MRCPI Sean S O'sullivan

MSc Clare M Loane
email: c.loane@imperial.ac.uk

PhD Andrew D Lawrence
email: lawrencead@cardiff.ac.uk

MD Andrew H Evans
email: andrewhevans@yahoo.com

PhD Paola Piccini
email: paola.piccini@imperial.ac.uk

FMedAcadSci Andrew J Lees
email: alees@ion.ucl.ac.uk

O ' Sullivan

Sleep disturbance and impulsive-compulsive behaviours in Parkinson's Disease

Keywords: Parkinson's disease, Sleep disorders, Bipolar Spectrum Disorders, Nonmotor symptoms, Impulse control disorders

Objectives: Impulsive-compulsive behaviours (ICBs) in Parkinson's Disease (PD) have been anecdotally linked with impaired sleep. We investigate measures of sleep in PD patients with and without ICBs, and in healthy controls.

Methods

: We compare Parkinsonian features, measures of depression, anxiety and mania, and sleep disturbance in 30 PD patients with ICBs (PD+ICB), 62 PD patients without ICBs (PD -ICB), and 48 healthy controls. Results: PD+ICB patients had younger age of PD onset, took more dopamine replacement therapy (DRT), had worse sleep, and elevated anxiety, depression and mania scores. Using multiple linear regression analyses, the total anxiety and depression scores, and presence of ICBs were the only variables associated with poorer sleep in PD. Conclusions: PD+ICB patients may show enhanced psychomotor effects of DRT that may in turn contribute to poor sleep quality. Sleep disturbance should be specifically queried in PD+ICB patients.

Introduction

Although Parkinson's disease (PD) is defined by bradykinesia, rigidity and tremor at rest, a fragmented sleep pattern with insomnia is a common complaint, affecting approximately 66% of patients in one study. [START_REF] Tandberg | A community-based study of sleep disorders in patients with Parkinson's disease[END_REF] Impulsive-compulsive behaviours (ICBs) are increasingly recognized as a complication of dopamine replacement therapy (DRT), including pathological gambling (PG), hypersexuality (HS),compulsive shopping (CS), binge eating (BE), punding, and the dopamine dysregulation syndrome (DDS) . [START_REF] Voon | Medication-related impulse control and repetitive behaviors in Parkinson disease[END_REF] [3] [START_REF] Evans | Punding in Parkinson's disease: its relation to the dopamine dysregulation syndrome[END_REF] Although the aetiology of ICBs remains to be clarified, a "global sensitization" of appetitive behaviours has been proposed. [START_REF] Lawrence | Compulsive use of dopamine replacement therapy in Parkinson's disease: reward systems gone awry?[END_REF] Anecdotal evidence links ICBs, DRT and impaired sleep. For example, DRT induces restlessness, insomnia, nightmares and vivid dreaming, [START_REF] Lawrence | Compulsive use of dopamine replacement therapy in Parkinson's disease: reward systems gone awry?[END_REF] [START_REF] Nausieda | Sleep disruption in the course of chronic levodopa therapy: an early feature of the levodopa psychosis[END_REF] and dopamine is now known to play a prominent role in promoting wakefulness. [START_REF] Boutrel | What keeps us awake: the neuropharmacology of stimulants and wakefulness-promoting medications[END_REF] This study is the first to specifically investigate sleep disturbances in PD patients with ICBs and tests the hypothesis that higher levels of DRT, ICB diagnosis and/or mood, are related to sleep disturbance.

Methods

Outpatients attending a specialist PD clinic were assessed by the lead author for the presence of ICBs in a semi-structured interview using proposed criteria. [START_REF] Lawrence | Compulsive use of dopamine replacement therapy in Parkinson's disease: reward systems gone awry?[END_REF] [2] [START_REF] Evans | Punding in Parkinson's disease: its relation to the dopamine dysregulation syndrome[END_REF] REM sleep behaviour disorder (RBD) was identified during the clinical interview, according to International Classification of Sleep Disorders criteria. [START_REF]American Sleep Disorders Association: International classification of sleep disorders, revised: diagnostic and coding manual[END_REF] Patients were screened using the Mini-Mental State Examination (MMSE) and 15 who scored below 26 were excluded because of the requirement to complete self-report scales.

The Unified PD Rating Scale (UPDRS) part 3 was rated in the "on" state by the treating physician. Calculation of a daily L-dopa equivalent dose (LED) dose for each patient was based on theoretical equivalence of dopamine agonists to L-dopa. [START_REF] Evans | Punding in Parkinson's disease: its relation to the dopamine dysregulation syndrome[END_REF] Healthy controls without PD or dementia were identified from partners or friends of participating patients attending the outpatient department.

Questionnaires. Participants who provided written informed consent to protocols approved by the UCLH Trust local ethics committee were given a series of questionnaires to complete in their own time and return in a reply-paid envelope.

These included the PD Sleep Scale (PDSS), [START_REF] Chaudhuri | The Parkinson's disease sleep scale: a new instrument for assessing sleep and nocturnal disability in Parkinson's disease[END_REF] the Mood Disorder Questionnaire (MDQ), [START_REF] Hirschfeld | Development and validation of a screening instrument for bipolar spectrum disorder: the Mood Disorder Questionnaire[END_REF] and The Hospital Anxiety and Depression Scale (HADS). [START_REF] Zigmond | The hospital anxiety and depression scale[END_REF]. The MDQ is a patient-completed bipolar disorder screen, with 73% sensitivity and 90% specificity in the identification of bipolar spectrum disorders. [START_REF] Hirschfeld | Development and validation of a screening instrument for bipolar spectrum disorder: the Mood Disorder Questionnaire[END_REF] Data analysis. Data were analyzed with SPSS 14, SPSS Inc., Chicago, IL.

Medians/means were compared using Krusall-Wallis, Mann-Whitney U tests (MWU), one-way analyses of variance (ANOVA), or Student t test where appropriate.

Post-hoc multiple comparisons within a variable using ANOVA were corrected by the Bonferroni method. Kolmogorov-Smirnof tests showed that PDSS and MDQ scores were not normally distributed. Levene's test showed that all variables tested exhibited homogeneity of variance. Univariate correlations between PDSS score and variables such as total anxiety score of HADS, total depression score of HADS, total score of the MDQ symptoms, daily L-dopa amounts used, and daily LED of dopamine agonists used were made using Spearman's or Pearson's correlation as appropriate.

Factors were entered in a multiple linear regression model to identify clinical features that were independently associated with total PDSS scores. Backward entry method was used, with the probability for stepwise removal set at 0.1. To check the consistency of the model a forward approach with a probability of stepwise entry of 0.05 was also used. Variables entered in the statistical model with total PDSS score as the dependent variable included age, gender, combined total anxiety + depression score of HADS (due to significant univariate correlation between the two HADS subscales, these results were not entered separately), total score of the MDQ symptoms, daily L-dopa amounts used, daily LED of dopamine agonists used, and the presence of documented ICBs.

Results

Participant demographics and PD history

One hundred and thirty three patients with PD received questionnaires, and 41 either did not consent or did not return the questionnaires. Thirty patients who completed the questionnaires were identified as ICBs (PD+ICB), and many of these exhibited more than one ICB. These included 15 with punding, 12 with hypersexuality, 11 with pathological gambling, 8 with compulsive shopping and 8 with binge eating. Forty eight healthy controls were also included. (Table 1). Antidepressant and/or anxiolytic were used in 28.6% of PD+ICB (28.6%), and 16.1% of PD-ICB patients, (Chi-square = 1.2, df = 1, p=0.28,). 1).

No participants had a diagnosis of bipolar mood disorder. 30% of PD + ICB, 3.2% of the PD-ICB, (Chi-square = 13.8, df = 1, p< 0.0005), and 4.2% of healthy controls scored above the recommended cut-off for bipolar spectrum disorder on the MDQ. [START_REF] Hirschfeld | Development and validation of a screening instrument for bipolar spectrum disorder: the Mood Disorder Questionnaire[END_REF] No clinical differences were seen when comparing PD patients with and without RBD.

Predictors of sleep disturbance in PD

In the combined PD group, there was a negative correlation between sleep impairment PD+ICB patients had higher rates of mood disturbance, which could potentially directly impact sleep quality. In PD, the association between sleep disturbances and mood disorders is well established. [START_REF] Martinez-Martin | SCOPA-sleep and PDSS: two scales for assessment of sleep disorder in Parkinson's disease[END_REF] [14] PD patients had increased anxiety and depression than controls, with PD+ICB more so than PD-ICB. Consistent with previous reports, [START_REF] Borek | Mood and sleep in Parkinson's disease[END_REF] we found a correlation between poor sleep scores and depression and anxiety severity.

(
The PD + ICB patients had higher MDQ scores than the PD-ICB patients; although the MDQ has not been validated for use in PD. None of the included PD participants fulfilled diagnostic criteria for bipolar mood disorder. However, in our study 29% of PD + ICB, 3.2% of the PD -ICB group, and 4.2% of healthy controls scored above the recommended cut-off for bipolar spectrum disorder. [START_REF] Hirschfeld | Development and validation of a screening instrument for bipolar spectrum disorder: the Mood Disorder Questionnaire[END_REF] A recent large study of bipolar disorders including sub-threshold bipolar disorder found a lifetime prevalence of 4.4% amongst the general population. [START_REF] Merikangas | Lifetime and 12-month prevalence of bipolar spectrum disorder in the National Comorbidity Survey replication[END_REF] The increased frequency of manic symptoms in the PD patients may relate to medication-effects which have been recognised since the introduction of L-dopa. [START_REF] Ryback | Manic response to levodopa therapy. Report of a case[END_REF] An association between medicationinduced hypomania and pathological gambling in PD has been described, [START_REF] Voon | Medication-related impulse control and repetitive behaviors in Parkinson disease[END_REF] in contrast with patients developing ICBs after being treated with dopaminergic medications for restless legs syndrome, where none describe medication-induced mania. [START_REF] Driver-Dunckley | Gambling and increased sexual desire with dopaminergic medications in restless legs syndrome[END_REF] As with any work involving multiple comparisons, caution is required regarding the risk of false positive results.

