Midori Yajima

Arnold Van Huis

Janice Jiggins

Life history analysis of AIDS-affected households in rice and cassava-based farming communities in northern Malawi

Keywords: Malawi, HIV and AIDS, rural households, life history, impact level word; Malawi, HIV and AIDS, impact level, rural households, life history

Life history analysis of HIV/AIDS-affected households in rice and cassava-based farming communities in northern Malawi

Introduction

AIDS is the leading cause of death in the age group 15-49 years in Malawi (Malawi National AIDS Commission 2003). The adult HIV prevalence was estimated in 2007 to be 12% overall, 11% in rural and 16% in urban areas (Office of the President and Cabinet 2007), i.e. it has become a generalized epidemic (U.S. Census Bureau 2000). In 2006, less than half of the population (44% male and 20% female) showed acceptance of people living with HIV and AIDS, and people generally were reluctant to disclose their serostatus (Office of the President and Cabinet and Republic of Malawi 2007).

The 'New Variant Famine' hypothesis (de Waal and Whiteside 2003;[START_REF] Arrehag | New variant famine' revisited: chronic vulnerability in rural Africa[END_REF] argues that the AIDS epidemic is a major contributing factor in food insecurity in southern Africa.

The hypothesis identified four causal factors: labour shortage, asset and skill loss, increasing burden of care for the sick and orphans, and malnutrition [START_REF] De Waal | New variant famine: AIDS and food crisis in southern Africa[END_REF]. The situation is regarded as an exceptional challenge for food security and overall welfare of the affected (de Waal and Tumushabe 2003; [START_REF] Arrehag | New variant famine' revisited: chronic vulnerability in rural Africa[END_REF]). AIDS has negatively impacted agricultural production, impairing food security, leading to malnutrition of children and adults [START_REF] Barnett | The social and economic impact of HIV/AIDS on farming systems and livelihoods in rural Africa: Some experience and lessons from Uganda, Tanzania and Zambia[END_REF][START_REF] Topouzis | The Implications of HIV/AIDS for Household Food Security in Africa, United Nations Economic Commission for Africa, Food Security and Sustainable Development Division[END_REF][START_REF] Piwoz | HIV/AIDS and Nutrition: A Review of the Literature and Recommendations for Nutritional Care and Support in Sub-Saharan Africa[END_REF][START_REF] Baylies | The Impact of AIDS on Rural Households in Africa: A Shock Like Any Other?[END_REF][START_REF] Shah | Impact of HIV/AIDS on agricultural productivity and rural livelihoods in the Central Region of Malawi[END_REF][START_REF] De Waal | New variant famine: AIDS and food crisis in southern Africa[END_REF][START_REF] Müller | HIV/AIDS and Agriculture in Sub-Saharan Africa: Impact on Farming Systems, Agricultural Practices and Rural Livelihoods -An Overview and Annotated Bibliography[END_REF]). In Malawi, many small-scale farmers depend on income from ganyu (casual hired labour) to bridge the seasonal food gap. Hence, AIDS-affected households tend to neglect their own gardens, creating a so-called 'downward spiral' [START_REF] Bryceson | Sustainable rural livelihoods in Sub-Saharan Africa: Sustaining what and for how long[END_REF][START_REF] Drinkwater | HIV/AIDS and agararian change in southern Africa[END_REF], and examples of such phenomena have been reported [START_REF] Robson | AIDS and Food Insecurity: 'New Variant Famine' in Malawi?[END_REF]. Change in and death of spouses and displacement of orphaned children, leading to labour exploitation and trafficking [START_REF] Drinkwater | HIV/AIDS and agararian change in southern Africa[END_REF], accelerates the erosion of small-scale farming communities' economic competence and traditional social unity.

The concept of 'social immunity' is defined as the 'ability of a collective of people, specifically the extended family, to mitigate the impact of an affliction' [START_REF] Mtika | The AIDS Epidemic in Malawi and its Threat to Household Food Security[END_REF]. The concept indicates that, when a household is struck by AIDS, it first receives assistance from the immediate and extended family members. As the gravity increases until it reaches the threshold level, however, the traditional support and social network system becomes depleted [START_REF] Mtika | The AIDS Epidemic in Malawi and its Threat to Household Food Security[END_REF][START_REF] Beraho | Living with AIDS in Uganda: Impacts on banana-farming households in two districts[END_REF].

The objective of this study is to understand the impact of AIDS in three livelihood systems, experiencing different levels of AIDS-related impacts. By eliciting life history narratives, the study explores people's responses and changing perceptions of HIV and AIDS. The findings are examined in light of the 'New Variant Famine' hypothesis in order to examine local responses to the impact on the basis of livelihood systems that support food security. By examining the threshold of 'social immunity' to adverse impacts of a range of risks, insight is gained into coping strategies that challenge the pessimism of the hypothesis. The implications open up new opportunities for public health programming. Since the first cases of AIDS were reported in the 1980s in Malawi, as of 2009 the respondents had lived through the transitional period: the growing public and private acknowledgement of the epidemic.

Methods

Research setting

The study was conducted in Chilumba (Karonga District, northern Malawi) from April 2007to October 2008. Surveys conducted in 1999[START_REF] Mtika | The AIDS Epidemic in Malawi and its Threat to Household Food Security[END_REF] showed the female HIV prevalence in Karonga District as 15.0% [START_REF] Crampin | Trends and measurement of HIV prevalence in northern Malawi[END_REF], while the estimated figure for rural Malawi was 12% (Malawi National AIDS Commission 2004).

