Louise Sherlock
email: lsherlock@rvc.ac.uk

Theo G M Demmers

Allen E Goodship

Ian D Mccarthy

Christopher Michael Wathes

Miss Louise Sherlock

The relationship between physical

Keywords: Behaviour, Broilers, Lighting, Welfare

INTRODUCTION

Background

25

In the UK, over 800 million chickens are produced annually (Defra, 2007), reaching a slaughter weight of over 2 kg within 6 weeks, well before sexual maturity (approximately 18 weeks) and skeletal maturity (approximately 23-27 weeks) [START_REF] Latimer | Postnatal growth of the chicken skeleton[END_REF][START_REF] Rath | Factors regulating bone maturity and strength in poultry[END_REF]. This fast growth rate is associated with poor leg health that can lead to lameness [START_REF] Kestin | Relationships 15 in broiler chickens between lameness, liveweight, growth rate and age[END_REF][START_REF] Sanotra | Monitoring leg problems in broilers: A 20 survey of commercial broiler production in Denmark[END_REF], which has been demonstrated to be detrimental to the bird's welfare [START_REF] Mcgeown | Effect of carprofen on lameness in broiler chickens[END_REF] and likely to infringe FAWC's Five Freedoms (www.fawc.org.uk). Large-scale surveys of 5 lameness using gait scoring techniques in commercial broiler flocks have estimated that about 27% of birds have an abnormal gait of sufficient severity for the birds' welfare to be compromised [START_REF] Kestin | Prevalence of leg weakness in broiler chickens and its relationship with 20 genotype[END_REF][START_REF] Knowles | Leg disorders in broiler chickens: Prevalence, risk factors and 25 prevention[END_REF].

Causes of poor leg health and lameness

There may be many causes of lameness in a broiler chicken, with the aetiology 10 generally classified as developmental, degenerative or infectious [START_REF] Bradshaw | A review of the aetiology and pathology of leg weakness in broilers in relation to welfare[END_REF]. Developmental abnormalities include varus-valgus deformation (VVD) at the intertarsal joint, one of the most common leg distortions in broilers (EC, 2000), and abnormal rotation within the tibiotarsus or femur. The most widespread degenerative disorder in commercial broilers is contact dermatitis, while infectious disorders are 15 suggested to cause the most severe cases of lameness [START_REF] Kestin | Leg weakness in broiler chickens, a review of studies using gait scoring[END_REF].

Developmental deformities may cause walking difficulties in the birds and increase the risk of injury during catching, causing downgrading or even rejection by the processor [START_REF] Aviagen | Leg health in broilers[END_REF]. The underlying cause of these conditions is poorly understood but high growth rate leading to altered load bearing, lack of activity and 20 genetic factors are possible candidates [START_REF] Duff | Abnormal angulation/torsion of the pelvic appendicular skeleton in broiler fowl: Morphological and radiological findings[END_REF][START_REF] Bradshaw | A review of the aetiology and pathology of leg weakness in broilers in relation to welfare[END_REF].

Contact dermatitis is a widespread problem in European broiler production (EC, 2000), with lesions occuring on areas of the body in prolonged contact with the litter, most commonly on the feet (foot pad or podo-dermatitis) but also on the hocks, commonly described as hock burns, and breast muscle, particularly in heavier and lame 25 birds (Kristensen et al., 2006b). In a survey of 28 broiler flocks in Denmark [START_REF] Sanotra | Monitoring leg problems in broilers: A 20 survey of commercial broiler production in Denmark[END_REF] the prevalence of podo-dermatitis was 42%, while others suggest it may be lower at 11.1% in the UK [START_REF] Haslam | Factors affecting the prevalence of foot pad dermatitis, hock burn and breast burn in broiler chicken[END_REF]. Proper litter management and the absence of pre-existing leg conditions that cause birds to sit for long periods can reduce the incidence and severity of contact dermatitis.

One possible reason for poor leg health in broilers is reduced bone quality.

Although the dimensions of the tibiotarsus in selected (modern) strains of broilers fed ad libitum are correct for load support, the bone itself is weak. The cortical bone shows 5 high porosity and low mineral content [START_REF] Thorp | Relationships between the bone 40 pathologies, ash and mineral content of long bones in 35-day-old broiler chickens[END_REF]Williams et al., 2000;Corr et al., 2003a;Williams et al., 2004), which may be due to rapid bone deposition in the outer layers to increase width to support the increasing mass of the bird but with insufficient time to allow infilling by osteoblasts due to the birds' rapid growth rate (Williams et al., 2004), although others found that reducing growth rate did 10 not improve bone quality [START_REF] Leterrier | Reducing growth rate of broiler chickens with a low energy diet does not improve cortical bone quality[END_REF].

Welfare concerns

Broilers from selected strains and fed ad libitum often show a number of morphological changes compared with those of relaxed (i.e. randomly bred) strains (Corr et al., 2003a). The result of this altered morphology is a change in walking in an attempt to 15 increase stability (Corr et al., 2003b). This altered gait is considered inefficient and would rapidly tire birds, giving an explanation for the low levels of activity seen in all broilers.

Sound broilers spend, on average, 76% of their time lying down, but in lame birds this increases significantly to 86% [START_REF] Weeks | The behaviour of broiler chickens and its modification by lameness[END_REF]. Time spent walking at 20 slaughter-weight is significantly shortened from 3.3% to 1.5% in the worst cases of lameness. Sound birds also spend more time standing idle, standing preening and standing eating [START_REF] Weeks | The behaviour of broiler chickens and its modification by lameness[END_REF].

Poor leg health may affect the bird in several ways, with particular concern given to the possibility of pain. McGeown and colleagues (1999) showed that 25 moderately lame birds would complete an obstacle course faster when given an analgesic, suggesting that in this case birds' walking speed was affected by pain from their gait abnormality, rather than the abnormality itself. Lame birds will also self-select an analgesic [START_REF] Danbury | Self-selection of the analgesic drug carprofen by lame broiler chickens[END_REF]. However, others have found that administration of an analgesic does not change the walking behaviour of broilers [START_REF] Corr | A force plate study of avian gait[END_REF]; thus, the presence of pain arising from poor leg health and its role in walking ability remains contentious. Nevertheless, the differences in behaviour exhibited between sound and lame birds may indicate poor welfare caused in ways other than pain.

5

Alleviation of poor leg health through husbandry

Changes in husbandry such as stocking density or feeding regime can alleviate some causes of poor leg health [START_REF] Su | Meal feeding is more effective than early feed restriction at reducing the prevalence of leg weakness in broiler chickens[END_REF][START_REF] Sorensen | Effects of age and stocking density on leg weakness in broiler chickens[END_REF], as can selective breeding.

Increasing the activity of a broiler chicken has been shown to improve walking 10 ability and influence bone development [START_REF] Reiter | Effect of the distance between feeder and drinker on behaviour and leg disorders of broilers[END_REF] and could be achieved by increasing the distance between feeders and drinkers [START_REF] Reiter | Effect of the distance between feeder and drinker on behaviour and leg disorders of broilers[END_REF] or by the addition of barriers [START_REF] Bizeray | Influence of increased environmental complexity on leg condition, performance, and level of fearfulness in broilers[END_REF]. These practices increase the thickness and density of the cortical bone and the diameter of the tibiotarsus diaphysis through the deposition of bone in response to the imposed stresses, as explained by 15 Wolff's Law (1892). Increased exercise also reduces the prevalence of abnormalities in the physis and physeal vasculature of bone extremities from 40 to 18% [START_REF] Thorp | Effect of exercise on the vascular pattern in the bone extremities of broiler fowl[END_REF].

