Ingrid Zwertvaegher

Aude Lamare

Jean-Paul Paul Douzals

Paolo Balsari

Paolo Marucco

Marco Grella

Amedeo Caffini

Nikos Mylonas

Donald Dekeyser

Dieter Foqué

David Nuyttens
email: david.nuyttens@ilvo.vlaanderen.be

Boom sprayer optimizations for bed-grown carrots at different growth stages based on spray distribution and droplet characteristics

Keywords: bed spray application, nozzle configuration, nozzle type, droplet size, droplet velocity, air support

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Since January 2014, growers in the European Union are obliged to implement the principles of integrated pest management (IPM). 1 These principles aim to minimise environmental, economic and health risks due to the use of plant protection products (PPP), by combining various biological, physical, cultural and chemical techniques to manage all classes of pests. Within the H2020-project OPTIMA (OPTimised

Integrated pest MAnagement for precise detection and control of plant diseases in perennial crops and

open-field vegetables, www.optima-h2020.eu), an environmentally friendly IPM framework for Alternaria leaf blight in carrots, downy mildew in vineyards, and apple scab in orchards is developed by providing a holistic approach which includes major elements related to integrated disease management, such as precision spraying techniques, as well as the use of novel bio-PPPs, disease prediction models, and spectral disease detection systems. The overall goal is to integrate those elements and develop three prototype smart sprayers in collaboration with sprayer manufacturers.

The main goal in all spray applications is to obtain an adequate coverage and uniform pesticide deposition on the target in order to provide sufficient efficacy against the target pest. [START_REF] Gil | Field validation of DOSAVINA, a decision support system to determine the optimal volume rate for pesticide application in vineyards[END_REF] Pesticide losses and unsatisfactory uniformity of distribution should be avoided as much as possible as they reduce the effectiveness of spraying and increase environmental contamination as well as costs. [START_REF] Vercruysse | Off target ground deposits from spraying a semi-dwarf orchard[END_REF][START_REF] Višacki | Effects of sprayer boom height and operating pressure on the spray uniformity and distribution model development[END_REF][START_REF] Forney | Laboratory and full boom-based investigation of nozzle setup error effects on flow, pressure, and spray pattern distribution[END_REF] For bed-grown crops, ideally the spray is applied evenly to the bed, and in particular to the target zone width depending on the crop growth stage, while no spray is applied to the paths in between the beds to avoid losses, [START_REF] Holterman | Optimizing sprayer boom design for bed-grown crops[END_REF] unless herbicides are applied.

As spray deposition and drift are affected by the spray and droplet characteristics, including droplet size and velocity distribution, the volume distribution pattern, and the entrained air characteristics, [START_REF] Miller | Effects of formulation on spray nozzle performance for applications from ground-based boom sprayers[END_REF][START_REF] Nuyttens | Comparison between indirect and direct spray drift assessment methods[END_REF] and droplet size determines the biological efficacy of the applied pesticide, [START_REF] Jensen | Biological efficacy of herbicides and fungicides applied with low-drift and twin-fluid nozzles[END_REF][START_REF] Brown | Efficacy of four corn (Zea mays L.) herbicides when applied with flat fan and air induction nozzles[END_REF][START_REF] Nuyttens | Effect of nozzle type, size and pressure on spray droplet characteristics[END_REF][START_REF] Vajs | Comparison of the effectiveness of standard and drift-reducing nozzles for control of some winter wheat diseases[END_REF][START_REF] De Cock | Investigation on optimal spray properties for ground based agricultural applications using deposition and retention models[END_REF][START_REF] Ferguson | Effect of spray droplet size on herbicide efficacy on four winter annual grasses[END_REF] the nozzle-pressure combination greatly determines the efficacy of the application process. This paper therefore focuses on the use of various nozzle types and configurations, and of variable nozzle spacing and height as possible optimizations of a smart sprayer for bed-grown carrots. The goal is to define optimal settings, in terms of spray distribution and reduced spray losses, of an air-assisted smart sprayer for bed-grown carrots with variable nozzle spacing at different target zone widths (depending on the growth stage of the crop) in comparison to the reference conventional horizontal boom sprayer.

MATERIALS AND METHODS

Planting system and crop characteristics

Beds of 1.83 m wide, containing 3 rows of carrots per bed, and an inter-bed distance of 0.5 m, thus resulting in a total distance of 2.33 m between carrot beds, were considered. This design matches the pilot fields in the southwest of France where at a later stage of the OPTIMA project field trials will be conducted using the developed smart sprayer. A schematic presentation of the design is given in Figure 1. In total, 9 beds of 2.33 m can be sprayed using a 21 m horizontal spray boom (holding 42 nozzles at 0.5 m nozzle spacing). In France, around four to five treatments against Alternaria are performed in carrots during a growing season. The first treatment is generally applied around BBCH 14 -16 (i.e. 4 th till 6 th true leaf unfolded). At that time, about 50% of the inter-row is covered by foliage. By BBCH 18 -19, the entire inter-row is covered by foliage (S. Bellalou, personal communication). Based on the carrot plant design (Fig. 1) and growth stages (with more developed canopies at full growth stage compared to early growth stage), target zone widths ranging from 1.2 to 2.2 m (at incremental steps of 0.2 m) were studied.

Sprayer configurations, nozzle types and spray settings

A horizontal spray boom application with TeeJet XR 110 04 nozzles at a spray pressure of 300 kPa, 0.5 m spray boom height and 0.5 m nozzle spacing, without air support, at 12.0 km h -1 , corresponding to 158 L ha -1 , was considered as reference condition. Studied possible carrot sprayer optimizations included the use of reduced spray volume nozzles (ISO 02 vs ISO 04 nozzles), the use of drift reducing nozzles (air inclusion AI vs standard XR nozzles), and bed spray applications instead of broadcast applications, by using off-center and/or narrow angle nozzles (80° vs 110°). In addition, the use of air support was also considered as optimization, and the effect of air support on the droplet characteristics is described. An overview of the nozzles and settings selected and tested as possible optimizations is given in Table 1. Theoretical application rates are expressed as L ha -1 of total ground area. The total ground area includes the carrot beds and the space between the beds.

Spray distribution

Prior to the spray distribution and droplet characteristics experiments, the flow rate of the nozzles was determined using a nozzle test bench (ITEQ, Belgium) at the Spray Technology Laboratory of Flanders Research Institute for Agricultural, Fisheries and Food (ILVO, Belgium). Every nozzle was tested three times at a spray pressure of 300 kPa. The nozzles with the lowest mean deviation from the nominal flow rate were selected for further experiments. Per off-center nozzle type, 6 nozzles were tested and 2 were selected for further experiments, of the other nozzle types (i.e. XR and AI), 12 nozzles were tested and 4 were selected for further experiments.

To achieve maximal and uniform deposition on the canopy (i.e. target zone) and to have minimal losses between the beds, optimal nozzle spacings and heights for bed spray applications were determined using a spray scanner. Spray depositions and losses for 6 target zone widths (1.2, 1.4, 1.6, 1.8, 2.0, 2.2 m range) were assessed for a distance between carrot beds of 2.33 m (Fig. 1).

The spray distribution of 13 nozzle configurations, consisting of different nozzle types (standard flat fan, air inclusion, off-center), nozzle size (ISO 02, 04), spray angle (80°, 110°), and number of nozzles (3 or 4 nozzles per bed) was determined. An overview of the tested configurations is given in Table 2.

For every nozzle configuration, measurements were performed at several nozzle spacing and height combinations, but within each test, nozzle spacing and height were kept equal as this is more practical for the farmers in real field conditions. The configurations with four nozzles were tested in a range from 0.35 to 0.65 m nozzle spacing/height, while those with three nozzles were performed from 0.40 to 0.65 m, all at incremental steps of 50 mm. Heights of 0.7 m and higher were not considered due to increased risk of drift. In addition, a broadcast application with XR 110 04 nozzles at 0.5 m spray height and 0.5 m nozzle spacing was tested as reference. In total, 81 different combinations were tested.

