- Open Access
Food-related exposure to systemic pesticides and pesticides from transgenic plants: evaluation of aquatic test strategies
Environmental Sciences Europe volume 31, Article number: 87 (2019)
The aquatic Environmental Risk Assessment (ERA) for pesticides relies on standardized experimental protocols focusing on exposure via the water phase or the sediment. Systemic pesticides (e.g., neonicotinoids) or pesticides produced in transgenic plants (e.g., Bt proteins) can be introduced into aquatic ecosystems as part of plant residues. Consequently, they may be taken up by organisms as part of their diet. Here, we analyzed (i) whether standardized aquatic ecotoxicological test guidelines consider an exposure route via food and (ii) whether these tests can be easily modified to take this exposure route into account. From the 156 existing test guidelines, only those for fish and amphibians partly consider a potential route of uptake via food. From the remaining invertebrate guidelines, those focussing on chronic endpoints may be most suitable to cover this exposure path. We suggest assessing the food-related effects of systemic pesticides in a dose-dependent manner using standardized guidelines or methods developed from peer-reviewed literature. For transgenic plants, spiking uncontaminated leaf material with increasing concentrations of the test substances would allow to test for dose responses. After adaption to oral uptake, standard test guidelines currently available for the ERA appear, in principle, suitable for testing effects of systemic pesticides and transgenic plants.
The current aquatic Environmental Risk Assessment (ERA) for pesticides involves a tiered approach of acute and chronic exposure scenarios under well-controlled laboratory, semi-field or field conditions . On this basis, a predicted no-effect concentration (PNEC) is derived by dividing the concentration of the product (i.e., its active ingredient) causing a defined effect in the respective environmental medium by a so-called assessment factor. The latter usually decreases with increasing biological complexity of the experimental design assuming that more complex systems do more likely represent the actual situation in the field reducing uncertainty [sensu 2].
In the course of this process, environmental risk assessors rely amongst others on standardized experimental protocols—especially during the first tier. These protocols have been developed to assess potential adverse effects caused by an exposure via the water phase [e.g., 3] or the sediment [e.g., 4]. The use of systemic pesticides or transgenic plants in agriculture, however, expands the relevant exposure pathways for some species that need to be considered during ERA. These exposure pathways relate to the presence of toxicants in the plant’s tissues. Systemic insecticides are absorbed via cuticula or root system and distributed in the plant . In transgenic plants, insecticidal proteins such as Cry or Vip proteins that originating from Bacillus thuringiensis (thus, often summarized as Bt proteins in the following)  or RNAi, which silence vital genes in the target organism, can be translated from the modified genes [7,8,9,10]. These options are the most relevant genetically modified organism applications for pest control. Both systemic pesticides and insecticidal proteins or RNAi from transgenic plants are designed to cause damage in target organisms after ingestion as some pests such as the larval stages of stem borers cannot be exposed to pesticides by direct spraying. For the risk assessment, the presence of these toxicants in the plant tissue implies that they can be introduced into aquatic ecosystems [11,12,13,14,15] and may subsequently leach into the water phase [12, 13, 16] or be taken up by detritivores as part of their diet [11, 16, 17]. Despite the fact that negative effects in aquatic invertebrates such as stone-, crane-, or caddisflies, and amphipods from the consumption of plant material have been described for both systemic insecticides [16,17,18,19] and Bt plants , the uptake of these toxicants with the diet seems not properly covered in standardized experimental protocols currently available and used in regulatory risk assessment.
The present study provides in a first step a detailed evaluation of pathways relevant to assess effects on aquatic life caused by systemic pesticides and Bt protein or RNAi from transgenic plants. In a second step, the suitability of current standardized experimental protocols to address these pathways is analyzed, experimental adaptation that may be needed is discussed, and suggestions for the development of target orientated experimental designs are provided.