[insert here Figure 1: map] The Tumbuka are the dominant ethnic group in the area. The Tumbuka are patrilineal [START_REF] Tew | Peoples of the Lake Nyasa Region[END_REF], and they practise high material transfers for their mutual support through their kinship [START_REF] Mtika | Family Transfers in a Subsistence Economy and under a High Incidence of HIV/AIDS: The Case of Rural Malawi[END_REF]. The Uliwa trading centre is the second-largest township after Ekwendeni along the 'northern corridor', offering a convenient stopover point for traders and long-distance truck drivers. At Chilumba Jetty, steamer lines make a weekly stop on their way to and from Tanzania and southern Malawi.

Sampling strategy

The respondents were purposively selected from three locations with different livelihood systems (Fig. 1):

(1) Mwandovi village: cassava as a subsistence crop, fishing for food and income;

(2) Kachere village: cassava and maize as subsistence crops, some grow rice and tobacco for cash income;

(3) Hara Irrigation Scheme: rice as a cash crop, supplemented by cassava and maize.

Ten respondents from each location (N=30) were selected, using a combination of random and reputational sampling; i.e., 15 of the respondents had participated in a related study on their agricultural practices and 15 were recruited through referral by local key informants who had been asked to identify those whom they thought might be experiencing particular hardships.

Data collection

The main information presented here is based on repeat (2-3 rounds) interviews with 30 individuals in three locations (10 in each location, Figure 1). Research assistants were hired locally, though from outside the community, and were trained in active listening techniques to encourage in-depth discussion of sensitive issues. The data comprise a narration based on in-depth interviews (of 3 hours or more), recorded using a digital audio recorder followed by translation and transcription. The chronologies were constructed to verify the information in the narration. This was followed by a semi-structured interview focussing on HIV and AIDS [START_REF] Deshpande | Training Manual on the Biographic Narrative Methods: Interview and Analytical Procedures[END_REF]. Responses were recorded in the local language (ChiTumbuka) with the respondents' consent; written notes were maintained in English.

All identities are expressed in pseudonyms.

Assessing the impact levels

We developed the following guidelines to estimate the level of AIDS impact among our respondents.

Group A (primary impact level): Respondents reported that they had had positive results from the blood test or their previous partners or their former spouses were known AIDS cases.

Group B (secondary impact level):

Respondents did not report their own serostatus, but reported known AIDS cases in their family trees.

Group C (no evident impact):

Respondents reported that either they had had a blood test with negative results, or did not report their serostatus but had no known AIDS cases in their family trees.

Data analysis procedure

All narrations (N=30) were coded using qualitative analysis software (Atlas.ti, version 5.0, Scientific Software Development 2004). The frequencies of codes were analysed using SPSS (version 16.0, SPSS 2007) and Excel (Microsoft 2007). Follow-up interviews were analysed in Excel.

Categorising codes into conceptual clusters

The concepts recorded in the narratives were coded with the help of Atlas.ti. Thereafter, each code was categorised into five conceptual clusters: family structure, health problems, risk exposure, shortages/hardships and social organisation/capital (Table 1).

[insert here Table 1] The clustering of codes was based on the life history narrations, personal communication with local informants, and field observation. During the clustering process, it was noted that some codes could be allocated to more than one category. For example, 'death' concerns family structure, but also could be included under health problems, hardships, and risk exposure.

Likewise, 'advice' could either be bad, which could be allocated to risk exposure, or good, which could be counted as social capital. The distinctions were sometimes obvious from the context, but when it was unclear, the codes were allocated in all possible categories; hence one code could be counted more than once.

Results

Respondents' profile

The 30 respondents comprised 19 women and 11 men, ranging from 27 to 80 years, with an average of 49 years. All respondents had married: 17 respondents were in monogamous marriages, 6 had been widowed, 5 were in polygynous marriages (involving 1-5 co-wives), and 2 had divorced. All respondents had attended school ranging from 1 to 12 years, to standard 7 in primary education on average.

AIDS impact levels

Based on our guidelines, we allocated the 30 respondents into three levels of AIDS impacts:

primary level, secondary level, and no evident impact. Their impact level, locations, gender, and livelihood systems are given in Table 2. 2 here] The data used to provisionally assess the 'impact level' of the respondents, namely the self-reported serostatus, and AIDS deaths within the family, could not be objectively verified, and self-reporting cannot be assumed a priori to be accurate when there is stigma and discrimination.

Therefore, the information presented here inevitably carries some degree of uncertainty and ambiguity.

Frequently cited concepts in the narrations

In total, there were 113 concepts recorded as codes during the analysis of the life histories by Atlas.ti, with citation frequencies ranging from 205 to 1 (Table 1). 'Children' was the most frequently cited, followed by in descending order, 'school', 'death', 'money', 'marrying', 'travelling', 'husband', 'father', 'farming', and 'health problem'. The conceptual cluster with the highest frequency was "family structure" (1,066 times), followed by "risk exposure" (825), "social organisation/capital" (778), "shortages/hardships" (583), and "health problems" (387).

New terminologies related to HIV and AIDS

The use of new, euphemistic expressions was observed in the life histories. AIDS was implied for instance by the following: "Matenda gha sono" (current disease), "Matenda gha boma" (government disease), "Akulwala kanthukala" (he/ she is suffering from 'that thing'), "Alikutola kanthukala" (he/ she has contracted 'that thing'). The research assistants in each case sought to clarify the exact meanings. For example, 'government disease' is used to indicate that AIDS is prevalent in every corner of the country -in all districts, sectors and levels -similar to the government structure (Manzunda 15/09/2008).