The spray distribution measurements were performed indoor at ILVO's Spray Technology Laboratory, according to ISO 5682-2 [START_REF]Equipment for crop protection -Spraying equipment -Part 2: Test methods to assess the horizontal transverse distribution for hydraulic sprayers[END_REF] . The spray scanner set-up consisted of a 0.8 m wide, channelled, sloping scanner with 0.1 m grooves and calibrated collecting tubes by AAMS-Salvarani (Maldegem, Belgium), running over a frame underneath a fixed 12 m 'ideal' spray boom. For this experiment, a short spray boom with variable nozzle spacing was constructed and mounted on the fixed spray boom. The center of the short spray boom was positioned above a channel partition, thus forming the zero-point position corresponding to the middle of the bed. The reciever-unit with 0.1 m wide grooves collected the liquid sprayed with the short spray boom during a known time interval, as described by Zwertvaegher et al. [START_REF] Zwertvaegher | Development and implementation of a laboratory spray device and rainfall simulator for retention research using small amounts of agroformulations[END_REF] .

The flow rates (L min -1 ) achieved from the spray scanner measurements, which are basically time measurements as also described by Višacki et al. [START_REF] Višacki | Effects of sprayer boom height and operating pressure on the spray uniformity and distribution model development[END_REF] , were recalculated to spray volume (L ha -1 ) based on a driving speed of 12 km h -1 . For each target zone width and nozzle configuration, following variables were calculated, taking into account possible overlap between sprays of neighboring beds: minimum spray volume in target zone (L ha -1 ), maximum spray volume in target zone (L ha -1 ), average spray volume in target zone (L ha -1 ), percentage of spray volume in target zone (%), percentage of losses outside the target zone (%), and Coefficient of variation (CV) of the spray distribution in the target zone (%).

The following criteria were used to select appropriate spray configurations and nozzle spacing/height combinations for the different target zone widths:

-Criterion 1: CV in target zone < 12%, to guarantee a uniform deposition in the target zone, -Criterion 2: Losses outside target zone < 17%, to minimise losses out of the target zone.

If both criteria were fulfilled, the configuration at this nozzle spacing and height was considered appropriate for that target zone width. Provided that multiple combinations were appropriate for the same target zone width, the combination with the highest minimum spray volume (L ha -1 ) in the target zone was selected. The thresholds were selected as such so that at least one spray configuration per target zone width met the criteria. Although chosen arbitrary, the uniformity threshold is close to those specified by the inspection of sprayers in use, i.e. the CV of the transverse distribution should not exceed 10% for broadcast spray applications. [START_REF]ISO 16122-2 -Agricultural and forestry machinery -Inspection of sprayers in use -Part 2: Horizontal boom sprayers[END_REF][START_REF]EN 13790-1 -Agricultural machinery -Sprayers -Inspection of sprayers in use -Part 1: Field crop sprayers[END_REF]

Droplet size and velocity characteristics

Droplet size and velocity characteristics were obtained at ILVO using a Phase Doppler Particle Analyser (PDPA) laser-based measuring set-up, as described by Nuyttens et al. [START_REF] Nuyttens | Effect of nozzle type, size and pressure on spray droplet characteristics[END_REF] . The used PDPA laser was a PowersSight PDPA one dimensional system (TSI, Minneapolis). With this one-dimensional system, velocity measurements were limited to the dominant vertical direction. When a droplet passes through a small sampling volume, formed by two intersecting laser beams, light is scattered by refraction. From the light scattering characteristics, droplet sizes and velocities are obtained. All measurements were performed at a distance of 0.5 m below the nozzle(s), and repeated three times. Rectangular scan profiles were used. All measurements were carried out along the horizontal long axis of the spray fan. All nozzles (Table 1) were tested without air support using a single nozzle set-up.

Based on the spray distribution measurements (see 2.3 Spray distribution), the 4 nozzle types of the most appropriate nozzle configurations, i.e. XR8004, XR8002, AI11004, and AI8004, were also tested with air support at 4 different settings (Table 1), using the Caffini Air Wing (Caffini s.p.a., Palù Verona, Italy) and ILVO fan (Ventomatic, Merelbeke, Belgium). Measurements were performed with the test set-up at fan frequencies corresponding to Caffini sprayer fan speeds of 0, 1400, 1750 and 2000 rpm and air speeds 0, 1.2, 1.6, and 1.9 m s -1 at 0.5 m below the air outlet. So in total, 16 nozzle-air support combinations (4 nozzle types x 4 air support settings) were tested. Following characteristics were calculated:

(1) BCPC -BCPC spray quality class based on droplet size;

(2) Dv0.5volume median diameter (VMD, µm) below which smaller droplets constitute 50% of the spray volume;

(3) Dv0.1, Dv0.9volume diameter (µm) below which smaller droplets constitute respectively 10% and 90% of the total volume;

(4) V100proportion of total volume (%) of droplets smaller than 100 µm in diameter;

(5) vv0.50droplet velocity (m s -1 ) below which slower droplets constitute 50% of the total spray volume; vavgarithmetic average droplet velocity (m s -1 ).

To test the effect of air support on the droplet characteristics, a short spray boom with multiple nozzles was used in order to sample droplets at different positions in the spray fan, as the position of the nozzles relative to the air holes should be fixed and comparable to in-field conditions for realistic measurements.

Due to the restricted movement of the air support system, using a single mobile nozzle would result in a misalignment between nozzle position and air hole, leading to incorrect results.

RESULTS AND DISCUSSION

Spray distribution

The spray distribution results showed that the configurations with 3 nozzles did not meet the criteria, and were therefore not appropriate, not even for the smallest target zone width of 1.2 m, as either the CV and/or the losses were too high (criterion 1 and 2, respectively). In total, 4 nozzle configurations were appropriate for all target zone widths (from 1.2 to 2.2 m), i.e. XR8004/XR8004/XR8004/XR8004, AIUB8504/AI11004/AI11004/AIUB8504, AI8004/AI8004/AI8004/AI8004, and XR8002/XR8002/XR8002/XR8002. Table 3 tabulates the spray distribution characteristics of these configurations at the most appropriate nozzle spacing/height combinations for the different target zone widths. As an example, the spray distribution patterns and characteristics of AIUB8504/AI11004/AI11004/AIUB8504 and the broadcast application with XR11004 nozzles, both at 0.5 m nozzle spacing/height, for a target zone width of 1.6 m, are presented in Figure 2a andb, respectively. Per target zone, the losses outside the target zone (%) and the CV (%) and the average of the applied dose (%), i.e. the average spray volume in the target zone relative to the theoretical spray volume per ha of total ground area (Table 1), expressed as %, are given in Figure 3 for the reference broadcast application and the 4 most appropriate nozzle configurations, i.e.

XR8004/XR8004/XR8004/XR8004, AIUB8504/AI11004/AI11004/AIUB8504, AI8004/AI8004/AI8004/AI8004, and XR8002/XR8002/XR8002/XR8002.

With lowest variation (CV) inside the target zone and lowest relative losses outside the target zone, while maintaining a high average spray volume in the target zone, the overall best ISO 04 configuration is AIUB8504/AI11004/AI11004/AIUB8504 for target zone widths from 1.2 to 1.6 m, as indicated by Table 3 and Figure 3. For a target zone width of 2.2 m, a nozzle spacing/height combination of 0.7 m might be more appropriate with this configuration, however, as reported earlier, this spacing/height combination was not tested due to increased risk of drift. [START_REF] Nuyttens | The influence of operator-controlled variables on spray drift from field crop sprayers[END_REF] However, compared to AIUB8504/AI11004/AI11004/AIUB8504, configurations XR8004/XR8004/XR8004/XR8004, AI8004/AI8004/AI8004/AI8004, and XR8002/XR8002/XR8002/XR8002 have the advantage that only one nozzle type can be used along the spray boom, and as no off-center nozzles are needed, they are less expensive and less sensitive to deviations in spray line and boom movements. For target zones up to 1.8 m, losses were always highest for the broadcast application (10 to 36% higher than the bed spray configurations), thus denoting a clear advantage for bed spray applications at these target zones.