Identification of relevant exposure pathways
Pesticides considered as systemic can be taken up by the crop plant and are distributed within this plant (see Fig. 1; ). Besides spraying or drenching on agricultural fields, which leads to an exposure pattern in aquatic ecosystems dominated by spray drift and surface run-off , seed treatment is another prevailing type of application . This means that the seeds are coated with pesticides prior to planting and from there, the pesticides are released into the soil where they are taken up by plant roots  and distributed within the plant during growth [cf. 23]. However, not the entire amount of pesticides associated with the seeds is taken up by the crop plant and parts of it may remain in the soil and, although subject to degradation , may accumulate over time . From soils treated with pesticide via this pathway, non-target plants from adjacent (non-agricultural) ecosystems may also take up these pesticides . As pollen from target plants may carry a pesticide load , deposition of pollen may be another exposure path for systemic insecticides. Following the harvest or abscission , plant residues, and leaf litter , which may contain measurable concentrations of the systemic pesticides , can be transferred into freshwater ecosystems via wind or surface run-off. Leaching of the pesticides retained in the plant biomass may expose and affect organisms feeding on the suspended or deposited pollen and leaf material (detritus) [16, 17]. Since the sowing of seeds coated with systemic pesticides is not considered as a pesticide application procedure, regulating measures such as obligatory distances from surface waters do not apply and surface waters may be directly exposed to seed-coating toxicants during sowing. Together pollen, plant residues and coated seed represent—in addition to exposure to the pure toxin in the water phase—additional exposure paths, which should be considered in the ERA.
With the exception of seed coating and spray drift, exposure pathways of systemic insecticides and Bt proteins from GM crops are largely similar [see 11, 12, 14]. Figure 1 provides an overview of exposure pathways of aquatic systems to systemic insecticides and insecticidal Bt proteins from GM crops.
Those pathways are, however, not explicitly considered in the testing regimes of either the ERA of pesticides [cf. 28] or transgenic plants [14, 29]. Since test methods to evaluate effects from Bt plants on non-target organisms are derived from pesticide testing , it is sensible to evaluate the currently employed test guidelines for their general suitability to cover the exposure pathway of toxicants via plant material.
Toxic mode of action
The mode of toxic action of chemical pesticides is described in detail, for instance in Stenersen , which is, however, not necessarily determined by the pesticides systemic or non-systemic nature. The major difference between systemic and non-systemic substances is the potential of systemic pesticides to be taken up by and distributed within the treated plants . This might lead to the overlooked exposure pathway for non-target species via the food .
Similar to systemic insecticides, Cry or Vip proteins are present within the plant tissues and designed to express toxicity after being ingested by the target organism. Compared to systemic pesticides, Bt proteins or RNAi approaches for pest control are supposed to be more specific and have a tighter spectrum of insecticidal activity. This specificity can be influenced by a number of processes such as the proteolytically transformation of Cry proteins to toxins in the insect gut and binding to receptors (such as aminopeptidases, involved in cell adhesion and digestion) in the midgut . Although the specificity of Bt proteins is challenged [33, 34] and the molecular mechanisms of the modes of action of Bt proteins are not fully understood [35, 36], differences in the activity spectrum of systemic insecticides, Bt proteins and even more so for RNAi constructs are obvious. Despite the joint potential to expose non-target species via their food, systemic pesticides and Bt proteins may require a different testing strategy to adequately reflect their respective characteristics. For systemic pesticides, for instance, it may be sufficient to understand the consequence of the food-related exposure path relative to the classical water phase exposure of sensitive aquatic species.
For the identification of potentially sensitive species, research and regulation can rely on published data though the overwhelming majority of publications only deal with waterborne exposure [see as an example 17]. For Bt proteins in contrast, studies identifying sensitive aquatic species are very limited .
Standardized test guidelines
To date, standardized test guidelines have not been developed specifically for systemic pesticides or transgenic plants . To evaluate, therefore, the existing standardized test guidelines for their current and potential ability to cover the exposure paths via food, the respective documents used during the ERA of pesticides were compiled. The present study exclusively focuses on test protocols for freshwater organisms including amphibians.
In detail, approved guidelines—or those being in the progress for standardization—published by the Organization for Economic Co-operation and Development (OECD), the Official Journal of the European Communities (EU), the International Organization for Standardization (ISO), the American Society of Testing and Materials (ASTM), the Ministry of Agriculture, Forestry and Fisheries (MAFF) and the Environmental Protection Series published by Environment Canada (EC) provided the basis for the present evaluation. In addition, guidelines published by Office of Chemical Safety and Pollution Prevention (OCSPP) within the United States Environmental Protection Agency (EPA) were taken into account. For each of the 156 test guidelines considered (see Additional file 1: Table S1), the taxon of the test species as well as the test design, including the assessed endpoints, the study duration and the experimental conditions were abstracted (Table 1). On this basis, the suitability of the guidelines to cover the exposure path via the food was evaluated. Where possible, we suggest amendments to or alterations in the guideline that allow integrations of food intake as an exposure path.
