By JEFF BARNARD
The Associated Press
GRANTS PASS, Ore. — Looking high and low, Robbin Thorp can no longer find a species of bumblebee that just five years ago was plentiful in northwestern California and southwestern Oregon.
Thorp, an emeritus professor of entomology from the University of California, Davis, found one solitary worker last year along a remote mountain trail in the Siskiyou Mountains but hasn't been able to locate any this year.
moreCentre d'Etude et de Recherche du Me´dicament de
Normandie, 5 rue Vaubenard, 14032 Caen cedex, France,
Laboratoire Evolution, Ge´nomes, Spe´ciation, CNRS-UPR9034,
Ba timent 13, Avenue de la Terrasse, F-91198 Gif-sur-Yvette,
France, and Laboratoire de Zoologie, INRA,
86600 Lusignan, France
The procedure to assess the risk posed by systemic insecticides to honey bees follows the European Directives and depends on the determination of the Hazard Quotient (HQ), though this parameter is not adapted to these molecules. This paper describes a new approach to assess more specifically the risk posed by systemic insecticides to honey bees with the example of imidacloprid (Gaucho).
This approach is based on the new and existing chemical substances Directive in which levels of exposure (PEC, Predicted Exposure Concentration) and toxicity (PNEC, Predicted No Effect Concentration) are compared. PECs are determined for different categories of honey bees in relation to the amounts of contaminated pollen and nectar they might consume. PNECs are calculated from data on acute, chronic, and sublethal toxicities of imidacloprid to honey bees, to which selected assessment factors are applied. Results highlight a risk for all categories of honey bees, in particular for hive bees. These data are discussed in the light of field observations made on honey bee mortalities and disappearances. New perspectives are given to better determine the risk posed by systemic insecticides to honey bees.
IntroductionIn the European Union, formulated pesticides are registered by the European Council Directive (EC-91/414) and the risk posed by these molecules to honey bees is directly assessed by the European and Mediterranean Plant Protection Organization (EPPO) guidelines No. 170
(1).These guidelines propose methods for evaluating side effects of agrochemical products on honey bees. The approach is based on a 3 tier assessment scheme comprising early studies in laboratory conditions, followed by semi-field studies, and completed by field studies. According to this Directive, and to the decision making scheme attached to the EPPO guidelines
(2)., moving from tier 1 (laboratory studies) to tier 2 (semifield studies) depends on a trigger criterion, the Hazard Quotient (HQ ) field application rate/oral or contact Lethal Dose (LD50)). When the calculated value ofHQis higher than a threshold of 50, further studies are required. This threshold is derived from data which only consider spray applications on honey bees
(3).In the case of plants treated by systemic insecticides, honey beesmaybe at risk via contaminated pollen and nectar
(4). The contamination of nectar by sprayed systemic insecticides has been long documented (5), whereas little information is available on systemic formulations applied in soils and on seeds. Published data deal mainly with aldicarb, a carbamate substance used for the protection of various cultures (6). More recently, several authors supplied data on the presence of imidacloprid, a neonicotinoid systemic insecticide, in nectar and pollen of treated plants (7). Generally, systemic insecticides provide the treated plant with a permanent protection from soil invertebrates and sucking insects (8). Applied in soils andonseeds, they degrade slowly over time and disperse in all the plant tissues during its growth. Therefore, using the field application rate of active substance as an exposure parameter to assess the risk posed by systemic insecticides to honey bees is not sensible. Unlike sprayed insecticides, which have a short-lasting action on plants, systemic insecticides are persistent. Moreover, these molecules, detected at low concentrations in the pollen and nectar of treated plants, are more likely to affect honey bees by acute, chronic, and sublethal intoxications (9) rather than by acute intoxications alone.
In this paper, we propose a new approach to determine the risk posed by systemic insecticides to honey bees. It is based on the European Technical Guidance Directive (TGD)that assesses the impact of new (793/93 and 1488/94/CE legislations) and existing chemical substances (EC-67/54/8 and EEC-93/67 Directives) on ecosystems (10). This approach is applied to imidacloprid, which is a good study case because it has been extensively studied and presents a lot of experimental data.
Materials and MethodsA group of experts, namely the Scientific and Technical Committee (CST), was nominated in 2001 by the French Ministry of Agriculture to assess the risk posed by imidacloprid to honey bees. This committee examined all studies, delivered up to July 2004 by the Ministry of Agriculture, on the toxicity of imidacloprid to honey bees (7). This paper refers to some of the work achieved by this committee. For many wildlife species, the standard practice in pesticide regulation (91/414 EEC) is to determine a toxicity exposure ratio (TER) and to compare it to a threshold (a safety factor) that aims at protecting these species. In this paper,weused thePEC/PNECratio (predicted environmental concentration/predictednoeffect concentration) which aims at protecting ecosystems (10). Honey bees (unlike most other species) live in coloniesanddependoneach other for survival. Such interdependent relationships define the honey bees' colony as a superorganism (11). The functioning of a superorganism is similar to that of an ecosystem in the sense that each unit (temporal castes in a colony and species in an ecosystem) is essential to sustain the system as a whole (12). Moreover, considering their role as pollinators (13), honey bees represent a good model to assess the risk of insecticides to pollinators and to protect many plant species that rely on these organisms, through pollination, to reproduce.
According to the PEC/PNEC approach and with the example of imidacloprid, we determined PECs with the Corresponding authors. Phone:(33/0) 2 31 56 59 10 (M.-P.H.),
(33/0-1) 69 82 37 17 (A.R.); fax: (33/0) 2 31 93 11 88 (M.-P.H.),
(33/0-1) 69 82 37 36 (A.R.); e- mail: MHalm@cermn.unicaen.fr or
Rotrais@pge.cnrs-gif.fr.
Centre d'Etude et de Recherche du Me´dicament de Normandie.
Laboratoire Evolution, Ge´nomes, Spe´ciation, CNRS.
§ Laboratoire de Zoologie, INRA.
Environ. Sci. Technol. 2006, 40, 2448-2454
2448 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 7, 2006 10.1021/es051392i CCC: $33.50 2006 American Chemical Society Published on Web 03/07/2006
known concentrations of imidacloprid found in the contaminated pollens (sunflower and maize) and nectar (sunflower) consumed by honey bees, and we determined PNECs with the data derived from studies on acute, chronic, and sublethal toxicities of imidacloprid to honeybees. Criteria for the Validation of Data. To determine the concentration of imidacloprid in pollens and nectar issued from imidacloprid seed-dressed plants and the honey bees' exposure to imidacloprid, the CST validated the data of all the studies that met the following requirements in terms of sampling procedure, chemical analyses, and toxicity testing. For the sampling procedure, studies needed to describe thoroughly the methods used (sampling location, pesticide treatments history) and gather sufficient samples, both qualitatively and quantitatively. Qualitatively, data obtained from pollens collected directly on anthers of treated flowers, rather than in pollen traps, were kept and validated because the concentration of imidacloprid in pollens of traps is highly variable and depends on the environment (i.e., the amount of plants treated by systemic insecticides) (14). Quantitatively, the CST retained the value of a minimum of 10 samples to enable statistics (means and standard deviations). Samples coming from different experiments and locations, but presenting similar protocols, were grouped together to get a minimum of 10 samples.
For the chemical analyses, given the high toxicity of imidacloprid,wevalidated studies that detected the molecule the most accurately as possible, that is by high performance liquid chromatography (HPLC) coupled tomassspectrometry (MS) (15) and using an appropriate limit of quantification (LOQ ) 1 íg/kg), limit of detection (LOD 0.5íg/kg), and sample weight (10 g). The only study that used a radioactivity method coupled with thin-layer chromatography (TLC) and automated multiple development(AMD)techniques (16) was validated too because it allowed a clear identification of imidacloprid, unlike less specific methods such as the derivation and gas chromatography (GC) (17, 18). To determine the toxicity of imidacloprid to honey bees, studies apply standardized tests designed by the OECD guidelines (19, 20). Such tests are developed in laboratory conditions to assess oral (19) and topical (20) acute toxicities of pesticides (and other chemicals) to adult worker honey bees. These laboratory tests follow the EPPO guidelines No. 170 (1) and the recommendations made by the International Commission for Plant-Bee Relationships (ICPBR). While these guidelines propose methods to test oral and topical acute toxicities, there are currently no standardized tests to study chronic and sublethal toxicities of pesticides to honey bees. Nevertheless, a few studies have investigated the impact of imidacloprid on honey bees by chronic and sublethal intoxications in laboratory, semi-field, and field conditions. To assess these studies, we referred to the EPPO guidelines because they present guidelines for semi-field and field experiments (1).