Another method found to be effective in encouraging activity in broiler chickens is providing step changes in light intensity (Kristensen et al., 2006a). The 20 colour and source of artificial light can also affect activity; low-frequency fluorescent lighting lowers activity compared with high-frequency [START_REF] Boshouwers | Responses of broiler chickens to high-frequency and low-frequency fluorescent light[END_REF] and activity is higher in red than in blue light both at the same intensity, and at higher intensities [START_REF] Prayitno | The effects of color and intensity of light on behavior and leg disorders in broiler chickens[END_REF]. Gait abnormalities were also reduced with red light.

The aim of this experiment was to investigate the relationship between physical 25 activity and leg health of broiler chickens on a semi-commercial scale, while also exploring the effect of step changes in light intensity on individual activity within a

MATERIALS AND METHODS

Animals, housing and husbandry

Three batches of Ross 308 broilers (approximately 2128 chicks 'as hatched' per batch) 5 were obtained at d-old from a commercial hatchery (P D Hooks, Bampton) and used in a large scale multi-factorial experiment. On arrival, the chicks were distributed randomly between eight identical rooms, each holding 266 birds. Within each batch 40 sexed chicks -20 of each sex -were marked for later identification then evenly distributed between two of the rooms. At 9 d old, 6 birds of each sex from these marked 10 birds in both rooms were assigned one of 12 symbols. These were sprayed with black stock marker for identification by overhead cameras. The marks were refreshed once or twice weekly as required. These individuals became the 'focal' birds for further detailed studies of behaviour.

The eight 19.6 m 2 rooms were filled with wood shavings to a depth of at least normal crude protein content and the feed quantity was 5% below the normal allowance to encourage slower growth and extend the experimental period.

Room temperature was reduced according to recommended guidelines [START_REF] Aviagen | Broiler management manual[END_REF]. Humidity was maintained at approximately 70% relative humidity or higher using a misting system. Animal health was monitored twice daily by a skilled 5 stockman and severely lame birds were culled humanely. Excluding the focal birds, the overall mortalities to 49 d were as follows; Batch 1: 3.7%, Batch 2: 5.5% and Batch 3: 2.3%. The higher mortality of the second batch was due to a yolk sac infection.

Experimental design 10

The experiment started on d 14 and finished on d 44 when the focal birds were killed by cervical dislocation.

The main experiment, the results of which will be reported separately, comprised a 2 3 multi-factorial design with three replicates (batches) of each treatment. This investigated the response of broilers to step-wise changes in humidity, illuminance 15 and feed quantity. In our study, however, the only treatment under consideration was the step-wise change in light intensity. In each batch this treatment was applied in one room, to which the focal birds were assigned in addition to the control room.

Step-wise changes in light intensity

The illuminance of the control room was kept at 10 lx throughout the photoperiod. The 20 light intensity treatment comprised step-wise changes in light intensity alternating between an illuminance of 10 lx and 200 lx throughout the photoperiod, as used in our previous research (Kristensen et al., 2006a). There were 4 step changes each day (i.e. dim to bright and vice versa, twice) as illustrated in Figure 1. The illuminance immediately following dawn alternated each day, to balance the experimental design. Video recordings were made of the focal birds when they were 2, 4 and 6 weeks of age.

25

Experimental procedures

Activity of the focal birds

Recording took place between 04:00 and 05:00, 09:00 and 10:00 and 14:00 and 15:00, covering a steady light state, one feed and 20 minutes after a step change in light intensity. This schedule also allowed time for inspection by the stockman without influencing the observed birds' activity. The activity was quantified by counting the 5 number of gridlines crossed by an individual every 5 minutes over an hour using a grid of 36 squares placed over the video image on a VDU. After initial analysis, more detailed investigation was required, so the bird's activity during a period of 5 minutes immediately following a step up in illuminance (at 10.18 am) was also examined by the same method when the birds were 4, 5 and 6 weeks of age.

10

Leg health

Measurements of leg health were taken while the birds were alive and post mortem.

During the experimental period the focal birds were assessed weekly using gait scoring [START_REF] Kestin | Prevalence of leg weakness in broiler chickens and its relationship with 20 genotype[END_REF][START_REF] Garner | Reliability and validity of a modified gait scoring system and its use in assessing tibial dyschondroplasia in broilers[END_REF], the prevalence and severity of any hock and foot pad burns, and the presence or absence of leg deformity (rotation and varus-valgus 15 deformation). To measure gait score (GS), the selected bird was herded, its movement observed and scored on a 6-point scale (0 -completely sound bird; 5 -unable to stand).

However, in this study any birds of gait scores 4 and 5 were culled on ethical grounds.

Hock burns were scored 0 -no mark, 1 -reddening, 2 -mild scabbing (<10% hock with lesion) and 3 -severe scabbing (>10% hock with lesion). Foot pad burns were scored 0 20 -no mark, 1 -mild lesion (<5mm) and 2 -severe lesion (>5mm) [START_REF] Dawkins | Chicken welfare is influenced more by housing conditions than by stocking density[END_REF]. At the end of the experimental period, the focal birds were killed by cervical dislocation and both legs removed at the coxofemoral joint. They were then stored at minus 20°C until analysis. The mid-physis of the tibiotarsus in both legs was scanned 5 using a pQCT (peripheral quantitative computed tomography) scanner (StraTec XCT 2000; Pforzheim, Germany) and a loop function performed for a measure of cortical bone density and thickness and area moments of inertia. Five slices, each 1 mm apart, were taken and the mean calculated.

Data analysis 10

While the majority of analysis involved data from all weeks from all batches, the more detailed investigation of activity and leg health was undertaken only on the final batch of birds at 2, 4 and 6 weeks of age due to the time taken to collect and analyse this data.

Minitab version 15 was used for all analysis.

The relationship between gait score and weight was determined using a non-15 parametric correlation coefficient (Spearman's). The differences in weight between sexes and treatments were examined in a General Linear Model (GLM). Chi squared analysis was used to compare the distribution of gait score and other leg health measurements between treatments. Where expected frequencies of cells were less than 5, Fisher's exact test was used.

20

Hourly activity data were transformed by square root to conform to the assumptions of a parametric test. A GLM was then used to analyse the activity data against age, time of day and light treatment and the interactions therein.

The activity data obtained over the 5 minutes immediately following a step up in light intensity could not be transformed fully so were analysed using the non-25 parametric Kruskal Wallis test for activity against treatment and activity against age. A two-way ANOVA was then used with logged activity data to check interactions A GLM was used to analyse transformed daily activity data against weight, age, sex, gait score and hock burn score. PQCT data from both legs were then analysed using paired t-tests to determine whether the density and thickness of the cortical bone 5 or the moments of inertia of the tibiotarsus differed significantly between the right and left. A multivariate GLM was then performed of cortical density and thickness and both horizontal and vertical moments of inertia against gait score, sex, mean activity and final weight.

RESULTS 10

Light treatment effects on leg health and production

Figure 2 shows the relationship found between gait score and mean weight across the growing period for all batches and ages (2 to 6 weeks). Gait score and weight were significantly correlated (Spearman's r = 0.7, P = 0.01); heavier birds had a poorer gait.

Mean weights differed between sexes (F = 45.8, P < 0.005) at slaughter at 6 weeks; 15 males -2.28 ± 0.38 kg and females -2.04 ± 0.22 kg. Batch also affected weight (F = 19.9, P < 0.005).

Light treatment did not affect mean weekly weight (P = 0.736) and had no significant effect on the overall numbers of sound (GS 0) or lame (GS 1-3) birds neither across individual gait scores (χ 2 = 0.668, P = 0.881) nor when grouped into sound vs. 20 lame categories (χ 2 = 0.445, P = 0.505). The majority (68.6%, n = 70) of birds displayed only slight gait impairment (GS 0 or 1) in both light treatments at week 6.