Analyses of standardized test guidelines
General characteristics of the guidelines
Most of the 156 evaluated test guidelines (Additional file 1: Table S1) describe single-species laboratory tests (148), whereas four are laboratory microcosms studies. Two recommend outdoor mesocosm or in situ studies, respectively, and two documents cover actual field studies (Table 1). Nine test methods evaluate the bioaccumulation of chemicals. As some guidelines describe test procedures for more than one species or trophic level, the overall sum of the described test procedures is exceeding the number of the evaluated test methods. We evaluated for each systematic group whether the test organisms’ route of exposure occurs via the water phase or the ingestion of potentially contaminated food. For reasons of completeness, we also included autotrophic organisms in our evaluation, since they may act as carrier of contaminant entry into the food web. Moreover, we provide an overview on the guidelines major route of exposure (Table 1) and the number of test guidelines per systematic group that feeds the test species during testing (Fig. 2) providing a potential avenue for adaptation to cover an exposure pathway via the food.
Algae, cyanobacteria and macrophytes
In total, 27 test guidelines have been identified that describe test procedures for algae, cyanobacteria and macrophytes, of which 24 are focusing on laboratory experiments with an acute or chronic experimental duration (Additional file 1: Table S1) ranging from 2 to 14 days. As neither algae nor cyanobacteria or macrophytes actively ingest plant residues containing either systemic pesticides or Bt proteins, the main exposure path for primary producers is via the water phase and hence as a result of spray drift and run-off from agricultural fields or leaching of the pesticides or Bt proteins from the plant residues. It should, however, not be ignored that the systemic pesticides or Bt proteins can adsorb to the surface of algae, cyanobacteria and macrophytes that are finally consumed by higher trophic levels not covered in these test guidelines. This can ultimately provide an additional exposure path for these stressors along the aquatic food web.
Bacteria and microorganisms
Standardized experimental guidance for the testing of bacteria and microorganisms is available in 15 different documents (Additional file 1: Table S1), while six of these guidelines refer to the utilization of representatives from the class of Gammaproteobacteria (Pseudomonas putida, P. phosphoreum and Salmonella typhimurium) and the remaining nine refer to an undefined composition of microorganisms summarized as “activated sludge”. The recommended test duration is between less than 1 h and 3 days, while mainly growth and respiration are assessed. Similar to algae, cyanobacteria and macrophytes, these microorganisms may mainly experience an exposure towards the systemic pesticides and Bt proteins via the water phase.
However, some microorganisms such as aquatic fungi, which are not covered by any of the test guidelines used during the ERA , can colonize allochthonous organic material  including crop plant residues. As a result of their activity (e.g., growth, release of enzymes) structural carbohydrates are degraded , which likely increases the leaching rate of systemic pesticides and Bt proteins into the water. Thus, the exposure concentrations may be higher for leaf-associated relative to pelagic microorganisms. In this context, one group of systemic pesticides, namely systemic fungicides, may be a particular concern as they can directly affect the leaf-colonizing fungal community with knock-on effects for the whole heterotrophic food web [sensu 41, 42] [but see 43].
The next higher trophic level is represented by the rotifer Brachionus calyciflorus, which is the recommended test species in three single-species test guidelines (Additional file 1: Table S1) covering acute and chronic exposure durations of up to 48 h as well as one of the various rotifer test species in two guidelines employing laboratory microcosms with a duration of up to 63 days. As the B. calyciflorus is mainly feeding on algae , an exposure to systemic pesticides and Bt proteins via the ingestion of food seems relatively irrelevant, although pesticides can be taken up in algae and delivered to rotifers after ingestion. However, since B. calyciflorus is a suspension feeder , it is feasible that the species ingests pollen or fine particles from crop plant residues when present in the water column. Hence, amending the test guidelines to also consider potential implications of systemic pesticides and Bt proteins contained in these plant parts may be feasible and could assist ERA (Table 2). In this context, Bøhn et al. [45, 46] have developed a procedure to generate fine particles from leaf material, which ensures a lower sedimentation rate of the particles, and hence represents a worst-case scenario for the test species.