PEC Estimates. By definition, a PEC corresponds to the amount of pesticides a honey bee might be exposed to, either by ingestion or contact. In this paper, with the example of imidacloprid and honey bees, we only considered oral exposures because data on topical exposures are scant. We can estimate honey bees' exposure to both contaminated pollens (sunflower and maize) and nectar (sunflower) with (i) the known and validated concentrations of imidacloprid found in contaminated pollens and nectars, and (ii) the amount of contaminated pollen and nectar consumed by different categories of honey bees (21).
(i) The amount of imidacloprid present in the food of honey bees is directly related to the environement. For example, if a hive is located near extensive cultures of maize andsunflower plants treated by imidacloprid, the proportions of pollen and/or nectar that might be contaminated by imidacloprid are expected to be high. Since the relative proportions of contaminated food, versus uncontaminated food, consumed by honey bees are unknown, we considered 5 different levels of contamination ranging from 20% (a low level of contamination) to 100% (the highest level of contamination) (Table 1), although the latter case might rarely occur in natural conditions.
(ii) The amount of contaminated food consumed by different categories of honey bees depends on the amount of food the bees require to achieve particular tasks within the colony. Among them, Rortais et al. (21) considered the categories that are potentially the most exposed to imidacloprid: those that achieve the most costly tasks in terms of energy and which consume the highest amounts of pollen for their development. Therefore, for the calculation of the PEC, the following categories of honey bees were considered: the worker larvae which consume pollen and nectar for their development over about 5 days; the drone larvae which consume pollen and nectar for their development over about 6.5 days; the nurses which consume pollen over a period of 10 days and nectar and/or honey to maintain the nest temperature at 34 C over the entire brood attendance period, lasting about 8 days; the wax-producing bees which consume nectar during the period of maximum wax production, lasting about 6 days; the winter bees which consume nectar and honey to maintain the nest temperature at viable TABLE 1. Estimated PECs for Different Categories of Honey Bees to Imidacloprida categories of bees sugarb pollenb
imidacloprid(20%)
imidacloprid
(40%)
imidacloprid
(60%)
imidacloprid
(80%)
imidacloprid
(100%)
worker larvae
(d ) 5)
59 5 41 82 124 165 206
drone larvae
(d ) 6.5)
98 N. A.c 63 126 189 252 315
nurses
(d ) 10 for pollen and
d ) 8 for nectar)
272-400 65 219-301 438-602 656-903 875-1204 1094-1505
wax-producing bees
(d ) 6)
108 69 139 208 278 347
winter bees
(d ) 90)
792 509 1017 1526 2034 2543
nectar foragers
(d ) 7)
224-899 144-577 288-1155 431-1732 575-2310 719-2887
pollen foragers
(d ) 7)
73-109 47-70 94-140 140-210 187-280 234-350
a Estimated amounts of food (sugar and pollen in mg) and imidacloprid (in pg) consumed per bee over d days, with different levels of food contamination (%). b Data from ref 21. c N. A.: No available data. VOL. 40, NO. 7, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2449 temperature during winter, lasting about 90 days in temperate regions; and the nectar and pollen foragers which consume nectar and/or honey to cover their daily flight expenses. As a forager life span is highly variable (between 1 and 3 weeks), the amount of food consumed by a forager to collect food has been estimated over a minimal period of one week. Honey Bees' Exposure to Contaminated Sunflower and/or Maize Pollens. The honey bee's exposure to contaminated pollens collected on treated plants is determined by the following equation: Honey Bees' Exposure to Contaminated Sunflower Nectar. The nectar brought back to the colony is consumed by honey bees, either rapidly as it is or later on as honey when it is stored. The relative amounts of nectar and honey consumed
by honey bees are unknown. However, the amounts of sugar contained in sunflower honey and nectar are known and are, on average,80%and 59%, respectively (22, 23). Therefore, the amounts of sunflower nectar and/or honey consumed by honey bees can be determined by their sugar consumption, in relation to their energy requirement (21). As a result, for every milligram of sugar required, a honey bee will have to consume 1.25mgof sunflower honey or 1.69mgof sunflower nectar. Therefore, a honey bee's exposure to contaminated sunflower nectar can be determined by the following equation:
Honey Bees' Exposure to Contaminated Sunflower and/or Maize Pollens and to Contaminated Sunflower Nectar. The honey bee's exposure to contaminated sunflower and/or maize pollens and to contaminated sunflower nectar is summarized as follows:
PNEC Estimates. By definition, a PNEC corresponds to the amount of substances that will have no impact on ecosystems. For numerous substances, the pool of data is usually too limited to predict their effects on ecosystems. In such circumstances, empirically derived assessment factors must be applied. These assessment factors allow the prediction of a concentration below which an unacceptable effect will most likely not occur. The size of these assessment factors incorporates various uncertainties due to extrapolations from single-species laboratory data to a multi-species ecosystem, in particular uncertainties due to intra- and inter-laboratory variations in toxicity data, intra- and inter-species variations, short-term to long-term toxicity extrapolations, and from laboratory data to field impact studies. For the terrestrial compartment, the size of the assessment factors depends on the confidence we have on the representativeness of the toxicity data. For example, the size of these factors is reduced when more data become available at various trophic levels and for several taxonomic groups.
Based on these parameters and in relation to the experimental conditions, the TGD determines various assessment factors. In laboratory conditions, for short-term toxicity tests (LD50) and for one trophic level, a factor 1000 is used, for long-term toxicity tests and for several trophic levels with known NOEC, a factor 100 is applied, and for additional long-term toxicity tests of two or three trophic levels of known NOEC, the factors 50 and 10, respectively, are selected. In field conditions, an assessment factor is determined case by case (10).
The approach presented in this paper consisted in finding appropriate PNECs for honey bees derived from PNECs designed for ecosystems. ThesePNECvalues were estimated with the available data obtained from studies on oral acute, chronic, and sublethal toxicities of imidacloprid to honey bees. These values were derived from the lowest validated toxicities (LD50, lowest observed effect concentration (LOEC), or no observed effect concentration (NOEC)) to which assessment factors are applied. This new approach is specifically adapted to honey bees because it allows the assessment of both colonies and individuals. These factors had to be determined case by case, following the standard approach used by the TGD. Every time new data enabled us to reduce extrapolations (chronic toxicity data in relation to acute toxicity data), the assessment factors were generally reduced by a factor 10.
PEC/PNEC Estimates. The hazard posed by new substances to organisms is determined by the PEC/PNEC ratio. When this ratio is over 1, it highlights an intoxication risk for honey bees, whereas when it is below 1, it indicates no risk. According to the TGD (10), this ratio is obtained and derived from acute toxicity data, but when a risk is found, the ratio is re-calibrated withnewdata obtained inmorerepresentative conditions. For honey bees, the PECs were determined with all the available scientific data found on honey bees' food consumptions because there were sufficient data, whereas the PNECs required more data. Therefore, following theTGD procedure, PNECs were derived from acute toxicity data. When a risk was highlighted, a new PEC/PNEC ratio was determined with data obtained from chronic toxicity studies. If the new ratio remained over 1, a final PEC/PNEC ratio was then calculated with new data coming from sublethal field toxicity studies. This final ratio is the most representative ratio of the natural conditions of a honey bees' colony. Results
PEC Estimates. (i) In pollen collected directly on the anthers of flowers, the concentrations of imidacloprid found in treated sunflower and maize plants are 3.3 and 3.5 íg/kg, respectively (24, 25), or on average 3.4 íg/kg for both pollen types. The concentration of imidacloprid found in treated sunflower nectar is 1.9 íg/kg (25). (ii) Based on the estimated amounts of pollen and nectar consumed by honey bees over several days of activity (21), the potential amounts of imidacloprid ingested by honey bees were determined (Table 1). PNECEstimates. There is currently no test and no toxicity data for larvae. For this category of honey bees, PNECs were derived from the toxicity data obtained in adult workers. Table 2 shows the PNECs determined in adults and derived from acute, chronic, and sublethal toxicity data, to which specific assessment factors were applied.
From acute toxicity data: the lowest validated LD50 (48 h) is 3.7 ng of imidacloprid per bee (26). According to the TGD (10), the assessment factor for acute toxicity data is 1000. However, the toxicity of imidacloprid was determined by several studies which tested models belonging to the same species, and found similar results. As these data present very few uncertainties, an assessment factor of 100 was applied. Therefore, the validated PNEC becomes 3.7/100 ) 37 pg/ bee.