Although the PD+ICB group were using more DRT than the PD-ICB patients, notably neither the daily amount of L-dopa nor dopamine agonist medication use correlated with altered sleep scores or RBD frequency. These results suggest that it is not simply the amount of DRT that causes poor sleep quality. This finding differs from previous reviews which have suggested an association between DRT and nightmares, [START_REF] Pagel | Drug induced nightmares--an etiology based review[END_REF] and RBD [START_REF] Gjerstad | Occurrence and clinical correlates of REM sleep behaviour disorder in patients with Parkinson's disease over time[END_REF] The presence of ICBs was identified as being independently associated with sleep disturbance. This suggests that PD+ICB patients may be more sensitive to the psychomotor stimulant-arousal effects of DRT, which in turn contributes to poorer sleep. Punding behaviours, may contribute to poor sleep quality as a consequence of persistence of these stereotyped behaviours throughout the night at the expense of a normal sleep pattern.[4] However, we found no difference when comparing sleep scores in PD+ICB patients with or without punding (p =0.7, MWU) This is consistent with animal models, showing an effect of dopamine on wakefulness independent of motor stereotypies. [START_REF] Wisor | Dopaminergic role in stimulantinduced wakefulness[END_REF] Alternatively, sleep disturbance in PD + ICB might relate to underlying impulsive personality traits, which are risk factors for developing ICBs, [START_REF] Evans | Factors influencing susceptibility to O'Sullivan 18 compulsive dopaminergic drug use in Parkinson disease[END_REF] and which are linked with insomnia independent of DRT use. [START_REF] Schmidt | Facets of impulsivity are differentially linked to insomnia: evidence from an exploratory study[END_REF] Sleep disruption has also been suggested to heighten psychomotor sensitization effects, [START_REF] Frussa-Filho | Paradoxical sleep deprivation potentiates amphetamine-induced behavioural sensitization by increasing its conditioned component[END_REF] which could impact on the expression of ICDs in PD. Further work is needed in this area, and we conclude that sleep disturbance should be specifically queried in PD+ICB patients.

Statement:

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in JNNP and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence (http://jnnp.bmjjournals.com//ifora/licence.pdf).

Figure :

 : Figure: PD Sleep Scale total scores

Acknowledgements:

  This work was supported by funding from the Reta Lila Weston Institute and the UK Parkinson's Disease Society. The study sponsors had no role in study design; in the collection, analysis, and interpretation of data; or in the writing of the report. The authors are grateful for the help received from participants in this study. Dr Constantinos Kallis provided statistical advice. The authors have no financial disclosures to make.

Table 1 . Participant details, sleep and mood results

 1

		PD total group	PD + ICB	PD -ICB	Healthy controls	Statistic (P value)
	N	92	30	62	48
						2
	M:F	70:22	24:6	46:16	39:9	χ	=0.88, df = 2, (p=0.6)
	Age	64.4 ± 9.9	58.9 ± 8.5* ‡	66.4 ± 9.7* †	57.9 ± 10.6 † ‡	ANOVA F (2,134) = 11.6, (p
						<0.0005)
						* 0.003	†<0.0005	‡ = 1.0
	Age PD onset.	52.3 ± 12.7	46.2 ± 10.1*	55.8± 12. 0*	N/A	* t =3.6, df = 66, (p= 0.001)
	Durati on of PD (years)	10.5 ± 6.7	11.5 ± 5.9*	9.5 ± 7. 0*	N/A	*t= 1.2, df = 67, (p= 0. 2)
	H + Y on medications	2 (2-3)	3 (2.1-3)*	2 (2-3)*	N/A	* MWU = 67, *(p = 0.04)
	Median (interquartile range)
	Current L-dopa dose/day (mg)	597 ± 443	701 ± 508*	543 ± 399*	N/A	* t =-1.4, df = 73, ( p= 0.16)
	Current DA LEU dose/day (mg)	90, ( 0-262)	201, ( 0-284)*	0, (0-201)*	N/A	* MWU = 555, (p= 0.12)
	Median (interquartile range)
	Combined L-dopa + DA LEU/day (mg).	764 ± 548	981 ± 651*	645 ± 443*	N/A	*t=-2.5, df = 75,( p= 0.018)
							2
	RBD diagnosed	19/55 (34.5%)	7 /18* (38.9%)	12 /37* (32.4%)	Not included	* χ	=0.03, df = 1, (p=0.9)
	HADS anxiety score	15.0 ± 3.6	16.9 ± 3.9¥ †	14.3 ± 3.2¥*	13.9 ± 3.9* †	ANOVA F (2,128) = 6.9
						(p=0.001)
						* = 1.0	¥ = 0.005 † = 0.002
	HADS depressi on score	13.2 ± 3.1	14.5 ± 3.5¥ †	12.8 ± 2.7¥*	9.9 ± 2. 9 †*	ANOVA F (2,129) = 23.7
						(p <0.0005)
						*= <0. 0005	¥ = 0.053
						†<0.0005
	Mania symptom score	3, (1-7)	6, (3-8) †¥	3, (1-6) *¥	2, (0-5) * †	Kruskall-Wallis Chi Square =
	Median (interquartile range)					13, df = 2, (p= 0.002)
						* MWU (p=0.6)
						¥ MWU (p=0.002)
						† MWU (p=0.001)

Means are compared with t-tests, except where indicated All values are mean +/-SD except where indicated ANOVA = analysis of variance. Post-hoc multiple comparisons within a variable using ANOVA were corrected by the Bonferroni method.

  longer history or those in who decisions to reduce DRT have been made because of cognitive or psychotic problems. Previous studies have found that age does not correlate with PDSS scores,[START_REF] Gomez-Esteban | Sleep complaints and their relation with drug treatment in patients suffering from Parkinson's disease[END_REF] but normal aging is accompanied by decreased ability to initiate and maintain sleep, and a decrease in the proportion of the more restorative slow-wave sleep and rapid eye movement sleep.[START_REF] Espiritu | Aging-related sleep changes[END_REF] Age differences alone therefore, cannot explain why the PD+ICB group have a poorer quality of sleep than PD-ICB patients.

	total PDSS score) and anxiety (Pearson Correlation coefficient = -0.39, p<0.00005),
	and depression (Pearson Correlation coefficient = -0.34, p=0.001). No correlations
	were seen between PDSS totals and variables such as age, gender, or DRT amounts.
	In all univariate analyses, the non-significant correlation coefficients were all < |0.22|)
	Using multiple linear regression analyses, the presence of ICBs (p=0.045) and
	combined HADS anxiety + depression totals (p=0.068) were the only variables
	associated with sleep disturbance in PD (p=0.004).
	Discussion
	PD patients with ICBs have poorer sleep quality, compared to PD patients without
	ICBs and healthy controls. Several clinical differences were found between the PD +
	ICB and PD-ICB groups including age and medication use but are unlikely to account
	for the observed poor sleep quality. PD + ICB patients were on average 8 years

younger at time of study despite a similar PD duration in both groups. The exclusion of patients scoring <26 on the MMSE might disproportionately exclude older patients, those with a

This work was supported by funding from the Reta Lila Weston Institute and the UK Parkinson's Disease Society. The authors have no financial disclosures to make.


Ms R Chadwick

Dr Vidya Nadig

Dr Nicholas S Oscroft

Dr John M Shneerson

Dr Ian E Smith

Weaning from prolonged invasive ventilation in motor neurone disease: analysis of outcomes and survival

Keywords: Motor neurone disease, respiratory failure, ventilator weaning

Introduction: Non-invasive ventilation (NIV) improves prognosis in patients with

Motor Neurone Disease (MND) in the absence of major bulbar involvement. However, some experience a rapid and unexpected decline in respiratory function and may undergo emergency tracheal intubation. Weaning from invasive ventilation can be difficult and reported independence from invasive ventilation is uncommon with poor prognosis. The outcomes of patients with MND referred to a specialist weaning service following emergency tracheal intubation were examined and compared with MND patients electively initiating NIV.

Methods: A case note review was performed on all patients with MND invasively ventilated and referred to a specialist weaning service between 1992 and 2007.

Outcomes were compared with those electively commenced on NIV during the same period.

Results: Thirty patients were referred for weaning from invasive ventilation which was started in 17 before MND was diagnosed. Fourteen patients (47%) were weaned from invasive ventilation but still required NIV, 13 failed to wean and three died.

Seventeen were discharged home from hospital. Median survival from tracheal intubation was 13.7 months (95% CI 0-30.8) for those previously diagnosed and 7.2 months (95% CI 5.1-9.4) for those not previously known to have MND. Comparison with patients initiated electively on NIV demonstrated similar survival estimates to that from emergency intubation (median 9.4 (95% CI 6.9 -12.0) vs. 7.8 (95% CI 2.6 -12.9) months respectively).