Critical events in the search for livelihood

For all respondents, the death of spouses, guardians, and close-kin were expressed as critical events. Migration, marriage and divorce (including acquiring co-wives by polygynous husbands), dropping out of school (due to lack of money to pay fees, unplanned pregnancy, violence at school, etc.), and their own sickness or that of family members were also important events. It was observed that respondents in the secondary and the non-affected category tended to show stronger fear about their risks as compared to those in the primary category, who felt themselves to have become empowered through sharing knowledge and experiences in support organisations.

For the 'affected' respondents in the primary category, learning about their own seropositive status was identified as an important turning point in their life history, leading to behavioural changes (e.g., selective use of condoms), and taking part in support organisations. The overall decisions concerning family planning remained unchanged for 6 out of 11 respondents in the primary category. Of the other five, three were widows, one was not married but had a distant partner, and, in the fifth case, the husband was staying with a second wife in a distant town. In other words, all respondents with primary impact who are in legitimate marriages are choosing to continue having children after knowing their serostatus. Some are receiving assistance from support organisations and government, such as money or advice on nutrition and sexual practices. All the respondents in the primary category said that they would not take the children out of school to help with the farm work, commenting that this would disturb the children's future.

Analysis

Analysis of conceptual clusters

The findings from the Atlas.ti analysis were examined for the three variables of gender, livelihood systems and impact levels, as shown in Figure 2.

[insert Figure B,C The concepts categorised in the 'Social organisation/capital' cluster, were cited more often by the respondents in Mwandovi village and Hara Irrigation Scheme compared to Kachere village. The respondents in the two locations base their livelihoods on a rather homogeneous enterprise involving collective activities, possibly leading to relative resilience in social capital. Other contributing factors could be the presence of Hara Water Users Association in Hara Irrigation Scheme, and Farmer Field Schools (FFS) in both locations. These might constitute an enabling environment for new social relationships to emerge. In contrast, the location of Kachere village, due to its proximity to the Uliwa trading centre, on the one hand offers wider opportunities for income, but on the other hand may result in more diversified livelihood strategies and individualised decision-making behaviours compared to the other two, irrespective of AIDS impacts. A more diversified livelihood system with weaker foundations for social organisation could aggravate social immunity.

Implications of new terminologies, language metaphors

During the life history narrations, respondents reported that perceptions related to HIV and AIDS had changed over the past few years. For example, among those in the 'affected' categories, a readiness to discuss AIDS-related topics and challenges was noted, linked to their experience of deaths in the family. Other contributing factors were the increased availability of VCT and ART in the study area and observation of the consequences of certain types of behaviour within their community. The emerging metaphors on the one hand could indicate the magnitude of the AIDS epidemic; however, it could also imply that AIDS is gradually becoming a social norm. The fact that the notion of AIDS could be less stigmatizing also merits a careful attention in prevention programming and psychosocial care for children.

Critical events, life choices

It was observed that learning about their serostatus worked as a strong motive for some to join groups for assistance, to open up to discussion, and reducing their stigma. The respondents in 'affected' categories seemed to be developing higher thresholds of social immunity and demonstrating higher confidence with the help of, or by taking up leading roles in, AIDS support Of all the conceptual codes in their narratives, "children" was the most frequently cited. Children were considered as important assets, a significant household workforce, and an investment in future welfare. "School" was the second most frequently cited and the most frequent one in the cluster of social capital. The strong perception of children and schooling as beneficial, enhanced by increased risk awareness and perhaps also because of a certain fatalism induced by the already unstable nature of their livelihoods could be a reason why the 'affected' respondents were positively continuing to plan their lives.

Discussion

Changing environments

While AIDS poses an enormous socioeconomic challenge, other equally challenging issues coexist in rural Malawian livelihoods, conferring social instability and uncertainties over an individual's lifetime. Deaths from a variety of causes already were among the most serious threats in rural Malawi prior to the onset of the epidemic [START_REF] Mtika | Social and cultural relations in economic action: the embeddedness of food security in rural Malawi amidst the AIDS epidemic[END_REF]. These contexts, together with the importance of children and efforts to minimize migration (Ansell and Young 2004), need to be recognised in public health programming.