From Chronic Toxicity Data. In laboratory conditions, the lowest validated value was LD50 (10 d) ) 0.012 ng/bee (27). As this value was obtained from a long-term experiment, it seemed appropriate to apply the same assessment factor as the one used for a long-termNOECexperiment, which is 100 (10). However, this factor is used to determine a PNEC for PEC1 (pg) ) Validated concentration of imidacloprid found in sunflower and/or maize pollens (íg/kg) _ Amount of pollens consumed by honey bees (mg) _ Levels of contamination found in pollen (%) PEC2 (pg) ) Validated concentration of imidacloprid found in sunflower nectar (íg/kg) _ Amount of sugar consumed by honey bees (mg) _ 1.69 Levels of contamination found in sunflower nectar (%) PEC (pg) ) PEC1 (pg) + PEC2 (pg)
2450 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 7, 2006 all the taxonomic groups of an ecosystem. To adjust this factor to one taxonomic group (the honey bees), we applied a factor 10. This factor includes all variations found among and within taxonomic groups (inter- and intra-species variations). Therefore, the validated PNEC becomes 0.012/ 10 ) 1.2 pg/bee.
From Sublethal Toxicity Data. In laboratory, semi-field, and field conditions, one or several administered doses might induce behavioral modifications among treated honey bees. When administering a unique oral dose of imidacloprid to honey bees for the testing of the knockdown effect, the lowest validated NOEC was 0.94 ng/bee (28). As this value was obtained from a short-term experiment, an assessment factor of 100 should have been applied (TGD). However, this value does not correspond to a LD50; it is a dose that has no impact on honey bees. Moreover, the measured effect is a sublethal effect. Therefore, we applied an assessment factor of 50. The validated PNEC becomes 0.94/50 ) 18.8 pg/bee. When administering several oral doses of imidacloprid to honey bees for the testing of the proboscis extension reflex (PER), the lowest validated concentration, after a 10 day experiment, was 0.2 ng/bee (29). This value corresponds to aNOECbased on the testing of sublethal effects after a long-term intoxication. Therefore, we applied an assessment factor of 10. The validated PNEC becomes 0.2/10 ) 20 pg/bee. In semi-field conditions, a LOEC (5 d) of 0.075 ng/bee was validated for the testing of the time spent feeding on contaminated syrup (30). As this study was conducted in the natural conditions of foragers, an assessment factor of 10 was applied (TGD, 10). The validated PNEC becomes 0.075/ 10 ) 7.5 pg/bee.
In field conditions, a lowest NOEC (10 d) of 0.25 ng/bee was validated for the testing of dances (31). Studies conducted in field conditions present similar conditions to those found in the natural environment of honey bees. Therefore, an assessment factor of 1 should be applied (TGD, 10), but we selected an assessment factor of 5 because the setting of the feeders is artificial. Therefore, the validated PNEC becomes 0.25/5 ) 50 pg/bee.
PEC/PNECEstimates. According to the previously defined assessment factors, and whatever the level of food contamination is, all the investigated categories of honey bees presented an intoxication risk to imidacloprid (Figure 1). The PEC/PNEC ratio was the highest for winter bees and nurses (between 10and100)andthe lowest for pollen foragers and larvae (between 1 and 10). Discussion
The PEC/PNEC derived from the calculation of honey bees' exposure to which appropriate assessment factors were applied show that the risk posed by imidacloprid is alarming for all categories of honey bees. These ratios are all over 1, and greater in adult hive bees than in any other categories of bees. Whatever the validated toxicity data are, the determined PNECs are in a limited range of values (between 1.2 and 50 pg/bee). These estimates are in agreement with observations made in regions of extensive sunflower and maize cultures, which report a decrease in honey production since the launching of imidacloprid on sunflower plants in 1994 (32), and several behavioral dysfunctions, foragers disappearances, and great honey bee mortalities in summer, during the blossoming of maize and sunflower plants, and after winter, when all sunflower and maize pollens have been consumed by colonies.
In areas of extensive sunflower and maize cultures treated by imidacloprid, all categories of honey bees, whatever their age is, are at risk of intoxication. In such a situation, honey bees are most likely to bring back food that is contaminated by imidacloprid, and the observed effects might relate to either acute, chronic, or sublethal intoxications, all inducing the death of honey bees.
In areas where sunflower and maize cultures treated by imidacloprid are less abundant, honey bees might be less intoxicated because they might consume a mixture of contaminated and uncontaminated food. In this situation, honey bees are most likely to be intoxicated by sublethal doses, rather than by acute or chronic doses, which might have lethal consequences at the individual and colony levels. At sublethal doses, pesticides are known to have profound impacts on the colony, in particular on the honey bees' longevity (34), the brood production (35, 36), the development of hypopharyngeal glands (37), and the egg laying (38). Imidacloprid is known to affect the honey bees' cognitive behaviors such as the proboscis extension reflex PER (33). Learning and memorization in honey bees' tasks are very important. For example, a forager that is disorientated might get lost and eventually die. In the case of massive foragers' intoxications, the colony is likely to be greatly affected. In an experiment under tunnels,Vandameet al. (39) exposed honey bees to deltamethrin at a sublethal dose that is 20-fold lower than the registered dose at which foragers are expected to be exposed to in the environment. They found that 54% of the treated bees were disoriented and took flight toward the sun. The authors concluded that such sublethal effects may be the cause of the symptom called the "disappearance bee disease" by beekeepers who observed colonies' weakening without finding dead bees close to the hives. This hypothesis was formerly raised by other scientists (40, 41). Imidacloprid can also affect honey bees by chronic intoxications. In the long run, a repeated ingestion of low doses of imidacloprid could cause immunodeficiency and diseases in honey bees. The impairment of the bees' immunity system is a nonspecific mechanism (42). For TABLE 2. PNECs for Oral intoxications of Honey Bees to Imidacloprid toxicities
experimental conditions (imidacloprid intakes) doses (ng/bee) observed variables assessment factors PNEC (pg/bee) acute laboratory (one determined dose) LD50 (48 h) ) 3.7 survival (% mortality) 100 37 chronic laboratory+ (one determined dose) LD50 (10 d) ) 0.012 survival (% mortality) 10 1.2 sublethal laboratory (one determined dose) NOEC ) 0.94 behavioral dysfunction (knockdown effect) 50 20 laboratory (several determined doses) NOEC ) 0.2 behavioral dysfunction (proboscis extension reflex) 10 20 semi-fields (at feeders, several determined doses) LOEC ) 0.075 behavioral dysfunction (feeding) 10 7.5 fieldsa (at feeders, several determined doses/on plants) NOEC ) 0.25 behavioral dysfunction (dances) 5 50 a Feeders contained syrup contaminated by imidacloprid. In field conditions, feeders were placed near hives to reinforce the observed effects of imidacloprid seed-dressed plants on honey bees. VOL. 40, NO. 7, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2451 example, sublethal concentrations of malathion result in higher invasions of treated colonies by the wax moth (43). In some cases, no honey bee troubles were observed by beekeepers, but no scientific study has ever confirmed these FIGURE 1. Hazard posed by imidacloprid to different categories of honey bees feeding on various proportions of contaminated food: estimated PEC/PNEC ratios derived from (A) acute toxicity data, (B) chronic toxicity data, and (C) sublethal toxicity data obtained in field conditions for foragers and in laboratory conditions for all the other categories of honey bees (a risk is highlighted when ratios are greater than 1). 2452 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 7, 2006 observations. The presence of untreated or very little treated areas near hives, and the presence of compensatory phenomena (increase of brood development, replacement of dead foragers) with no visible harmful consequences for the colony may occur and explain the absence of any observed troubles.When assessing the risk posed by systemic insecticides, the HQ does not take into account several idiosyncrasic parameters such as persistence in soils, presence in pollen and nectar, and transport in the air. The calculation of the Toxicity Exposure Ratios (TER) (ratio between a toxicological end point and a PEC), regularly used in the risk assessment of pesticides to organisms (mainly vertebratesandnematods), could take into account such crucial parameters. However, for social invertebrates such as honey bees, the use of the new and existing chemical substances approach (herein the PEC/PNEC ratio) should be more appropriate than the use of the TER because it enables the protection of the whole colony.ThePEC/PNECratio could then be re-calibratedwhen moredataonimidacloprid andonother systemic insecticides are available.
For hive bees (nurses and winter bees), the PNECs could be refined when more data are available on the mechanisms of a colony's regulation (e.g., brood development) in field and semi-field conditions. For larvae, exposures were derived from data obtained on adult toxicities in order to obtain an indicative and comparative value. Given that larvae are more or less sensitive than adults to chemicals (4, 44), more studies need to determine accurately their exposure risk to imidacloprid and to other systemic insecticides.
Wecould not investigate topical exposures of imidacloprid to honey bees because there are not enough data available onthismodeof exposure. However, honey bees' intoxications by topical exposures should not be discarded. For examples, foragers might get contaminated by contaminated dust particles during sowing operations (45).
The impact of systemic insecticides on honey bees is not limited to the impact of the parent compound; it also includes exposures to its metabolites. In the case of imidacloprid, some metabolites (e.g., olefin, which is twice more toxic than imidacloprid) are found to be very toxic to honey bees (9, 46, 47) and some of them are detected at low concentrations (between 0.3 and 1 íg/kg) in rape pollen and nectar (48). However, to investigate in further detail the impact of metabolites on honey bees, their concentrations in other types of pollen and nectar must be determined.