However, the broadcast application losses decreased with increasing target zone and at 2.0 m target zone the losses were comparable to those of the bed spray applications (13 to 17%). Over a 2.2 m target zone, only configuration AIUB8504/AI11004/AI11004/AIUB8504 had lower losses than the broadcast application (7 vs 8%). The four bed spray applications had similar losses at all target zones, ranging from 16% at 1.2 m to 7% at 2.2 m. The CV, which is a measure of uniformity, was lowest for the broadcast application at all target zones (0.6 to 5.3% lower than the bed spray configurations), indicating that the most uniform spray applications were obtained with this configuration. However, this is at the expense of higher losses outside the target zone and lower applied doses in the target zone. Although the average applied dose (relative to the theoretical application rate, Depending on the canopy growth stage and thus the target zone width, the bed spray configurations at their most appropriate nozzle spacing/height combinations may also reduce spray drift because lower spray boom heights also reduce spray drift. [START_REF] Jong | Effect of sprayer boom height on spray drift[END_REF][START_REF] Balsari | Field-crop-sprayer potential drift measured using test bench: Effects of boom height and nozzle type[END_REF] Reducing boom height generally results in less uniform spray distributions, but this negative effect was buffered by the narrower nozzle spacings used in this study, as also reported by Azimi et al. [START_REF] Azimi | Nozzle spray distribution for pesticide application[END_REF] . The four most optimal bed spray configurations and the reference broadcast application were further tested for spray deposition and potential spray drift in the OPTIMA project, as described in Douzals et al. [START_REF] Douzals | Optimization of spray application on bed-grown vegetables -On-going developments within OPTIMA project[END_REF] Based on the spray distribution patterns of single nozzles at different boom heights, models could be build to design and select the most optimal set-ups of nozzles on a sprayer boom for bed-grown crops, as illustrated by Holterman et al. [START_REF] Holterman | Optimizing sprayer boom design for bed-grown crops[END_REF] Their model simulated spray patterns while varying nozzle types, nozzle spacing and the position and angling of end nozzles based on single nozzle spray patterns. The authors concluded that, although the number of possible designs is extremely large, relatively few met the user definable criteria concerning bed width, edge width and uniformity of depositions.

Droplet size and velocity characteristics

Droplet characteristics without air support

The cumulative volumetric droplet size and velocity distribution of the different nozzles spraying at 300 kPa, 0.5 m spray boom height, and without air support are presented in Figure 4. An overview of the most important droplet size and velocity characteristics, as well as the BCPC spray quality class, [START_REF] Southcombe | The international (BCPC) spray classification system including a drift potential factor[END_REF] is given in Table S1 ). These findings are in agreement with those of other authors who also reported the coarsest droplet size spectrum for air injection nozzles, followed by standard flat-fan nozzles, and who reported generally coarser droplet size spectra with larger ISO nozzle sizes. [START_REF] Nuyttens | Effect of nozzle type, size and pressure on spray droplet characteristics[END_REF][START_REF] Ferguson | Effect of spray droplet size on herbicide efficacy on four winter annual grasses[END_REF][START_REF] Etheridge | Characterization of the spray droplet spectra and patterns of four Venturi-type drift reduction nozzles[END_REF][START_REF] Nuyttens | Droplet size and velocity characteristics of agricultural sprays[END_REF][START_REF] Zwertvaegher | The impact and retention of spray droplets on a horizontal hydrophobic surface[END_REF][START_REF] Creech | Influence of herbicide active ingredient, nozzle type, orifice size, spray pressure, and carrier volume rate on spray droplet size characteristics[END_REF][START_REF] Vulgarakis Minov | Spray droplet characterization from a single nozzle by high speed image analysis using an in-focus droplet criterion[END_REF] With regard to droplet velocity (Figure 4b and Table S1), the standard ISO 02 nozzles showed the lowest volumetric median droplet velocity (vv0.5 = 2.7, 2.9, and 4.8 m/s for UB 85 02, XR 110 02, and XR 80 02). Within the ISO 04 nozzles, the standard nozzle type always generated higher volumetric median droplet velocities than the air inclusion type, in increasing order of AI 110 04, XR 110 04, AIUB 85 04, UB 85 04, AI 80 04, and XR 80 04 (vv0.5 = 5.1, 5.5, 5.8, 6.0, 6.5, and 8.3 m s -1 ). Nuyttens et al. [START_REF] Nuyttens | Droplet size and velocity characteristics of agricultural sprays[END_REF] also found that bigger ISO nozzle sizes correspond with significantly higher droplet velocity characteristics for all nozzle types. Vulgarakis Minov et al. [START_REF] Vulgarakis Minov | Spray droplet characterization from a single nozzle by high speed image analysis using an in-focus droplet criterion[END_REF] also observed higher droplet velocities with standard flat fan nozzles compared to air inclusion nozzles measured using a high speed image system. For the same droplet size, flat-fan nozzles produced higher average vertical droplet velocities than air inclusion nozzles, for the same ISO nozzle size and spray pressure, as can be seen in Figure 5, and as also reported by Nuyttens et al. [START_REF] Nuyttens | Droplet size and velocity characteristics of agricultural sprays[END_REF] . The results furthermore show a clear effect of spray angle with higher average velocities for 80° nozzles compared to 110° nozzles.

Droplet characteristics, in particular droplet size, are very important factors related to spray drift and biological efficacy. Smaller droplets are more sensitive to evaporation and drift, because, due to their lower velocity, they remain in the air longer before deposition. [START_REF] De Cock | Investigation on optimal spray properties for ground based agricultural applications using deposition and retention models[END_REF][START_REF] Matthews | Pesticide application methods[END_REF] A common approach to reduce drift is to shift the droplet size spectrum towards coarser droplets. However, coarser droplets can result in relatively low degree of target surface coverage and may shatter or bounce of the target. [START_REF] De Cock | Investigation on optimal spray properties for ground based agricultural applications using deposition and retention models[END_REF][START_REF] Massinon | Spray droplet impaction outcomes for different plant species and spray formulations[END_REF] On the other hand, larger droplets are more likely to collide with the target surface as they are less likely to deviate from their initial path when there are changes in the direction of air due to an object. By contrast, very small droplets follow almost exactly the streamlines of air flowing around an encountered object. [START_REF] Spilman | Spray impaction, retention and adhesion: an introduction to basic characteristics[END_REF] The trade-off between spray deposition and drift, emphasizes the need for optimal droplet size distribution and effective drift control practices, such as the use of air support.

Droplet characteristics with air support

The cumulative volumetric droplet size and velocity distribution of the nozzles AI 80 04, AI 110 04, XR 80 02, and XR 80 04 spraying at 300 kPa, 0.5 m spray boom height, without (0 rpm) and with air support (1400, 1750, 2000 rpm) are presented in Figure 6, respectively. An overview of the most important droplet size and velocity characteristics is given in Table S2. [START_REF] Nuyttens | The effect of air support on droplet characteristics and spray drift[END_REF] also reported only a limited effect of air support on droplet size, but they found a more important and significant increase in droplet velocities with air support. In addition, the effect of air support on droplet velocity was found to be more important for larger nozzle heights. [START_REF] Nuyttens | The effect of air support on droplet characteristics and spray drift[END_REF] In this study, the volumetric median droplet velocity increased with increasing air support within nozzle type, except for XR 80 04, although even than velocities were considerably higher with than without air support (vv0.5 = 6.9, 7.1, 7.9, 8.4 m s -1 for AI 80 04, 5.5, 6.2, 7.1, 7.4 m s -1 for AI 110 04, 5.5, 6.4, 7.2, 7.9 m s -1 for XR 80 02, and 8.4, 11.2, 11.0, 11.4 m s -1 for XR 80 04 at 0, 1400, 1750, and 2000 rpm, respectively). Although these measurements were more or less static, and therefore the air stream would interact less with the spray fan than compared to field conditions where the sprayer drives at larger speeds, a similar trend of increased droplet velocities with increased air support is to be expected in the field. An increase in vertical droplet velocity induced by air support on boom sprayers reduces the time of flight and thus the risk of drift. In addition, the forced airstream under the spray boom directs the spray towards the target and blows the spray droplets into the crops, thus resulting in drift reduction, [START_REF] Jong | Effect of sprayer boom height on spray drift[END_REF][START_REF] Nuyttens | The effect of air support on droplet characteristics and spray drift[END_REF] and improved deposition on the target. [START_REF] Van De Zande | Spray distribution when spraying potatoes with a conventional or an air-assisted field boom sprayer[END_REF] The increase in droplet velocity by means of air support was found to have the highest impact on the amount of spray drift for finer sprays, as especially small droplets quickly lose momentum imparted by the nozzle system and tend to quickly adopt the speed and direction of the ambient airflow in situations without air support. [START_REF] Nuyttens | The effect of air support on droplet characteristics and spray drift[END_REF] However, drift reducing techniques, such as air support, can also lead to increased soil deposition underneath the crop canopy and consequently shift the risk to water contamination by leaching through the soil. [START_REF] Van De Zande | Spray distribution when spraying potatoes with a conventional or an air-assisted field boom sprayer[END_REF] It is therefore important to also consider soil deposition when studying the effect of air support. A combination of air support and adjusted spray boom height depending on the canopy growth stage and target zone, as discussed above, could result in even better drift reduction on bed-grown crops, as lower spray boom height generally reduces spray drift and the effect of air support on drift reduction increased when sprayer boom height was reduced. [START_REF] Jong | Effect of sprayer boom height on spray drift[END_REF][START_REF] Van De Zande | Classification of spray applications for driftability, to protect surface water[END_REF] The effect of air support and adjusted nozzle spacing and boom height on potential spray drift reduction and canopy and soil deposition on early stage and full grown carrots in lab trials is discussed in Douzals et al. [START_REF] Douzals | Optimization of spray application on bed-grown vegetables -On-going developments within OPTIMA project[END_REF]