Data from the final week of study from all batches (n = 70) showed that neither the prevalence nor severity of any leg health measure was affected by light treatment.

Table 1 shows two examples: rotation of the tibiotarsus (Fisher's exact test: P = 0.149) 25 and hock burns (χ 2 = 5.5, P = 0.064), where the latter could be considered a trend for the step change treatment to reduce the prevalence of the highest severity of hock burns.

Light treatment effects on activity

The GLM analysis showed that light treatment did not affect the mean activity of focal birds at any age (n = 24, F = 12.3, P = 0.069), although this could be considered a trend with the control light treatment producing higher activities than the step change. There was no interaction between light treatment and age or time. Activity decreased with age 5 in both light treatments (Figure 3). Time and age interacted significantly to affect activity levels in both light treatments (Figure 4; F = 25.5, P = 0.004); as age increased, the difference in activity between times decreased. Table 2 shows the average hourly activity (represented by grid lines crossed) at each age across the focal birds.

Given the trend for activity of the focal birds to be higher in the control room 10 than with a step change in light intensity, the period (5 minutes) immediately following a step up in illuminance was examined to test the hypothesis that step change birds were more active during this period then became less active than the control birds at other times (i.e. those used in the original study). Treatment did not affect activity (P = 0.572), while age remained highly significant (F = 10.71, P < 0.001) and there was 15 again no significant interaction between the two factors.

Individual activity and leg health

The GLM analysis showed that none of sex, gait score, hock burn score or weight affected daily activity of focal birds. The only significant result was found with age (F = 5.97, P = 0.004); i.e. younger birds were more active than older birds.

20

Data from bird 11 were removed as this individual had a large influence, being an anomaly outwith the rest of the data due to the bird's low weight. Table 3 shows the descriptive data for cortical density and area moments of inertia in each leg as well as the final weight of the birds. The statistical analysis demonstrated that there was no difference between the right and left legs of the birds for any value; cortical bone 25 density (P = 0.461), thickness (P = 0.232) horizontal plane area moment of inertia (P = 0.319), vertical plane area moment of inertia (P = 0.977). Therefore, for subsequent analysis only data from the right legs were used. Females showed lower moments of inertia (lower resistance to bending) for both horizontal (F = 5.0, P = 0.04) and vertical (F = 13.8, P = 0.002) planes of the tibiotarsus; females: 142.1 ± 24.7 and 163.1 ± 26.2 mm 4 and males: 198.3 ± 46.4 and 233.0 ± 35.2 mm 4 . Final weight also increased the horizontal plane moment of inertia (F = 15.7, P = 0.001), but this is likely to be somewhat confounded by sex (females weighed less than males at slaughter age, 6 5 weeks). No variable had a significant effect on cortical density or thickness. The R 2 value was high at 0.98.

DISCUSSION

Effects of step-wise changes in light intensity on leg health and production

The correlation seen between gait score and weight was expected. Other studies have 10 reported a strong positive correlation between gait score and body weight [START_REF] Kestin | Prevalence of leg weakness in broiler chickens and its relationship with 20 genotype[END_REF][START_REF] Sorensen | The effect of photoperiod:scotoperiod on leg weakness in broiler chickens[END_REF][START_REF] Vestergaard | Relationships between leg disorders and changes in the behaviour of broiler chickens[END_REF][START_REF] Kestin | Relationships 15 in broiler chickens between lameness, liveweight, growth rate and age[END_REF]Kristensen et al., 2006a), although this is sometimes confounded when severely lame birds (scores 4 and 5) are included since an inability to feed causes a loss in body weight. At a slaughter age of 6 weeks, a broiler chicken's skeleton is not mature, so 15 heavier birds put more strain on soft bones and less fully formed joints. Complete ossification does not occur in the chicken until around 25 weeks [START_REF] Latimer | Postnatal growth of the chicken skeleton[END_REF][START_REF] Rath | Factors regulating bone maturity and strength in poultry[END_REF]. The rapid growth rate of modern strains of broilers leads to less dense and poorly mineralised cortical bone (Williams et al., 2000;Williams et al., 2004), which could increase the risk of fractures and leg deformations (varus-valgus or rotation).

20

Heavier birds may also tire faster, either from their sheer weight or from an altered morphology, so are reluctant to walk.

The light treatment had no significant effect on overall gait score although there was a trend for the number of birds with the highest severity of hock burn to be reduced with the step-wise changes in light intensity. This may have been due to a reduction in 25 time spent sitting or a drier litter with reduced ammonia content. A study on turkeys found that the incidence of leg abnormalities, as observed by gait and deformities at the intertarsal joint, was significantly reduced in high intensity light regimes (Hester et al., 1983). However, Kristensen and colleagues showed that broiler leg health was unaffected by light intensity (Kristensen et al., 2006b). Overall, our light treatment had no significant effect on the leg health of broilers.

Activity and light treatment

There was insufficient evidence to support the original hypothesis that the step-wise 5 changes in light intensity increases the activity of individual birds. On the basis of previous work (Kristensen et al., 2006a), we had expected activity to be greater when the step-wise changes were implemented, but lighting treatment had no significant effect on the activity of individuals; indeed there was a trend for control birds to be more active than those in the step change lighting treatment. This is contrary to the 10 results of Kristensen et al. who found that a similar step-wise change in light intensity resulted in greater activity of broilers during the periods of bright intensity (Kristensen et al., 2006a), but is similar to a study in which male turkeys were less active during the period of higher light intensity [START_REF] Hester | Effect of light intensity, litter source, and litter management on the incidence of leg abnormalities and performance of male turkeys[END_REF]. There are several explanations for these conflicting findings. Firstly Kristensen et al. (2006) only used 4 broiler chickens 15 in each group, compared to much larger flock of 266 birds (of which 12 were the focal birds). There is some evidence of greater jostling between individuals at higher stocking densities [START_REF] Dawkins | Chicken welfare is influenced more by housing conditions than by stocking density[END_REF], which may have affected the activity of the birds preventing similar results to be seen, and broilers have also been observed to walk longer distances at lower densities [START_REF] Lewis | Locomotion of broiler chickens in floor 45 pens[END_REF]. Secondly, it may be the 20 case that birds response with a spurt of activity to the intensity change per se rather than the absolute intensity. This was tested by observing activity immediately after a step change in intensity, but there was no significant difference between treatments.