Exposures to imidacloprid were estimated by assuming that the molecule is stable in the hive because it is stored in a dark environment. However, the transformation of pollen and nectar into bee bread and honey, respectively, imply the action of several enzymes that might change the stability of imidacloprid. Therefore, the concentration of imidacloprid in the stored food (bee bread and honey) should be measured to test its stability in the hive over time.Honeybees' exposures to contaminated sunflower nectar were determined with data issued from one study (25). To confirm and generalize the trend found, it is necessary to conduct more studies (i.e., the concentration of imidacloprid in nectar coming from other varieties of sunflower and from other melliferous plants). The method and the assessment factors proposed in this paper could be re-calibrated when more data are available. Although the determination of the LD50 (48 h) is readily obtained for the calculation of HQ, its representativeness in testing the survival of a honey bee colony is arguable. To assess the risk posed to honey bees, chronic and sublethal toxicity tests must be conducted systematically, especially in the case of systemic insecticides which have a long-lasting action. To achieve these tests, standardized protocols are required and could be elaborated on the grounds of existing experimental studies which have investigated the chronic impacts of systemic insecticides on honey bees (9, 49, 50), as well as their sublethal effects on the behavior (41, 47, 51-53) and physiology (38, 54, 55) of these organisms. Based on the risk assessment method used for terrestrial organisms, this method is original. It includes the assessment of several important parameters such as the following: (i) The detection and measurement of the amount of an active ingredient present in the various substrates used by honey bees. These measures are not statutorily requested. (ii) The development of various scenarios of honey bees' exposure to the active ingredient. These scenarios better predict the risk posed by systemic insecticides to honey bees because they take into account the biology and particular requirements of a honey bees'colony (21). (iii) The use of novel and validated methods for the assessment of lethal and sublethal honey bees' intoxications. (iv) The use of assessment factors, when experimental designs are tightly related to environmental conditions. This approach is usually applied to assess the risk of chemical substances.
AcknowledgmentsAuthors M.P.H. and A.R. contributed equally to this work.
The validation of the data used in this study on imidacloprid
was realized in concert with a group of experts nominated
by the French Ministry of Agriculture and known as the
"Comite´ Scientifique et Technique (CST) de l'Etude multifactorielle
des troubles des abeilles". M.P.H. and A.R. were
financed by the French Ministry of Agriculture to give
scientific support to the CST.
Note Added after ASAP Publication
An incorrect reference was cited in Table 1 in the version
published ASAP March 7, 2006; the corrected version was
published March 8, 2006.
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Agnès RORTAISa*, Gérard ARNOLDa, Marie-Pierre HALMb, Frédérique TOUFFET-BRIENSb a Laboratoire Populations, Génétique et Évolution, CNRS UPR 9034, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France b Centre d'Études et de Recherche Sur le Médicament de Normandie, Université de Caen, 5 rue Vaubénard, 14032 Caen Cedex, France Received 15 December 2003 - Revised 4 May 2004 - Accepted 21 May 2004 Published online 16 March 2005 Abstract - The hazard posed to honeybees by systemic insecticides is determined by toxicity tests that are designed to study the effects of insecticides applied on the aerial parts of plants, but are not adapted to systemic substances used as soil or seed treatments. Based on the available data found in the literature, this paper proposes modes of honeybees exposure to systemic insecticides by estimating their pollen and nectar consumption.
Estimates are given for larvae and for the categories of adults which consume the highest amounts of - pollen, the nurse bees, and - nectar, the wax-producing bees, the brood attending bees, the winter bees, and the foraging bees. As a case study, we illustrate these estimates with the example of imidacloprid because its concentrations in sunflower nectar and in sunflower and maize pollens of seed-dressed plants have been precisely determined, and because its levels of lethal, sublethal, acute, and chronic toxicities have been extensively investigated.
Apis mellifera / systemic insecticide / exposure / imidacloprid / nectar / pollen
1. INTRODUCTIONTo be launched on the market, pesticide products need to be granted an authorisation. In this process, tests are required to ensure that these chemicals do not present any harm to pollinators, in particular to honeybees. In Europe, these tests follow the EPPO No. 170 guideline, adopted by the European Plant Protection Organisation (EPPO, 2001). These tests present methods for studying the toxicity and hazard of pesticides to honeybees in laboratory, semifield (cages or tunnels) and field conditions. This toxicity corresponds to the single dose of insecticide, administered by ingestion or contact that kills half of a treated group of bees in 24 h or 48 h (LD50, expressed in weight of active ingredient per bee). The toxicity risk of pesticides is commonly estimated with the Hazard Quotient (HQ = application rate/ LD50, EPPO, 2001). This quotient is adapted to pesticides sprayed on plants (i.e. carbamates, organophosphates, organochlorates, pyrethroids), but not to those applied to soil or seeds. Most pesticides sprayed on the surface of the plant have a rapid and residual action of a few hours to a few days, whereas systemic insecticides penetrate into the plant, including melliferous and polleniferous plants, and protect it all through its development from soil invertebrates and in some cases from sucking insects (Elbert et al., 1991). The relevant * Corresponding author: rortais@pge.cnrs-gif.fr 1 Manuscript editor: Jean-Noël Tasei 72 A. Rortais et al. parameter to consider for examining honeybees exposure to the active substance is the contamination of nectar and pollen instead of the application dose to soil or seeds. Therefore, in the case of systemic insecticides, the official HQ should not be used. Moreover, the regulatory testing guidelines do not take into account the potential persistence of these molecules in plants and do not specify which laboratory tests should be performed to estimate the possible lethal or sublethal impacts on bees due to chronic exposures occurring after several days of repetitive ingestion of (or contact with) a given insecticide. Insecticides sprayed on plants can be toxic to foraging honeybees when they are in contact with treated plants (Koch and Weisser, 1997) and when they fly through the adsorption of contaminated dust particles (Prier et al., 2001). Honeybees can also intoxicate the whole colony by bringing contaminated pollen and nectar back to the hive (Bos and Masson, 1983; Villa et al., 2000). However, the risk of any chemical transfer into the hive is greater with systemic insecticides (Waller et al., 1984). Moreover, in comparison with the majority of the insecticides of the old generation, the toxicities of these new molecules and their metabolites are very high, and although they are detected in pollens and nectars at low concentrations (Schmuck et al., 2001; Bonmatin et al., 2003), their hazard on bees might not be negligible. When honeybees consume small amounts of pesticides, they might exhibit sublethal toxic effects. Such impacts might affect honeybees by disrupting their cognitive capacities (i.e. the learning and orientation abilities) and behaviours (i.e. the collection of food). In such conditions, a forager might not be able to return to the hive and, as it relies on the colony for its survival, might die within a few hours. Therefore, the initial sublethal effect might eventually become lethal to honeybees. Among systemic insecticides, one neurotoxic molecule, imidacloprid, acting on nicotinic receptors, is widely used. It is commonly used as a seed treatment (formulation Gaucho ®) for the protection of maize and sunflower crops. For the last few years, honeybees have been dying in huge numbers and colonies have been declining dramatically, in particular in regions where large areas of sunflower crops are treated by systemic insecticides (Belzunces and Tasei, 1997). Beekeepers and scientists suspect that these chemicals are responsible for these troubles (Vermandère, 2002; Bonmatin et al., 2003). Considering the essential role of honeybees in honey production and pollination (Williams, 1994), this lack of assessment poses a serious problem that needs to be quickly solved. The objective of this study was to describe different possible modes of honeybees exposure to systemic insecticides by estimating their individual consumption in contaminated pollen and nectar. To achieve this goal, we used the available data presented in the literature to estimate the total amount of pollen and nectar consumed by different categories of honeybees, and to determine their possible exposure to systemic insecticides when all the food consumed is contaminated by these molecules. As an example, we considered the case of imidacloprid because its concentrations in pollens of Gaucho® seed-dressed sunflower and maize plants and in nectar of Gaucho® seeddressed sunflower plants are known, and because its toxicity to honeybees has been determined (Schmuck et al., 2001; Bonmatin et al., 2001, 2002; CST, 2003).
2. AMOUNT OF FOOD BROUGHT BACK TO THE HIVEHoneybees supply the colony with nectar and pollen collected at varying distances from the hive. Currently, it is considered that 95% of the foraging activity of honeybees extends up to 6 km away from the hive, but honeybees might forage up to 12 km from the hive (von Frisch, 1967; Seeley, 1985; Winston, 1987), which implies that they might visit plants over large areas of several tens of km2.