In the case of Malawi, inequality in accessing ART roll-out based on region, gender and age has been reported, indicating specific need for improved access in the northern region, and for men and children [START_REF] Makwiza | Monitoring equity and health systems in the provision of anti-retroviral therapy (ART)[END_REF]. The access rate of ART in Karonga District was reported to be only 3% of the total projected number of HIV positive patients as of December 2005 [START_REF] Makwiza | Equity and Health System Strengthening in ART roll out: An analysis from literature review of experiences from east and southern Africa[END_REF]). In addition, there are concerns about ART such as drug resistance [START_REF] Deeks | Treatment of antiretroviral-drug-resistant HIV-1 infection[END_REF][START_REF] Calmy | Simplifying and adapting antiretroviral treatment in resource-poor settings: a necessary step to scaling-up[END_REF][START_REF] Clavel | HIV Drug Resistance[END_REF], adverse effects including hepatic steatosis [START_REF] Carr | Toxicity of Antiretroviral Therapy and Implications for Drug Development[END_REF], adherence and scaling up in resource-poor settings (World Health Organization 2002;[START_REF] Manzi | High acceptability of voluntary counselling and HIV-testing but unacceptable loss to follow up in a prevention of mother-to-child HIV transmission programme in rural Malawi: scaling-up requires a different way of acting[END_REF]. In addition to debates on equity in accessing HIV and AIDS-related health services [START_REF] Farmer | Immodest Claims of Causality. Infections and Inequalities: The Modern Plagues[END_REF][START_REF] Greener | The Impact of HIV/AIDS on Poverty and Inequality. The Macroeconomics of HIV/AIDS M. Haacker[END_REF][START_REF] Whyte | Treating AIDS: Dilemmas of unequal access in Uganda[END_REF]), financing, institutional support, and monitoring to facilitate ART roll-out remain as major challenges [START_REF] Makwiza | Equity and Health System Strengthening in ART roll out: An analysis from literature review of experiences from east and southern Africa[END_REF]). Nevertheless, the availability of VCT and ART has shown positive impact in the research area. This development is strongly supported by NGOs (such as NAPHAM) and local initiatives (such as locally established CBOs). For example, some respondents in the 'affected' category reported that they were monitoring their CD4 count, following dietary recommendations and the ART instructions (if they were on therapy), and urging others to go for VCT. These respondents were willing to discuss AIDS-related experiences with confidence. The importance of children expressed in the narratives further emphasises the need for expansion of ART programmes to children as well as increased access for children to VCT services.

Changing perceptions

The AIDS epidemic has stimulated reflection on traditional social, sexual norms and behaviours [START_REF] Kaler | My Girlfriends Could Fill A Yanu-Yanu Bus": Rural Malawian Men's Claims About Their Own Serostatus[END_REF][START_REF] Smith | Perceptions of risk and strategies for prevention: responses to HIV/AIDS in rural Malawi[END_REF]. In this study, access to VCT and ART services and the presence of social organisations is reported to trigger perceptual changes that help reduce stigma and an opening-up of public discussion. While those in the secondary (indirect) and especially in the non-affected impact categories tended to show fear and anxiety about their future, those in the primary (direct) impact category, as well as some in the secondary, seemed to be empowered by sharing experiences in the new social organisations triggered by the epidemic, and were starting to feel positive about their lives. This discussion leads to a new hypothesis: while traditional social immunity through extended families is collapsing, a higher threshold of social immunity could emerge through social organisation, starting, counter-intuitively, among those with higher exposure. It is these individuals -who might seem the most impacted -who are becoming involved in social organisation or community-based support groups for counselling and care, so that they have access to social institutions that are helping them to cope. We hypothesise that this

Conclusion

Our life history analysis provides four recommendations that are important for public health programming:

-Recognising the wide range of risks which coexist with the risk of HIV infection.

-Learning about one's own seropositive status as an important turning point in terms of behavioural changes.

-Recognising VCT and ART services and community-based organisations as supportive of behavioural changes.

-Supporting children and schooling opportunity among AIDS-affected communities as core elements.

Affected individuals, who gain an understanding of their risk exposure and strengthen their individual control over the risks by sharing knowledge and experiences through social organisation, could play central roles in emerging social organisations that over time could re-create social capacity and check the downward spiral proposed by the 'New Variant Famine' hypothesis. To promote this shift and to confer a higher level of 'social immunity', investments in expanding access to VCT and ART services, and assistance to community-based organisations would be essential. Codes 2)

F

Family structure:

28 codes children( 205) death( 149) marrying( 118) mother( 118) husband( 98) father( 89) siblings( 44) brother(32) co-wives(32) parents( 32) relatives( 31) childcare( 23) divorce( 22) sister( 21) wife( 19) unplanned pregnancy( 17) orphans(15) step children( 12) grandchildren(9) step mother(8) child fosterage( 7) grandparents( 7) guardians( 6) partnership( 6) uncle( 6) polylgyny(5) family planning(1) step-father(1)

Health problems:

18 codes death(149) health problem(79) HIV/AIDS(25) hospital( 17) herbalist( 15) serostatus( 15) caring the sick( 14) mental problem( 13) HCT( 11) ART( 10) medication( 10) nutrition( 8) doctor(5) frequent illness( 5) malaria( 4) TB(3) HBC(3) weight loss(1)

Risk exposure:

29 codes death( 149) money( 142) travelling( 115) employment(73) stop school( 59) business( 58) advice( 31) divorce( 22) conflict( 21) 'bad' behaviour(20) unplanned pregnancy( 17) violence( 16) teacher( 16) orphans(15) illicit behaviour( 14) police( 7) poverty( 7) imprisonment( 6) unfaithfulness( 6) witchcraft( 6) military( 5 18) CBO( 17) government( 17) hobby( 16) FFS( 12) HCT( 11) friends(8) farmers club(7) volunteer work( 7) fishing( 6) guardians( 6) partnership( 6) leadership(6) political party(5) choir( 5 C (no evident impact): He or she reported that either they had had a blood test with negative results

or did not report their serostatus but had no known AIDS cases in their family trees.