Conclusion:

The prognosis in MND following acute respiratory failure and intubation is not always complete ventilator dependence if patients are offered a comprehensive weaning programme.

INTRODUCTION

Motor neurone disease (MND) is a progressive neurodegenerative disorder that usually leads to death within 2-4 years. Ventilatory failure is the most common cause of death, caused in varying degrees by respiratory muscle weakness, unsafe swallowing and poor cough. [START_REF] Mustfa | Respiratory muscle assessment in motor neurone disease[END_REF] Non-invasive positive pressure ventilation (NIV) can alleviate symptoms, improve quality of life [START_REF] Bourke | Effects of non-invasive ventilation on survival and quality of life in patients with amyotrophic lateral sclerosis: A randomised controlled trial[END_REF] and prolong survival in MND patients, [START_REF] Bourke | Effects of non-invasive ventilation on survival and quality of life in patients with amyotrophic lateral sclerosis: A randomised controlled trial[END_REF][START_REF] Berlowitz | A retrospective analysis of sleep quality and survival with domiciliary ventilatory support in motor neuron disease[END_REF] particularly in those without severe bulbar dysfunction. However, the initial symptoms of respiratory failure may be subtle and difficult to detect. [START_REF] Mustfa | Respiratory muscle assessment in motor neurone disease[END_REF] Some patients experience a rapid and unexpected decline in respiratory function, frequently in the context of respiratory tract infection, and may be invasively ventilated, even before a diagnosis of MND is established. [START_REF] Chen | Motor neuron disease presenting as acute respiratory failure: a clinical and pathological study[END_REF][START_REF] Bradley | Outcome of ventilatory support for acute respiratory failure in motor neurone disease[END_REF] Case series have reported that weaning from invasive ventilation can be difficult, independence from invasive ventilation is rarely achieved and prognosis poor. [START_REF] Chen | Motor neuron disease presenting as acute respiratory failure: a clinical and pathological study[END_REF][START_REF] Bradley | Outcome of ventilatory support for acute respiratory failure in motor neurone disease[END_REF] Therefore an emphasis has been placed on the palliation of symptoms. [START_REF] Bradley | Outcome of ventilatory support for acute respiratory failure in motor neurone disease[END_REF] In contrast, the active management of these patients including attempted weaning from invasive ventilation and treatment of associated complications, such as intercurrent infection with physiotherapy, antibiotics and bronchial toilet using bronchoscopy has not been reported.

In this article, the outcomes of 30 patients with MND, referred to a specialist weaning service, who were intubated and ventilated for acute respiratory failure are presented.

The aim was to examine whether differences in outcomes justify elective NIV being considered best practice [START_REF] Bourke | Effects of non-invasive ventilation on survival and quality of life in patients with amyotrophic lateral sclerosis: A randomised controlled trial[END_REF] while intubated patients are considered for a more palliative approach due to their reported prognosis. [START_REF] Bradley | Outcome of ventilatory support for acute respiratory failure in motor neurone disease[END_REF]

METHODS

A retrospective case note review was performed on all patients with a diagnosis of MND referred to a specialist weaning centre (Respiratory Support and Sleep Centre (RSSC), Papworth Hospital) between 1 st January 1992 and 31 st December 2007. The diagnosis of MND was confirmed by a consultant neurologist and appropriate investigations including electrophysiology. The outcomes of patients invasively ventilated and referred for weaning were compared with those of patients referred for consideration of elective NIV during the same time period. In addition patients with a known diagnosis of MND prior to intubation were compared with those without a diagnosis at intubation.

In those invasively ventilated the demographics, duration of symptoms, duration of tracheostomy, length of stay at referring Intensive Care Unit (ICU) and RSSC, ventilation mode on arrival and discharge from the RSSC and discharge destination were recorded. Patients were categorised as having severe bulbar involvement if their swallow was deemed unsafe. Outcome measures were successful weaning from invasive ventilation, survival to RSSC and subsequent hospital discharge, and longterm survival. Successful weaning was defined as the withdrawal of invasive ventilation, but included continuing support with NIV. Patients with a tracheostomy for airway suction only were regarded as weaned.

The National Health Service Strategic Tracing Service tracked patients and survival was compared from the date of assisted ventilation between patients referred for weaning and those electively initiated on NIV. Patients were followed up to the 31st December 2007 or death, whichever occurred first.

Statistical analysis

Data were analysed using SPSS 16.0 for Windows (Chicago, IL). Normal distribution was established using the Kolmogorov-Smirnov test. Parametric data were compared using independent t-tests and non-parametric data compared using the Mann-Whitney U test. Kaplan-Meier survival analysis was performed to estimate survival; the log rank test was used to compare groups. A p value of < 0.05 was considered statistically significant.

RESULTS

Thirty (five female) MND patients were admitted for ventilatory weaning between January 1992 and December 2007. All were ventilated via tracheostomy. Twelve had identifiable acute precipitants to ventilatory failure, 9 with pulmonary sepsis. Thirteen patients (43%) had a diagnosis of MND prior to ICU admission (diagnosed patients (DP)) and seventeen (57%) did not (undiagnosed patients (UP)). Fourteen (47%) were weaned from invasive ventilation onto NIV (13 required only nocturnal support).

Three died during their admission on the RSSC. Thirteen failed to wean with 9 requiring continuous ventilatory support and 4 nocturnal support only via tracheostomy.

Seventeen patients were discharged home and they were significantly younger than the 13 who died in hospital (mean 60 (SD 14) vs 70 (6) years, p = 0.02). The overall oneyear survival rate from tracheal intubation was 43.3%. Of the 27 patients discharged from the RSSC, survival was not significantly different if ventilated via tracheostomy or NIV (mean 14.3 (95% CI 5.5-23.1) vs.14.0 (95% CI 5.9-22.1) months respectively).

Ten of the 14 weaned to NIV (71%) and 7 of 13 (54%) not weaned were ultimately discharged home. The median survival from tracheal intubation was 7.8 (95% CI 2.6 -12.9) months across the whole cohort. The DP group survived longer (median 13.7 (95% CI 0 -30.8) months) than the UP group (median 7.2 (95% CI 5.1 -9.4) months) a non significant difference (log rank test p = 0.68). Data comparing the UP with DP groups are presented in Table 1. Fifteen patients had severe bulbar involvement. Five (33%) of these were weaned to NIV in contrast to nine (60%) of those without marked bulbar disease. Eight (62%) of the patients who died in hospital had severe bulbar disease although overall there was no significant difference in survival between those with or without severe bulbar involvement.

During the period examined 332 patients with MND (209 male) were referred electively for ventilatory assessment and 126 patients (88 male) initiated domiciliary NIV. Their mean age was 63.8 (9.9) years. Figure 1 illustrates the survival times of the patients, referred for weaning and elective NIV, from the initiation of assisted ventilation. The median survival in the group referred for weaning was 7.8 (95% CI 2.6 -12.9) months, this was not significantly different from that for patients electively initiated on NIV which was 9.4 (95% CI 6.9 -12.0) months. Successful weaning removes the complications and cost of prolonged invasive mechanical ventilation. There was no difference in mortality between those weaned and those who continued with tracheostomy ventilation but a higher percentage of those weaned to NIV were discharged home. The majority of the weaned patients only required NIV overnight. We have no data on quality of life in patients weaned to NIV but previous reports suggest it is improved with NIV compared to tracheostomy ventilation [START_REF] Cazzolli | Home mechanical ventilation for amyotrophic lateral sclerosis: nasal compared to tracheostomy-intermittent positive pressure ventilation[END_REF] and better if cared for at home. [START_REF] Moss | Patients with amyotrophic lateral sclerosis receiving long-term mechanical ventilation: Advance care planning and outcomes[END_REF] We compared the survival of patients referred for weaning to that of patients who were electively initiated on NIV and found that survival from the date that assisted ventilation was initiated was similar. Survival after the initiation of NIV in our study (median 9.4 months) was equivalent to that of previous studies of MND patients who were tolerant of NIV (median 8 -15 months). [START_REF] Bourke | Effects of non-invasive ventilation on survival and quality of life in patients with amyotrophic lateral sclerosis: A randomised controlled trial[END_REF][START_REF] Peysson | Factors predicting survival following noninvasive ventilation in amyotrophic lateral sclerosis[END_REF][START_REF] Kleopa | Bipap improves survival and rate of pulmonary function decline in patients with ALS[END_REF][START_REF] Aboussouan | Effect of noninvasive positivepressure ventilation on survival in amyotrophic lateral sclerosis[END_REF] Survival is significantly worse in patients who are unable to tolerate NIV. [START_REF] Lo Coco | Noninvasive positive-pressure ventilation in ALS: predictors of tolerance and survival[END_REF] Berlowitz and colleages report a survival advantage of tracheostomy ventilation compared to NIV and no ventilatory support. [START_REF] Berlowitz | A retrospective analysis of sleep quality and survival with domiciliary ventilatory support in motor neuron disease[END_REF] However, survival was calculated from symptom onset and tracheostomy ventilation was initiated electively, making comparisons with our study difficult.