2.1. PollenPollen foragers collect pollen on flowers and bring it back to the colony by making and carrying on their posterior legs two pollen pellets. The amount of pollen collected per colony and per year is in the range of a few tens of kilos to about 55 kg (Louveaux, 1968; Seeley, 1985; Winston, 1987). This pollen is composed of a mixture of different plants present in variable numbers, reflecting the floral composition of Honeybees exposure to systemic insecticides 73 the environment of the hive. In areas of extensive cultures of polleniferous crops, large amounts of pollens coming from these plants might be brought back to the colony. For example, honeybees can collect 10 to 20 kg of sunflower or maize pollen per year, and sometimes even more (Odoux et al., 2004). During the flowering time of these plants, which lasts between 1 and 1.5 months, sunflower and maize pollens can represent up to 80-90% of the total weight of all pollen types collected by honeybees (Odoux et al., 2004).
2.2. NectarNectar foragers bring nectar back to the colony, which might either be quickly consumed or consumed later after being transformed into honey by water evaporation and by changes in sugar composition. Nectar, depending on its floral origin, contains between 5-80% of sugar and honey contains in average 80% of sugar (Crane, 1975). For sunflower plants, nectar contains on average 40% of sugar (Pham Delègue and Bonjean, 1983). Therefore, if the annual honey requirement for a honeybee colony is about 60-80 kg (Seeley, 1985; Winston, 1987), the total amount of nectar collected by bees each year might be in the range of a few hundreds of kilos per colony. As we do not know the bees' differential consumption of nectar and honey, we related their sugar consumption depending on whether they consume nectar or honey. With the example of sunflower, when a honeybee requires 1 mg of sugar, it will have to consume either 2.5 mg of fresh sunflower nectar or 1.25 mg of sunflower honey.
3. NECTAR AND POLLEN CONSUMPTIONIn the literature, some estimates of honeybees
consumption of pollen and nectar are
available. These estimates are presented below
and summarised in Table I.
During their development, honeybees go
through two stages during which they feed: the
larval and adult stages. They utilize the proteins
contained in pollen to insure their development
and growth, and they require the sugar contained
in nectar (or honey) to cover their energetic
expenses.
Table I. Estimated amounts of sugar (contained in nectar or honey), pollen and imidacloprid consumed by
larvae during their development over N days (N = 5 days for workers and N = 6.5 days for drones) and by
adults over a period of N days of activity (N = 10 days for nurses, N = 6 days for wax producing bees, N =
8 days for brood attending bees, N = 90 days for winter bees and N = 7 days for foraging bees). The
amount of imidacloprid consumed by honeybees is determined by the following equivalence: 1 mg of sugar
contained in nectar or honey = 4.75 pg of imidacloprid and 1 mg of pollen = 3.4 pg of imidacloprid in
nectar and pollen coming from Gaucho® seed-dressed plants. N.A. = no data available.
Categories of bees Estimated amounts of food (sugar and pollen) and imidacloprid consumed
per bee over N days
Sugar (mg) Pollen (mg) Imidacloprid (ng)
Larvae
Workers 59.4 5.4 0.3
Drones 98.2 (N.A.) 0.5
Nurses - 65 0.2
Hive bees
Wax-producing bees 108 - 0.5
Brood attending bees 272-400 - 1.3-1.9
Winter bees 792 - 3.8
Foraging
bees
Nectar foragers 224-898.8 - 1.1-4.3
Pollen foragers 72.8-109.2 - 0.3-0.5
74 A. Rortais et al.
Larvae consume royal jelly, produced by
nurses, which contains honey and pollen
(Haydak, 1943, 1968, 1970; Kunert and
Crailsheim, 1988; Malone et al., 2002).
Among adult honeybees, nurses consume
pollen during the first 8 to 10 days of their life,
to develop their hypopharyngeal and mandibular
glands and to produce some of the larval
food (Maurizio, 1954; Crailsheim et al., 1992;
Hrassnigg and Crailsheim, 1998a). However,
under certain circumstances, they might consume
pollen until the age of 18 days (Hrassnigg
and Crailsheim, 1998b).
Adult honeybees consume nectar to perform
various tasks. Among these tasks, some require
more energy and sugar than others.
Maximal exposure to systemic insecticides
are expected among honeybees that consume
the greatest amounts of contaminated pollen
and nectar. Large amounts of pollen are consumed
by nurses, and to a less extent by larvae,
whereas large amounts of nectar are consumed
by wax-producing bees, brood attending bees,
"winter" bees, and foragers. In this study, we
focused only on these categories of honeybees.
A worker larva is fed over 5 days and after this feeding stage weighs, on average 150 mg (Jay, 1963). During the first 3 days of its development, it consumes about 30 mg of food (Nelson, 1924), and during the next 2 days, about 120 mg. The latter estimate is based on the results of Bishop (1961), who demonstrated that most of the food consumed by the larva contributes to its gain of weight within these 2 days. The sugar content of the food of a worker larva is 18% during the first 3 days, and 45%, during the following 2 days (Planta, 1888 cited by Haydak, 1968). Therefore, a worker larva will consume a total of 59.4 mg of sugar in 5 days; that is, 5.4 mg of sugar within the first 3 days and 54 mg of sugar within the last 2 days. It will also consume 5.4 mg of pollen between the 3rd and 5th day of its development (Babendreier et al., 2004).
3.2. Drone larvaeDrone larvae are fed over 6.5 days and after this feeding stage weigh on average 340 mg (Jay, 1963). The development of a drone larva has a similar growth pattern to that of a worker larva (Thrasyvoulou and Benton, 1982). The precise amount of food consumed by a drone larva is not known, but it might be deduced from that of a worker larva. The sugar content of the food of a drone larva is 9.6% during the first 3 days of its development and 38.5% during the following 2 days (Planta, 1888 cited by Haydak, 1968). Therefore, during the first 5 days of its development, a drone larva will consume a total of 49.1 mg of sugar, 2.9 mg within the first 3 days and 46.2 mg over the next 2 days. During the last 1.5 days, the amount of food consumed by a drone larva is probably the same as the amount consumed earlier, since a drone larva increases its weight by a factor of two during this short period of time. Therefore, a drone larva will consume a total of about 98.2 mg of sugar in 6.5 days. The pollen consumption of drone larvae has never been determined.
3.3. Nurse beesWithin a period of 10 days, the total amount of pollen consumed by a nurse bee is on average 65 mg (Pain and Maugenet, 1966; Crailsheim et al., 1992). However, during this period, honeybees could consume up to 12 mg of pollen within one single day (Pain and Maugenet 1966; Crailsheim et al., 1992).
3.4. Wax-producing beesThe production of wax by a honeybee colony varies greatly, depending on various factors (i.e. blossoming, nectar flow, season, outside temperature, number of young waxproducing bees, gathering of nectar and pollen, etc.) (Hepburn, 1986). However it is generally accepted that the amount of sugar consumed per unit weight of beeswax produced is on average 6:1, notwithstanding racial, seasonal, and colony density variations (Tokuda, 1955; Hepburn et al., 1984). Over the period of maximum wax production, lasting about 6 days, a wax producing bee produces 3 mg of wax per day (Taranov, 1959; Hepburn et al., 1984), requiring 18 mg of sugar per day, or a total of 108 mg of sugar in 6 days. Honeybees exposure to systemic insecticides 75
3.5. Brood attending beesFrom April to October, brood attending bees require energy to maintain the brood temperature at about 34 °C (Simpson, 1961; Seeley and Heinrich, 1981; Heinrich, 1985). During this period and in temperate climates temperatures average 15-20 °C, outside the hive. In such conditions, a brood attending bee will consume between 34 mg (at 20 °C) and 50 mg (at 15 °C) of sugar per day (Free and Spencer-Booth, 1958; Simpson, 1961) and a total of 272- 400 mg of sugar over the entire brood attendance period, lasting about 8 days.
3.6. Winter beesIn temperate regions, "winter" bees require energy to maintain the nest temperature at 5- 8 °C (in the periphery) and 15-20 °C (in the centre) (Winston, 1987). During winter, lasting about 3 months in temperate regions, a honeybee colony composed of about 20,000 of bees will consume on average 20 kg of honey (Farrar, 1952, 1960; Johansson and Johansson, 1969). Therefore, a "winter" honeybee requires about 8.8 mg of sugar per day (equivalent to 11 mg of honey) and a total of about 792 mg of sugar over the entire winter period. This average is a broad estimate which does not take into account any natural variations (periods of low consumption alternated with periods of high consumption in relation to external temperature variations) that might occur during this long period.
