The effects of fenoxycarb in a chronic Oomen feeding test – results of a ringtest

Background - The Oomen bee brood feeding test (Oomen et al. 1992)6 is recommended by the EFSA (2013)2 as one method to investigate potential effects of plant protection products on honeybee brood (Apis mellifera L.), with the ‘brood termination rate’ as the key endpoint. In 2013 the test method of Oomen was adapted to a chronic feeding scenario including current methods (OECD GD 75, improvements by Pistorius et al. 20127) and was subsequently ring-tested in 2013 and 2014.Results - The results were compared to data of acute feeding studies. Overall the obtained results of the chronic Oomen feeding studies indicated that the design is a robust and reliable test method with low brood termination rates in the control and a sufficient exposure of the brood to the reference item.Conclusion - Nevertheless, based on current experiences and recent publications adaptations are proposed concerning dosing of the test item, assessment intervals and methodology (digital brood assessments). Moreover the test method was compared to the bee brood test according to OECD GD 75 and several advantages were pointed out.Keywords: honeybees, chronic Oomen bee brood feeding test, ring-test, EFS

2.1 Detailed brood evaluation under field conditions: advantages and disadvantages

Bee brood studies under semi-field conditions according to OECD GD 75 display a strong variability of the brood termination rates (BTRs) as the key endpoint. Therefore, the ICP-PR Bee Brood Group considered the performance of EPPO 170 field studies using the OECD GD 75 bee brood evaluation as one option to achieve more reliable BTR data. This approach was envisaged already some years ago and used for several years. However, broader data sets supporting the benefit of this combined methodology were still lacking.The analysis of current field studies performed since 2012 indicate that control BTRs were approximately half the size compared to values observed under semi-field conditions. Moreover, results give a strong indication that the BTR values under field conditions are more reliable and less variabel. Therefore, the combined method is a valuable tool to investigate potential effects of a plant protection product on the bee brood to refine the risk under realistic exposure conditions.Bee brood studies under semi-field conditions according to OECD GD 75 display a strong variability of the brood termination rates (BTRs) as the key endpoint. Therefore, the ICP-PR Bee Brood Group considered the performance of EPPO 170 field studies using the OECD GD 75 bee brood evaluation as one option to achieve more reliable BTR data. This approach was envisaged already some years ago and used for several years. However, broader data sets supporting the benefit of this combined methodology were still lacking.The analysis of current field studies performed since 2012 indicate that control BTRs were approximately half the size compared to values observed under semi-field conditions. Moreover, results give a strong indication that the BTR values under field conditions are more reliable and less variabel. Therefore, the combined method is a valuable tool to investigate potential effects of a plant protection product on the bee brood to refine the risk under realistic exposure conditions

Honeybee brood testing under semi-field and field conditions according to Oomen and OECD GD 75: is there a difference of the brood termination rate?