CONCLUSION

In light of the optimization of a smart sprayer for bed-grown carrots within the H2020-project OPTIMA, the use of various nozzle types and configurations, variable nozzle spacing and height, and air-support was presented in this study. Four bed spray configurations, i.e. XR8004/XR8004/XR8004/XR8004, AIUB8504/AI11004/AI11004/AIUB8504, AI8004/AI8004/AI8004/AI8004, and XR8002/XR8002/XR8002/XR8002, were identified that clearly show an added value compared to a standard broadcast application for spraying different target zone widths (1.2 to 1.8 m) with high uniformity (CV < 12%) and minimal losses out of the target zone (< 17%), using the correct nozzle spacing/height depending on the carrot growth stage. At later crop stages, when the canopy is more closed and the bed is covered with foliage (target zone of 2.0 -2.2 m), the broadcast application might still be the most suitable spray application. Bed spraying and adjusting the target zone width to the leaf foliage (cultivar, growth stage, planting system) can thus reduce the use of PPP's by reductions in application volume or dosage compared to broadcast applications up to a certain target zone width. In general, reducing the boom height in combination with narrower nozzle spacing, as done in this study with the bed spray applications for smaller target zone widths, may aid in decreasing spray drift. Nozzle type had an important effect on the droplet size and velocity spectra. For the same nozzle size and spray pressure, air inclusion nozzles produced larger but slower droplets than standard flat-fan nozzles, potentially reducing spray drift. Air support increased the droplect velocities but only had a very limited effect on droplet size. This paper shows that laboratory measurements of spray distribution and droplet characteristics can aid in selecting the most optimal spray settings for bed spray applications of different 360

target

Figure 1 .

 1 Figure 1. Schematic of the carrot bed design at early (left) and full growth stage (right), with indication

Figure 2 .Figure 3 .

 23 Figure 2. Spray distribution pattern of (a) AIUB8504/AI11004/AI11004/AIUB8504 and (b) a broadcast

Figure 4 .Figure 5 .

 45 Figure 4. (a) Cumulative volumetric droplet size distribution and (b) cumulative volumetric droplet

Figure 6 .

 6 (a) Cumulative volumetric droplet size distribution and (b) cumulative volumetric droplet 482 velocity distribution for AI 80 04, AI 110 04, XR 80 02, and XR 80 04 spraying at 300 kPa, 0.5 m 483 spray height, without (0 rpm) and with air support (1400, 1750, 2000 rpm). 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840

  Table1) in the target zone was always around 100% for the broadcast application, it was considerably lower (11 to 67%) than compared to the bed spray applications for target zones from 1.2 to 1.8 m, indicating lower depositions in those target zones for the broadcast application. This demonstrates the added value of the bed spray These values indicate a good uniformity within the target zone for the bed spray applications, especially considering the threshold value has primarily been defined for broadcast applications. As suggested by the overall low CV in the target zone and the lower losses outside the target zone, the broadcast application might still be the most suitable spray application at later crop stages, when the canopy is more developped and more closed and the bed is covered with foliage (target zone of 2.0 -2.2 m).

	from 2.0 to 2.2 m, the average applied doses were comparable for all configurations, ranging from 99 to
	105%. Configurations XR8004/XR8004/XR8004/XR8004 and AI8004/AI8004/AI8004/AI8004 had
	the highest CV (8.7 to 10.8%), i.e. lowest uniformity, followed by XR8002/XR8002/XR8002/XR8002
	and AIUB8504/AI11004/AI11004/AIUB8504, except for the target zone of 2.2 m. For the 2.2 m target
	zone, AI8004/AI8004/AI8004/AI8004 and AIUB8504/AI11004/AI11004/AIUB8504 had the highest

applications since potential dosage or application rate savings can be obtained. Considering the example from Figure

2

, configuration AIUB8504/AI11004/AI11004/AIUB8504 resulted in an average spray volume of 173 L ha -1 within the target zone of 1.6 m, whereas a theoretical application rate of 135 L ha -1 ground area was determined for a boom sprayer with 36 ISO 04 nozzles (4 nozzles per bed) at 12 km h -1 driving speed and a spray pressure of 300 kPa. The latter is already a 14% reduction in theoretical application rate compared to a broadcast application of 158 L ha -1 with 42 XR11004 nozzles at the same driving speed and spray pressure. The increased on-target deposition of 173 L ha -1 indicates that even lower spray volumes or dosages could be applied with the bed spray configurations at adjusted nozzle spacing/height while maintaining the same bio-efficacy as for the reference broadcast application. Indirectly these reductions would also result in lower losses and spray drift. Variable rate application methods could also be used to obtain the desired, reduced application rate or dosage. At target zones CV (over 8%). The CV in the target zone of the bed spray applications are almost always below the 10% threshold value stated in ISO 16122-2

[START_REF]ISO 16122-2 -Agricultural and forestry machinery -Inspection of sprayers in use -Part 2: Horizontal boom sprayers[END_REF]

and EN 13790-1

[START_REF]EN 13790-1 -Agricultural machinery -Sprayers -Inspection of sprayers in use -Part 1: Field crop sprayers[END_REF]

, which should not be exceeded by standard horizontal boom sprayers.

  As for the measurements with a single nozzle without air support, air inclusion nozzles generated the coarsest droplet size spectrum, followed by the standard ISO 04 nozzle, and the standard ISO 02 nozzle. Within nozzle type, VMD was slightly higher with air support compared to without air support, except for XR 80 02, but no clear trends

	were visible (VMD = 457, 473, 472, 467 µm for AI 80 04, 445, 456, 455, 456 µm for AI11004, 320,
	337, 336, 337 µm for XR 80 04, and 262, 264, 261, 263 µm for XR 80 02 at 0, 1400, 1750, and 2000

rpm, respectively). Nuyttens et al.

Table 1 .

 1 zone widths. 361 Overview of nozzles and settings selected and tested as possible optimizations. 448 Theoretical application rate at 12 km h -1 , expressed as L ha -1 of total ground area ‡ Broadcast application with 42 nozzles on a 21 m spray boom § Bed spray application with 36 nozzles (4 nozzles per bed, incl. 2 off-centre nozzles) on a 21 m spray boom ¶ Bed spray application with 36 nozzles (4 nozzles per bed) on a 21 m spray boom † † No / Yes = tested without air support and with air support set at 0, 1400, 1750, 2000 rpm 449

	Technique	Nozzle type + size	Spray pressure (kPa)	Nozzle flow rate (L min -1 )	Appl. rate (L ha -1 ) †	Air support † †
	Reference nozzle	TeeJet XR 110 04	300	1.58	158 ‡	No
	Reduced volume nozzle	TeeJet XR 110 02	300	0.79	79 ‡	No
	Drift reducing nozzle	TeeJet AI 110 04	300	1.58	158 ‡	No / Yes
	Off-center reference nozzle	TeeJet UB 85 04	300	1.58	135 §	No
	Off-center reduced volume nozzle	TeeJet UB 85 02	300	0.79	101 §	No
	Off-center drift reducing nozzle	TeeJet AIUB 85 04	300	1.58	135 §	No
	Narrow angle, reference nozzle	TeeJet XR 80 04	300	1.58	135 ¶	No / Yes
	Narrow angle, reduced volume nozzle	TeeJet XR 80 02	300	0.79	68 ¶	No / Yes
	Narrow angle, drift reducing nozzle	TeeJet AI 80 04	300	1.58	135 ¶	No / Yes
	†

Table 2 .