3.7. Pollen and nectar foraging beesPollen and nectar foragers require about 8- 12 mg of sugar per hour of flight (Balderrama et al., 1992). Nectar foragers achieve 10 trips/ day on average, of about 30 to 80 min each (Winston, 1987), with a maximum of 150 trips/ day (Ribbands, 1953), and pollen foragers achieve 10 trips/day on average, of 10 minutes each (Winston, 1987). If we assume that during 1 h of activity foragers spend 80% of this time flying and 20% foraging, for which the energetic cost is not known, nectar and pollen foragers will spend between 4-10.7 hours/day and 1.3 hours/day, respectively, for flight activities alone. The lifetime of a forager is highly variable and related in particular to its foraging intensity and activity (Winston, 1987), but on average might vary between one and three weeks. To realise their flights, nectar and pollen foragers will consume between 32-128.4 and 10.4-15.6 mg of sugar per day or a total of 224-898.8 and 72.8-109.2 mg of sugar per week, respectively. During flights, foragers might perform stationary flights, which are energetically very costly (Nachtigall et al., 1989), but the time spent in these flights is not known. While collecting pollen or nectar, foragers get their body covered by pollen (Parker, 1981). Foragers are also in contact with pollen while making and carrying pellets back to the hive (Louveaux, 1958). Therefore, a topical exposure of foragers to contaminated pollen cannot be excluded, though it is difficult to estimate.
4. THE EXAMPLE OF IMIDACLOPRID4.1. Pollen and nectar contamination
by imidacloprid
4.1.1. PollenPollen contamination by imidacloprid can be determined in two types of pollens: the pollen present in flowers and collected by foragers, and the pollen pellets harvested by beekeepers in pollen traps. - Pollen collected on flowers: the level of contamination of this type of pollen is related to the systemic property of the molecule. In Gaucho seed-dressed sunflower and maize plants it is about 3.4 g of imidacloprid per kilo of pollens (Bonmatin et al., 2001; Schmuck et al., 2001). - Pollen pellets in traps: pollen traps are installed at the hive entrance to catch some of the pollen pellets brought back by pollen foragers. For this reason, the pollen sampled in pollen traps is a mixture of different kinds of pollens collected in the foraging area of honeybees. The level of contamination found in pollen pellets varies in relation to the environment of the colony where they are collected (Charvet et al., 2003). If this environment contains many plots of treated plants, the level of contamination found in 76 A. Rortais et al. the pollen pellets will reach that of the pollens collected in treated flowers. In contrast, if this environment contains few treated plots, the mean concentration of insecticides found in pollen pellets will be lower. For example, the concentrations of imidacloprid found in sunflower and maize pollens collected in the pollen traps of some particular hives were 2.2 and 0.75 g/kg, respectively, or about 1.5 and 4.5 times less, respectively, than the concentration of imidacloprid found in the same types of pollen collected in flowers (Bonmatin et al., 2001, 2002). Therefore, the level of contamination found in pollen pellets cannot be generalised, whereas that of pollens collected on flowers gives a more accurate estimate of the maximal honeybees exposure to contaminated pollens.
4.1.2. Nectar and honeyTo cover their energy requirements, honeybees consume either freshly collected nectar or stored honey. - Fresh nectar: the level of contamination of nectar is directly related to the systemic property of the molecule. For example, in Gaucho® seed-dressed sunflower plants, it is 1.9 g of imidacloprid per kilo of nectar (Schmuck et al., 2001), or 4.75 pg of imidacloprid per milligram of sugar contained in sunflower nectar. - Honey: the level of contamination of honey by imidacloprid, with a limit of detection that is sufficiently low, has not been determined yet. However, the persistence of this molecule in acid environments (Agritox, 2004) and the low pH value of honey suggest that the imidacloprid contained in fresh nectar might not be degraded in honey at least over several months. Further investigation is required to confirm this assumption.
4.2. Estimated amounts of imidaclopridbrought back to the colony through contaminated nectar and pollen To determine the exact amount of imidacloprid brought back to the colony, it is necessary to know the total amount of contaminated nectar and pollen collected by honeybees. In the case of Gaucho seed-dressed maize and sunflower plants, the annual quantity of imidacloprid brought back to the hive is 34 g for every 10 kilos of sunflower or maize pollen and 19 g for every 10 kilos of sunflower nectar brought back to the hive by honeybees. However, previous studies tend to demonstrate that foraging bees reduce their visit to syrup feeders when they are contaminated by imidacloprid at concentrations of 3 g/kg (Colin et al., 2004), 24 g/kg (Decourtye, 2002), and 100 g/kg (Kirchner, 1999). This phenomenon might be due to a decrease in the effectiveness of the dances produced by honeybees at the hive to recruit foragers for food collecting (Kirchner, 1999; Decourtye, 2002). Therefore, if honeybees visit treated plants, they might collect and bring back to the hive less nectar than if they visit untreated plants. In such conditions, the amounts of nectar and honey stored in the hive by honeybees should decrease, whereas the amount of pollen stored at the hive might not be affected. However, no experimental study has ever confirmed that this phenomenon occurs with nectar and pollen collected on treated plants.
4.3. Honeybees exposure to imidaclopridBased on the estimated amounts of pollen and nectar consumed by honeybees over several days of activity, the potential amounts of imidacloprid ingested by honeybees can be determined (Tab. I). As the relative proportions of contaminated and uncontaminated food consumed by honeybees cannot be determined, we considered the case of a food that is 100% contaminated by imidacloprid. Such a case might occur in natural conditions (extensive treated cultures, e.g.), though lower exposure cases might also take place when honeybees consume a mixture of contaminated and uncontaminated food.
4.3.1. Worker larvaeIf a worker larva is fed contaminated nectar and pollen, it will consume a total of about 0.3 ng of imidacloprid within the first 5 days of its development as follows: about 0.28 ng of imidacloprid through nectar and about 0.02 ng of imidacloprid through pollen. Honeybees exposure to systemic insecticides 77
4.3.2. Drone larvaeIf a drone larva is fed contaminated nectar, it will consume about 0.5 ng of imidacloprid within the first 6.5 days of its development. The total amount of pollen consumed by drone larvae is not known and, therefore, it is not possible to estimate the oral exposure of a drone larva to contaminated pollen.
4.3.3. Nurse beesIf a nurse bee feeds on contaminated pollen, it will consume up to a maximum of 40.8 pg of imidacloprid within one day of intensive feeding and a total of about 0.2 ng in 10 days.
4.3.4. Wax-producing beesIf a wax-producing bee feeds on contaminated nectar, it will consume 85.5 pg of imidacloprid per day during the period of maximum wax production, lasting about 6 days, or a total of about 0.5 ng in 6 days.
4.3.5. Brood attending beesIf a brood attending bee feeds on contaminated nectar, it will consume between 161.5- 237.5 pg of imidacloprid per day or a total of about 1.3-1.9 ng in 8 days of brood attendance.
4.3.6. Winter beesIf a winter bee feeds on contaminated nectar, it will consume 41.8 pg of imidacloprid per day, or a total of about 3.8 ng during winter, lasting about 3 months.
4.3.7. Nectar and pollen foraging beesIf a nectar foraging bee feeds on contaminated nectar, it will consume 152-609.9 pg of imidacloprid per day or a total of about 1- 4.3 ng per week of foraging activity. If a pollen foraging bee feeds on contaminated nectar (for its flight energy requirement), it will consume 49.4-74.1 pg of imidacloprid per day or a total of about 0.3-0.5 ng per week of foraging activity.
5. DISCUSSION AND CONCLUSIONThis paper highlights the potential hazard of systemic insecticides to honeybees through contaminated pollen and nectar. This phenomenon has previously been reported but never quantified (Villa et al., 2000). In this study, some estimates are given based on the available data found in the literature on pollen and nectar consumptions of different categories of honeybees. Assuming that this food is contaminated by systemic insecticides, the amount of insecticide consumed by each of these categories of honeybees and their potential exposure to these molecules can be estimated. In regions of extensive cultures treated by systemic insecticides, honeybees might bring high amounts of contaminated pollen and nectar back to the colony. For example, in the case of sunflower and maize crops, which are attractive plants to honeybees, 10-20 kg of sunflower pollen and 10-20 kg of maize pollen might be stored at the hive every year during the flowering time of these plants (Odoux et al., 2004). Sunflower nectar is also known to be very attractive to honeybees, with honey production averaging at best 80 kg/year, corresponding to some hundreds of kilos of nectar brought back to the colony every year (Vermandère, 2002). However, since 1994, in regions of extensive sunflower cultures, sunflower honey yield has been dramatically declining (Belzunces and Tasei, 1997).