Individual activity and leg health

Rath and colleagues have surmised that bone density can be used as an index of bone 25 strength [START_REF] Rath | Factors regulating bone maturity and strength in poultry[END_REF], since tibiotarsus breaking strength and bone density are significantly correlated [START_REF] Frost | Research note: Current methods used in determination and evaluation of tibia strength: A correlation study involving birds fed various levels of cholecalciferol[END_REF]. Therefore in our study cortical density was used as a proxy measure for bone strength. There was insufficient evidence to support the original hypothesis that increased activity promotes good leg health, as measured in terms of gait, the prevalence of hock burn, cortical bone density and thickness and the moments of inertia of the tibiotarsus. This is contrary to the findings of [START_REF] Reiter | Effect of the distance between feeder and drinker on behaviour and leg disorders of broilers[END_REF], who showed that active birds had thicker and denser cortical bone in the tibiotarsus compared with less active controls. However, the former had 5 been trained to run 100 m in 20 minutes daily on a treadmill, whereas the controls moved freely around their home pen (a 1 x 1m enclosure with 8 birds per m 2 ). The distance travelled by these trained birds was mostly higher than the hourly average activity of the focal birds in our experiment, which ranged from approximately 140.6 m at two weeks of age to 46.1 m at 6 weeks (estimated from the average distance between 10 grid lines and the number crossed). It is a strong possibility that the activity of our broilers, even in those considered more active, was insufficient to affect cortical density or shape of the tibiotarsus. An alternative explanation is that treadmill-based exercise imposes a greater dynamic load than normal walking, enlarging the circumference of the tibiotarsus; however this would not explain the increased bone density. Reiter and 15 colleagues reported that by training the fast growing strain of birds, the reduction in activity normally seen at three weeks of age was delayed until 6 weeks. While the final liveweight was unaffected by exercise, perhaps the high growth rate typical of broilers was delayed enough to allow sufficient infilling by osteoblasts, a process suggested by Williams et al. (2004). The mean cortical densities of the birds in this study are higher 20 than those reported by Reiter et al., but this can be explained by the location of scanning used in the latter study, namely at the proximal end of the tibiotarsus rather than at the mid point used here. However, both measures of cortical density are much lower than those found in comparable studies of slower growing broiler breeders (1160 mg/cc at a liveweight of 0.99 kg at five weeks of age, and 1250 mg/cc at 2.5kg at 15 25 weeks of age [START_REF] Rath | Factors regulating bone maturity and strength in poultry[END_REF]). The cortical density of the tibiotarsus of adult layers is also much higher at 1350mg/cc (Zhang and Coon, 1997). The significance of sex on the shape of the tibiotarsus may be related to the change in width of the bone in response to increased loading, i.e. mass, in the males. Our results indicate that the fast growing, young broiler chickens used in meat production have weak legs for their body size that, despite an increased width to reduce bending, may nonetheless put them at a greater risk of leg fractures and leg deformations.

Conclusions

5

In this study, carried out in a semi-commercial setting in contrast to other studies of its kind, a novel lighting regime of step-wise changes in light intensity did not affect performance, leg health or activity of broiler chickens. There was no association between an individual's activity and its leg health, measured in terms of gait, the prevalence of hock burn, tibiotarsus cortical bone density and thickness and shape.

10

Although this latter finding is counter intuitive, one plausible explanation is that modern, fast growing strains of broiler chickens do not take sufficient exercise to produce a difference in bone quality when given the choice.

F

  Our hypothesis was that step-wise changes in light intensity would increase individual activity thereby improving leg health.

15 7. 5

 155 cm and warmed to a temperature of 30 o C at a relative humidity of about 70% prior to the chicks' arrival. The birds were kept under normal commercial husbandry conditions until 49 d old when they weighed approximately 2.5 kg, equivalent to a stocking density of 34 kg m -2 . For the first 7 d, fluorescent lighting was provided on a 23:1h cycle of light: dark with lights on at 01.00 and a dawn and dusk period each of 30 20 minutes. From day 7 onwards, this cycle changed to 18:6h of light: dark, with lights on at 03.00 and the same dawn and dusk period. Illuminance was dependent upon the experimental treatment.Birds were provided with ad libitum access to water and were fed on a standard, 4-stage commercial diet (ABN, Peterborough), provided in 4 meals per d at 25 03:00, 09:00, 15:00 and 20:00. Chicks were first provided with a starter crumb until dy 11, a starter pellet from d 12 to 23, grower pellets from dy 24 to 44 and a withdrawal pellet ration between d 45 and 49. The diet was formulated to provide 90% of the

Figure

  Figure 1 near here

  Leg deformities were assessed by both varus-valgus deformation and abnormal rotation of the legs at the intertarsal joint. The bird was inverted, ventral side towards handler, and held by the legs below the intertarsal joint. Varus-valgus deformation and 25 rotation were assessed subjectively with scores assigned as follows: 0 for straight, 1 for not straight (varus -angle of >22 o where legs meet; valgus -angle of >/= 30 o between legs; rotation -pads face each other >15 o )[START_REF] Dawkins | Chicken welfare is influenced more by housing conditions than by stocking density[END_REF]. The presence or toes was also noted. Body weight was also measured after leg assessment prior to returning the individual to the flock.

  age, although attention was paid to statistical significance (P) to ensure agreement with the results of the non-parametric tests.

Figures

  Figures 3, 4 near here

Figure 2 .Figure 3 .

 23 Figure 2. Relationship between mean weight and gait score at all ages (weeks 2 to 6).

Figure 4 .

 4 Figure 4. The interaction between age and time in relation to median activity of focal birds in Batch 3 (n=24).

Table 1

 1

	near here

Table 2

 2

	near here

Table 3

 3

	near here

Table 1 .

 1 . & COON, C. N. (1997) The relationship of various tibia bone measurements in hens. Poultry Science, 76, 1698-1701. Prevalence of two measures of leg health of focal birds from batches 1-3 (n=70) at 6 weeks of age against light treatments

	o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

Table 2 .

 2 Mean hourly activity across focal birds at each age (n = 24)

	Age of birds	Mean hourly activity	Std. deviation
	5	(weeks)	(grids crossed) (+/-95% CI)
	2		163.5 ± 16.77	39.72
	4		77.0 ± 10.57	25.04
	6		53.6 ± 7.55	17.89

Table 3 .

 3 Descriptive data from right (RL) and left (LL) legs of Batch 3 focal birds (n=23)Figure 1.Step changes employed for illuminance.

	5
	10
	15
	20
	25

E-mail: br.poultsci@bbsrc.ac.uk URL: http://mc.manuscriptcentral.com/cbps British Poultry Science

ACKNOWLEDGEMENTS

Funding was provided by the BBSRC. Support was provided by Professor Claire 15 Wathes, John Lowe and staff from the RVC's Biological Services Unit and Professor Goodship's research group.


Ruediger Helling

Katja Kutschbach

Thomas Joachim Simat

Investigations on the migration behaviour of silicone moulds in contact with different foodstuffs

Keywords: Migration, Packaging -food simulants, Packaging -overall migration, Rubber Food Types: Infant formulae, Meat Silicones, polydimethylsiloxanes, 1 H-NMR, migration, foodstuffs

Investigations on the migration behaviour of silicone moulds

Introduction

During the last decade flexible silicone moulds have achieved significant market share and are widely used in households as well as in commercial food production. Many advantages such as a non-sticky surface, flexible form and low weight as well as the general low toxicity of migrating polysiloxanes led to a broad acceptance of silicone materials in food contact situations (Wacker 2008). On the other hand, silicone elastomers are not very inert when in contact with fatty or oily foodstuffs and may show rather high migration compared to metal moulds (Forrest andSidwell 2005, Helling et al. 2009). Different authors have described the interaction between silicone elastomers and foodstuffs.

Earlier studies focused on the amount of volatile organic compounds as an indicator for potential high migration [START_REF] Bannister | Studies of cyclic and linear poly(dimethylsiloxanes) : 6. Effect of heat[END_REF][START_REF] Lund | Safety of food contact silicone rubber: Liberation of volatile compounds from soothers and teats[END_REF]. A limit of 0.5 % for the release of volatile organic compounds was introduced to differ between tempered and nontempered materials and to avoid excessive migration (BfR recommendation XV). Later on gravimetric migration studies using the official substitute simulants for olive oil ethanol (95%), isooctane and Tenax were performed (Forrest andSidwell 2005, Meuwly et al. 2005).