 2 Nozzle configurations tested for spray distributions. 450

	Configuration	Spray pressure (kPa)	Nozzle spacing & height (m)
	XR 110 04 †	300	0.5
	UB 85 04 / XR 110 04 / UB 85 04	300	0.4 -0.45 -0.5 -0.55 -0.6 -0.65
	UB 85 04 / XR 80 04 / UB 85 04	300	0.4 -0.45 -0.5 -0.55 -0.6 -0.65
	XR 80 04 / XR 80 04 / XR 80 04	300	0.4 -0.45 -0.5 -0.55 -0.6 -0.65
	UB 85 04 / XR 110 04 / XR 110 04 / UB 85 04	300	0.35 -0.4 -0.45 -0.5 -0.55 -0.6 -0.65
	UB 85 04 / XR 80 04 / XR 80 04 / UB 85 04	300	0.35 -0.4 -0.45 -0.5 -0.55 -0.6 -0.65
	XR 80 04 / XR 80 04 / XR 80 04 / XR 80 04	300	0.35 -0.4 -0.45 -0.5 -0.55 -0.6 -0.65
	AI UB 85 04 / AI 110 04 / AI 110 04 / AI UB 85 04	300	0.35 -0.4 -0.45 -0.5 -0.55 -0.6 -0.65
	AI UB 85 04 / AI 80 04 / AI 80 04 / AI UB 85 04	300	0.35 -0.4 -0.45 -0.5 -0.55 -0.6 -0.65
	AI 80 04 / AI 80 04 / AI 80 04 / AI 80 04	300	0.35 -0.4 -0.45 -0.5 -0.55 -0.6 -0.65
	XR 110 04 / XR 110 04 / XR 110 04 / XR 110 04	300	0.35 -0.4 -0.45 -0.5 -0.55 -0.6 -0.65
	AI 110 04 / AI 110 04 / AI 110 04 / AI 110 04	300	0.35 -0.4 -0.45 -0.5 -0.55 -0.6 -0.65
	XR 80 02 / XR 80 02 / XR 80 02 / XR 80 02	300	0.35 -0.4 -0.45 -0.5 -0.55 -0.6 -0.65
	† Reference broadcast application, spray distribution of 12 nozzles measured
	451

Table 3 .

 3 Spray distribution characteristics of the broadcast application (XR 110 04) and the 4 most appropriate bed spray configurations at the most optimal 452 nozzle spacing/height combinations for different target zone widths of 1.2, 1.4 , 1.6, 1.8, 2.0, and 2.2 m. 453

	Configuration	Spray distribution characteristic	1.2	1.4	Target zone width (m) 1.6 1.8	2.0	2.2
	Broadcast application -XR 110 04	Nozzle spacing/height (m)	0.5	0.5	0.5	0.5	0.5	0.5
		Min. spray volume (L ha -1 )	144	144	144	144	144	144
		Max. spray volume (L ha -1 )	171	171	171	176	176	176
		Avg. spray volume (L ha -1 )	158	158	158	159	159	159
		Spray volume in target zone (%)	49.9	58.2	66.6	75.2	83.5	91.8
		Losses (%)	50.1	41.8	33.4	24.8	16.5	8.2
		CV (%)	5.8	5.6	5.3	5.8	5.5	5.4
	XR 80 04/XR 80 04/XR 80 04/XR 80 04	Nozzle spacing/height (m)	0.35	0.4	0.45	0.5	0.55	0.6
		Min. spray volume (L ha -1 )	181	147	132	115	96	121
		Max. spray volume (L ha -1 )	247	214	184	176	158	161
		Avg. spray volume (L ha -1 )	220	189	166	150	138	135
		Spray volume in target zone (%)	84.5	85.9	87.0	87.8	84.9	91.0
		Losses (%)	15.5	14.1	13.0	14.0	15.1	9.0
		CV (%)	9.2	9.4	8.7	10.2	10.8	6.2
	AIUB 85 04/AI 110 04/AI 110 04/AIUB 85 04 Nozzle spacing/height (m)	0.4	0.45	0.5	0.6	0.65	0.65
		Min. spray volume (L ha -1 )	206	180	153	129	123	118
		Max. spray volume (L ha -1 )	252	222	191	169	157	157
		Avg. spray volume (L ha -1 )	225	200	173	151	140	139
		Spray volume in target zone (%)	85.5	88.5	91.0	85.5	85.5	92,9
		Losses (%)	14.5	11.5	9.0	14.5	14.5	7.1
		CV (%)	6.6	6.2	5.9	7.0	7.1	8.0
	AI 80 04/AI 80 04/AI 80 04/AI 80 04	Nozzle spacing/height (m)	0.35	0.4	0.45	0.5	0.55	0.6
		Min. spray volume (L ha -1 )	197	160	148	126	104	117
		Max. spray volume (L ha -1 )	252	220	194	176	165	155
		Avg. spray volume (L ha -1 )	224	191	172	152	141	135
		Spray volume in target zone (%)	85.5	86.6	88.2	89.1	87.3	90.5
		Losses (%)	14.5	13.4	11.8	11.5	12.7	9.5
		CV (%)	9.4	9.8	9.6	9.4	10.8	8.2
	XR 80 02/XR 80 02/XR 80 02/XR 80 02	Nozzle spacing/height (m)	0.35	0.4	0.45	0.5	0.55	0.6

Table S2 .

 S2 Droplet size and velocity characteristics Dv0.1, Dv0.5, Dv0.9, V100, vv0.50, vavg (average ± SD) of the 4 nozzle types tested with air support (0, 1400, 1750, 2000 rpm).

	Nozzle type	Air support (rpm)	Pressure (kPa)	Dv0.1 (µm)	Dv0.5 (µm)	Dv0.9 (µm)	V100 (%)	vv0.50 (m s -1 )	vavg (m s -1 )
	AI 80 04	0	300	275.2 ± 2.5	456.7 ± 6.0	731.1 ± 6.3	0.2 ± 0.0	6.9 ± 0.1	3.9 ± 0.1
	AI 80 04	1400	300	275.4 ± 2.5	473.9 ± 3.9	734.7 ± 4.8	0.2 ± 0.0	7.1 ± 0.2	4.9 ± 0.2
	AI 80 04	1750	300	272.7 ± 4.5	471.5 ± 7.9	733.9 ± 13.0	0.2 ± 0.0	7.9 ± 0.0	6.1 ± 0.1
	AI 80 04	2000	300	273.7 ± 8.3	466.9 ± 2.8	740.4 ± 4.1	0.2 ± 0.1	8.4 ± 0.1	6.9 ± 0.1
	AI 110 04	0	300	266.6 ± 4.9	445.4 ± 7.2	712.9 ± 4.0	0.2 ± 0.0	5.5 ± 0.1	3.3 ± 0.1
	AI 110 04	1400	300	264.6 ± 1.2	456.2 ± 0.8	714.9 ± 4.8	0.3 ± 0.0	6.2 ± 0.3	4.9 ± 0.4

ACKNOWLEDGEMENTS

This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 773718 (OPTIMA-project).

Supporting information

Table S1. BCPC class and droplet size and velocity characteristics Dv0.1, Dv0.5, Dv0.9, V100, vv0.50, vavg (average ± SD) of the 9 nozzle types tested without air support.

Nozzle type


Dominique Turck

Jean-Louis Bresson

Barbara Burlingame

Tara Dean

Susan Fairweather-Tait

Marina Heinonen

Karen Ildico

Inge Mangelsdor

Harry J Mcardle

Androniki Naska

Monika Neuh € Auser-Berthold

Gra _ Zyna Nowicka

Kristina Pentieva

Yolanda Sanz

Anders Sj

Martin Stern

Daniel Tom

Henk Van Loveren

Marco Vinceti

Peter Willatts

Ambroise Martin

J J ) Strain

Alfonso Siani

Inge Mangelsdorf

Hendrik Van Loveren

Hendrik Van Loveren

normal endotheliumdependent vasodilation: evaluation of a health claim pursuant to

Keywords: tea, flavanols, tea, endothelium, vasodilation, health claim

Following an application from Unilever NV, submitted for authorisation of a health claim pursuant to Article 13(5) of Regulation (EC) No 1924/2006 via the Competent Authority of Ireland, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion on the scientific substantiation of a health claim related to black tea and maintenance of normal endotheliumdependent vasodilation. The scope of the application was proposed to fall under a health claim based on newly developed scientific evidence. The food proposed by the applicant as the subject of the health claim is black tea beverages, either freshly prepared or reconstituted from water extract powders of black tea, characterised by the content of flavanols (expressed as catechins plus theaflavins) of at least 30 mg per 200 mL serving. The Panel considers that black tea characterised by the content of flavanols (expressed as catechins plus theaflavins) is sufficiently characterised. The claimed effect proposed by the applicant is 'improvement of endothelium-dependent vasodilation'. The Panel considers that maintenance of normal endothelium-dependent vasodilation is a beneficial physiological effect. Of the five human intervention studies provided on the chronic effect of black tea consumption on endothelium-dependent vasodilation, two investigated the effect after regular consumption of black tea for a sufficiently long time period (i.e. at least 4 weeks). These two studies did not allow an effect of black tea on endothelium-dependent vasodilation to be established. The Panel concludes that a cause and effect relationship has not been established between the consumption of black tea and maintenance of normal endothelium-dependent vasodilation.