Honeybees might consume several milligrams of pollen (Pain and Maugenet, 1966; Crailsheim et al., 1992; Badendreier et al., 2004) and several tens of milligrams of nectar per day (Farrar, 1952, 1960; Free and Spencer- Booth, 1958; Simpson, 1961; Johansson and Johansson, 1969; Balderrama et al., 1992). Nurses, which require high amounts of protein for the development of their hypopharyngial and mandibular glands and to produce some of the larval food (royal jelly), might consume up to 65 mg of pollen in 10 days (Maurizio, 1954; Crailsheim et al., 1992; Hrassnigg and Crailsheim, 1998). Foragers, wax-producing bees and heat-producing bees, which perform high energetic tasks, require large amounts of sugar contained in nectar. With the example of sunflower plants, nectar foragers might consume 80-321 mg of nectar (equivalent to 32- 128.4 mg of sugar) per day and between 560 mg 78 A. Rortais et al. and 2.25 g of nectar (equivalent to 224- 898.8 mg of sugar) in a week of foraging activity.
These estimates suggest that, if honeybee colonies are placed in environments containing melliferous and polleniferous crops treated by systemic insecticides, large amounts of insecticides might be brought back to the hive and thereafter consumed by colonies. These estimates are based on a case of a maximal exposure of honeybees to systemic insecticides; that is honeybees consuming pollen and nectar that are 100% contaminated by systemic insecticides. This relates to the case of colonies placed near extensive cultures treated by systemic insecticides. In regions of less extensive cultures, honeybees might consume a mixture of contaminated and uncontaminated food and therefore less systemic insecticides, but it is impossible to precisely estimate these amounts.
The estimated amounts of nectar and pollen consumed by different categories of honeybees allow the determination of the maximal amounts of insecticides consumed by each of these bees. In this paper, we illustrated these modes of exposure with the example of imidacloprid (formulation Gaucho®), but they might be used to describe the impact of any other systemic insecticides and their metabolites on honeybees, providing that their concentration in pollens and nectars are known. For example, the toxicity of the metabolites of imidacloprid (Suchail et al., 2001; Nauen et al., 2001; Decourtye et al., 2003), suggest that these molecules might also have an impact on honeybees.
In this paper, we focused on oral exposure of honeybees to systemic insecticides, but some possible modes of topical exposure also should be investigated. The data in the literature is insufficient to develop this type of exposure on different categories of bees, although larvae might be topically exposed through contaminated nectar. Larvae under 3 days old float in an excessive amount of food containing nectar (Haydak, 1970). If this nectar is contaminated by systemic insecticides, larvae might be exposed to these molecules by contact. Some new tests have been proposed to estimate the larvae exposure. Among them, field (Oomen et al., 1992) and semi-field (Leyman et al., 1999; Tornier, 1999) tests do not appear appropriate since larval exposure cannot be controlled, whereas laboratory tests (Malone et al., 2002; Brødsgaard et al., 2003) might be used to estimate adequately the larvae exposure. The different modes of oral exposure presented in this paper, with the example of imidacloprid, might be used to determine the impact of other systemic insecticides on honeybees.
Gaucho® seed-dressed sunflower and maize plants contain on average 3.4 g of imidacloprid per kilo of pollen. Nurses, which consume the highest amounts of pollen of any other category of honeybees, might be exposed to 0.2 ng of imidacloprid after 10 consecutive feeding days. In Gaucho® seed-dressed sunflower plants, nectar contains 1.9 g/kg of imidacloprid and nectar foragers, might be exposed to about 0.15-0.61 ng of imidacloprid per day, or to 1.1- 4.3 ng in a week of foraging activity. The lethal toxicity of imidacloprid is in the range of a few picogramms after repetitive ingestions of this insecticide over a minimum period of 8 days (Suchail et al., 2001) to 3.7 ng after a unique ingestion of this insecticide in one or 2 days (Schmuck et al., 2001; Agritox, 2004). Imidacloprid might also induce sublethal effects that might affect bees. In particular, it might modify the learning and orientation abilities of honeybees at concentrations as low as 0.1 ng/bee (Guez et al., 2001) and 1.25 ng/bee (Lambin et al., 2001). However, these results might vary according to honeybees age (Guez et al., 2001), race (Suchail et al., 2000), colony (Suchail et al., 2001), and season (Decourtye et al., 2003).
When comparing the known toxicity doses for imidacloprid to the estimated amounts of imidacloprid consumed by different categories of honeybees, we find out that honeybees are potentially exposed to lethal and sublethal doses. However, it has to be kept on mind that our estimates are based on a case of maximal exposure that might reflect the case of colonies placed in regions of extensive treated cultures. Colonies placed in regions of less extensive treated cultures might, more probably, be exposed to sublethal doses. However, in this last situation, the impact of systemic insecticides on honeybees should not be underestimated since some sublethal effects may induce bee losses in particular if physiological troubles and disorientation of foragers are concerned. Honeybees exposure to systemic insecticides 79 This paper should give some input for the setting of a new risk assessment procedure adapted to these systemic molecules now widely used. In particular, European regulatory guidelines should provide a HQ and its specific threshold, adapted to systemic insecticides, along with test methods taking into account chronic and sublethal effects caused by low doses to honey bee adults and larvae.
These new regulatory tests need to assess the toxicity of these molecules and their metabolites. They might be elaborated on the grounds of existing experimental studies which have investigated the chronic impacts of these molecules on honeybees (Stoner et al., 1982; Suchail et al., 2001; Moncharmont et al., 2003), as well as their sublethal effects on honeybees behaviour (Cox and Wilson, 1984; Johansen, 1984; Taylor et al., 1987; Decourtye et al., 2003; Thompson, 2003) and physiology (Bounias et al., 1985; Bendahou et al., 1999; Papaefthimiou and Theophilidis, 2001).
The data provided by these new toxicity tests, combined with the different modes of honeybee exposure developed in this paper, might be used to assess the risk of systemic insecticides according to the approach proposed by the European Commission (technical guidance document on risk assessment in support of the Commission Directive 93/67/EEC on risk assessment for new notified substances, Commission Regulation (EC) No. 1488/94 on risk assessment for existing substances, and Directive 98/8/EC of the European Parliament and Council). In this approach, developed for aquatic organisms, the risk might be estimated by calculating the ratio PEC/PNEC (Predicted Environmental Concentration/Predicted Non Effect Concentration, which is the concentration below which unacceptable effects on organisms will most likely not occur). The values of the PNEC might be refined with the recent results found on the chronic and sublethal toxicity of systemic insecticides associated with an appropriate safety factor. The values of the PEC, usually derived from available measured data and/or from a model of calculation, might be derived from the modes of honeybees exposure presented in this paper, and new risk assessments of systemic insecticides on honey bees can be developed (Halm et al., unpublished data).
Production of food and drink water without plant protection compounds and without damaging of bee colonies is possible! The normally used plant protection strategies of the offices show only, that the official advisers don't want think , integrated or biological'. They are not willing to work with other strategies, this even though the most damaging fungus, bacteria, insects, mites can killed easy and extensive. Unfortunately is the most important point to milk the cow of the plant protection research, but not willing solving the problem and to distribute the money under friends with the same mind and thinking.
As a help for all only plant protection scientists and unfortunately not scientist plant producer the definition for "integrated und biological production":
"Integrated and biological production" but only under the specially observation and including of the bee colonies. It is a wonder that some plant protection compounds are still used. It is wondering too, that the most of the offices of the agriculture service don't look for the bee colonies and only a few look for the bees alone.
If it is possible to believe the politics of agriculture from the public and from the companies and from the farmer organisations, then the most of the farmers would produce integrated. What is "integrated production"?
Hoffmann et al (1985) defined "integrated plant protection" following: integrated pest management is a method, by using all economic, ecologic and toxicological acceptable methods are used, to bring all damaging organism under the economic damaging threshold, by the consciously using of the natural factors. It is also this, that the necessary corrections are to do in the system under preservation and reactivation or changing natural proceedings with a minimum on cost and the possibility of a combination well tolerated measures instead of only one effective method.
In the agriculture practise by the 'integrated production' is by using from plant protection compounds the first point the treating of ladybird and others (coccinella, chrysoperla, phytoseiidae) with care and without damaging. But unfortunately the bee colonies as a life partnership are not considered. It would be from very important if the bee colonies would be included as a life partnership as the first insect in the 'integrated plant production'. It is not to tolerate, that agriculture animals are damaged in the field or in their stable or hive o if they loos their orientation. It should not be too, that different plant protection compounds collected to different times and their relation chips are not observed and not be included in the legal registration by plant protection compounds. By economical view of a farm with bee colonies as the important insect and as a agriculture animal is by looking for the lost honey from honeydew or flowers in the cereals after using of herbicides, insecticides the use of this not economical acceptable.
It is not to understand, that the farmers don't use more often the slaked lime, because it is possible to kill with the very cheap manure slaked lime (Ca(OH)2) as a solution all epiphytical living bacteria, fungus, mites and insects. After the treatment in few hours the slaked lime is changed in to normal lime (CaCO3) how it is used as substitution for food. A other application technique is necessary for a better covering of the leaves and to get a better success. By the economical view this would be the best, because the slaked lime manure is normally used in winter time and now spitted over the hole year. But with out this, it would be the best too, if the hours for the advisers for the counting from aphids and mites had to pay from the farmers.