These studies only gave a first indication of the different temperature profiles in foodstuffs and simulants and of the different types of diffusion in simulants compared to foodstuffs such as cake, which change from nearly liquid to solid during the baking process. Using GCtechniques, linear and cyclic siloxane oligomers were identified as the main constituents of the migrate. [START_REF] Meuwly | Heat stability and migration from silicone baking moulds[END_REF] used Tenax to demonstrate the strong temperature dependence of siloxane migration, and postulated a formation of low molecular weight oligomers during repeated heating and thus a regeneration of migrating compounds. GC-MS was discussed as a suitable method to determine the siloxane migration into complex food such as pizza. However, only a poor recovery of mid and high molecular weight oligomers [START_REF] Meuwly | Migration of siloxane oligomers in foodstuffs from silicone baking moulds[END_REF], Helling et al. 2009) could be achieved. Until now the only described method for a reliable quantification of migrating siloxane oligomers in fatty foodstuffs is the 1 H-NMR spectroscopy (Helling et al. 2009), despite various other methods having been described for biological samples such as tissues [START_REF] Čavić-Vlasak | Detection and characterization of poly(dimethyl)siloxanes in biological tissues by GC/AED and GC/MS[END_REF][START_REF] Mccamey | Determination of silicone in fats and oils by electrothermal atomic absorption spectrometry with in-furnace air oxidation[END_REF], Peters et al. 1995[START_REF] Kennan | Methods for Detecting Silicones in Biological Matrixes[END_REF], Dorn and Skelly 1994[START_REF] Biggs | Determination of Silicon Compounds by Gradient Liquid Chromatographic Separation with Direct Current Plasma Atomic Emission Spectrometric Detection[END_REF].

A 1 H-NMR spectroscopic method was developed and validated for determination of the migration of organosiloxanes from silicone materials into cake. It was also demonstrated, that the overall migrate as determined by gravimetric methods consists to more than 90% of organosiloxanes. Using this method it was shown that the results of migration experiments in simulants according to the time and temperature conditions as given in the plastics Directive (82/711/EEC) differ strongly from the siloxane amounts measured in food (Helling et al. 2009). Estimation of the siloxane content in food is essential for the correct assessment of the individual moulds. Especially for cake the overall migration limit of 60 mg kg -1 food or 10 mg sdm -1 , as defined in the Resolution ResAP (2004) 5 and in the Swiss legislation EDI, was respected -even in cases where migration experiments with simulants showed migration results were above the limit. In contrast to the migration studies carried out on simulants, the migration value in cake after multiple use of the mould tended to be almost constant, Moreover all test cakes baked in differently shaped moulds contained similar siloxane amounts which may result from a somewhat longer baking time needed in case of larger moulds, and thus equalizing the more advantageous volume/surface ratio (Helling et al. 2009).

Less surprisingly, migration was found to rise with increasing fat content of a comparable cake or pizza and also with baking time and temperature (Helling et al. 2009[START_REF] Meuwly | Migration of siloxane oligomers in foodstuffs from silicone baking moulds[END_REF]).

Despite the initial data concerning siloxane migration in food, some questions remain: What could be a worst case scenario for migration into food and are the migration limits still respected? Are the tempering process and the previously introduced BfR limit for volatile organic compounds useful and necessary to avoid excessive migration values? Does a formation of siloxane oligomers happen during repeated and long heating process? How should silicone moulds for a long term use be assessed from a hygiene point of view? To find an answer to all these questions and to demonstrate the influence of proper tempering of silicone materials, more studies on silicone moulds were carried out, using again the 1 H-NMR spectroscopic method to determine siloxane migration into food. Again, the focus was only on the overall migration analyzed via the NMR measured siloxane amount which is almost equal to the overall migration. Meatloaf as well as a milk based dessert (crème brulée) were chosen as possible critical foodstuffs and instant baby milk was used for additional testing of teats.

Anthrachinone was introduced as an internal standard to have the opportunity to quantify the triglyceride content in the multiple used moulds [START_REF] Malz | Quantitative NMR-Spektroskopie als Referenzverfahren in der analytischen Chemie[END_REF].

Materials and Methods

Silicone moulds

The silicone moulds used from various manufacturers were all bought on the German market except the moulds for the tempering study, which were provided by Wacker AG, Burghausen Germany. More detailed investigations concerning different food type experiments were carried out with a commercial 12 cups muffin silicone mould (volume: 95.8 cm³, inner surface: 0.86 sdm, thickness: 1.0 mm, Pt catalyzed crosslinked) and a box shaped silicone mould (volume: 1300 cm³, inner surface: 5.8 sdm, thickness: 1.2 mm, no information on crosslinking agents), both bought on the German market as well. The tempered / non-tempered moulds had a round shape (volume: 300 cm³, inner surface: 2.5 sdm, thickness: 1.5 mm, Pt-catalyst for crosslinking). Teats (tempered and non-tempered, Ptcatalysed) have been donated by MAPA GmbH (Zeven, Germany).

All samples were first washed with warm water (45-50°C) and household detergent, as recommended by the manufacturers.

Chemicals

All chemicals were obtained from Sigma-Aldrich Germany at analytical grade or higher. All food ingredients used for the baking studies were of commercial quality.

Migration into food simulants ethanol 95 % and ethanol 50 % 95 % ethanol and and 50 % ethanol were used as substitute simulants. The test conditions were based on common time-and-temperature conditions used for preparing the relevant foodstuffs and were selected according to Council Directive 82/711/EEC chapter 3 N o 1 and table 4. As baking moulds are intended for repeated usage, the first, second and third overall migrate were determined in all cases (Commission Directive 2002/72/EC).

The tests were carried out as a 2fold determination as described in part 14 of the European Standard EN 1186:2002: For migration tests with ethanol/water 95/5 or 50/50 (v/v), the intact samples were brimfully filled with the preheated solvent (60°C). In all cases, only the inside surface area of the moulds was assumed as contact area. Time measurement was already started when the samples were put into the oven, although the examination temperature of 60°C was regained later. An exposure time of 75 to 180 min depending of the size of the moulds was respectively chosen. The solvent was evaporated to determine the overall migration by the gravimetric standard method (EN 1186(EN :2002)).

Migration into foodstuffs

The studies were performed with creamed cake with 21 % fat (as described previously by Helling et al. 2009) and meat loaf.

Creamed cake, 21 % total fat (Das Backbuch, 1967) The dough contained 125 g sunflower oil, 100 g egg (homogenized), 125 g sugar, 250 g wheat flour, 8 g baking powder and 50 g water.

Meat loaf, 14 -19% total fat depending on the meat used 500 g minced meat, 125 g onions, 75 g breadcrumbs, 30 g cheese and 3 g salt were carefully mixed. The samples were filled to the brim with dough or meat paste and then put into the preheated oven at 175°C. After a suitable baking time (25 min for the small muffin moulds, 35 min for the larger round shaped mould, 40 min for the box shaped mould), the samples were removed from the oven. The cakes or meatloafs (including the leaked water / fat) were removed from the moulds and each one was put into a beaker.

In case of experiments 4, 6 and 8 of the 10 fold baking experiment with meatloaf the leaked sauce (fat and water) was decanted and sauce and meat have been analysed both independently. For the blank samples, the dough or meat paste was filled into a Teflon coated metal baking mould.

Crème brulèe (milk-based sweet)

Ingredients: 395 g cream, 80 g sugar, 70 g egg yolk, 8 g vanilla aromatised sugar.

Vanilla sugar and cream were cooked and added to a mixture of sugar and egg yolk. After intensive mixing, the mixture was poured into the mould and heated in a water bath in an oven at 175°C for 40 min. After half the time of heating the moulds were covered with aluminium foil to avoid excessive browning. After the heating period the moulds were allowed to cool for 2 hours at 7°C.