A total of 47 guidelines were identified that describe standardized ecotoxicological methods to assess the effects of stressors on crustaceans (Additional file 1: Table S1) involving study durations of up to 65 days. The largest number of guidelines is available for the filter-feeding representatives of the family Daphniidae (Crustacea: Branchiopoda). Also, for daphnids, acute and chronic experimental designs are available. The descriptions for acute toxicity tests are nearly identical irrespective of which institution has published the document. Although this indicates a high consensus regarding the optimal experimental design, the current acute toxicity test guidelines exclusively focus on the exposure via the water phase and even avoid feeding the test species during the course of the experiment. This procedure is similar to the guidelines describing acute testing procedures for the two families Atyidae (i.e., Neocaridina denticulata and Paratya compressa improvisa) and Thamnocephalidae (i.e., Thamnocephalus platyurus) [e.g., 47, 48], which feed amongst others on algae. However, during chronic testing, daphnids are fed with algae (Fig. 2), which are non-selectively ingested by the filter-feeding test species if retained by their filter apparatus . Therefore, and similar to the situation for the rotifers, it is feasible to modify the testing regime that either pollen or fine particles originating from the crop plant residues can be ingested and tested (Table 2). In fact, Bøhn et al. [45, 46] have fed daphnids fine particles originating from genetically modified maize plants expressing the insecticidal protein Cry1Ab and compared their response with the isogenic counterpart. Although the reproduction of Daphnia magna was substantially lower relative to a control situation where algae served as food, the study indicated a negative impact of the Cry1Ab proteins on the reproductive performance of daphnids . These insights were supported by follow-up studies showing adverse effects in the survival, fecundity and population growth rate of D. magna when fed with fine particles generated from genetically modified maize [46, 50]. These studies, hence, indicate the general feasibility to modify the experimental design of chronic tests with daphnids to cope with the challenges specific for the assessment of systemic pesticides and Bt proteins [see also 51] contained in plant material. At the same time, they also highlight that commonly employed validity criteria regarding the reproductive output may not be met if fine particles are provided as food source, as they usually exhibit a relatively low nutritious quality [45, 50].
In contrast to the filter feeders, representatives of the families Gammaridae (e.g., Gammarus pulex or G. fossarum), Dogielinotidae (i.e. Hyalella azteca) and Cyprididae (i.e., Heterocypris incongruens) are leaf-shredding organisms (more generally detritivores) or omnivores contributing substantially to the decomposition of allochthonous organic material—including crop plant residues—in freshwater ecosystems [e.g., 52–54]. Therefore, an adaptation of the experimental designs detailed in the respective acute and chronic guidelines may be an ecologically relevant and regulatory sensible procedure (Table 2). The available test guidelines either exclude feeding (i.e., Gammaridae) (Fig. 1) or use artificial food such as fish flakes (i.e., Dogielinotidae). In this context, Li et al.  demonstrated no substantial effects of Cry proteins extracted from GK-12 transgenic cottonseeds and spiked to the water or sediment using the (sub)acute 10-day sediment and 4-day water-only exposure with H. azteca. In the light of the exposure path of systemic pesticides and Bt proteins, which is expected to occur via the food, leaf material containing these stressors should be offered. At the same time, extending the exposure duration (from acute to chronic exposures) as a result of the expected sublethal effects [18, 56] should be considered. This extension is particularly relevant for the guidelines addressing the toxicity for Gammaridae after a short-term exposure of 4 days (Tables 1 and 2). Besides reproduction, which is according to current guidelines exclusively assessed for H. azteca over a study duration of 42 days, sublethal responses such as the feeding rate on leaf material can be quantified for both Hyalella [cf. 57] and Gammarus [cf. 17, 19, 58] already after a subacute exposure of 7 days. However, chronic exposure durations of several weeks with regular water exchanges and food renewal should be preferred, which would allow for a meaningful assessment of additional endpoints related to the energy availability and processing of the test species over longer study durations and may be particularly valuable for fungicides . By following, for instance, the design of Bundschuh et al. , the feeding rate, feces production, assimilation, growth as well as the energy reserves of the test species may be determined that would foster the scientific understanding regarding the physiological reaction of organisms towards different types of chemical stress. This would likely increase the level of protection provided by the ERA. Finally, a procedure, which allows for the assessment of multiple sublethal energy processing related endpoints—as suggested here and below—seems particularly sensible for Bt proteins with unknown consequences for non-target species .