  At the time of the research, services for voluntary counselling and testing (VCT) and antiretroviral therapy (ART) were becoming accessible in Malawi. Between 2005 and June 2007, ART coverage fold countrywide: from 38,000 to 114,000 (Office of the President and Cabinet and Republic of Malawi 2007). In 2006, 21% of those with advanced HIV infections had access to ART in Malawi (World Health Organization 2008). However, access to medical and psychosocial support for children affected by AIDS was relatively low in the northern Malawi in 2006 (Office of the President and Cabinet and Republic of Malawi 2007).

  here] Only 'Social organisation/capital' in Kachere village (Figure 2-B) scored significantly low compared to the other two locations (p≤0.05). The frequency of conceptual clusters recorded during the narrations associated neither with gender nor impact levels.

  receiving therapy. These collective actions are therefore gradually taking up the conventional roles of extended families by providing support to the affected. Their life histories demonstrate awareness and control over their fate and in some cases, reaching out to others, to form new social networks that replace or augment support formerly provided through the family.

  reduce risks and might eventually open up possibilities for new livelihood opportunities.

  money(142) hardship(63) stop school(59) food(36) domestic work (22) conflict(15) clothes(15) caring for the sick(14) accident(11) house(8) hunger(8) nutrition(8) poverty(7) dowry(6) discrimination(3) funeral(3) labour loss(3) drought(2) stigma(2

  was based on interpretation of life history narrations, personal communication with local

Figure 1

 1 FIGURE LEGENDS

Table 1 . Conceptual clustering of codes cited during the life history narrations

 1

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

Acknowledgements

The authors wish to thank the local collaborators at Bunda College, University of Malawi and agricultural extension staff in Chilumba. The paper is an outcome of PhD research project at Wageningen University, funded by Japan International Cooperation Agency, Foundation for Advanced Studies on International Development, Murata Science Foundation, and Matsushita International Foundation.

(2) Kachere village

Key

(1) Mwandovi Village: cassava as a subsistence crop, fishing for food and income

(2) Kachere Village: cassava as a subsistence crop, some grow rice and tobacco for cash income

(3) Hara Irrigation Scheme: based on cash income from rice production A (primary level): He or she reported that they had had positive results from the blood test, or their previous partners or their former spouses were known AIDS cases. assumed as 'people living with HIV/AIDS': PLWHA B (secondary level): He or she did not report their own serostatus, but reported known AIDS cases in their family trees.


Riitta ; Koivikko
email: riitta.koivikko@jrc.ec.europa.eu

Sarah Pastorelli

Ana Rodríguez-Bernaldo De Quirós

Rafael Paseiro-Cerrato

Perfecto Paseiro

Catherine Simoneau

Rodríguez-Bernaldo De Quirós

Rapid multi-analyte quantification of benzophenone, 4-methylbenzophenone and related derivatives

Keywords: Chromatography -HPLC, Extraction, Chromatography -GC/MS, Regulations Packaging -inks, Food contact materials, Packaging recycling

especially for its combined analysis with others. We present an HPLC method with diode 2 array detector to simultaneously determine the levels of BP, 4-MBP as well as 7 other 3 possible derivatives from secondary packaging for food applications. The method was 4 tested and applied to 46 samples of paperboard for secondary packaging collected both 5 from supermarkets and directly from a paperboard supplier. In addition, a survey was 6 conducted on recycled paperboard (n= [START_REF] Macdougall | Guidelines for data acquisition and data quality evaluation in 20 environmental chemistry[END_REF]) collected from a supplier, to evaluate the 7 background quantity of BP and other derivatives in recycled board. The most abundant 8 photo-initiator found in the survey was BP, in 61% of samples, and 4-MBP was found in 9 30% of the samples. It seems that these compounds are used to replace one another. 10 Other derivatives were found in minor quantities. Traces of BP were also found in 42% 11 of the samples of recycled, unprinted board. 12 13 Keywords: benzophenone; 4-methylbenzophenone; photo-initiators; recycled board 14

Running head: Multi-analyte quantification of photo-initiators 15

Introduction 1

German authorities recently reported a high migration of 4-methylbenzophenone (4-2 MBP) from packaging into cereals using the Rapid Alert System for Food and Feed 3 (RASFF). Following this alert, the European Commission asked the European Safety 4 Authority to evaluate whether 4-MBP should be included in the current tolerable daily 5 intake (TDI) for benzophenone (BP) and hydroxybenzophenone. The risk of the presence 6 of 4-MBP was also to be evaluated (EFSA 2009a). Later, the Scientific Panel on food 7 contact materials, enzymes, flavourings and processing aids (CEF) published a scientific 8 opinion on its toxicity, concluding that short term consumption of cereals containing 4-9 MBP at observed levels should not constitute a risk to the population. However, if the 10 consumption of the compound is continuous, more data on the occurrence as well as 11 proper toxicity data of the compound is needed (EFSA 2009b). 12 Photo-initiators are used in inks hardened with ultraviolet (UV) light (Anderson 13 and Castle 2003). Their benefits include reduced drying times compared to water-based 14 coatings, allowing a continuous production process [START_REF] Anderson | Benzophenone in cartonboard packaging materials and 2 the factors that influence its migration into food[END_REF]. As UV 15 inks usually do not contain any traditional organic solvents, they are considered 16 environmentally friendly (Papilloud and Baudraz 2002). UV inks are used to produce an 17 appealing glossy cover to the printed packaging, especially for paperboard, and high 18 amounts of photo-initiators are used for this purpose in the ink preparation. One of the 19 most known photo-initiators is BP. It has been determined to migrate from printed board 20 into the food as it is neither totally used up during the printing process, nor removed 21 afterwards, nor irreversibly bound into the print film layer. The migration of photo-22 initiators from the packaging to the food through the vapour phase is possible even from 23 BP is 0.6 mg kg -1 for its use as an additive in plastics. Recently, due to the high levels 14 detected, the European Food Safety Authority (EFSA) published recommendations and 15 the Standing Committee for the Food Chain and Animal Health endorsed a limitation to 16 0.6 mg kg -1 for the sum of BP and 4-MBP (SANCO 2009). 17