Instant baby milk, 4.0 % fat

22g commercial instant baby milk powder was dissolved in 135 ml bidistilled water at 50°C. Teats were incubated in the preheated milk for 6 hours at 40°C. (A longer test time could not be chosen because the instant milk was not stable for a longer time period.)

Soxhlet extraction

The foodstuffs were homogenized and an aliquot (approx. 12 g of the cake, 5 g of the meat loaf and 3 g of the Crème brulèe) was mixed thoroughly with sodium sulphate (Na 2 SO 4 ) in a mortar (approx. 15 g for cake and meatloaf, approx. 40 g for Crème brulèe and instant milk).

The dry powder was transferred quantitatively into a Soxhlet cartridge and extracted for 6 h with 150 ml diethyl ether in a Soxhlet extractor to isolate the fat matrix, including the siloxanes. At the end of the extraction, the diethyl ether was removed from the roundbottomed flask by distillation. Before and after extraction, the flask was dried repeatedly for Sodium sulphate had to be annealed for 3 h at 550 °C in a muffle furnace prior to use to remove siloxane impurities left over from manufacturing. Soxhlet cartridges and filter papers (used to clean the mortar and pestle and for filtration of the CDCl 3 solution, respectively) were extracted thoroughly in a Soxhlet extractor with diethyl ether prior to use.

For recovery experiments instead of the minced meat, tartare was mixed with in total 17 % lard, containing a known amount of polysiloxanes.

NMR measurement of fat extracts

For NMR measurement, 400 mg of the extracted fat were dissolved in 800 µl CDCl 3, filtered through silicone free paper and transferred into a NMR tube. The 1 H-NMR spectra were recorded at 500 MHz on a DRX 500 P NMR spectrometer (Bruker). Parameter settings were as follows: temperature: 22°C, number of scans: 128, spectral width: 10330 Hz, acquisition time: 3.17 s, resolution: 0.158 Hz per point, frequency sample rotation: 20 Hz, pulse delay: 1 s, pulse angle: 30°. Tetramethylsilane (TMS) was not added as a position standard for the chemical shift. Instead of this, the solvent signal (δ(CHCl 3 ) = 7.25 ppm in relation to TMS) was used to ascertain the position of resonance bands.

Calculation of the siloxane content using the triglyceride signal as an internal standard was described previously (Helling et al. 2009).

Determination of the amount of extractable substances of the box shaped mould

8 g of the silicone elastomer were cut into small pieces (3 x 3 mm) and extracted with diethyl ether for 10 hours using a soxhlett unit. The residue was dried at 103°C for 2 hours, weighted and 200 mg sunflower oil was added as an internal standard. The mixture was dissolved in 1 ml CDCl 3 and used for NMR measurement. The amount of extractable dimethyl siloxanes was 1.9 %.

Complete extraction of the intact mould

The box shaped mould was cleaned after the 10fold baking experiment with meatloaf and filled completely with ethanol. (Diethyl ether was not used to avoid irreversible swelling of the mould that would probably influence the following migration experiments.) Incubation was done at 60°C for 24hours, evaporated ethanol was subsequently refilled. After 24 hours an aliquot of 50 ml ethanol was concentrated to 5 ml. To 800 µl of this ethanol extract 100 µl of internal standard anthrachinone (5 mg ml -1 in CDCl 3 ) were added and the amount of siloxane was calculated using the same principle as described for the triglyceride as an internal standard. Anthrachinone was used according to [START_REF] Malz | Quantitative NMR-Spektroskopie als Referenzverfahren in der analytischen Chemie[END_REF] because of the incorporated fat in the mould which required the use of another internal standard.

The mould was incubated for another 24 hours, and an aliquot was taken again to determine the siloxane content but no additional extraction could be detected. In total 2.08 g dimethyl siloxanes were extracted with ethanol from the already used mould. The sum of the ethanol extracted siloxanes added to the sum of all 10 previously performed migration experiments with meatloaf is in good accordance with the extract determined from a new mould using diethyl ether.

Determination of the incorporated fat of the silicone elastomer

Approx. 50 cm 2 of the silicone elastomer were cut into small pieces (3 x 3 mm) and extracted with diethyl ether for 10 hours using a soxhlett unit. The residue was dried at 103°C for 2 hours, weighted and 100 µl of internal standard anthrachinone (5 mg ml -1 in CDCl 3 ) were added. The mixture was dissolved in 1 ml CDCl 3 and used for NMR measurement.

Calculation of the amount of triglycerides was done using the protons at C1 and C3 of the glycerin backbone as described for the determination of siloxanes with triglyceride as an internal standard.

Volatile organic compounds

To determine the amount of volatile organic compounds 10 g of the elastomer were cut into pieces of 1 x 2 cm and conditioned over dried CaCl 2 for 48 hours. Afterwards 10 g were weighted with a precision of 0.1 mg into a glass bowl and heated for 4 hours at 200°C, conditioned in an exsiccator and weighted again. The loss of weight calculated in percent gives the amount of volatile organic compounds.

Results and discussion

Meatloaf

The method used was validated previously for muffins (Helling et al. 2009) but a sufficient recovery rate was demonstrated again for meatloaf as described in the materials and method part and found to be at (103.1 ± 3.4) %. A blank sample of a meatloaf baked in a coated metal mould amounted to 1.9 mg siloxane per kg. This may result from the foodstuffs used as ingredients. Pork meat can be especially contaminated during industrial processing when exposed to a certain amount of siloxane. All equipment and chemicals used were shown to be almost free of siloxane contamination.

In a first experiment the influence of the baking time was investigated for meatloafs. Three pieces of meatloaf were baked in the same cup-sized mould one after another at a temperature of 175°C for 25 or 45 min. Both foodstuffs differed in the grade of browning but were visually appealing and tasty. In the baking series at 25 min migration tended to increase or stay constant at a level between 55 and 92 mg kg -1 , whereas the migration experiments at 45 min resulted in a much higher but decreasing migration from 177 to 135 mg kg -1 . Baking time seems not only to influence the level of migration but also the migration behaviour during multiple use.

"[Insert Figure 1 about here]" An identical mould was used in a previous study for muffins (Helling et al. 2009). The actual experiment with meatloaf resulted in migration values 5 to 18 fold higher compared to those achieved for muffins and cake in the previous study. It is noticeable that the migration values are much higher for meatloaf, although its fat content is somewhat lower. Since it is not typical to prepare meatloaf in a muffin mould, a box shaped mould with a volume of 1300 ml was used for further experiments. To compare the results again with bakery products a creamed cake was baked twice in an identical box shaped mould and gave migration values in the expected range at both 10.0 mg kg -1 (1.9 mg sdm -1 ). The overall migration limit was exceeded again when preparing a meatloaf in this mould (87.8 mg kg -1 and 15.2 mg sdm -1 ).

The impressive migration compared to cake may be explained by the leaking fat which extracts siloxane oligomers much more effectively than dough and may also affect a swelling of the silicone material. A baking sequence was performed to the 10 th migrate. For experiment 4, 6 and 8 the leaking fat was separated from the meatloaf and both analysed separately. The amount of siloxane oligomers in the liquid fat was at 1330, 1088 and 813 mg kg -1 and therefore about 30-fold above the amount measured in the meat (41, 40 and 26 mg kg -1 ).

A slight decrease in the migration values was observed from the 1 st to 10 th migrate. If judged according to the 3 rd migrate as claimed for plastics (Commission Directive 2002/72/EC), the investigated mould exceeded the overall migration limit as defined in the Resolution ResAP ( 2004) 5 on silicones used for food contact applications. On the other hand, rejecting the leaked fat before consuming the food gives a good opportunity to avoid higher contaminated meals.