The high weaning success rate in the present series could be attributable to selection bias in the referral of patients to a specialist weaning centre. No patients were declined by the RSSC. It is possible that younger patients with less advanced disease were more likely to be referred. All patients had survived prolonged ventilation at the referring ICU, and most were medically stable. However invasive ventilation for over 14 days is associated with increased mortality, [START_REF] Brassard | Correlation of APACHE scores with duration of mechanical ventilation in patients with respiratory failure[END_REF] and the condition of the patients was poor enough to prohibit weaning by conventional techniques. Seventeen patients were diagnosed with MND following intubation. Compared to the 13 patients with an established diagnosis they were older at symptom onset, had a shorter time from first symptom to respiratory failure, took longer to wean from invasive ventilation and had poorer survival. It may be that older patients with rapidly progressive disease and a known diagnosis were not offered invasive ventilation or not referred for weaning.

During weaning the aim should be to optimize treatment of all associated conditions and infections, provide manual and mechanically assisted cough therapy if required, address nutrition, communication, and psychological aspects of care in addition to reviewing medications and ventilatory requirements. [START_REF] Smith | A progressive care programme for prolonged ventilatory failure: analysis of outcome[END_REF] With this approach 57% of patients referred were discharged home compared to 17% in a previous series. [START_REF] Bradley | Outcome of ventilatory support for acute respiratory failure in motor neurone disease[END_REF] Although weaning is possible and outcomes reasonable, the close monitoring of respiratory status in patients known to have MND could avoid invasive ventilation.

The acute use of NIV may also be important as this has been shown to reduce intubation rates, length of ICU stay and mortality. [START_REF] Vianello | Non-invasive ventilatory approach to treatment of acute respiratory failure in neuromuscular disorders. A comparison with endotracheal intubation[END_REF] Our results show that a more positive approach can be justified in the management of patients with MND following tracheal intubation and ventilation. Many patients can be successfully weaned onto NIV and discharged home with survival overall comparable to that for patients electively initiating NIV.

Table 1 All

 1

			DP Group	UP Group	p-value
		(n = 30)	(n = 13)	(n = 17)
	Age at onset of symptoms,	64.2 (12.0)	56.7 (14.1)	70.0 (5.5)	0.006
	years
	Age at intubation of trachea,	67.1 (8.0)	62.0 (7.8)	71.0 (5.8)	0.001
	years
	Median time (IQR) between	12.9 (18.0)	21.2 (38.8)	9.1 (10.4)	0.02
	first symptoms and respiratory
	failure, months
	Length of stay in ICU,	40.3 (37.8)	28.1 (40.4) 49.6 (34.0)	0.1
	days
	Length of stay on the RSSC,	33.6 (23.9)	27.9 (15.7)	37.9 (28.3)	0.2
	days
	Weaned from invasive	14	6	8	0.02
	ventilation
	Time to wean,	51.4 (38.1)	25.8 (10.3)	70.5 (40.6)	0.03
	days

DISCUSSION

Our findings show that, in contrast to previously published case series,

[START_REF] Chen | Motor neuron disease presenting as acute respiratory failure: a clinical and pathological study[END_REF][START_REF] Bradley | Outcome of ventilatory support for acute respiratory failure in motor neurone disease[END_REF][START_REF] Shoesmith | Prognosis of amyotrophic lateral sclerosis with respiratory onset[END_REF]

14 from 30 patients with MND were successfully weaned from tracheostomy ventilation, although all had continuing requirements for NIV. Survival to hospital discharge overall was 57% with one year survival of 43.3%. Survival from the initiation of ventilatory support was similar for patients treated with emergency intubation and those started on elective NIV.

ACKNOWLEDGEMENTS None

COMPETING INTERESTS None

FUNDING

The charity DeNDRoN (Dementias and Neurodegenerative Diseases Research Network) paid part of the first author's salary but had no input into data collection, analysis or manuscript preparation.

COPY LICENCE STATEMENT

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in Journal of Neurology,

Neurosurgery & Psychiatry and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence (http://jnnp.bmj.com/ifora/licence.pdf).


Victoria Singh-Curry

Paresh Malhotra

Simon F Farmer

Prof Masud Husain
email: m.husain@ion.ucl.ac.uk

Attention deficits following ADEM ameliorated by guanfacine

Keywords: acute disseminated encephalomyelitis, neglect, noradrenaline, sustained attention, thalamus

We report here the case of a patient with severe deficits in arousal and sustained attention, associated with hemispatial neglect. These impairments were secondary to acute disseminated encephalomyelitis (ADEM), with bilateral involvement of the medial nuclei and pulvinar of the thalamus. Treatment with the noradrenergic agonist guanfacine, previously used for attention deficits in ADHD and stroke, was associated with a significant amelioration of both the spatial and sustained attention impairments in neglect. Guanfacine may prove to be a useful tool in the treatment of disorders of attention associated with neurological conditions.

Acute disseminated encephalomyelitis (ADEM) is a relatively rare neuroinflammatory disorder associated with multifocal lesions, frequently preceded by a viral prodrome. It is usually a monophasic illness, with many patients recovering well, although up to half followed up long-term have been reported to have persistent neurological deficits. [START_REF] Schwarz | Acute disseminated encephalomyelitis: a follow-up study of 40 adult patients[END_REF] Magnetic resonance imaging (MRI) usually reveals asymmetrical subcortical white matter involvement. However, the deep gray matter nuclei, such as the thalamus and basal ganglia may also be affected. [START_REF] Bernarding | Diffusion-and perfusion-weighted MR imaging in a patient with acute demyelinating encephalomyelitis (ADEM)[END_REF] There are few studies documenting the cognitive and neuropsychological sequelae of ADEM, [START_REF] Sunnerhagen | Rehabilitation problems after acute disseminated encephalomyelitis: Four cases[END_REF][START_REF] Hollinger | Acute disseminated encephalomyelitis in adults: a reappraisal of clinical, CSF, EEG, and MRI findings[END_REF] with most focusing on motor disabilities. Here, we report a patient with thalamic lesions secondary to ADEM, causing persistent left-sided hemispatial neglect (failure to attend to stimuli in contralesional space) and difficulty sustaining attention. These deficits were subsequently ameliorated by administration of the noradrenergic agonist guanfacine, a drug that has been shown to have positive effects in children with attention deficit/hyperactivity disorder, [START_REF] Biederman | A randomized, double-blind, placebo-controlled study of guanfacine extended release in children and adolescents with attention-deficit/hyperactivity disorder[END_REF] as well as sustained and spatial attention deficits in some stroke patients with neglect. [START_REF] Malhotra | Noradrenergic modulation of space exploration in visual neglect[END_REF]

Case Report

A 38 year-old male presented with a right-sided facial droop and hemiparesis following a two-week prodrome of headache, fever, cough and right hemisensory symptoms. Soon after admission, he developed tonic-clonic seizures, necessitating treatment with phenytoin, intubation and ventilation. MRI revealed patchy signal changes in the thalamus, cerebellum, temporal and occipital lobes bilaterally, while MR angiogram revealed normal extra and intra-cranial blood flow. Cerebrospinal fluid examination, vasculitic blood screen and transoesophageal echocardiogram were normal.

Electroencephalography demonstrated features consistent with encephalopathy and a diagnosis of ADEM was made. He subsequently received two courses of intravenous methylprednisolone, intravenous immunoglobulin, plasma exchange and antibiotics. He was also anticoagulated for a deep venous thrombosis and required surgical treatment for an associated compartment syndrome.

The patient remained in intensive care, due to persistent epileptiform activity, for four months, when seizures were stabilised on a regimen of levetiracetam 2g, phenytoin 700mg and prednisolone 30mg. He was then transferred to a rehabilitation unit, at which time he had a tetraparesis, with predominant left-sided weakness.

Neuropsychological testing also revealed significant cognitive impairments, including left-sided neglect (with intact visual fields on confrontation), reduced arousal and difficulty sustaining attention. In addition, there were significant impairments in verbal memory (chance performance on the short and easy Recognition Memory Test for verbal material and low average performance on the Wechsler Adult Intelligence Scale-Revised (WAIS-R) Digit Span subtest) and naming (5/15 correct on Graded Naming Test). There was also evidence of dysfunction on tests of executive function (concrete performance on Proverb Interpretation and perseveration on Single Letter Reading and the Similarities subtest of the WAIS-R). On the basis of educational and occupational background, he was estimated to have been functioning in at least the superior range premorbidly and had therefore suffered severe intellectual deterioration.

Admission for reassessment occurred two years later, at which time anticonvulsant medication consisted of levetiracetam 750mg and gabapentin 300mg both twice daily (the total daily dose of levetiracetam was 1250mg at 6 months follow-up, with a further reduction to 1000mg 10 months later). Cranial nerve examination was normal, except for a mild left-sided facial weakness. Examination of the limbs revealed a severe hemiparesis with increased tone on the left and a pyramidal distribution of weakness, worse distally.

The limb reflexes were all brisk with bilaterally extensor plantar responses. There was severe left hemispatial neglect and impairments in sustained attention and arousal, with the patient spending 20 hours a day in bed due to drowsiness. A decision was taken to trial the alpha-2-noradrenergic agonist guanfacine, with the hypothesis that this might improve these cognitive impairments.

Assessment measures

MRI was repeated to determine the extent of lesions (Fig. 1A). Two standard bedside neuropsychological tests were used to assess neglect while a computerised task was used to measure the deficit in sustained attention. The neglect tests used were line cancellation, in which subjects are instructed to cancel all the lines they can find (total 40) distributed across an A4 sheet of paper and bisection of 17cm horizontal lines, where the mean deviation rightwards from centre was taken from three attempts. The computerised task which has been used previously, [START_REF] Malhotra | Role of right posterior parietal cortex in maintaining attention to spatial locations over time[END_REF] was presented on a Dell Latitude D820 laptop computer, and entailed the subject depressing the central button on a response box (RB-530 Cedrus Corp.), whenever an infrequently occurring black circle (8mm diameter) occurred. The circle remained on the screen for 1 second and was presented on a grey background with interstimulus intervals of 1-7s. 100 stimuli were presented over a total period of eight minutes.