As the paperboard of secondary packaging is widely printed with the UV inks, it 18 is important to bear in mind that the most commonly used raw material for paperboard is 19 recycled and there the levels of photo-initiators, like BP, might persist or even 20 accumulate [START_REF] Anderson | Benzophenone in cartonboard packaging materials and 2 the factors that influence its migration into food[END_REF]

Materials and methods 4

Packaging and food samples 5

The samples for the study were collected from the supermarkets (with the food, n = 22) 6 and some packages were acquired from the industrial production lines prior to 7 introducing the foodstuff (n = 24) (Table 1). As paperboard for secondary packages is 8 commonly made from recycled material, a survey was also conducted with recycled, 9 unprinted board material (n = 19). 10 11

Chemicals and standards 12

Acetonitrile (ACN, CAS n 75-05-8) was supplied both from Carlo Erba and Advanced 13 Biotech and methanol (CAS n 67-56-1) was supplied from Sigma-Aldrich. Solvents used 14 were high purity grade. Ultrapure water was prepared with Milli-Q filter system 15 (Millipore, Bedford, MA, USA). 16

Standards 4-hydroxybenzophenone 98% (CAS n 1137-42-4), methyl-2-17 benzoylbenzoate 97% (606-28-0), 2-hydroxybenzophenone 99% (117-99-7), 4-methyl 18 benzophenone (4-MBP) 99% (134-84-9), 4-benzoylbiphenyl (PBZ) 99% (2128-93-0), 19 4,4'-bis(diethylamino)benzophenone 99+% (90-93-7), and 2-ethylhexyl-4-20 (dimethylamino)benzoate 98% (21245-02-3) were obtained from Aldrich and 21 benzophenone (BP) ≥99% (119-61-9) was supplied by Fluka. Benzophenone acrylate 22 (BPacr) was obtained directly from a supplier (CAS 59626-79-8). 23

Standard solutions 2

A stock solutions (2000 µg mL -1 of ACN) of 4-hydroxybenzophenone, methyl-2-3 benzoylbenzoate, 2-hydroxybenzophenone, 4,4'-bis(diethylamino)benzophenone and 2-4 ethylhexyl-4-(dimethylamino)benzoate we prepared and diluted into the intermediate 5 standard solutions (100 µg mL -1 of ACN). Also the stock solutions of BP, 4-MBP and 6 PBZ (900 µg mL -1 of ACN) as well as of BPacr (2.4 mg mL -1 of ACN) were prepared and 7 diluted into the intermediate standard solutions (180 µg mL -1 of ACN; for BPacr 120 and 8 360 µg mL -1 of ACN). The calibration curves were obtained with further dilutions (in 9 ACN) from the intermediate standard solution in the ranges of 4.5-117 µg mL -1 (8-14 10 points) for BP, 4-MBP and PBZ and 12-250 µg mL -1 (11 points) for BPacr. For methanol 11 studies, separate standard solutions were made into methanol. Solutions were stored 12 refrigerated in the dark. 13 14

Sample preparation 15

The board material from the food packages, and in some cases the food as well, were 16 analyzed with modifications of methods previously used for BP and other photo-initiators 17 For some samples (see Table 1), the content of photo-initiators in the food was 1 also analyzed. The food was homogenized and a portion (m = 1 or 4 g) was placed into a 2 glass container and extracted tightly-closed with acetonitrile (V = 3 mL) for 24 h at 70°C. 3

The extract was cooled down to room temperature, filtered through 0.45 µm PTFE filter 4 and analyzed with HPLC-DAD. 5

Primarily, the paperboard extractions were done with acetonitrile (V = 20 ml) as 6 described above, but the use of methanol (V = 20 ml) as extraction solvent was also 7 tested. 8 9

Chromatographic conditions 10

HPLC-UV 11

Analytical chromatographic analyses were performed with different HPLC systems using 12 a method adopted and modified from a previously published method for benzophenone 13 The binary mobile phase consisted primarily of acetonitrile and water. The 20 gradient elution was from 50% acetonitrile in water (v/v) to 100% acetonitrile in 10 min 21 (linear); 100% acetonitrile was kept for 5 min (isocratic), back to 50% acetonitrile in 1 22 min (linear) and it was kept for 4 min (isocratic) before the next run. The total run time 23 and the calibration curves were done separately for each compound and instrument using 2 the appropriate wavelength (see Table 2 for precise numbers). The flow rate was 1.0 mL 3 min -1 and the injection volume 50 µL. 4

Additional experiments were done by replacing acetonitrile with methanol, 5 otherwise keeping the same method parameters. The samples for this experiment were 6 chosen among the total 46 samples included to the survey (samples in which BP or 4-7 MBP was present) and further identified with letters A-J (as only the difference between 8 two solvents was under interest). With these samples (A-J), we also studied the stability 9 of the samples. 10 11