"[Insert Figure 3 about here]"

Although migration from 1 st to 10 th experiment shows a slight tendency to decrease, there are much greater variations between successive steps. The main reason is the variation in the fat content of the used meat, which effects the resulting migration quite strongly. This is difficult to avoid because minced meat standardized in fat was not available.

Because there was high migration even in the 10 th experiment, it was of great interest, whether the migration would decrease to negligible values or siloxane oligomers would arise during long term use because of thermal and oxidative stress as described by [START_REF] Meuwly | Heat stability and migration from silicone baking moulds[END_REF].

Total extraction of a similar mould with diethyl ether resulted in 1.9% or 2.57 g extractable siloxanes per mould. The sum of all ten migration experiments yielded 460 mg (18 % of the totally extractable siloxanes). To shorten the ongoing experiment, further baking was stopped and the previously used mould was filled with ethanol and incubated at 60°C until no further siloxanes could be extracted. At the end of this extraction the mould became somewhat rough At this point a 2fold baking experiment with the same meatloaf recipe was performed to demonstrate if siloxane chains were cracked and new oligomers were built and released. In both cases no migration of siloxanes could be detected (limit of detection 1 mg kg -1 ).

Compared to the migration values during the first 10 baking cycles, it can be concluded that no significant formation of low molecular weight and freely available siloxanes occurred. Furthermore it must be pointed out that during the last baking procedure, the mould became even more frangible and started leaking. No further use was possible. It can be speculated that low and mid-size cyclic siloxanes act as plasticizers and are of some importance for the functionality and the elasticity of the polymer. At least if all those oligomers are migrated the mould might no longer be useable. Significant new formation of oligomers did not occur.

Milk products

To assess another kind of food, a milk-based dessert (crème brulèe) was prepared. Again the recovery rate was proven before and found to be lower than for cake and meatloaf but still to be analytically suitable (88.5 %). The limit of detection for this dessert was determined at 2.4 mg kg -1 . It has to be mentioned that the time of preparation for crème brulèe in the moulds is clearly longer than for the other investigated foodstuffs. But due to the high water content, the preparation procedure (water bath) and the fat in water emulsion type, the temperature at the contact surface between mould and food will not rise above 100°C. There are obviously no significant amounts of free fat despite the pretty high fat content of 27 % in total.

A 3 fold determination of each of 5 subsequent migration experiments (one after another) was done. In all cases, except the 3 rd migrate, the amount of migrated siloxane was below the limit of detection (2.4 mg kg -1 ). For the 3 rd migrate, the limit of detection was exceeded slightly but no reliable quantification could be done. Similar results were obtained for silicone teats incubated in instant baby milk for 6 hours at 40°C. Again no migration above the limit of detection of 2.4 mg kg -1 could be detected using the described NMR-method.

Thus, due to the already investigated foodstuffs muffin and cakes, pizza, meat loaf, milk based sweets and food [START_REF] Meuwly | Migration of siloxane oligomers in foodstuffs from silicone baking moulds[END_REF], Helling et al. 2009) it can be concluded, that exceeding the overall migration limit may only occur in case of foodstuffs with free available and liquid fat at temperatures well above 100°C as for instance meatloaf and soufflés / gratin.

To compare once more the situation in food and simulants, it was investigated whether the very low migration into milk-based food was adequately simulated by the use of an ethanol / water mixture (50 %) as a simulant for milk as introduced by Directive 2007/19/EEC. 7 moulds were analyzed gravimetrically using the substitute simulants 95 % ethanol and 50 % ethanol. Table 1 lists the results: In some cases migration into 50 % ethanol was not detectable, in other cases only a low migration compared to ethanol 95 % and tenax was measured. In contrast to the other simulants for fatty foodstuffs, the migration limit of 10 mg sdm -1 was never reached or exceeded. This accords strongly with the already described study with instant milk and milk-based sweets and in this special case confirms the suitability of 50 % ethanol as a simulant.

"[Insert Table 1 about here]"

Influence of the tempering process on migration properties

Tempered and the corresponding non-tempered silicone moulds were received from Wacker Chemie AG (Burghausen, Germany) to demonstrate the influence of the tempering on the overall migration in both cake and meat loaf.

The amount of volatile organic compounds is often used as a parameter to check adequate tempering (RASFF) and is regulated within BfR-recommendation XV to a limit of 0.5 %. This is based on the demand that volatile compounds should be eliminated before use of the moulds. Otherwise they could both migrate or evaporate and recondensate on the surface of the food.

The non-tempered moulds showed an amount of 1.11 % volatile organic compounds, which is clearly above the BfR limit, whereas the tempered moulds yielded only very low 0.07 %. Figure 5 shows a comparison of the tempered and non-tempered moulds for cake and meatloaf. Surprisingly, the overall migration in creamed cake in the non-tempered mould was for all 3 migration experiments 8 -9 fold higher than in the tempered mould, resulting in a first time measured migration well above 60 mg kg -1 for cake. In contrast to the previous studies with tempered forms from the market, a decrease of the migration from 1 st to 3 rd experiment was observed also for creamed cake.

Earlier investigation programs of the official food control authority in

Preparation of meatloaf in the untempered moulds also resulted in very high migration values up to 281 mg kg -1 but only 2.5 to 3 fold higher as measured in the tempered moulds. Thus, appropriate tempering seems to be essential to achieve low migration values for silicone baking moulds. Analyzing the amount of volatile organic compounds according to BfR recommendation XV can give some reliable hints of what migration values should be expected. But from this single experiment it cannot be stated whether the actual limit of 0.5 % for volatile organic compounds is low enough to avoid excessive migration or not. As our results for 201 moulds from the German market (analyzed from 2004 -2009) indicate, lower values than 0.5% could be easily achieved.

"[Insert Figure 6 about here]"

Enrichment of fat in the silicone material

In all baking experiments migration of fat originating from the food into the silicone polymer was observed. After some baking cycles fat sweated out, even on the outside of the moulds used. Moulds which were not used for a while and stored at room temperature for about six months exhibited a typical and unpleasant odour of oxidised (rancid) fat. As to be expected, extensive washing and cleaning could not remove the incorporated fat from the moulds. To estimate what amount of fat was incorporated into the polymer, diethyl ether extraction and quantification of the fat using again 1 H-NMR spectroscopy was done for 2 different moulds, both after repeated preparation of cake or meat loaf.

The obtained values were in the range from 3.5 to 5.9 g fat per kg elastomer and did not show a significant difference between the moulds used for cake or meatloaf. Outside this range and with the highest fat content, the mould used for the long-term baking experiment with meatloaf (including total extraction of the siloxanes) the following was found: 8.0 g kg -1 fat was measured in the polymer. Because all free available substances were extracted with ethanol before the baking process, it can be assumed that the polymer had more capacity to swell and incorporate the leaking fat.

Values of about 0.5 % free fat in used silicone moulds raises the question of the hygiene of those moulds. Not only do migrating siloxanes need to be given attention but so should the migrating fat from previously prepared food. A regular change of moulds used seems advisable. Due to the typical odour of rancid fat, it should be possible to determine the last time a mould should be used even without any analytical measurement.

Conclusion

Different types of food were prepared in silicone moulds of different sizes and shapes. The main result is that the type of food and availability of fat is the most important criteria for the amount of migration. The total fat content is only of relevance if similar foodstuffs are compared. In our studies meatloaf with a total fat content of about 17 % was the worst case in the field of migration and the only food to exceed the overall migration limit as stated in the Resolution ResAP ( 2004) 5 on silicones used for food contact applications or in the Swiss legislation EDI. In contrast creamed cake with a similar baking time and temperature and with up to 30 % fat or a milk-based dessert (crème brulèe) with even longer heating time and 27 % fat showed a much lower or even non detectable migration. This applied both for small and larger moulds with a very different surface / volume ratio.