By the "integrated plant protection" is a threshold of damaging for weeds in cereals (40 dicotyle and 40 monocotyle / m2 by 250 cereals plants / m2) in relating to nutrient concurrent.
Against a lot of weed is done:- harrowed, hoed, weeded
- Bring out calcium cyanamid
- Using herbicides
- Cereals killed before harvesting with all round herbicides.
In stead of the use of the all round herbicides like glyphosate is in Australia for fast drying from plants KOH the lye of K2O (chief ingredient of the ash from wood) used, which functioned with the fog technique of BELATEC too.
In Austria and Slovenia is in recent times to dry grass heat exchangers and pumps used. This technique is cheap / kg dry substance and would be god too for cereals and raps as hole plants.
By this drying technique the weed would not be negative and the straw would be a better food for cows and horses or could used as energy.
www.bioheu.comwww.heutrocknung.com
www.belatec.ch
The same is by the agriculture science, only a few are working with the influence of the radiation of the earth and the effect of the food.
I you belief that the "bio production" would be better, so you are wrong. Unfortunately the "bio" farmers use naturally insecticides and copper and other compounds which influence the bees and bee colonies and bee products negative.
The most of the alternatives I couldn't test by the public institutes, it was only possible in the own farm or orchard. But unfortunately a lot of bio organisations are against easy methods too, because they want sell their bio productions and they are afraid that to much farmers could change their production in bio. Unfortunately the bio organisations use still plant protection compounds like naturally pyretroides and copper. I am not principle against bio" my parents were many years too member of a bio organisation, but I have something against the dogma and the religion bio" Bio had to produce without residues in the environment and in all bee products. It is not important how many god mites are living on the leaves if the bad mites can easy killed with slaked lime.
The use of plant protection compounds are not necessary. The everlasting offices of plant protection and other friendly offices are against this. But there is a question, get this officers and institutes their money only from the public, they use money for students from companies too!
For myself I am angry, that official office are for plant protection compounds, which are damaging bee colonies. This by showing from me, that it is function only with manures and physical influence. For this the article "slaked lime the healthiness bringer for the nature!?", which I wrote for the Austrian beekeeper- fruit grower- organisations and for the public.
By the specific use of manures for the leaves by using a specific technique and by using physical machines against the radiation from the earth, I needed since 2001 any g plant protection compound. By this treatment of the plants the bees (colonies) are not damaged if the treatment is done after the bees are in their hive. As a farmer it is better for spraying in comparing to other insecticides because it is possible to spray the hole night and not only after the bees are in their hive and before 23 a clock! By economical view it is the best!
By this the honey bees bring any systemic fungicides, any antibiotics into the bee products. The bee colonies will not be damaged by insecticides of the new generation. My manures are compounds, which are too natural compounds in the food and allowed too as substitute in the food in the EU!
The lime strategy is underlined by the publication of the office of plant nutrition, but is shows too, that the most plant protection officers are not willed to think integrated!
Oh how nice would be, if our official public institutes would not think so chemical industry friendly and would have a honoured codex as scientists. As a officer it was not allowed for me to do such exercise...
I am sure, that the most public officers in the plant protection institutes and other officers by the agriculture officers are in future always god friends to the chemical industry, because they know, that their job have any existence right after the new knowledge. So they work with fear by the farmers and are leaving like dealers! Some of this officers want to have, that the use of slaked lime had to recognized as a plant protection compounds and not more as manure. But in the EU it is a traditional compound and manure and it allowed to use. I ask in this point too, where are standing this officers, do they tell the science or are they fighting only for their job?
Please let us cut unnecessary things, let us start by the plant protection for the help of the tax payers, farmers, beekeepers and the environment. A subvention per ha, which is very hard to pay after a larger EU, was only told something for the farmers. The really winner were the chemical plant protection companies, because the costs for the plant protection compounds are in the high like the subvention per ha.
Literatur:Literature:
Hoffmann G M, Nienhaus F, Schönbeck F, Weltzien H C, Wilbert H (1985) Lehrbuch der Phytomedizin. Paul Parey 2. Auflage, 1-488.
"slaked lime the healthiness bringer for the nature!?"
In all agriculture, forest culture, fruit culture literature the writers come to the result, that all year
there are 300 kg - 800 kg burned lime CaO / ha on sandy earth and until to 2000 kg burned lime CaO / ha by glay earth.
Normally by fertilizing the manure is brought out crystalline and in the earth fixed by the moisture. Only 1.7 g slaked lime a soluble in 1 l water H2O, with this there is then a pH from 12.4. The normal farmer or fruit grower use 400-500 l water H2O / m high of the plants. By the using of slaked lime Ca(OH)2 as a substitution from plant protection compounds are only 1 kg Ca(OH)2 / ha necessary and by fruit cultures by a high from 2-2.5 m ca. 2 kg. So a normal agriculture culture could sprayed 600 times and a fruit culture 300 times per year. By using tenfold more 60 or 30 spraying times are possible without a changing of the pH in the earth.
By using a better spraying or fog technique it is possible to kill the most of the epiphytic leaving bacteria, fungus, mites and insects similar to the use of contact bactericides, fungicides, acaricides, insecticides.
By thinking, that by changing of the technique for bringing out manure or fertilizers, the use of plant protection compounds, which influence negative the environment, is unnecessary, than it is not to understand, that we have farmers, officers and politics, which are fighting for the use of antibiotics and other the environment negative influencing compounds in the opposite to the slaked lime, which is cheap.
Because the same slaked lime had to bring out as manure, so only an other termination is afford like the termination of plant protection compounds!
Slaked lime give any residues in the harvested products (cereals, oilseeds, vegetables, fruits, honey) because slaked lime is in few hours chemical changed by the CO2 from the air to carbonate lime which is a normal nutrient substitution by the food and is very god for better bones.
Please notice too following literature from the officinal office for plant nutriation."Bestimmung des Kalkbedarfs von Acker und Grünlandböden"(2000) von
Dr. sc. M. Kerschberger, Jena; Dr. B. Deller, Karlsruhe; LD U. Hege, Freising; Dr. J. Heyn, Kassel; Dr. H.-E. Kape,
Rostock; Prof. Dr. O. Krause, Jena; Dipl.-Ing. J. Pollehn, Köln; Dr. M. J. Rex, Mühlheim; Dr. K. Severin, Hannover
http://www.vdlufa.de/vd_00.htm?4
Dr. Friedhelm Berger
The Independent, UK, by Geoffrey Lean, 9 Oct 2005 (zitiert von GENET-news, archive: http://www.genet-info.org/) GM crops contaminate the countryside for up to 15 years after they have been harvested, startling new government research shows.
The findings cast a cloud over the prospects of growing the modified crops in Britain, suggesting that farmers who try them out for one season will find fields blighted for a decade and a half.
Financed by GM companies and Margaret Beckett's Department of the Environment, Food and Rural Affairs, the report effectively torpedoes the Government's strategy for introducing GM oilseed rape to this country. Ministers have stipulated that the crops should not be grown until rules are worked out to enable them to "co-exist" with conventional ones. But the research shows that this is effectively impossible.
The study, published by the Royal Society, examined five sites across England and Scotland where modified oilseed rape has been cultivated, and found significant amounts of GM plants growing even after the sites had been returned to ordinary crops. It concludes that the research reveals "a potentially serious problem associated with the temporal persistence of rape seeds in soil."
The researchers found that nine years after a single modified crop, an average of two GM rape plants would grow in every square metre of an affected field. After 15 years, this came down to one plant per square metre - still enough to break the EC limits on permissible GM contamination.
Last night Pete Riley, the director of GM Freeze, said; "It is becoming clearer and clearer that it is going to be impossible to grow GM crops in Britain."
GM crops contaminate the countryside for up to 15 years after they have been harvested, startling new government research shows. The findings cast a cloud over the prospects of growing the modified crops in Britain, suggesting that farmers who try them out for one season will find fields blighted for a decade and a half.
Financed by GM companies and Margaret Beckett's Department of the Environment, Food and Rural Affairs, the report effectively torpedoes the Government's strategy for introducing GM oilseed rape to this country. Ministers have stipulated that the crops should not be grown until rules are worked out to enable them to "co-exist" with conventional ones. But the research shows that this is effectively impossible.
The study, published by the Royal Society, examined five sites across England and Scotland where modified oilseed rape has been cultivated, and found significant amounts of GM plants growing even after the sites had been returned to ordinary crops. It concludes that the research reveals "a potentially serious problem associated with the temporal persistence of rape seeds in soil."
GENET European NGO Network on Genetic EngineeringHartmut MEYER (Mr)