Summary

Following an application from Unilever NV, submitted for authorisation of a health claim pursuant to Article 13(5) of Regulation (EC) No 1924/2006 via the Competent Authority of Ireland, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion on the scientific substantiation of a health claim related to black tea and maintenance of normal endotheliumdependent vasodilation.

The scope of the application was proposed to fall under a health claim based on newly developed scientific evidence.

The general approach of the NDA Panel for the evaluation of health claims applications is outlined in the EFSA general guidance for stakeholders on health claim applications and the guidance on the scientific requirements for health claims related to antioxidants, oxidative damage and cardiovascular health.

The food proposed by the applicant as the subject of the health claim is black tea beverages, either freshly prepared or reconstituted from water extract powders of black tea, characterised by the content of flavanols (expressed as catechins plus theaflavins) of at least 30 mg per 200 mL serving. The Panel considers that black tea characterised by the content of flavanols (expressed as catechins plus theaflavins) of ≥ 30 mg/200 mL serving, which is the subject of the health claim, is sufficiently characterised.

The claimed effect proposed by the applicant is 'improvement of endothelium-dependent vasodilation'. The proposed target population is 'adults in the general population'. The Panel considers that maintenance of normal endothelium-dependent vasodilation is a beneficial physiological effect.

The applicant identified as directly pertinent to the claim five human intervention studies on the effect of black tea consumption on endothelium-dependent vasodilation measured in fasting conditions after regular consumption of black tea (chronic effect) and four human intervention studies on the acute effect of black tea consumption on endothelium-dependent vasodilation after regular consumption of black tea (acute-in-chronic effect). In addition, the applicant provided eight human intervention studies on the acute effects of tea beverages on endothelium-dependent vasodilation as supportive evidence. The applicant also submitted meta-analyses of the chronic, acute-in-chronic studies and acute studies, both individually and combined.

Out of the five human intervention studies provided on the chronic effect of black tea consumption on endothelium-dependent vasodilation, only two investigated the effect after regular consumption of black tea for a sufficiently long time period (i.e. at least four weeks).

In one of these studies, an effect of black tea consumption not only on endothelium-dependent vasodilation but also on endothelium-independent vasodilation was observed. Therefore, this study did not allow conclusions to be drawn on whether the effects of black tea consumption on vasodilation were endothelium dependent or mediated via endothelium-independent pathways. The Panel considers that no conclusions can be drawn from this study on the effect of black tea consumption on endothelium-dependent vasodilation. The second study in which black tea was consumed for 6 months did not show an effect of black tea consumption on endothelium-dependent vasodilation.

In the absence of studies allowing an effect of black tea consumption to be established on endothelium-dependent vasodilation in fasting conditions in healthy individuals after regular consumption of the food for at least 4 weeks, the Panel considered that studies of shorter duration, on an acute effect of black tea consumption with or without preceding continued consumption of the food, or in patients, as well as the meta-analysis provided by the applicant cannot be used for the scientific substantiation of the claim.

In weighing the evidence, the Panel took into account that the two studies which were provided by the applicant and which investigated the effect of black tea consumption on endothelium-dependent flow-mediated dilation in fasting conditions after regular consumption of the food for at least four weeks did not allow an effect of black tea on endothelium-dependent vasodilation to be established.

On the basis of the data provided, the Panel concludes that a cause and effect relationship has not been established between the consumption of black tea and maintenance of normal endotheliumdependent vasodilation. According to Article 18 of this Regulation, an application for inclusion in the Community list of permitted claims referred to in Article 13(3) shall be submitted by the applicant to the national competent authority of a Member State, which will make the application and any supplementary information supplied by the applicant available to the European Food Safety Authority (EFSA).

Table of contents

Interpretation of the Terms of Reference

EFSA is requested to evaluate the scientific data submitted by the applicant in accordance with Article 16(3) of Regulation (EC) No 1924/2006. On the basis of that evaluation, EFSA will issue an opinion on the scientific substantiation of a health claim related to: black tea and improvement of endothelium-related vasodilation.

The present opinion does not constitute, and cannot be construed as, an authorisation for the marketing of black tea, a positive assessment of their safety, nor a decision on whether black tea is, or is not, classified as a foodstuff. It should be noted that such an assessment is not foreseen in the framework of Regulation (EC) No 1924No /2006. . It should also be highlighted that the scope, the proposed wording of the claim and the conditions of use as proposed by the applicant may be subject to changes, pending the outcome of the authorisation procedure foreseen in Article 18(4) of Regulation (EC) No 1924/2006.

2.

Data and methodologies

Data

Information provided by the applicant Food/constituent as stated by the applicant According to the applicant, the food for which the health claim is made is 'catechins and theaflavins found in beverages prepared from the leaves of Camellia sinensis (L.) Kuntze'. Following EFSA's request for clarification, the applicant indicated that the food for which the health claim is made is 'black tea beverages characterised by the content of flavanols (expressed as catechins plus theaflavins)'.

Health relationship as claimed by the applicant

According to the applicant, the claimed effect relates to: 'improvement of endothelium-dependent vasodilation'.

Mechanism by which the food/constituent could exert the claimed effect as proposed by the applicant

The applicant claims that 'the available evidence demonstrates that flow-mediated dilation is increased acutely after consumption of tea flavanols and this effect is still present after regular consumption for longer periods of time'.

Wording of the health claim as proposed by the applicant

The applicant has proposed the following wording for the health claim: 'improves endotheliumdependent vasodilation which contributes to healthy blood flow'.

Specific conditions of use as proposed by the applicant

According to the applicant, the target population for the intended health claim is adults in the general population and a daily intake of ~160 mg tea flavanols (approx. 1,090 mL (5-6 servings) of black tea) is required to obtain a physiologically relevant increase of 1% in flow-mediated dilation (FMD).

Data provided by the applicant

Health claim application on tea flavanols and improvement of endothelium-related vasodilatation pursuant to Article 13.5 of Regulation 1924/2006, presented in a common and structured format as outlined in the Scientific and technical guidance for the preparation and presentation of applications for authorisation of health claims (EFSA NDA Panel, 2011a).

As outlined in the General guidance for stakeholders on health claim applications (EFSA NDA Panel, 2016), it is the responsibility of the applicant to provide the totality of the available evidence.

Methodologies

The general approach of the Dietetic Products, Nutrition and Allergies (NDA) Panel for the evaluation of health claims applications is outlined in the EFSA general guidance for stakeholders on health claim applications (EFSA NDA Panel, 2016).

The scientific requirements for health claims related to antioxidants, oxidative damage and cardiovascular health are outlined in a specific EFSA guidance (EFSA NDA Panel, 2011b).

The application contains data claimed as confidential: a) Unpublished consumer research. b) Analytical technique for the determination of the flavin content of black and green teas. c) Unpublished compositional data of a range of black and green teas.

Assessment

Characterisation of the food/constituent

The food proposed by the applicant as the subject of the health claim is 'catechins and theaflavins found in beverages prepared from the leaves of Camellia sinensis (L.) Kuntze'.

The main catechins in tea include (-)-epigallocatechin gallate, (-)-epigallocatechin, (-)-epicatechin-3-O-gallate, (-)-epicatechin, (+)-gallocatechin and (+)-catechin. The major theaflavins are theaflavin, theaflavin-3-O-gallate, theaflavin-3'-O-gallate and theaflavin-3,3'-O-digallate. Black tea also contains thearubigins, believed to constitute about 60% of black tea solids [START_REF] Haslam | Thoughts on thearubigins[END_REF].

Upon a request from EFSA to clarify whether the claim refers to tea flavanols (including catechins, theaflavins and thearubigins), to a fixed combination of catechins and theaflavins in tea or to tea beverages made from the leaves of Camellia sinensis (L.) Kuntze characterised by their content in catechins and theaflavins, the applicant replied that the food that is the subject of the claim is black tea beverages, either freshly prepared or reconstituted from water extract powders of black tea, characterised by the content of flavanols (expressed as catechins plus theaflavins) of at least 30 mg per 200 mL serving.

Catechins and theaflavins can be measured in foods by well-established methods.