Responses quicker than 100ms were classified as anticipations, and therefore as commission errors, as were responses occurring more than 1600ms after target onset.

Perceptual sensitivity, or d-prime (d'), which is derived from signal detection theory [START_REF] Stanislaw | Calculation of signal detection theory measures[END_REF] and takes into account commission as well as omission errors, was the behavioural outcome measure of this task and calculated according to the formula below:

d' = Φ -1 (H) -Φ -1 (F)
Where H is the hit rate and F is the false alarm rate. The higher this value, the better the subject was at accurately detecting targets.

These tests were performed on two consecutive days prior to commencing guanfacine, as well as at several time points after its introduction. The dose of guanfacine was titrated up slowly over three days in the following increments: 0.5mg, 1mg and 2mg as single daily doses. The first on-guanfacine testing session was performed the day 2mg was reached.

An additional testing session off-guanfacine was performed 2 months later (due to initial difficulty obtaining the drug locally) at which point the patient had not received guanfacine for 2 weeks. Three on-guanfacine testing sessions were performed 6 months after the initial commencement of 2mg of guanfacine daily and a final session occurred a further 10 months later. Therefore, in total, the patient underwent three testing sessions off and five on guanfacine. Testing was always performed during the afternoon.

Guanfacine has previously been well tolerated in studies investigating its efficacy in children and adolescents with attention deficit/hyperactivity disorder, however, the most commonly experienced side effects include drowsiness, sedation and dizziness. [START_REF] Biederman | A randomized, double-blind, placebo-controlled study of guanfacine extended release in children and adolescents with attention-deficit/hyperactivity disorder[END_REF][START_REF] Hunt | An open trial of guanfacine in the treatment of attention-deficit hyperactivity disorder[END_REF] The patient reported here did not experience any side effects.

Results

Figure 1A demonstrates the thalamic lesions on T2-weighted MRI, along with performance on attention tests on and off guanfacine. The thalamic lesions localise to the medial thalamic nuclei (including the mediodorsal nucleus) and pulvinar on the left and to the pulvinar on the right. [START_REF] Schmahmann | Vascular syndromes of the thalamus[END_REF] Lesions of the right pulvinar have previously been linked to the pathogenesis of neglect, [START_REF] Karnath | The subcortical anatomy of human spatial neglect: putamen, caudate nucleus and pulvinar[END_REF] while the patient's remaining small lesions (in the cerebellum, temporal and occipital lobes) all lie outside areas commonly associated with the syndrome, such as the inferior parietal lobe, temporoparietal junction and inferior frontal lobe. [START_REF] Mort | The anatomy of visual neglect[END_REF][START_REF] Husain | Visual neglect associated with frontal lobe infarction[END_REF] All three measures used to assess attention deficits improved on guanfacine. Permutation testing was used to investigate whether these effects were statistically significant. This established procedure has specifically been used in single-case designs and works by considering all possible recombinations of the data. [START_REF] Todman | Randomisation in single-case experimental designs[END_REF] The purpose of considering these recombinations or permutations is to attempt to account for fluctuations in assessment scores which may occur over time, unrelated to the effects of treatment.

First, the difference between the actual baseline and treatment means is calculated. Then the mean difference for every other possible combination of data if the treatment had been introduced at different time points in the data set is computed. In other words, imagine that you were given the results without knowing when the patient was on or off drug. One could look at all the different permutations of being on and off drug and calculate the mean difference in performance for each of these permutations.

If the actual difference between on-and off-treatment means is greater than that for any other combination, it is possible to calculate how often this would happen by chance. In general, the actual difference between means will be statistically significant at the 5% level, if this difference falls in the 5% most extreme differences in the distribution of possible recombinations of the data. [START_REF] Todman | Randomisation in single-case experimental designs[END_REF] In this case, the total number of permutations for our three off-and five on-treatment observations is 56. Therefore, if the actual difference between off-and on-treatment means is greater than the mean difference for all the other possible recombinations of the dataset, the probability of this occurring purely by chance is 1/56=0.018. This analysis revealed that rightward deviation on line bisection reduced significantly (Fig 1B ) after commencing guanfacine (p=0.018), demonstrating clear amelioration of the spatial bias most characteristic of neglect. The number of items identified on line cancellation also increased (Fig 1C), although this did not reach statistical significance (p=0.071). On the other hand, accurate target detection (perceptual sensitivity) over the eight minute computerised sustained attention task was significantly enhanced (p=0.018, Fig. 1D), revealing that, in addition to ameliorating aspects of the spatial bias of neglect, guanfacine was able to improve the deficit in sustained attention.

Clinical observation was consistent with these data, with the patient's general level of alertness and arousal improving following introduction of guanfacine. Furthermore, after a period of 6 months on the drug his carers reported an "improvement in his awareness and conversation…" to the extent that he was able to "…contribute significantly to crossword puzzles and enjoy his music CDs". As a result of these persistent benefits the patient continues to take 2mg guanfacine daily.

Discussion

The case presented here demonstrated severe hemispatial neglect and impairments in sustained attention, associated with bilateral thalamic lesions caused by ADEM. Initiation of the noradrenergic agonist guanfacine led to improvement of these deficits, which persisted with continued guanfacine use. Thalamic lesions, especially those involving medial nuclei -including the medial pulvinar -are most frequently associated with deficits in arousal, which can be particularly severe following bilateral damage, such as can occur following stroke. [START_REF] Schmahmann | Vascular syndromes of the thalamus[END_REF] Hemispatial neglect is also often reported, again following strokes involving the medial thalamus and pulvinar, most frequently in the right hemisphere. [START_REF] Schmahmann | Vascular syndromes of the thalamus[END_REF][START_REF] Karnath | The subcortical anatomy of human spatial neglect: putamen, caudate nucleus and pulvinar[END_REF][START_REF] Watson | Thalamic neglect. Possible role of the medial thalamus and nucleus reticularis in behavior[END_REF] In contrast, these impairments have rarely been referred to in the literature as a sequela of ADEM. [START_REF] Sunnerhagen | Rehabilitation problems after acute disseminated encephalomyelitis: Four cases[END_REF] Difficulty with arousal or maintaining alertness, so that attention can be sustained on current goals, is increasingly accepted as a component of the neglect syndrome, capable in fact of predicting the severity of the spatial bias. [START_REF] Robertson | Auditory sustained attention is a marker of unilateral spatial neglect[END_REF] It has been proposed that arousal is dependent on noradrenergic input from the locus coeruleus to inferior parietal and frontal cortex, and indirectly to these regions via the thalamus. [START_REF] Watson | Thalamic neglect. Possible role of the medial thalamus and nucleus reticularis in behavior[END_REF] Damage to these regions can also be associated with neglect. [START_REF] Karnath | The subcortical anatomy of human spatial neglect: putamen, caudate nucleus and pulvinar[END_REF][START_REF] Mort | The anatomy of visual neglect[END_REF][START_REF] Husain | Visual neglect associated with frontal lobe infarction[END_REF] Further evidence of the intimate link between arousal and the spatial orientation of attention comes from rehabilitation studies. Alertness training can improve neglect, [START_REF] Sturm | Alertness-training in neglect: behavioral and imaging results[END_REF] while single doses of guanfacine have previously been shown to enhance sustained attention, in addition to ameliorating the spatial deficit. [START_REF] Malhotra | Noradrenergic modulation of space exploration in visual neglect[END_REF] Although continued guanfacine use has been demonstrated to be efficacious in the treatment of inattentiveness in children and adolescents with attention-deficit/hyperactivity disorder, [START_REF] Biederman | A randomized, double-blind, placebo-controlled study of guanfacine extended release in children and adolescents with attention-deficit/hyperactivity disorder[END_REF] this is the first demonstration of a persistent amelioration of at least some components of the spatial deficit of neglect with a noradrenergic agonist.

Figure 1 .

 1 Figure 1. Bilateral thalamic lesions associated with hemispatial neglect and impaired sustained attention, and amelioration with guanfacine. A, Bilateral thalamic lesions as demonstrated by T2-weighted MRI scanning. The red arrow indicates one of the left-sided lesions, involving the medial thalamic nuclei, including the medio-dorsal nucleus. There are also smaller adjacent lesions in the pulvinar (blue arrow). The yellow arrow indicates the right-sided lesion, which is also in the pulvinar. B, Line bisection. The rightward deviation on bisecting 17 cm lines decreased significantly after commencement of guanfacine (deviation given in mm). C, Line cancellation. The total number of lines cancelled by the patient increased on 2mg guanfacine. D, Perceptual sensitivity on the sustained attention task improved significantly on guanfacine treatment.Pre: pre-guanfacine Post 1: initial assessment after commencement of 2mg guanfacine Off: assessment at 2 months after guanfacine had been ceased for 2 weeks Post 2: assessments at 6 months after initiation of guanfacine Post 3: assessment at 16 months after initiation of guanfacine

Acknowledgments

This research is supported by The Wellcome Trust and the NIHR CBRC at UCLH/UCL.

Licence Statement

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in Journal of Neurology, Neurosurgery & Psychiatry and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence.

Competing interests:

None declared.