The class of insects is represented by 18 guidelines (Additional file 1: Table S1), of which one guideline describes a procedure for a 21-day-lasting sediment spiked survival and growth assay with Hexagenia sp. (Emphemeridae) and 14 focus on the family of Chironomidae with acute (48 h) and chronic (up to 65 days) test designs, including full life-cycle tests. The diversity of endpoints that can be assessed by employing these guidelines includes besides mortality also emergence rate, developmental rate, time to emergence, fecundity, fertility in the parental generation as well as the sex ratio in the parental and filial generation. Against this background, the aquatic insect community composition (e.g., Ephemeroptera, Plecoptera and Trichoptera) is an endpoint that responded to leaf litter containing insecticidal Bt proteins  and individual insect species showed a reduced feeding when offered leaf material containing the systemic pesticides [16, 19] or were exposed to Bt proteins [55, 63]. Consequently, insects are likely responsive and sensible test organisms to assess negative impacts of these stressors on aquatic ecosystems in general. Moreover, since chironomids and to some extent Hexagenia sp. are, similar to gammarids, detritivorous [64, 65], they represent a potentially suitable group of ecotoxicological test species for the ERA of systemic pesticides (particularly insecticides) and Bt proteins contained in crop plant residues; nevertheless, their test guidelines would need some modifications: Although the test organisms are regularly fed (Fig. 1)—at least during the chronic and full life-cycle tests—they usually receive ground artificial fish food (i.e. fish flakes), which needs to be replaced by the respective crop plant residue provided as fine particles (Table 2). As suggested above, the procedure detailed by Bøhn et al.  may provide an adequate guidance. It may also be desirable to assess the emerged insects for their energy reserves that may be affected by the food quality (i.e., the systemic pesticides and Bt proteins contained in crop plant residues) [sensu 65] and could result in implications in the filial generation. The guidance documents on full life-cycle tests with chironomids seem—due to the coverage of two subsequent populations—in general suitable for the characterization of sublethal environmental risks associated with the food-mediated exposure path of systemic pesticides and Bt proteins.
Besides the guidelines mentioned above, Ephemeroptera, Plecoptera, and Trichoptera are recommended as groups of test species in a total of ten, three and one test guideline, respectively. However, none of these guidelines have been developed specifically for any species belonging to these three orders and partly lack a clear description of the experimental design. In general, also these guidelines assess the sediment or waterborne exposure over up to 28 days while focusing on mortality, immobilization, growth and bioaccumulation as endpoints.
Seven test guidelines refer to molluscs as test species (Additional file 1: Table S1), while these are divided into guidelines using the class of Bivalvia or Gastropoda (i.e., the family of Physidae and Amnicolidae). The two guidelines using Gastropoda are acute laboratory test guidelines with an experimental duration range between 2 and 8 days without feeding. The water-only exposure experiments assess mortality and immobilization as endpoints. Furthermore, these test guidelines are not specific for Gastropoda as a broad range of species including fish, macroinvertebrates and amphibians are listed as appropriate test organisms. Five test guidelines—including one for in situ bioassays—suggest bivalves as test organisms. These guidelines recommend exposure durations from 24 h up to 30 days and focus on bioaccumulation, growth, mortality and immobilization as endpoints. An exposure of freshwater mussels towards Cry proteins was documented in agriculturally influenced ecosystems in Canada . Thus, their inclusion in a testing strategy seems sensible. Although molluscs cover a broad range of feeding strategies, including filter feeding of suspended fine particulate organic material , which may also be released by the decomposition of plant material potentially containing systemic pesticides or Bt proteins , the short study durations question the suitability of this protocol to adequately cover this particular exposure pathway. Nonetheless, by elongating the study duration and focusing mainly on sublethal, including developmental endpoints, as suggested by one guideline targeting in situ and, thus, field-orientated approaches may be a sensible advancement (Table 2).