The Panel notes that catechins are absorbed after oral consumption, whereas the absorption of theaflavins in the small intestine is low [START_REF] Mulder | Analysis of theaflavins in biological fluids using liquid chromatography-electrospray mass spectrometry[END_REF]. Theaflavins persist to the colon, where they undergo catabolism by gut microbiota [START_REF] Van Duynhoven | Metabolic fate of polyphenols in the human superorganism[END_REF][START_REF] Chen | The microbiota is essential for the generation of black tea theaflavins-derived metabolites[END_REF][START_REF] Del Rio | Dietary (poly)phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases[END_REF]. There is some evidence that the gut microbial catabolites of black tea polyphenols are recirculated into plasma [START_REF] Van Duynhoven | Rapid and sustained systemic circulation of conjugated gut microbial catabolites after single-dose black tea extract consumption[END_REF].

The Panel considers that black tea characterised by the content of flavanols (expressed as catechins plus theaflavins) of ≥ 30 mg/200 mL which is the subject of the health claim is sufficiently characterised.

Relevance of the claimed effect to human health

The claimed effect proposed by the applicant is 'improvement of endothelium-related vasodilation'. The proposed target population is 'adults in the general population'.

Improvement of endothelium-dependent (ED) vasodilation, which is a specific endothelial function, can be measured in vivo in humans using well-accepted methods. The capacity of blood vessels to respond to physical and chemical stimuli in the lumen confers the ability to self-regulate tone and to adjust blood flow and distribution in response to changes in the local environment. Many blood vessels respond to an increase in flow, or more precisely shear stress, by dilating. This phenomenon is designated as FMD. A principal mediator of FMD is endothelium-derived nitric oxide (NO). Endothelial denudation or treatment with a nitric oxide synthase (NOS) inhibitor abolishes FMD in a variety of arterial vessels. Endothelium-derived prostanoids and the putative endothelium-derived hyperpolarising factor have also been implicated as backup mechanisms mediating changes in arterial diameter in response to shear stress, so that there may be some redundancy in the system in order to ensure an appropriate response of blood vessels to shear stress. ED vasodilation contributes to the maintenance of an adequate blood flow to body cells and tissues.

ED vasodilation can be assessed in vivo at different points of the arterial tree using well-established methods (e.g. the FMD technique). Endothelium-independent vasodilation (EIVD) of the brachial artery induced by exogenous NO donors (e.g. after the sublingual administration of nitroglycerin) is also measured as control [START_REF] Corretti | Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force[END_REF][START_REF] Thijssen | Assessment of flow-mediated dilation in humans: a methodological and physiological guideline[END_REF].

The effect of a food/constituent on ED vasodilation can be expressed as changes in ED-FMD either in fasting conditions after regular consumption of the food/constituent, or as acute changes in ED-FMD occurring shortly after consumption of the food/constituent. A sustained increase in ED vasodilation in fasting conditions in response to an intervention (regular consumption of a food/constituent for at least four weeks) is a beneficial physiological effect.

Markers of plasma NO status (e.g. nitrite/nitrosyl species measured by reductive gas-phase chemiluminescence) can provide evidence on the mechanisms by which the food/constituent could exert the claimed effect, but cannot be used alone for the substantiation of the claim.

The Panel considers that maintenance of normal ED vasodilation is a beneficial physiological effect.

Scientific substantiation of the claimed effect

The applicant performed a literature search in Medline, Embase, Chemical Abstracts and Biosis (from starting date of the databases until July 2016) with the following key words: (tea or black tea or green tea or flavonoid or tea extract or tea component or tea solid or Camellia sinensis) and (flowmediated or flow mediated or FMD or endothelial function or endothelial dysfunction or endotheliumdependent or blood flow or arterial stiffness or vascular resistance or circulat* or micro-circulat* or microcirculat* or vasodilat* or dilat*). No restrictions were applied. Hand searches were also performed.

In the original application, the applicant had identified 15 published and three unpublished human intervention studies as well as one published and one unpublished meta-analysis as pertinent to the claim. These included studies investigating the effects of green tea, black tea or tea flavanols on ED-FMD. Following EFSA's request and the applicant's clarification that the food that is the subject of the claim is black tea beverages, the applicant identified as directly pertinent to the claim five human intervention studies on the effect of black tea consumption on ED-FMD measured in fasting conditions after regular consumption of black tea (chronic effect) (three published [START_REF] Duffy | Short-and long-term black tea consumption reverses endothelial dysfunction in patients with coronary artery disease[END_REF][START_REF] Hodgson | Acute effects of tea on fasting and postprandial vascular function and blood pressure in humans[END_REF][START_REF] Bohn | Effects of black tea on body composition and metabolic outcomes related to cardiovascular disease risk: a randomized controlled trial. Food & Function, 5, 1613-1620. Black tea and endothelium-dependent vasodilation www.efsa.europa.eu/efsajournal 9[END_REF], two unpublished (Mulder et al., 2013, unpublished;Mulder and Dutman, 2014, unpublished)) and four human intervention studies on the acute effect of black tea consumption on ED-FMD after regular consumption of black tea (acute-in-chronic effect) (two published [START_REF] Grassi | Black tea consumption dosedependently improves flow-mediated dilation in healthy males[END_REF][START_REF] Grassi | Black tea increases circulating endothelial progenitor cells and improves flow mediated dilatation counteracting deleterious effects from a fat load in hypertensive patients: a randomized controlled study[END_REF] and two unpublished (Mulder et al., 2013, unpublished;Mulder and Dutman, 2014, unpublished)). In addition, the applicant provided eight human intervention studies on the acute effects of tea beverages on ED-FMD [START_REF] Duffy | Short-and long-term black tea consumption reverses endothelial dysfunction in patients with coronary artery disease[END_REF][START_REF] Hodgson | Acute effects of tea on fasting and postprandial vascular function and blood pressure in humans[END_REF][START_REF] Ardalan | Black tea improves endothelial function in renal transplant recipients[END_REF][START_REF] Lorenz | Addition of milk prevents vascular protective effects of tea[END_REF][START_REF] Jochmann | The efficacy of black tea in ameliorating endothelial function is equivalent to that of green tea[END_REF]Mulder and Dutman, 2014, unpublished;Mulder et al., 2016, unpublished) as supportive evidence. The applicant also submitted meta-analyses of the chronic, acute-in-chronic studies and acute studies, both individually and combined.

The Panel notes that the study by [START_REF] Ardalan | Black tea improves endothelial function in renal transplant recipients[END_REF] was not randomised and consisted of a sequential administration of the test products. The Panel considers that no conclusions can be drawn from this study for the substantiation of the claim.

The Panel considers that human intervention studies in healthy individuals on the effect of black tea consumption on ED-FMD measured in fasting conditions after regular consumption of black tea of at least 4 weeks are directly pertinent to the claim, while studies of shorter duration, on the acute effects of black tea on ED-FMD with or without preceding regular consumption of the food or studies in patients can provide additional information, but are not sufficient on their own to establish an effect of the food on ED vasodilation.

Among the studies provided by the applicant, two investigated the effect of black tea consumption on ED-FMD measured in fasting conditions after regular consumption of black tea of at least 4 weeks. [START_REF] Hodgson | Regular ingestion of black tea improves brachial artery vasodilator function[END_REF] studied the effect of consuming black tea for 4 weeks in five servings per day (250 mL per serving; in total 1250 mL, estimation of the flavanol content provided by the applicant complying with the specifications for the food being the subject of the claim; claimed as confidential) in 21 subjects (10 in black tea group and 11 in hot water group, mean age ~59 years, 16 men), who were regular tea drinkers, in a randomised, single-blind (blinded to staff performing measurements), parallel design. The infusion time for black tea was one minute with constant movement. During the study period, subjects were instructed not to consume any caffeinated beverages other than the tea beverage to which they were assigned and not to change their habitual diet.

Measurements of endothelial function of the left brachial artery after an overnight fast were obtained at the end of the baseline period and at the end of the intervention period. EIVD of the brachial artery was measured following administration of 400 lg of sublingual glyceryl trinitrate (GTN) spray. Analysis of ED-FMD and of GTN-mediated dilation was carried out using a semi-automated edge-detection software system. The sample size of the study was sufficient to detect an absolute 1.5% improvement in FMD and an absolute 4.5% improvement in the response to GTN with an 80% power (a = 0.05).

General linear models were used to examine differences in post-intervention values after adjustment for baseline values. The Panel notes that baseline FMD was different between groups (mean AE SE: 5.1% AE 1.0% in the black tea group and 6.1 AE 0.9% in the hot water group).