GC-MS 12

The gas chromatograph system consisted of a Hewlett Packard 6890 Series gas 13 chromatograph equipped with an autosampler and a programmed split/splitless injector, 14 and a Hewlett Packard 5973 mass selective detector with MSD ChemStation D.02.00.275 15 software. The column was DB-5HT (5%-Phenyl-methylpolysiloxane, Agilent J&W, 30 m 16 × 0.25 mm i.d., 0.1 µm thickness). 17

The carrier gas was helium with flow of 1.3 ml min -1 . The injector temperature 18 was 250°C and the samples were injected with a split mode (ratio 1:40). The injection 19 volume was 1 µl. The oven was running with temperature programme: 100°C for 2 min, 20 followed by a rate of 10°C min -1 up to 250°C, which was held for 10 min. Finally, the HPLC method introduced here managed to separate BP, 4-MBP and 7 other 14 related derivatives within a rather short time of analysis (Figures 2 and3). The retention 15 time for BPacr overlapped with the PBZ and 4,4'-bis(diethylamino)benzophenone, but 16 the identification was possible because their UV spectra differed (Fig. 4). Further, BPacr 17

was not found to be present in the same samples as PBZ and 4, 4'-18 bis(diethylamino)benzophenone. With the current method, BPacr characteristically 19 creates a bunch of peaks in HPLC, which facilitates its identification. 20

The stability of the sample extracts was studied according to their contents of BP 21 and 4-MBP (Fig. 5). The extracts were analysed both immediately and after three to five 22 months of storage (in hermetically closed vials in 4°C or in room temperature). The 23 E) between two measurements (analyses done in duplicates in both times) when the 2 contents were 0.20 mg dm -2 or higher. Further, it could be seen that as the concentrations 3 of analytes decreased, the variation between immediate and after storage analyses 4 increased. 5

Calibration curves were constructed for BP, 4-MBP, PBZ and BPacr with 6 different HPLC systems (see Table 2). Detection (LOD) and quantification limits (LOQ) 7

were established according to the Guidelines of the American Chemical Society (ACS) 8 (MacDougall et al. 1980). BPacr is a mixture of oligomers of which it was possible to 9 create the calibration curve integrated over the cluster of peaks (Table 2, Fig. 3). 10 11

The effect of solvent in extraction and elution 12

Due to the recent shortage in the production and on the market of acetonitrile (Tullo 13 2008), we tested the possibility of replacing it with methanol during the extraction and 14 elution (samples were done in duplicates). The results were relatively similar for BP and 15 4-MBP. The retention times turned out to be a bit longer for all the standards with 16 methanol, but the measured concentrations (calculated from the peak areas) remained 17 quite stable. BP results achieved with the different extraction and elution solvents 18 differed on average 6% in respect to the peak areas (C, F, G), but more with some 19 samples (A: 22% and E: 62%). The measurements made for the five samples containing 20 4-MBP differed from 2.3 to 9.4% in respect to the peak areas. The most evident 21 difference between the solvents was the elution of BPacr. The cluster of peaks observed 22 with acetonitrile (Fig. 3) is lost when eluted with methanol; BPacr gave only one peak 23 and in elution, and as a solvent when preparing the standards, especially in the analysis of 2

BP. 3 4

Analysis of samples 5

The contents of benzophenone and derivatives 6

The most abundant UV-initiator found in this survey was BP. We found it in quantitative 7 levels in 27 samples (59%) of the total 46 samples, whereas 4-MBP was found in 14 8 samples (30%) (Table 1). It appeared that BP and 4-MBP were not usually present in the 9 same samples. When the level of BP is high in the sample, there is no (or only traces of) 10 4-MBP, and vice versa. It seems that these compounds are used to replace one another. 11 Measurable levels of PBZ were found in three samples, #11; 13 and 21, where the 12 levels of the compound were 97, 21 and 36 mg m -2 , respectively. Identification was 13 verified with GC-MS. In four samples, #14-17, we found BPacr in contents of 151.5; 14 792.6; 819.5 and 815.1 mg m -2 , respectively. The other benzophenone related derivatives 15 included in the analysis were not found in the products in the survey. 16 Some of the carton packages were printed with two different techniques. Part of 17 the package was printed with UV-ink to achieve a shiny result whereas another part was 18 printed with conventional water-based ink not containing photo-initiators. Theoretically, 19 this should have given rise to differences in the contents of photo-initiators in different 20 parts of packages. Contrary to assumptions, in spite of different printing ink types on 21 different parts of the package no differences could be seen between different parts of 22 In the survey of recycled, unprinted board material (n = 19), BP was found in 3 detectable levels in nearly half of the samples (42 %, 8 samples). The content of BP in 4 those samples varied from 0.57 to 3.99 mg m -2 (the board material varied from 2.78 to 18 5 mg kg -1 in weight). Other derivatives studied here were not found in measurable levels 6 from recycled, unprinted board material. 7

After finding high contents of BP in sample 1 and 4-MBP in sample 2, we also 8 analysed the products. Despite high contents of photo-initiators in the package and their 9 high migration ability, we found only very scarce levels of photo-initiators in the 10 homogenized product (below the SML (SANCO 2009)). Therefore, the plastic film used 11 as primary packaging in direct contact with the product was also analysed. The analyses 12 were done with the attenuated total reflection (ATR-IR) spectroscopy which is used for 13 surface analysis. The plastic film in sample 2 was found out to be polypropylene whereas 14 the plastic film in sample 1 was found to be consisted of different layers one side being 15 polyethylene terephthalate (PET) and the other side polypropylene (PP). Hence, it 16 seemed to be a multilayer material preventing the migration of BP. Multilayer material 17 consisting of PET/silic oxide/PP has been reported to prevent BP migration (Pastorelli et 18

al. 2008). 19 20

Additional drying step for inks including photo-initiators 21

The board samples acquired from the industrial production lines included samples from a 22 test of the different oven treatments for drying the UV ink after the printing process 23 3). The additional drying procedures were unable to push the printing process to 1 decreased levels of BP and 4-MBP. The most drastic treatment (Table 3, treatment IV) 2 with the most severe oven drying failed to produce lower levels of these photoinitiators. 3