According to current European regulations on the risk assessment of plant protection products, the risk on honey bee larvae or honey bee brood has to be addressed. If the assessment indicates, that a potential risk cannot be excluded based on data derived from laboratory studies, two higher-tier options are given by the EFSA bee Guidance Document to refine this under more realistic conditions: the Oomen bee brood feeding test and brood studies performed according to the OECD Guidance Document 75. Both study types focus on the brood termination rate (BTR) as the key endpoint. While the Oomen brood test investigates the brood development after the acute or chronic administration of a test item spiked sugar solution to unconfined colonies, brood studies according to OECD GD 75 are performed under semi-field confined exposure conditions and examine potential effects on the bee brood after the overspray of a bee attractive flowering crop. However, the evaluation of historical data from semi-field studies according to OECD GD 75 showed a strong variability of the BTR of pre-imaginal stages developing from marked eggs (BTReggs) in the control. As an alternative, field studies according to EPPO 170 which comprise bee brood evaluations according to OECD GD 75 were considered to produce more reliable termination data. The statistical analysis of available control data shows that Oomen feeding studies and bee brood studies performed under field conditions lead to significantly lower BTReggs of = 20% compared to semi-field bee brood studies for which a mean BTR of about 30% is observed. Moreover, studies with unconfined colonies show a high proportion of control replicates with BTReggs =30% and =40% indicating a higher reliability compared to semifield studies. A comparison of the possibilities and limitations of the three methods shows the strength of each method. In Oomen studies, the exposure of the brood and of the hive bees only can be regarded as artificial. However, the test concentrations can be adjusted to specific needs and to different feeding durations of at least one (acute) or 9 days (chronic). Furthermore, the absence of ‘caging effects’, the low dependency on climatic or crop conditions, the potential to test also herbicides which control dicotyledonous plants (since no crop plant is adversely affected by its mode of action) and an exposure period of at least nine days in chronic Oomen studies are crucial advantages. In contrast, the exposure scenarios of the two other methods are much more realistic and especially for semi-field studies a worst-case situation. Moreover, they also include exposure via pollen and exposure levels and durations, which strongly depend on the application rate and the flowering period of the treated crop. Whereas a dilution of plant protection product residues cannot be excluded during the exposure period in studies with unconfined colonies due to the shift to untreated flowering plants in the surrounding, this is not given for semi-field studies.According to current European regulations on the risk assessment of plant protection products, the risk on honey bee larvae or honey bee brood has to be addressed. If the assessment indicates, that a potential risk cannot be excluded based on data derived from laboratory studies, two higher-tier options are given by the EFSA bee Guidance Document to refine this under more realistic conditions: the Oomen bee brood feeding test and brood studies performed according to the OECD Guidance Document 75. Both study types focus on the brood termination rate (BTR) as the key endpoint. While the Oomen brood test investigates the brood development after the acute or chronic administration of a test item spiked sugar solution to unconfined colonies, brood studies according to OECD GD 75 are performed under semi-field confined exposure conditions and examine potential effects on the bee brood after the overspray of a bee attractive flowering crop. However, the evaluation of historical data from semi-field studies according to OECD GD 75 showed a strong variability of the BTR of pre-imaginal stages developing from marked eggs (BTReggs) in the control. As an alternative, field studies according to EPPO 170 which comprise bee brood evaluations according to OECD GD 75 were considered to produce more reliable termination data. The statistical analysis of available control data shows that Oomen feeding studies and bee brood studies performed under field conditions lead to significantly lower BTReggs of = 20% compared to semi-field bee brood studies for which a mean BTR of about 30% is observed. Moreover, studies with unconfined colonies show a high proportion of control replicates with BTReggs =30% and =40% indicating a higher reliability compared to semifield studies. A comparison of the possibilities and limitations of the three methods shows the strength of each method. In Oomen studies, the exposure of the brood and of the hive bees only can be regarded as artificial. However, the test concentrations can be adjusted to specific needs and to different feeding durations of at least one (acute) or 9 days (chronic). Furthermore, the absence of ‘caging effects’, the low dependency on climatic or crop conditions, the potential to test also herbicides which control dicotyledonous plants (since no crop plant is adversely affected by its mode of action) and an exposure period of at least nine days in chronic Oomen studies are crucial advantages. In contrast, the exposure scenarios of the two other methods are much more realistic and especially for semi-field studies a worst-case situation. Moreover, they also include exposure via pollen and exposure levels and durations, which strongly depend on the application rate and the flowering period of the treated crop. Whereas a dilution of plant protection product residues cannot be excluded during the exposure period in studies with unconfined colonies due to the shift to untreated flowering plants in the surrounding, this is not given for semi-field studies

Effectiveness of method improvements of OECD GD 75 – Evaluation by the ICP-PR Bee Brood Working Group