H J Van Der Fels-Klerx

Slge Burgers

Cjh Booij

H J Van Der Fels
email: ine.vanderfels@wur.nl

to predict deoxynivalenol in winter wheat in The Netherlands

Keywords: Mycotoxins, Mycotoxins -trichothecenes, Mycotoxins -fusarium Food Types: Cereals and grain deoxynivalenol, mycotoxins, winter wheat, predictive modeling mc.manuscriptcentral.com

published or not. The documents may come

Descriptive modeling to predict deoxynivalenol in winter wheat in The Netherlands

Introduction

Mycotoxins are secondary metabolites of fungi, some of which pose a threat to animal and human health. These toxins have been implicated as causative agents in a variety of animal disorders, such as depressed feed intake and feed refusal, decreased growth and feed efficiency, reproductive disorders, and skin and gastrointestinal irritation [START_REF] Diekman | Mycotoxins and reproduction in domestic livestock[END_REF][START_REF] Mello | Fusarium mycotoxins: a review of global implications for animal health, welfare and productivity[END_REF][START_REF] Morgavi | An historical overview of field disease outbreaks known or suspected to be caused by consumption of feeds contaminated with Fusarium toxins[END_REF][START_REF] Minervini | Zearalenone and reproductive function in farm animals[END_REF]. In human, these compounds have been associated with, amongst others, immunosurpression and cancer induction [START_REF] Creppy | Update of survey, regulation and toxic effects of mycotoxins in Europe[END_REF][START_REF] Smith | Role of mycotoxins in human and animal nutrition and health[END_REF].

Under favorable conditions, related to agronomical and environmental circumstances, fungi may colonize crops and produce mycotoxins. In wheat cultivation, the most prominent fungi are Fusarium spp., resulting in Fusarium Head Blight (FHB), one of the most important fungal diseases affecting all cropping areas of the world (Botallico and Perrone 2002;Champeil et al. 2004a;[START_REF] Osborne | Epidemiology of Fusarium head blight on small-grain cereals[END_REF]. FHB results into reduced wheat yield and quality, and consequent economic losses. One of the most common toxins produced by Fusarium spp. is deoxynivalenol (DON) [START_REF] Miller | Mycotoxins in small grains and maize: Old problems, new challenges[END_REF].

Besides the losses due to reduced wheat yield and quality, DON contaminated wheat has lower market value due to its potential health threats. Growing wheat is, therefore, faced with the challenge of keeping contamination with Fusarium spp. and related mycotoxins at the minimum. Application of Good Agricultural Practices (GAP) during cultivation, and Good Manufacturing Practices (GMP) and Hazard Analysis and Critical Control Point (HACCP) procedures during consecutive stages of the wheat feed and food supply chain will ensure that fungal infection and mycotoxin formation is as low as possible given the circumstances (CAC 2003;EC 2006). To enhance application of these practices and for further mycotoxin control, early insight into the expected DON contamination at harvest is useful to the various stakeholders However, when developing predictive models, the needs of the different stakeholders of the wheat chain regarding the intended use of the predictions and their timing should be considered. Farmers may use DON predictions around wheat flowering to underpin decision making on the application of fungicides, using the most accurate predictions available at that critical growing stage. Feed and food industry, including collectors, millers, and processors, may use DON predictions to focus their inspections and to decide on buying, routing and processing of batches. They may need the DON prediction as early as available in the season and up to harvest, considering that the prediction accuracy will increase as the growing season progresses. Food safety authorities are expected to improve inspection efficiency when using mycotoxin predictions in constructing their yearly monitoring plan.

Worldwide, several initiatives are going on to develop quantitative predictive models for DON levels in mature wheat, however, only a few have been published [START_REF] Schaafsma | Climatic models to predict occurrence of Fusarium toxins in wheat and maize[END_REF][START_REF] Prandini | Review of predictive models for Fusarium head blight and related mycotoxin contamination in wheat[END_REF]). The most well-known model is DONcast, developed in Canada, aimed to assist farmers in their decision to spray [START_REF] Hooker | Using weather variables preand post-heading to predict deoxynivalenol content in winter wheat[END_REF]. The model predicts DON levels at wheat heading, and is based on regression analyses of field data. The model is commercialized to provide region specific DON predictions, using input on climate and agronomy [START_REF] Schaafsma | Climatic models to predict occurrence of Fusarium toxins in wheat and maize[END_REF]. [START_REF] Rossi | A model estimating the risk of Fusarium head blight on wheat[END_REF] developed a mechanistic model, using experimental data, for (fungal infection and) accumulation of DON and zearalenole in wheat until harvest. The model is linked into a semi-quantitative system to estimate the risk for both mycotoxins [START_REF] Rossi | A decision support system for Fusarium head blight on small grain cereals[END_REF]. A neural network model has been developed to [START_REF] Klem | A neural network model for prediction of deoxynivalenol content in wheat grain based on weather data and preceding crop[END_REF]). For The Netherlands, a quantitative model to predict DON content in wheat at harvest has been developed recently [START_REF] Franz | Prediction of Deoxynivalenol content in Dutch winter wheat[END_REF]. Model development was based on regression analyses using six years of field data. The best performing model included several agronomical variables and various climatic variables up to 24 days after wheat heading. This model could be useful in practice for feed and food industry, and food safety authorities as -in contrary to farmers -these stakeholders do not necessarily need the model results around wheat heading. For disease management, the postheading period is too wide, as hourly meteorological forecasts may be reliable up to 7 to 10 days, but loose reliability after this period.

The main aim of the current study is to develop quantitative models to predict DON levels in mature winter wheat in The Netherlands for different groups of endusers; more specifically, a model for farmers focusing on the wheat flowering period, and a model for industry and authorities including the period up to harvest. The central hypothesis of this study is that considering weather data during the entire period of cultivation will increase the prediction accuracy.

Material and Methods

Data collection

In the years 2001 -2008, data 2002, 24 in 2003, 50 in 2004, 96 in 2005, 28 in 2006, 36 in 2007, and 114 in 2008. Information collected from these fields included: field location (zip code), wheat variety (VAR), cultivar resistance level (RES, levels 1-10), application of Fusarium fungicides (SPRAY, levels 0, 1, 2 times), and DON content (in µg kg -1 ) in samples of mature wheat. Spikes were collected several days before harvest at random throughout the field until 1 kg was obtained. The entire grain sample was sent to an independent private laboratory in The Netherlands. DON was extracted and quantified using a liquid-chromatography with double in-line mass spectrometry (LC-MS/MS) [START_REF] Spanjer | LC-MS/MS multi-method for mycotoxins after single extraction, with validation data for peanut, pistachio, wheat, maize, cornflakes, raisins and figs[END_REF], using a limit of quantification of 5 µg kg -1 . From the nearest meteorological station to each field, hourly weather data on temperature, rainfall and relative humidity was obtained for the entire observation period. Data from in total 29 meteorological stations were used. Part of the collected data was also used by [START_REF] Franz | Prediction of Deoxynivalenol content in Dutch winter wheat[END_REF].

Data processing

In case of a sample not exceeding the limit of quantification, the DON level of the particular sample was set at this limit (of 5 µg kg -1 ). The locations of the fields were divided into four regions, including North (N), West (W), South-East (SE) and North-West (NW), based on provinces (n=13) of The Netherlands, such that the four regions included a nearly equal number of provinces. For each record, the intensity of wheat production around the field, named "cereality" (CER), was calculated as the area of wheat grown divided by the total area. Field CER (in percentage per km 2 , 0-100%) was calculated using data from the Dutch Ministry of Agriculture, Nature and Food Quality. For the wheat fields, flowering dates were registered by the farmers. However, some data were missing and observations showed to not to be precise in all cases. Therefore, for each record, the flowering date (FD) -representing the middle of the critical wheat flowering period -was calculated as the day at which the sum of average daily temperatures since January 1 of the particular year reached 1265˚C. Also, for each record, the harvest date (HD) was calculated as the first date at which the sum of the average daily temperatures after the (calculated) FD reached 989˚C [START_REF] Lazar | Methodology of the MARS crop yield forecasting system. Volume 2. Agrometeorological data collection, processing and analysis[END_REF]. Both FD and HD are expressed as the number of days defined from January 1 of the particular year. From the calculated FD and HD, the length of the period (in number of days) between flowering and harvest (leFH, in days) was derived.

For the development of the two models, i.e., the model for the farmers (model A) and the model for industry and authorities (model B), climatic variables were constructed in five relevant time windows of the wheat growing season. For model A, these time windows run up to 10 days after FD, whereas for model B they run up to harvest. The time blocks were defined on a daily basis with days running from 12 AM the previous day to 11 AM the actual day. Time blocks used in model A -represented relative to FD (day 0) -were: day -17 to -10 (A0), day -10 to -3 (A1), day -3 to 0 (A2), day 0 to 3 (A3), day 3 to 10 (A4). For model B, the time blocks included day -17 to -10 (B0), day -10 to 0 (B1), day 0 to 10 (B2), day 10 to 10 days before HD (B3), 10 days before HD to HD (B4). Note, that the number of days in time block B3 may vary between the records. For each of these time blocks, the following climatic variables were calculated per record:

-average hourly temperature (Tavg in ˚C) -number of hours that temperature is 25˚C or higher (Th25) -sum of hourly rainfall (rain in mm) -total number of hours that relative humidity (RH) is 80% or higher (RHh80) -sum of hourly temperatures over the hours that RH is 85% or higher, provided this is the case for at least 2 consecutive hours (ThRH85_2) -sum of hourly temperatures over the hours that RH is 85% or higher, provided this is the case for at least 4 consecutive hours (ThRH85_4)

Model development and validation

In model development, a variety of statistical analyses was performed, including: descriptive statistics, and univariate and multiple regression analyses. All statistics were performed using GenStat 11 th Edition (GenStat Committee, Algorithm group Inc., see http://www.vsni.co.uk/), using a significance level of 0.05. Given the range of DON values (the response variable) and the expected increasing variation at higher levels of DON, it was assumed that DON follows a lognormal distribution. Therefore, all statistical analyses were performed on logtransformed data, i.e., the natural logarithm of DON (lnDON). [START_REF] Archdeacon | Correlation and regression analysis: a historian's guide[END_REF][START_REF] Gross | Lecture Notes in Statistics 175 -Linear Regression[END_REF]). Models with the highest R 2 adj and a low Mallows'C p that included only significant variables were selected. All the above described steps for the selection of variables were done for constructing model A and B separately, and using the entire dataset. However, three records that showed to result in a very high leverage trough all the modeling steps were not considered in the analyses.