Other invertebrate species
Only eight guidelines (Additional file 1: Table S1) with an experimental duration between 4 and 28 days are dedicated to the assessment of chemical effects on sediment-dwelling organisms from various families including the species Lumbriculus variegatus, Tubifex tubifex, Caenorhabditis elegans, Dugesia tigrina and Branchiura sowerbyi. The endpoints of these tests include one or more of the following: bioaccumulation, mortality, growth, reproduction, fertility and sex ratio. Moreover, most of the test guidelines require the provision of standardized food (mainly fish food). Since these test species are consuming microorganisms likely associated with detritus, it may be feasible to experimentally assess the implications of systemic pesticides and Bt proteins contained in crop plant residues using a similar approach as suggested above for the experiments with chironomids and daphnids.
With 55 test guidelines, the class of fish is the most frequently considered taxonomic group (Additional file 1: Table S1), while the study duration ranges from 24 h to a full life cycle of a species, which can last several months. These guidelines focus on endpoints such as mortality, development, growth, reproduction, hatching success, swimming behavior as well as bioaccumulation. These endpoints in combination with the partly relatively long study duration suggest these guidelines as a suitable starting point for the development of some guidance for the determination of environmental risks associated with systemic pesticides and Bt proteins introduced into aquatic systems together with plant material on fish. However, most of the fish species covered by these guidelines are carnivores and do not primarily rely on the ingestion of detritus. Together with the general call to reduce animal (mainly vertebrate) testing during the registration process of chemicals , fish experiments may not be considered as an approach substantially improving the ERA of these substances in future.
The class of amphibians is represented by seven guidelines mainly focusing on Xenopus laevis and Rana catesbeiana (Additional file 1: Table S1), which are during their larval stages (=tadpoles) feeding on pelagic microorganisms such as bacteria and algae . Although the recommended study duration (between 4 and 30 days) and the endpoints (i.e., development, growth, mortality) may be suitable to detect adverse effects caused by systemic pesticides and Bt proteins associated with plant material, the feeding ecology of the relevant species together with the ethical concerns regarding animal testing  suggest this group of organisms at the current stage as suboptimal to advance the ERA of these substances.
Improvement of test guidelines for testing systemic pesticides
As detailed above, the mode of toxic action of systemic pesticides is not expected to be substantially different from their non-systemic counterparts. However, the importance of the exposure pathway via the ingestion of contaminated food needs to be related to the exposure via the surrounding medium, namely water. Ideally, this should be realized using dose–response relationships allowing also for a direct inclusion in the ERA of these substances.
In a first step, plant material that contains increasing concentrations of these systemic pesticides should be generated. One feasible approach is to spike potted trees (or other plants such as maize or rapeseed) with different concentrations of a systemic pesticide either via soil or stem injection, while the application rates may be related to the recommendations of the respective producer . Later during the year, more precisely at the time of abscission, the leaf material can be collected directly from the trees and stored either frozen or air-dried until being used for ecotoxicological experiments. The concentrations of the systemic pesticides within the leaf material—or other plant tissue such as pollen—need to be verified via chemical analysis, likely involving accelerated solvent extraction and liquid chromatography tandem mass spectrometry techniques . In a second step, the acute and chronic toxicity of these plant residues can be estimated using the standard methods considering the amendments for the respective test organisms described above and in Table 2. From the evaluation of existing guidelines, however, it is apparent that only few test species can be classified as leaf-shredding invertebrates. Since shredders are the functional group which will be directly exposed to systemic pesticides contained in the plant material, we suggest expanding the list of test organisms with additional shredder species from insect taxa including trichopterans and plecopterans. These taxa are in the case of neonicotinoids also considerably more sensitive compared to standard crustacean test organism [71 but see 19]. The inclusion of insects as a further group appears also sensible since the enzyme activity, gut pH and other parameters may be deviating among representatives from different subphyla or classes , which may influence the digestion of the leaves, the release of the pesticide from the leaf material and ultimately their effects in the exposed organisms. Although, this physiological diversity in the group of insects complicates standard toxicity testing, their consideration during test development could inform about the magnitude of differences in sensitivity among subphyla or classes ultimately informing risk assessment.