Background: The OECD Guidance Document No. 75 (2007) is a method to investigate potential effects of plant protection products on the brood of honeybees (Apis mellifera L.), with the ‘brood termination rate’ (BTR, failure of individual eggs or larvae to develop) as the key endpoint. As in recent years a number of studies displayed a strong variability in BTRs, Pistorius et al. (2012) recommended some measures for improvements. First results in the season 2011 indicated that these measures led to lower BTRs and lower variability. The ICP-PR bee brood working group has evaluated the effectiveness of the recommended measures for improving the reliability of the method and the resulting BTRs and reports in this paper. Results: To evaluate the effectiveness of these measures a data analysis of a total of 62 studies was performed which were carried out in Germany and Switzerland between 2011 and 2014. Based on this analysis, the mean BTR in the control was 29.2% and this result did not display a distinct improvement compared to the historical data (34.7%) (Pistorius et al. 2012) and neither compared to the data of the bee brood ring-test (28.0%) (Schur et al. 2003). Moreover, the proportion of replicates (colonies) with BTRs ≤30% amounted to be 61.5% compared to 55.6% in the years before. And every 2nd study displayed BTRs >30% in two or more replicates. Also, the proportion of replicates (colonies) with BTRs ≤40% amounted to 76.9% compared to 68.3% in the years before and just 21.7% of the studies displayed BTRs >40% in two or more replicates.Conclusion: Overall, these findings highlight that the test method according to the OECD Guidance document in 2007was not be considerably improved with the recommended measures. But although the reliability of the method and a reliable interpretation regarding potential effects of a plant protection product (PPP) on bee brood was not given in all studies, it currently remains the only available test method to address the potential risk of a plant protection product on honeybee larval development in realistic worst-case (semi-field) exposure conditions. Among other factors, it is assumed that the limitations are most likely due to the confined semi-field conditions. Further work should investigate potential additional improvements in semi-field conditions and also brood termination rates in field conditions.Keywords: honeybees, bee brood test, OECD GD 75, brood termination rat

Precision farming – consideration of reduced exposure in the pollinator risk assessment

Observed declines in the distribution and abundance of various insect species have moved the topic of biodiversity and the protection of honey bees, an insect species of particular economic interest, into the focus of public attention. This also resulted in an increasing public pressure to reform the European agricultural policy and as part of this to minimise the amount of synthetic plant protection products used. In this context, so-called ‘precision farming’ offers a considerable potential for a reduced application of plant protection products by using precision application techniques that allow to adjust applications to the actual scale of target distribution within a field. Is however currently not possible to exactly quantify the subsequent decrease of exposure of non-target organisms. Focusing on honey bees, the authors are therefore in a first step proposing a field study design to quantify the direct and indirect exposure of honey bees and their colonies in relation to the ratio of treated to untreated field area and the application pattern used. Furthermore, parameters of the bee risk assessment scheme are discussed that could be suitable to describe exposure reduction by precision application.Observed declines in the distribution and abundance of various insect species have moved the topic of biodiversity and the protection of honey bees, an insect species of particular economic interest, into the focus of public attention. This also resulted in an increasing public pressure to reform the European agricultural policy and as part of this to minimise the amount of synthetic plant protection products used. In this context, so-called ‘precision farming’ offers a considerable potential for a reduced application of plant protection products by using precision application techniques that allow to adjust applications to the actual scale of target distribution within a field. Is however currently not possible to exactly quantify the subsequent decrease of exposure of non-target organisms. Focusing on honey bees, the authors are therefore in a first step proposing a field study design to quantify the direct and indirect exposure of honey bees and their colonies in relation to the ratio of treated to untreated field area and the application pattern used. Furthermore, parameters of the bee risk assessment scheme are discussed that could be suitable to describe exposure reduction by precision application

3.8 ‘Focal species’ – can this well-known concept in higher-tier risk assessments be an appropriate approach for solitary bees?