Once model A and B were selected, they were fitted to 90% of the records, and validated using the remaining 10% of the records. For each model, the two data subsets were constructed by drawing a random sample of 10% from each year-region combination from the entire dataset (thus resulting in a 10% subset for model A and a 10% subset for model B). The two 90% data subsets were checked for balanced number of records and DON levels over years, regions, cultivar and spraying levels.

Parameter estimates and standard errors (s.e.) were obtained by fitting the final models to their respective 90% data subsets. These final models were checked for data with high leverages, correlations between model variables, residuals (plots) and figures of the fitted and observed relationships between the individual model parameters and lnDON. Based on the two final models, predictions (with s.e.) of DON levels were calculated for the 10% data subsets. The correlation coefficients between these predicted values and the corresponding observed DON levels were calculated.

Also, the 95% confidence intervals of the predictions were calculated. 1). Yearly average values in 2001, 2002, 2004 and 2008 were around the overall average. In 2006, all samples were found to be below the limit of quantification.

Results

F

The distribution of observations over regions showed a comparable number of records in three regions (M, N, W), but a lower number in the S-region. Observed average DON levels in mature winter wheat were comparable for the regions M, W and S, being 424.5 µg kg -1 (5.0 -4600, n=170), 357.2 µg kg -1 (5.0 -3150, n=106) and 572.3 µg kg -1 (5.0 -1300, n=23), respectively. DON levels were somewhat higher in the N-region, averaging 681.0 µg kg -1 (5.0 -10400, n=126). DON levels were lower in case of spraying one or two times, as compared to no fungicide treatment; averages were 299.3 µg kg -1 (22.6 -1300, n=66) for two applications, 291.9 µg kg -1 (5.0 -3217, n=91) for one application, and 607.0 µg kg -1 (5.0 -10400, n=268) in case no fungicide was applied. In total 27 different wheat varieties were used, with 13 of them having less than 5 observations. Wheat resistance level (RES) ranged from 5.0 to 8.5 with a median of 6.5. Average DON values run from 682.5 µg kg -1 for a RES of 5.0, around 500 µg kg -1 for a RES of 6.5-7.5 (468.2 µg kg -1 and 507.7 µg kg -1 for a RES of 6.5 and 7.5, respectively), to a lowest value of 137.5 µg kg -1 for a RES of 8. Model A (flowering period) Within each of the five A time periods, the correlation between the variables RHh80, ThRH85_2 and ThRH85_4 showed to be very high.

Out of these three variables related to relative humidity, RHh80 is the simplest one to record and, therefore, this variable was chosen for further model building. The variables Tavg and Th25 also were strongly correlated with each other; correlation coefficient values ranged from 0.77 to 0.86 in the different A time blocks.

Correlations between the same climatic variables in different A time blocks in general

showed to be low. The highest correlation was found for Tavg in A2 and Tavg in A3 (r= 0.67); all other correlations were below 0.60. For model A, the best fitting multiple regression model included the following variables: REG, RES, SPRAY, FD, Tavg (with quadratic effects) in all A time blocks except for A2, RHh80 in A0 and A3, rain in A1 and A4 (with quadratic effect), and the interaction between rain in A1 and Tavg in A3. The results of fitting this model to its 90% data subset are presented in Table 2. All variables showed to be significant (p<0.05). The percentage of variance accounted (R 2 ) for was 64.4%, the standard error of observations was 0.87.

Model B (entire cultivation period)

In all B time periods, the correlation between ThRH85_2 and ThRH85_4 showed to be very high. This also accounted for each of these two variables and RHh80, except for the period B4. The correlation between Tavg and Th25 in individual B time periods was also high, varying from 0.77 to 0.96.

Correlations between the same climatic variables in different B time blocks were low; the highest value was found for Tavg in B0 and Tavg in B4, equalling 0.68. The interaction between rain in B1 and Tavg in B2 showed significantly to affect DON levels univariately.

For model B, the best fitting set of variables included: REG, RES, SPRAY, FD, leFH, rainfall and RHh80 in B0, and Tavg in B0, B1, B3 and B4 (with quadratic effects in B3 and B4). The results of fitting this model to its 90% data subset are presented in Table 3. The percentage variance accounted for (R 2 ) was 65.6%, the standard error of observations was 0.836. The correlation between leFH and Tavg in B3 was somewhat high (r= -0.72). However, both variables had a significant effect in the final model, and were included based on their logical relationship.

Model validation

Discussion

The two models constructed in the current study present the second attempt to predict DON levels in mature winter wheat in The Netherlands. Compared to the (basic) model of [START_REF] Franz | Prediction of Deoxynivalenol content in Dutch winter wheat[END_REF], the two current models are more specified to the intended user-groups, particularly wheat growers (model A) and other stakeholders, including feed and food industry and food safety authorities (model B). Model A includes climatic variables in a shorter period around wheat flowering, i.e., up to 10 days after FD, as compared to the basic model that used the period up to 24 days after wheat heading [START_REF] Franz | Prediction of Deoxynivalenol content in Dutch winter wheat[END_REF]. Unless this shorter period, the percentage of explained variance was higher (R 2 of 65% vs 59%). The agronomical variables in both models are comparable, as are the climatic variables, all being related to rainfall, This study showed that taking into account the entire period up to harvest (model B), as compared to flowering period (model A), increased the accuracy of the model predictions; the percentage explained variance of model B (R 2 =65.6%) was slightly higher, as compared to this metric for model A (R 2 =64.4%), and the standard error of observations was somewhat lower. The agronomical variables in models A and B are identical, however, model B also includes the length of the period from FD to HD. The longer this period the higher the DON levels at harvest. This effect did not dependent on effects of the actual FD and wheat resistance level. Given the negative relationship between FD and DON values, the (positive) effect of an increasing length from FD to HD may be related to extended conducive periods of humidity and high temperatures during mid summer.

Both in model A and B, climatic variables in the preflowering period relate to average temperature, rainfall and relative humidity. Both a higher rainfall and an increase in the number of hours that RH is 80% or more increase DON levels at harvest. A higher average temperature in the early preflowering period (-17 to -10 days FD) increases DON levels, but a higher average just before FD decreases DON levels. The latter finding may be related to the humid conditions that often go hand in hand with low temperatures and are conducive for fungal infection in the preflowering period. Climatic effects in the postflowering period relate to rainfall, temperature and relative humidity in case a 10 day period is considered (model A). However, temperature is the only significant factor when the entire postflowering period is taken into account (model B). These findings of climatic effects during the postflowering period correspond well to [START_REF] Franz | Prediction of Deoxynivalenol content in Dutch winter wheat[END_REF]. In model A, an interactive effect of the with a more profound effect of increasing temperature after FD (i.e., higher DON levels) in case of a higher rainfall before FD. In the two current models, the majority of the temperature related variables in the different periods of wheat growing showed

to have an optimal effect on DON levels at about 16-20 ˚C. The effects of the climatic variables in the two models of the current study reflect the ecological conditions of infection of Fusarium spp. in wheat and consequent DON formation, with increasing effects of moist (wet and humid) and warm conditions around flowering [START_REF] Osborne | Epidemiology of Fusarium head blight on small-grain cereals[END_REF].

In the construction of both models, the intensity of wheat production in the area around the wheat field (cereality) was considered in attempt to unravel the region effect on DON values, found in the current study and previously [START_REF] Hooker | Using weather variables preand post-heading to predict deoxynivalenol content in winter wheat[END_REF][START_REF] Franz | Prediction of Deoxynivalenol content in Dutch winter wheat[END_REF]. It was hypothesized that the more wheat produced in the area surrounding the wheat field, the higher the Fusarium spp. infection pressure.

However, cereality did not significantly affect DON levels at harvest and, obviously, the region effect is caused by other factors. These might include unanalyzed climatic factors such as precrop winter conditions, regional variations in farm management and/or in the genetic composition of Fusarium spp. These potential factors underlying the region effect should be further investigated.