The actual experimental design to test the effects of systemic pesticides depends also on the purpose of the study. If a dose–response relationship is to be established, the assessment of multiple concentrations is required and calls for rather short exposure durations to allow sufficient replication. Kreutzweiser et al.  indicates that at the recommended application rates of systemic pesticides, relatively low concentrations of the pesticide in the plant tissues can be anticipated. As a result, sublethal rather than lethal effects may be expected . To account for sublethal effects, other experimental designs may be applied: Quantification of feeding rates of leaf-shredding invertebrates on standardized and uncontaminated leaf material has been done in various studies [58, 60]. Replacing the uncontaminated leaf material with leaves from trees treated with different concentrations of systemic pesticides would allow for the estimation of the potential effects associated with the introduction of such leaves into aquatic ecosystems. However, systemic pesticides might leach from the leaf material into the surrounding medium generating a situation in which the test species is exposed to the test substance both via the water phase and its food [17, 19].
To disentangle the relative importance of both exposure pathways, a 2 × 2 factorial approach as proposed by Englert et al.  may be employed: The experimental design is composed of five treatments during one of which the test species receive leaves from an uncontaminated tree grown under the same conditions as those subjected to a treatment with systemic pesticides. In the second treatment, the test species will also receive leaves from the control trees but in the same replicate, an equivalent amount of leave material from a treated tree will be provided but inaccessible for the test species. This ensures the quantification of the effects caused by the leached pesticide. In a third treatment, the test species will be offered exclusively leaf material from a treated tree, while a continuous water exchange (flow through) will ensure that water phase concentrations, remain at negligible levels and thus enabling the quantification of effects caused via the ingested food exclusively. As this treatment likely deviates from the others with regard to the development of water quality parameters over time, a respective control treatment (fourth treatment) needs to be established relative to which the effects can be expressed. The fifth treatment, in which the test organism receives leaves from a treated tree and the medium will not be exchanged, ensures the exposure of test organisms to the systemic pesticide via both the water phase and food.
Improvement of test guidelines for testing Bt plant material
The estimation of potential environmental effects of Bt proteins has various experimental challenges that need to be solved during their ERA. One very important challenge is—in contrast to the testing of systemic insecticides—the establishment of a dose–response relationship. This appears difficult with any transgenic plant material such as tissues from Bt plants for several reasons: To date, the expression of Bt proteins in transgenic crops is in most cases governed by a constitutive promotor and Bt proteins are expressed in most, if not all, plant tissues. However, expression levels are usually tissue specific and vary with the growth stage [73, 74]. Although tissue specific concentrations are assumed to be rather constant at a given growth stage, the use of different genetic backgrounds and other factors [75, 76] can cause considerable variations in expression levels. Driven by the variation in Bt protein expression, GM plant parts that have an equal nutritious value and constant Bt protein concentrations can hardly be generated. At the same time, the maximum test concentration of Bt proteins within tissue is limited to the naturally expressed levels. Practically, worst-case assumptions including common safety factors similar to tests of chemical pesticides are difficult, if not impossible. Moreover, in Bt plants, the genetic modification may induce changes in the plant that go beyond the expression of genes initiating the production of Bt proteins. These changes may, for instance, influence the general nutritious quality for the organisms such as shredders in the aquatic ecosystem . Current EFSA guidelines for genetically modified organism  advise to use a near-isogenic line that should be similar to the genetically modified plant. Although this addresses the issue of a similar nutritious quality, the difficulty to establish dose–response relationships remains.