Preparing cake or milk-based desserts as well as the use of silicone teats for baby feeding will typically not lead to an excessive migration of siloxane oligomers, which exceed the overall migration limit. On the other hand, it must be concluded that most silicone moulds are not suitable for preparing meatloaf or gratins with larger portions of free available fat if judged according to the criteria of the Resolution ResAP ( 2004) 5. Preparation of roasts, gratins and similar foodstuffs should not be recommended or even better excluded within the declaration of silicone moulds. Although the overall migration limit will be exceeded in case of the preparation of those foodstuffs, no toxicolocigal concern exists so far.

Testing silicone moulds with simulants such as Tenax® instead of foods does not give a realistic scenario and is not suitable for judging legal compliance. Tests with simulants commonly focus on the total fat content of foodstuffs and suggest reducing factors to previous studies, the total fat content is only one of several parameters to look at and the amount of the overall migration depends strongly on the type of food, the kind of emulsion and further aspects. As mentioned in Article 3 of the framework Regulation (EC) 1935/2004, it is important to assess and overweight the amount of migrating substances in food, but not in simulants (except if it can be shown, that the simulants reflect the situation in the foodstuffs in a realistic manner).

In general, migration from silicone elastomers into food seems to be nearly constant in the beginning but slightly decreasing with ongoing use. No additional formation of low molecular weight silixone oligomers during repeated heating could be observed. This stands in contrast to the previously published work of [START_REF] Meuwly | Heat stability and migration from silicone baking moulds[END_REF] and requires further investigation.

Eventually the fat incorporated during the preparation of food acts as an oxygen scavenger, and thus prevents oxidative cleavage of the silicone chains during heating. Further studies that focus on this particular question are already in progress.

Adaqueate tempering was shown to be essential to guarantee a low migration for all types of food. Analyzing the amount of volatile organic compounds can give a good indication of the need for further migration studies to examine an individual mould.

Another critical point of silicone moulds compared to the traditional metal coated ones is the uptake of food originated fat, which can lead in some cases to organoleptic and hygienic faults. An exchange of odorous moulds is strongly recommended. n.d. -not detectable (the limit of detection and quantification for the gravimetric method is 0.5 mg dm -1 ).

  behaviour compared to the liquid simulants as well as Tenax®.

  until constant weight was reached. The weight of the lipid extract was determined accurately to 0.1 mg.

  "[Insert Figure2about here]"

  and frangible and the original blue colour faded out. "[Insert Figure4about here]"

  Saxony demonstrated that common silicone moulds have in general volatile organic compounds between 0.2 and 0.4 %. "[Insert Figure 5 about here]"

  fat content compared to olive oil or simulants. As shown in this and

F

  foodstuffs. Official Journal of the European Communities, L 372, 1985 December 315, p. 14-21 Council Directive 89/107/EEC of 21 December 1988 on the approximation of the laws of the Member States concerning food additives authorized for use in foodstuffs intended for human consumption. Official Journal of the European Communities, L 40, 1989 February 11, p. 27-33. Dorn SB, Skelly Frame EM. 1994. Development of a high-performance liquid chromatographic-inductively coupled plasma method for speciation and quantification of silicones: From silanols to polysiloxanes. Analyst 119. 1687-1694 Food Migrosure project. Modelling Migration from plastics into foodstuffs as a novel and cost efficient tool for estimation of consumer exposure from food contact material. Available from: http://www.foodmigrosure.com/. Accessed 2008, July 17. Forrest MJ, Holding SR, Howells D, Eardley M. 2006. The use of GCxGCTOFMS and LC-MS for the determination of migrants from silicone rubbers into food simulants and food products. Silicone Elastomers, 19-20 September 2006, Frankfurt Germany (oral contribution) Forrest MJ, Sidwell J. 2005. Chemical migration from silicones used in connection with food contact materials and articles (Food Standard Agency Contract Number A03046) GFK Panel Services Germany [internet]. Available from: http://www.gfk.com/imperia/md/content/ps_de/chart_der_woche/kw49_06_silikonbackforme n.pdf. Accessed 2007, April 15. Helling R, Mieth A, Altmann S, Simat TJ. 2009. Determination of the overall migration from silicone baking moulds into simulants and food using 1 H-NMR techniques. Food Additives and Contaminants 26/3. 395-407 . silicone -Problemlösungen im Haushalt. Available from: http://www.wacker.com. Accessed 2008, May 06.

Figure 1 .Figure 2 .

 12 Figure 1. Overall migration in dependence on the baking time of meat loaf prepared in a small round shaped silicone mould. Data given for first (M1), second (M2) and third (M3) baking process. All data are based on a 3fold determination.

Figure 3 .

 3 Figure3. Overall migration of meat loaf prepared 10 times (M 1-10) in a box shaped silicone mould. Differences in the overall migration occurred partly due to differences in the fat content of the meat used (14 -19 % fat). M 5 was the foodstuffs lowest in fat. For samples M 4, M 6 and M 8 sauce and meat loaf were analyzed separately and the sum used for calculation of the overall migration. For these, 20 g of sauce contained as much siloxanes as aprox. 640 g meat loaf. All together 0,46 g siloxanes were extracted with those 10 experiments.

Figure 4 .

 4 Figure 4. Picture of the bottom of the box shaped silicone mould after 10 migration experiments with meat loaf, complete extraction of remaining unbound siloxanes with ethanol and another baking procedure. The elastomers colour faded out, the elastomer itself became rough and frangible. During the next experiment the mould started leaking and could not be used further.

Figure 6 .

 6 Figure 5 a) Migration in creamed cake (21 % fat) b) Migration in meat loaf (aprox. 17 % fat); both 1 st -3 rd migration shown: dark columns represent the tempered moulds whereas the lighter columns stand for the untempered moulds

Table I .

 I Determination of the overall migration with ethanol 50% as a simulant for milk compared to ethanol 95% and Tenax as the most common simulants for fatty foodstuffs.

	Type	Migration with ethanol 50% [mg dm -1 ]	Migration with ethanol 95% [mg dm -1 ]	test conditions for ethanol (t / T)	Migration with Tenax [mg dm -1 ]	test conditions for Tenax (t / T)
		1st migrate 3rd migrate 1st migrate 3rd migrate		1st migrate 3rd migrate
	large mould for king`s cake A	0,6	0,5	36,5	15,7	180min 60°C	25,3	15,4	60min 175°C
	large mould for king`s cake B	n.d.	n.d.	30,6	24,9	180min 60°C	35,0	15,0	60min 175°C
	Madeleine type mould	4,6	3,2	42,0	14,3	75min 60°C	29,2	15,9	60min 175°C
	muffin mould A (6 cups)	n.d.	n.d.	18,2	22,2	75 min 60°C	20,0	14,2	25min 175°C
	muffin mould B (6 cups)	3,7	1,2	25,3	8,1	75 min 60°C	25,8	16,3	25min 175°C
	muffin mould C (6 cups)	1,4	1,3	58,6	25,1	75 min 60°C	17,5	10,0	25min 175°C
	muffin mould D (8 cups)	n.d.	n.d.	20,4	17,7	75min 60°C	11,3	5,9	25min 175°C

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Acknowledgement

The authors would like to thank the Centre Européen des Silicones (Brussels, Belgium) for financial support of the studies and helpful advice. We also gratefully acknowledge Dr.

Margit Gruner and Annett Rudolph (Technical University of Dresden, Institute of Organic Chemistry) for doing numerous NMR measurements.