Bumble bees and solitary bees have to be considered in addition to honey bees regarding environmental pollinator risk assessments. For solitary bees it is proposed to use Osmia cornuta (LATR., 1805) or O. bicornis (L., 1758) as test organisms. Whereas for higher-tier assessments, semi-field testing of solitary bees has been proved to obtain sound results, experience from current Osmia field studies show that exposure of adults and larvae is not necessarily the case due to the pronounced polylectic feeding behaviour. As an alternative refinement option the ‘focal species’ concept may be used, which is well-known as a kind of first step for higher tier bird and mammal risk assessments. This approach as it applies to solitary bees, as well as its needs, refinement options and limitations is presented.Bumble bees and solitary bees have to be considered in addition to honey bees regarding environmental pollinator risk assessments. For solitary bees it is proposed to use Osmia cornuta (LATR., 1805) or O. bicornis (L., 1758) as test organisms. Whereas for higher-tier assessments, semi-field testing of solitary bees has been proved to obtain sound results, experience from current Osmia field studies show that exposure of adults and larvae is not necessarily the case due to the pronounced polylectic feeding behaviour. As an alternative refinement option the ‘focal species’ concept may be used, which is well-known as a kind of first step for higher tier bird and mammal risk assessments. This approach as it applies to solitary bees, as well as its needs, refinement options and limitations is presented

3.9 Semi-field testing of the solitary bee Osmia bicornis (L., 1758) (Hymenoptera, Megachilidae) in flowering Phacelia tanacetifolia – Chances, improvements and limitations

Based on the proposed test design of the ICPPR non-Apis working group, a semi-field (tunnel) study was conducted with the solitary bee Osmia bicornis (L., 1758) in flowering Phacelia using tunnels. Untreated crop was used as a control, and the insect growth regulator fenoxycarb as a test item which is intended to be used as reference item for this study type. To improve the design and to enhance the informative value of such studies our method deviated in three points: placing the cocoons in the tubes of the nesting units, performing additional brood assessments in two to three day intervals and increasing the application rate of the test item.Overall the results indicated that the proposed test design is suitable to perform studies on O. bicornis in Phacelia under semi-field conditions.Data on the reproduction performance, brood termination and hatching rate of the progenies show low variability between the replicates in both treatment groups. No impact on the flight activity, mortality, reproduction performance and hatching success of the progenies was observed, but an increased brood termination rate of larvae produced within the first days after application was recorded; in particular, placing the cocoons in the tubes lead to higher proportions of nesting established females. Moreover, due to the assessment of the cell production in 2 to 3 day intervals, it is possible to analyse time dependent effects on the reproductive performance, brood termination and hatching rate which can be expected by the decreasing exposure in the course of the study. And finally, it is shown that fenoxycarb is not a suitable reference item for such studies.Based on the proposed test design of the ICPPR non-Apis working group, a semi-field (tunnel) study was conducted with the solitary bee Osmia bicornis (L., 1758) in flowering Phacelia using tunnels. Untreated crop was used as a control, and the insect growth regulator fenoxycarb as a test item which is intended to be used as reference item for this study type. To improve the design and to enhance the informative value of such studies our method deviated in three points: placing the cocoons in the tubes of the nesting units, performing additional brood assessments in two to three day intervals and increasing the application rate of the test item.Overall the results indicated that the proposed test design is suitable to perform studies on O. bicornis in Phacelia under semi-field conditions.Data on the reproduction performance, brood termination and hatching rate of the progenies show low variability between the replicates in both treatment groups. No impact on the flight activity, mortality, reproduction performance and hatching success of the progenies was observed, but an increased brood termination rate of larvae produced within the first days after application was recorded; in particular, placing the cocoons in the tubes lead to higher proportions of nesting established females. Moreover, due to the assessment of the cell production in 2 to 3 day intervals, it is possible to analyse time dependent effects on the reproductive performance, brood termination and hatching rate which can be expected by the decreasing exposure in the course of the study. And finally, it is shown that fenoxycarb is not a suitable reference item for such studies

3.3 ICP-PR Bee Brood Working Group – Variability of brood termination rates in reference to validity criteria and limited effectiveness of method improvement in honeybee semi-field studies (OECD GD 75)