During sensitivity testing of terrestrial non-target organisms microbially produced Bt proteins, instead of Bt plant material are frequently used . A similar procedure may be applied during aquatic ecotoxicology testing by spiking GM plant material with increasing Bt toxin levels of microbial origin. This procedure would allow for a dose response testing of the Bt toxin considering also the changes in the crop metabolism induced by the genetic modification. At the same time, this procedure requires the availability of a non-GM isoline. Food quality and the fact that not all Bt plants (e.g. maize) may allow aquatic organism to perform a full life cycle are critical in this respect. To address this challenge, we suggest spiking conditioned leaf material of a tree species such as black alder—a highly nutritious food source—with increasing and known concentrations of the Bt proteins of microbial origin during the testing of aquatic shredders [see e.g., 60, 80]. The Enzyme-Linked Immunosorbent Assay (ELISA) technique can be used to measure the concentrations of Bt proteins in plant tissues. Subsequently, the material can, in principal, be tested as detailed for the systemic pesticides, namely by offering the spiked material to leaf-shredding invertebrates and monitor their response. The approach, however, does not allow testing the GM plant as a whole  and the toxins of microbial origin may not be (toxicologically) identical to those produced by a GM plant. Further uncertainties remain as to whether the toxicant availability is comparable between spiking and regular GM plant treatment in which the toxins are contained within cells and not on the plant material surface. Nonetheless, the illustrated procedure would allow for an establishment of a dose–response relationship for an individual Bt protein or a defined mixture. Moreover, risk assessment procedures of genetically modified organisms in Europe  are based on a case-by-case approach calling for test organisms representative for the receiving environment. As Hilbeck et al.  provided a decision support system containing criteria for the selection of potential test species for aquatic GM testing, this will not be addressed in further detail in this document.
Although the experimental design suggested in the following still needs some verification and adjustments, it is recommended to offer the spiked leaf material of interest, e.g., to a caddisfly species for at least 6 weeks (ideally longer) under continuous aeration in a climate-controlled room ideally using a temperature gradient simulating the field situation. The caddisfly larvae may be kept in groups of around five individuals in glass vessels containing the preferred substrate of the selected test species, while each treatment should be replicated at least ten times ensuring an appropriate statistical power. At weekly intervals, the test item, namely the food of the test species, can be renewed, the feeding rate of the species determined [see 82] and the test medium, which may either be a standardized medium as, for instance, described in Borgmann  or stream water (the site where the organisms have been collected from), may be renewed. At the same time, the survival and the growth of the caddisfly larvae can be monitored. The latter will be determined by measuring the width of the head from a digital image, an endpoint correlating well with the biomass of the species  and providing a measure of the larval instar. At the termination of the experiments, the individuals may be analyzed for the energy reserves, which provides insights into the physiological implications caused by the ingestion of food containing Bt proteins. This endpoint was successfully established as a measure for stress caused by chronic exposures and allows inferences on the population development in the long run [e.g. 60]. Another option, which one could pursue is to assess the time until insect emergence, which is a sensitive endpoint  and indicative for implication in the subsidy of terrestrial ecosystems by aquatic resources [land–water coupling, 85]. Also, in the emerged adults, energy reserves can be determined which may allow for insights into potential effects in the reproductive output, and thus population development [sensu 86].
Differences in the exposure route, namely the uptake of the active ingredient as part of the food matrix, scientifically justify that the risk assessment of both systemic insecticides and transgenic insect resistant plants should include this path of exposure in their ERA prior to authorisation of the respective products. Significant challenges remain, which include the need to update or adjust the currently available test guidelines to provide a meaningful basis for ERA. Moreover, only few test species represent the functional group of shredders which are supposedly regularly exposed to potentially contaminated plant residues under field conditions. Thus, particularly for the ERA of Bt proteins from transgenic crops, further research is required to optimize test strategies and methods which would allow to assess the dose–responses relationships.
Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.
Environmental Risk Assessment
- Bt :
predicted no-effect concentration
ribonucleic acid interference
- Cry :
proteins crystal proteins
- VIP :
vasoactive intestinal peptide
Organization for Economic Co-operation and Development
International Organization for Standardization
American Society of Testing and Materials
Ministry of Agriculture, Forestry and Fisheries
Office of Chemical Safety and Pollution Prevention
Environmental Protection Agency
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Overview of the Test guidelines covered in this review. The Table carries information on the guideline number, the year of publication, its title, the level of testing and the systematic group assessed.
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Bundschuh, R., Bundschuh, M., Otto, M. et al. Food-related exposure to systemic pesticides and pesticides from transgenic plants: evaluation of aquatic test strategies. Environ Sci Eur 31, 87 (2019). https://doi.org/10.1186/s12302-019-0266-1
- Risk assessment
- Aquatic ecosystems
- Genetically modified organisms
- Bt protein