Several agronomical variables that are previously reported to affect DON contamination in mature winter wheat, particularly tillage practices and previous crop (Champeil et al. 2004a;[START_REF] Schaafsma | Effect of previous crop, tillage, field size, adjacent crop, and sampling direction on airborne propagules of Gibberella zeae/Fusarium graminearum, fusarium head blight severity, and deoxynivalenol accumulation in winter wheat[END_REF][START_REF] Beyer | Quantifying the effects of previous crop, tillage, cultivar and triazole fungicides on the deoxynivalenol content of wheat grain -A review[END_REF][START_REF] Eiblmeier | Risk evaluation of deoxynivalenol levels in Bavarian wheat from survey data[END_REF], were not considered in model construction, primarily because data on these two factors were not available. Secondly, variability in these two factors was (expected to be) small, making them not useful for model construction. In The Netherlands, generally, deep ploughing is applied and maize is not grown before wheat (unpublished data), hereby avoiding the conducive effects of growing grain maize as preceding crop and applying minimum or not tillage on DON contamination.

Would the models be applied to countries with other tillage and cropping practices, it is worthwhile to investigate the possible benefits of considering these variables in model construction, at least for the farmer's model. Industry and authorities often do not have data on precrop and tillage practices and therefore, may prefer one of the current models.

Corresponding to the two comparable previous modeling studies [START_REF] Hooker | Using weather variables preand post-heading to predict deoxynivalenol content in winter wheat[END_REF][START_REF] Franz | Prediction of Deoxynivalenol content in Dutch winter wheat[END_REF], the current study showed that DON levels in mature winter wheat can be predicted accurately using regression analyses of field data, and considering a narrow time window around wheat flowering. The predictive performance of models of the three studies, expressed by the percentage explained variation (R 2 ), is in the same order of magnitude, being 73% for DONcast in Canada [START_REF] Hooker | Using weather variables preand post-heading to predict deoxynivalenol content in winter wheat[END_REF], 64.4% for model A of this study, and 59% for the previous Dutch model [START_REF] Franz | Prediction of Deoxynivalenol content in Dutch winter wheat[END_REF]). The r-square reported for DONcast [START_REF] Hooker | Using weather variables preand post-heading to predict deoxynivalenol content in winter wheat[END_REF][START_REF] Hooker | The DONcast model: predicting deoxynivalenol (DON) in wheat[END_REF][START_REF] Schaafsma | Climatic models to predict occurrence of Fusarium toxins in wheat and maize[END_REF] is somewhat higher than this figure for the three Dutch models. This might -at least partly -be related to the more uniform conditions of wheat cultivation in Canada (climate, field size etc.) as compared to The Netherlands. A previous study showed that application of DONcast to the Dutch field data resulted in very poor performance [START_REF] Franz | Prediction of Deoxynivalenol content in Dutch winter wheat[END_REF], The construction of predictive models for mycotoxins in wheat and their statistical performance not only depends on the specific variables considered in the model building process but also on the quality, particularly the precision, of the underlying data used, e.g., the extent to which the nearest meteorological station represents the climate of the particular wheat field. In addition, the wheat samples and measured DON levels are known to be subject to effects of sampling error and characteristics of the analytical method (e.g., sensitivity, specitivity, detection limit) used (Josephs et al. 2001;[START_REF] Whitaker | Sampling wheat for deoxynivalenol[END_REF][START_REF] Whitaker | Standardisation of mycotoxin sampling procedures: An urgent necessity[END_REF]Champeil et al. 2004b). At least part of the model residues is due to the variation in the response variable (DON levels) and the explanatory (agronomical and climatic) variables caused by one or more of these effects. Improving the performance of the predictive models could, therefore, possibly be obtained by using data of even higher quality.

justifying
The input variables used in constructing the current models represents a combination of the underlying process of fungal infection of the wheat and mycotoxin formation in the different critical periods of wheat growing. Expertise on the ecology of these processes was used to construct the variables and the time blocks in model

  supply chain. As agronomical and climatic factors are assumed to affect mycotoxin formation during the entire growing season, prediction accuracy of DON in mature wheat is expected to increase with the progressing cultivation period.

  in mature wheat around flowering, using data from two fouryear field experiments in Czech. The best fitting model includes preceding crop and four climatic related variables

  Univariate regression analyses were run to test the relationships of the individual agronomical variables, being REG, VAR, RES, SPRAY, CER, FD and leFH, with lnDON. The variables that contributed significantly in explaining variation in the DON values were considered in further analyses. Pearson correlation coefficients were calculated for all combinations of the various climatic variables in the different time blocks. Climatic variables that were sufficiently uncorrelated (r<0.7) were taken into account in the further selection process. Effects of quadratic terms of the climatic variables in the different time periods and of relevant interactions between climatic variables were also tested univariately. The resulting agronomical and climatic variables, with relevant quadratic terms and interactions, were considered in the multiple regression analyses ('all possible subset selection' RSEARCH). The best set of variables was identified by evaluating the percentage variance accounted for (R 2 adj ) and the value of Mallows'C p

  models A and B to their respective 10% data subsets resulted in a correlation coefficient between predicted and observed DON levels of 0.84 and 0.78, respectively (n=41 records). The relationships between the predicted and observed DON levels are illustrated in Figures1 and 2for model A and B, respectively. All observed DON values were within the 95% confidence interval of the predicted values.

  temperature and relative humidity in the pre-and postflowering periods. The increased predictive power of model A of this study might have been due to the additional climatic variables considered -in particular the inclusion of quadratic effects and interactions -and/or the different distinction between days. In this study, days were considered to run from 12 AM the previous day to 11 AM the actual day such to include potential overnight effects of climate, specifically the effect of leaf wetness (dew) during the night and early morning on fungal development.

  postflowering temperature on DON at harvest was found,

  the development of models for this country. Taking into account the entire cultivation period (model B) as compared to the flowering period (model A) increased the accuracy of the DON predictions for The Netherlands, albeit this increase is rather small. Therefore, the intended end-users of model B (feed and food industry and/or as well prefer to use model A, providing the DON predictions much earlier in the season. The costs and benefits of the increased prediction accuracy against the additional time could be further investigated by application of both models into practice.

Figure 1 .

 1 FIGURE CAPTIONS

  5. CERranged from 1.6% to 62.5% at the highest, with an average of 17.8%. FD ranged from 119 at the lowest to 173 at the highest, it's median being 155. LeFH ranged from 30 to 76 days with a median value of 56. Observed DON content in mature wheat increased with a higher CER, an earlier FD, and an increased LeFH.Univariate analyses that showed year had a highly significant effect on DON levels, and solely accounted for 52.8 % of the variance (s.e. 1.02). However, year was excluded as a potential explanatory variable in further analyses as it was assumed its effects are due to the effects of other variables. Additionally, the variable year is not useful for making predictions for the future. Individual effects of REG, VAR, RES, SPRAY, FD, and leFH on DON levels also were significant and accounted for, respectively, 3.3%, 4.9%, 2.4%, 1.1%, 20.5%, and 30.1 % of the variation. The (slight) effect of CER was not significant. The variables VAR and RES are (by definition) related to each other, and given the low number of observations in some

	Regression analyses
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	11

VAR classes, this variable was excluded from further model building. Based on these results, agronomical variables included in constructing both models were: REG, RES, SPRAY, FD, added with leFH for model B.

Table 2 .

 2 development. When increased insight into the factors affecting the behavior of Summary of the final model A for prediction of DON levels in mature winter wheat in The Netherlands 1

	Fusarium spp and mycotoxin formation becomes available it should be included in
	the modeling process to further improve the definition of possible input variables and
	variable selection in model construction. Also, feedback from the end-users, applying

1

The model predicts ln(DON) concentrations (in µg kg -1 ). DON values (in µg kg -1 ) can be obtained by back transformation: e

(model A)

.

Table 3 .

 3 Summary of the final model B for prediction of DON levels in mature winter wheat in The Netherlands 1 The model predicts ln(DON) concentrations (in µg kg -1 ). DON values (in µg kg -1 ) can be obtained by back transformation using e(model B) . Model variables are defined in the body text of this manuscript. Parameter estimates for the factors REG and SPRAY are differences compared with their reference levels, being REG M and SPRAY 0, respectively.

	Model variable 2	Parameters	Standard error	Parameter
		estimate 2		significance
	Constant	-31.39	9.03	<0.001
	REG N	0.246	0.194	0.206
	REG W	-0.729	0.159	<0.001
	REG S	0.305	0.260	0.241
	SPRAY 1	0.688	0.165	<0.001
	SPRAY 0	0.842	0.155	<0.001
	Flowering date	-0.0647	0.0102	<0.001
	Resistance level	-0.4281	0.0463	<0.001
	Length FH	0.0888	0.0173	<0.001
	Rain B0	0.03869	0.00572	<0.001
	RHh80 B0	0.00633	0.00252	0.012
	Tavg B0	0.1076	0.0477	0.025
	Tavg B1	-0.0654	0.0402	0.104
	Tavg linear B3	2.614	0.965	0.007
	Tavg quadratic B3	-0.0781	0.0271	0.004
	Tavg linear B4	2.18	1.02	0.033
	Tavg quadratic B4	-0.0570	0.0277	0.040
	1

2

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Model variables are defined in the body text of this manuscript. Parameter estimates for the factors REG and SPRAY are differences compared with their reference levels, being REG M and SPRAY 0, respectively.

Acknowledgements

The research presented in this paper was financed by the Dutch Product and Food Safety Authority (VWA) and the Dutch Product Board for Arable Products (HPA).

The authors greatly acknowledge all persons involved in data collection: Harro Spits, Huub Schepers, Don Jansen, Cees Waalwijk, Plant Sciences Group (Wageningen UR). Paulien Adamse and Eelco Franz, RIKILT -Institute of Food Safety and Michel Uiterwijk, Alterra (Wageningn UR) are kindly thanked for assistance in data