Consumption of black tea resulted in a significant increase in FMD with an absolute change of 2.3% (95% CI 0.7 to 3.9%). In one subject in the tea group, a large increase in ED-FMD was observed. When this subject was removed from analysis, the increase in ED-FMD still remained significant.

The increase in the response to GTN was also significantly higher in the black tea group compared with the hot water group (mean AE SE: 22.1 AE 2.0% vs. 16.9 AE 0.9%, p = 0.03). Upon a request from EFSA to comment on the biological plausibility of the differential responses to GTN in the study groups, the applicant explained that the finding may potentially be related to the high dose of GTN used in this study (400 lg while 25 lg is the dose generally recommended), which, according to the applicant, elicits very large and therefore inherently noisier brachial artery dilation responses and might lead to spurious conclusions. The Panel notes that even if the dose of GTN used in the study might have led to more variable brachial artery dilation responses among individuals, this would have not been expected to be different between groups and cannot explain the differential response to GTN administration of the black tea and the hot water group.

The Panel notes that this study showed an effect of black tea consumption for 4 weeks on vasodilation. However, owing to the significant differences observed between groups on EIVD, the Panel notes that results of the study do not allow conclusions to be drawn on whether the effects of black tea consumption on vasodilation were ED or mediated via endothelium-independent pathways. The Panel considers that no conclusions can be drawn from this study on the effect of black tea consumption on ED vasodilation. [START_REF] Bohn | Effects of black tea on body composition and metabolic outcomes related to cardiovascular disease risk: a randomized controlled trial. Food & Function, 5, 1613-1620. Black tea and endothelium-dependent vasodilation www.efsa.europa.eu/efsajournal 9[END_REF] performed a randomised, two-arm, placebo-controlled, double-blind, 6-month parallel trial to investigate the long-term effects of daily consumption of black tea in 111 healthy subjects who were regular tea drinkers. The primary outcome was changes in 24 h systolic blood pressure, upon which also sample size calculations were based. ED-FMD was a secondary outcome.

The participants were randomly allocated to drink three servings of black tea prepared from black tea powder by adding 100-200 mL of hot water (containing 429 mg of polyphenols assessed as gallic acid equivalents and 96 mg of caffeine/day) or a placebo beverage containing no tea solids, but was matched in flavour and caffeine content. During the study period, subjects were asked to restrict the intake of tea, coffee, red wine, chocolate and fruit juices.

ED-FMD was measured at baseline and at 3 and 6 months of the intervention after overnight fasting and was assessed as maximum diameter (max. FMD diameter) and maximum dilation relative to vessel size (Peak FMD). Analysis was carried out using a semi-automated edge-detection software system. EIVD was not measured.

Data were analysed using a linear mixed model. Statistical analysis were performed on the per protocol population (77 participants (38 black tea group and 39 placebo group) mean age ~56 years, 50 females) that was defined as all participants who completed the 6-month study, had measurements performed for the relevant outcome and consumed at least 70% of the study product.

No statistically significant changes in ED-FMD were found after having consumed black tea for 3 and for 6 months.

The Panel notes that this study did not show an effect of black tea consumption on ED-FMD. The Panel notes that none of studies showed an effect of black tea consumption on ED vasodilation in fasting conditions in healthy individuals after regular consumption of the food for at least 4 weeks. In this context, studies of shorter duration (i.e. one week (Mulder et al., 2013, unpublished;Mulder and Dutman, 2014, unpublished;[START_REF] Grassi | Black tea increases circulating endothelial progenitor cells and improves flow mediated dilatation counteracting deleterious effects from a fat load in hypertensive patients: a randomized controlled study[END_REF]), on an acute effect of black tea consumption with or without preceding regular consumption of the food [START_REF] Lorenz | Addition of milk prevents vascular protective effects of tea[END_REF][START_REF] Jochmann | The efficacy of black tea in ameliorating endothelial function is equivalent to that of green tea[END_REF][START_REF] Grassi | Black tea consumption dosedependently improves flow-mediated dilation in healthy males[END_REF]Mulder et al., 2013, unpublished;Mulder and Dutman, 2014, unpublished;[START_REF] Grassi | Black tea increases circulating endothelial progenitor cells and improves flow mediated dilatation counteracting deleterious effects from a fat load in hypertensive patients: a randomized controlled study[END_REF]Mulder et al., 2016, unpublished), or in patients [START_REF] Duffy | Short-and long-term black tea consumption reverses endothelial dysfunction in patients with coronary artery disease[END_REF][START_REF] Hodgson | Acute effects of tea on fasting and postprandial vascular function and blood pressure in humans[END_REF], as well as the meta-analyses provided by the applicant cannot be used for the scientific substantiation of the claim.

Weighing of the evidence

In weighing the evidence, the Panel took into account that the two studies from which conclusions can be drawn for the effect of black tea consumption on ED-FMD in fasting conditions after regular consumption of the food for at least 4 weeks did not show effect of black tea on ED vasodilation.

The Panel concludes that a cause and effect relationship has not been established between the consumption of black tea and maintenance of normal ED vasodilation.

Conclusions

On the basis of the data presented, the Panel concludes that:

• The food/constituent, black tea characterised by the content of flavanols (expressed as catechins plus theaflavins) of ≥ 30 mg/200 mL which is the subject of the health claim, is sufficiently characterised.

• The claimed effect proposed by the applicant is 'improvement of endothelium-dependent vasodilation'. The target population proposed by the applicant is 'adults in general population'. Maintenance of normal ED vasodilation is a beneficial physiological effect.

• A cause and effect relationship has not been established between the consumption of black tea and maintenance of normal ED vasodilation.

Steps taken by EFSA

Health claim application on 'tea flavanols' and 'improvement of endothelium-dependent vasodilation' pursuant to Article 13(5) of Regulation (EC) No 1924/2006 (Claim serial No: 0458_IE). Submitted by Unilever N.V., Weena 455, 3013 AL Rotterdam, The Netherlands. 1) This application was received by EFSA on 16/5/2017.

2) The scope of the application was proposed to fall under a health claim based on newly developed scientific evidence.

3) The scientific evaluation procedure started on 27/6/2017. 4) On 6/9/2017, the Working Group on Claims of the NDA Panel agreed on a list of questions for the applicant to provide additional information to accompany the application. The scientific evaluation was suspended on 2/10/2017 and was restarted on 16/10/2017, in compliance with Article 18(3) of Regulation (EC) No 1924/2006. 5) During its meeting on 13/12/2017, the NDA Panel, having evaluated the data, adopted an opinion on the scientific substantiation of a health claim related to black tea and improvement of ED vasodilatation.

  Abstract.................................................................................................................................................. Summary................................................................................................................................................ 1. Introduction.................................................................................................................................... 1.1. Background and Terms of Reference as provided by the requestor...................................................... 1.2. Interpretation of the Terms of Reference........................................................................................... 2. Data and methodologies .................................................................................................................. 2.1. Data............................................................................................................................................... 2.2. Methodologies................................................................................................................................. 3. Assessment..................................................................................................................................... 3.1. Characterisation of the food/constituent............................................................................................ 3.2. Relevance of the claimed effect to human health............................................................................... 3.3. Scientific substantiation of the claimed effect .................................................................................... 4. Conclusions..................................................................................................................................... Steps taken by EFSA ............................................................................................................................... References.............................................................................................................................................. Abbreviations ..........................................................................................................................................

  of Reference as provided by the requestor Regulation (EC) No 1924/2006 1 harmonises the provisions that relate to nutrition and health claims, and establishes rules governing the Community authorisation of health claims made on foods. As a rule, health claims are prohibited unless they comply with the general and specific requirements of this Regulation, are authorised in accordance with this Regulation and are included in the lists of authorised claims provided for in Articles 13 and 14 thereof. In particular, Article 13(5) of this Regulation lays down provisions for the addition of claims (other than those referring to the reduction of disease risk and to children's development and health) which are based on newly developed scientific evidence, or which include a request for the protection of proprietary data, to the Community list of permitted claims referred to in Article 13(3).

www.efsa.europa.eu/efsajournal

Regulation (EC) No 1924/2006 of the European Parliament and of the Council of

December 2006 on nutrition and health claims made on foods.OJ L 404, 30.12.2006, p. 9-25.

18314732, 2018, 1, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2018.5138 by Cochrane France, Wiley Online Library on [05/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

EFSA Journal 2018;16(1):5138 18314732, 2018, 1, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2018.5138 by Cochrane France, Wiley Online Library on [05/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

EFSA Journal 2018;16(1):5138

Competent Authority of Ireland following an application by Unilever NV.