OECD Guidance Document 75 (2007) outlines a test method to assess effects of plant protection products (PPPs) on honeybee brood under semi-field conditions. The assessment of bee brood development is conducted by mapping cells containing eggs and following their development until emergence. Evaluated parameters are: brood termination rate (BTR), brood compensation index (CI) and brood index (BI). Due to high variability of BTRs within treatments and high control mortality in a number of studies no definite conclusions regarding effects on brood were possible in the past and studies needed to be repeated (Pistorius et al. 2012). To address this variance, effort was taken by ICP-PR and AG Bienenschutz to improve the method by further analyzing current and historical data considering possible influencing factors (Pistorius et al. 2012, Becker et al. 2015) to give recommendations for future testing. The main findings were that reliability of the test method was questionable and that further method improvement and data evaluation was required. Therefore in this paper data evaluation of studies conducted between 2014 and 2017 is carried out and potential key parameters influencing outcome of studies are given.To evaluate the improvement of the OECD 75 test method following the recommendations from 2015, a data analysis of 86 studies conducted in Germany, France, Spain and US was performed. The mean BTR value in the control group was 30.2% for studies conducted in Germany (mean of 61 studies), 19.4% in France (mean of 3 studies), 41.8% in Spain (mean of 5 studies) and 50.6% in US (mean of 17 studies). Results from Spain and US displayed higher BTRs in control compared to data from Germany. Evaluation of BTRs for Germany displayed only a slight improvement (historical value of 32.9%). Analysis of data shows a limitation of options to improve the method as no main driver for high variability of BTRs in the control group was found. The cause for low precision may be multifactorial and driven by “caging effect”. There are alternative test methods available to observe bee brood development, without confinement in the tunnels, under field conditions (Oomen et al. (1992), OECD GD 75 field test design). Therefore it is necessary to investigate differences between these open field methods and semi-field testing with regard to routes of exposure, residues in brood and brood mortality, to choose the most reliable and adequate testing method assessing potential effects of PPP on honeybee brood development.OECD Guidance Document 75 (2007) outlines a test method to assess effects of plant protection products (PPPs) on honeybee brood under semi-field conditions. The assessment of bee brood development is conducted by mapping cells containing eggs and following their development until emergence. Evaluated parameters are: brood termination rate (BTR), brood compensation index (CI) and brood index (BI). Due to high variability of BTRs within treatments and high control mortality in a number of studies no definite conclusions regarding effects on brood were possible in the past and studies needed to be repeated (Pistorius et al. 2012). To address this variance, effort was taken by ICP-PR and AG Bienenschutz to improve the method by further analyzing current and historical data considering possible influencing factors (Pistorius et al. 2012, Becker et al. 2015) to give recommendations for future testing. The main findings were that reliability of the test method was questionable and that further method improvement and data evaluation was required. Therefore in this paper data evaluation of studies conducted between 2014 and 2017 is carried out and potential key parameters influencing outcome of studies are given.To evaluate the improvement of the OECD 75 test method following the recommendations from 2015, a data analysis of 86 studies conducted in Germany, France, Spain and US was performed. The mean BTR value in the control group was 30.2% for studies conducted in Germany (mean of 61 studies), 19.4% in France (mean of 3 studies), 41.8% in Spain (mean of 5 studies) and 50.6% in US (mean of 17 studies). Results from Spain and US displayed higher BTRs in control compared to data from Germany. Evaluation of BTRs for Germany displayed only a slight improvement (historical value of 32.9%). Analysis of data shows a limitation of options to improve the method as no main driver for high variability of BTRs in the control group was found. The cause for low precision may be multifactorial and driven by “caging effect”. There are alternative test methods available to observe bee brood development, without confinement in the tunnels, under field conditions (Oomen et al. (1992), OECD GD 75 field test design). Therefore it is necessary to investigate differences between these open field methods and semi-field testing with regard to routes of exposure, residues in brood and brood mortality, to choose the most reliable and adequate testing method assessing potential effects of PPP on honeybee brood development