicity (based on evaluation of body weight and feed consumption) was observed on Northern bobwhite quails administered (via oral gavage) a nominal limit dose of 1700 mg Cry1B.34/kg body weight (representing Cry1B.34.1), 2000 mg Cry1B.61.1/kg body weight, or 2000 mg IPD083Cb/kg body weight compared to the negative control (Tables 7–9; Supplemental Tables 1–3). The NOECs for Northern bobwhite quails were 992.8 mg Cry1B.34.1/kg body weight (after adjusting Cry1B.34 for the molecular weight), 2000 mg Cry1B.61.1/kg body weight, and 2000 mg IPD083Cb/kg body weight (Tables 7–9) since there were no adverse effects detected. Based on these NOECs, the MOE for Northern bobwhite quails exposed to Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein in COR23134 soybean tissue was 1.0×, 1.5×, or 15.4× the worst-case EEC, respectively (Tables 7). Refinement to worst-case MOE was determined to be 2.2× or 4.1× the exposure for Cry1B.34.1 or Cry1B.61.1, respectively (Tables 7 and 8). Although the estimated MOEs were less than 10× the worst-case and refined exposure for Cry1B.34.1 and Cry1B.61.1, the general lack of adverse effect observed after exposing Northern bobwhite quails, a representative surrogate insectivorous bird, to a nominal limit dose of 1700 mg Cry1B.34/kg body weight (992.8 mg Cry1B.34.1/kg body weight) and 2000 mg Cry1B.61.1/kg body weight, combined with the vagile nature of insectivorous birds and the extremely conservative assumption (100% tri-tropic transfer of the insecticidal proteins without degradation or loss) used to derive the EEC (worst-case and refined) and MOE values, demonstrates that exposure to Cry1B.34.1 and Cry1B.61.1 via the cultivation of COR23134 soybean poses negligible risk to insectivorous birds. Additionally, the same mitigating factors previously discussed for insect predators and parasitoids would also apply to insectivorous birds (degradation or loss of the insecticidal proteins within the insect herbivore, availability of a variety of food choices for insect herbivores and insectivorous birds, etc.), further reducing the actual exposure of non-target insectivorous birds to Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein below the worst-case and refined EECs determined in this ERA. Therefore, the risk to non-target insectivorous birds from the cultivation of COR23134 soybean is considered negligible.

3.6. Granivorous Mammals

Granivorous mammals (e.g., wild rodents) may be exposed to the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins by feeding on COR23134 soybean seed. The exposure of these mammals to these insecticidal proteins was expressed as a daily dietary dose which accounts for food intake rate, body weight, and the concentration of Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein in the COR23134 seed. The food intake rate and body weight ratio for wild rodents consuming cereal seeds have previously been estimated to be 0.28 and 0.33 for the wood mouse (Apodemus sylvaticus) and harvest mouse, respectively.²⁶ Based on the maximum concentrations of Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins in COR23134 soybean seed, and the assumption that 100% of the wild mammalian diet contained COR23134 soybean seed and was consumed at the maximum food intake rate to body weight ratio (0.33), the worst-case EECs for wild granivorous mammals were determined to be 69.3 mg Cry1B.34.1/kg body weight, 7.3 mg Cry1B.61.1/kg body weight, and 6.3 mg IPD083Cb/kg body weight (Tables 7–9). No mortality or other evidence of acute oral toxicity (based on evaluation of body weight, clinical signs, and gross pathology) was observed on mice administered (via oral gavage) a nominal limit dose of 5000 mg Cry1B.34/kg body weight (representing Cry1B.34.1), 5000 mg Cry1B.61.1/kg body weight, or 5000 mg IPD083Cb/kg body weight compared to the negative control (Tables 7–9; Supplemental Tables S1–S3). Since no adverse effects were detected, the NOECs for mice were determined to be 2920 mg Cry1B.34.1/kg body weight (after adjusting Cry1B.34 for the molecular weight), 5000 mg Cry1B.61.1/kg body weight, and 5000 mg IPD083Cb/kg body weight (Tables 7–9). Based on these NOECs, the MOE for mice, a representative surrogate wild granivorous mammal, exposed to Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein in COR23134 soybean seed was 42.1×, 684.9×, or 793.6× the worst-case EEC, respectively (Tables 7–9). Although refined EECs were not estimated for the representative surrogate wild granivorous mammal due to the high MOEs established using worst-case assumptions (Tables 7–9), several factors (e.g., availability of a variety of food choices other than COR23134 soybean seed) would further reduce the actual exposure of non-target wild granivorous mammals to Cry1B.34.1, Cry1B.61.1, or IPD083Cb protein below the worst-case EEC. Therefore, the risk to non-target wild granivorous mammals from the cultivation of COR23134 soybean is considered negligible.

4. Conclusions

This ERA focused on the insecticidal proteins (Cry1B.34.1, Cry1B.61.1, and IPD083Cb) expressed in COR23134 soybean and assessed the potential exposure and hazard to various functional groups of NTOs. Worst-case EECs of the proteins (Cry1B.34.1, Cry1B.61.1, and IPD083Cb) in COR23134 soybean were determined using extremely conservative assumptions to reflect the upper limit of potential NTO exposure. Several factors that reduce actual NTO exposure in the field were considered and used to refine EECs, when needed, to reflect more realistic environmental conditions and ecological processes. Tier I laboratory hazard studies were conducted using representative surrogate test species at concentrations exceeding the worst-case EEC to assess for potential adverse effects of the insecticidal proteins on NTOs. The environmental risk was characterized by comparing the Tier I laboratory hazard study results to worst-case or refined EECs to establish the MOEs for the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins in COR23134 soybean. The findings from the exposure, hazard, and risk assessments conducted in this ERA demonstrate, with reasonable certainty, that the likelihood of COR23134 soybean adversely affecting NTOs at field exposure levels is extremely low and unlikely, providing evidence to support the null hypothesis. Therefore, the ecological effects of cultivating COR23134 soybean are likely to be beneficial and no worse than those derived from the cultivation of non-transgenic soybean.

Funding Statement

The authors declare that no funding was associated with the work featured in this article.

Disclosure Statement

The authors are employees of Corteva Agriscience^TM.

Supplementary Information

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21645698.2025.2572186