The content of BP was initially very low and its concentration was barely affected by the 4 treatments. The concentrations of 4-MBP were initially higher but none of the additional 5 treatments decreased the levels of 4-MBP even by half, the lowest achieved concentration 6 was still as high as 65% of the initial concentration. BPacr turned out to be most 7 abundant after treatment III (Table 3), the same treatment where 4-MBP levels were the 8 lowest which suggests that BPacr might be a product of other photoinitators. Following 9 these results, it can be claimed that additional oven drying after printing does not bring 10 any further benefits. 11 12

Conclusions 13

The described method allowed a rapid multi-analyte quantification of benzophenone, 4-14 methylbenzophenone and 7 other possible derivatives from paperboard secondary 15 packaging for food applications by HPLC-DAD. The survey was carried out with 46 16 samples of paperboard for secondary packaging. Results showed that BP and 4-MBP are 17 not usually present in the same packaging, but rather seem to replace one another. More 18 than half the samples included BP, 30% of samples included 4-MBP, 9% of the samples 19 included BPacr and 7% of the samples included PBZ. This survey indicates that, 20 generally a high concentration of one photo-initiator and traces of other possible 21 substitutes are found. Those traces might also exist as residual levels of formerly used 22 printing inks remaining on recycled paperboards, as demonstrated by the survey on non-23

  photo-initiators in the past, there is a fundamental lack of data on 4-MBP 1

  packaging, i.e. when the ink is not in direct contact with the food(Johns et 1 al. 2000, Anderson and Castle 2003, Triantafyllou et al. 2007, Pastorelli et al. 2008, 2 Rodríguez-Bernaldo de Quirós et al. 2009). The rate of the migration of photo-initiators 3 through the vapour phase is highly influenced by their boiling point and the vapour 4 pressure as well as by the type of the food package. The commonly used BP is known to 5 migrate easily through the vapour phase and it has been substituted with other derivatives 6 over the last few years (Pastorelli et al. 2008). 7 4-MBP is an example of a photo-initiator used either instead or, more rarely, 8 together with BP (EFSA 2009a). At the present time, printing inks and/or cardboard 9 boxes are not covered by specific European legislation on food contact materials. 10 Nevertheless, the use of printing inks has to comply with the general rules of Regulation 11 (EC) No 1935/2004 and with good manufacturing practice as laid down in Commission 12 Regulation (EC) No 2023/2006 (EFSA 2009a). The specific migration limit (SML) for 13

(

  Pastorelli et al. 2008, Sanches-Silva et al. 2008a). Two different samples of 0.5 dm 2 18 were prepared from each packaging material. Each sample was cut into small pieces, 19 placed into a glass container and extracted tightly-closed with acetonitrile (V = 20 mL) 20 for 24 h at 70°C. The extract was cooled down to room temperature, filtered through 0.45 21 µm PTFE filter and analyzed with HPLC-DAD. 22

  only (Pastorelli et al. 2008). 14 The used HPLC was an Agilent technologies 1200 series degasser and diode array 15 detector equipped with Hewlett Packard 1100 series binary pump and autosampler. The 16 software was Chemstation (2001-2007, Rev.B.03.01[137]). The column was an Eclipse 17 XDB-C18 (250 × 4.6 mm i.d.; 5 µm; Agilent Technologies). The tests were performed 18 separately for each system with different standards and calibration curves. 19

  . The detection wavelengths varied according to the HPLC system used 1

1 0 7 8

 17 Total run time 21was 27 min. The mass spectra were obtained with a mass selective detector under 22 at a voltage of 70 eV and data acquisition was at a scan rate of described here, it was possible to simultaneously analyse the 5 contents of benzophenone and 4-methylbenzophenone as well as seven related 6 derivatives.Optimization of the extraction and chromatographic methods and the method 9 validation 10The single-solvent extraction of samples was carried out for 24 h at 70 °C which 11 has previously proved to provide complete extraction of BP(Pastorelli et al. 2008). 12 Different elution programmes were tested for the selected column and compounds. 13

  BP and 4-MBP varied only up to 4.6 % (variation of BP in sample 1

  It is accordingly recommended to use the same solvent both for extraction 1

  gives further proof of the migration of BP (Rodríguez-Bernaldo de Quirós 1 et al. 2009). 2

FoodFoodFoodFigure 1 . 4 Figure 2 . 7 Figure 3 . 9 Figure 4 .Figure 5 .

 14273945 photo-initiators to use and/or avoid belongs to the whole production line including the 6

  Chemical structures of the photo-initiators BP (1) and 4-MBP (2). 173x79mm (96 x 96 DPI) BPacr, PBZ and 4,4'-bis(diethylamino)benzophenone. 152x123mm (150 x 150 DPI) The content of (a) BP and (b) 4-MBP in samples A-J. Grey bars represent analyses done immediately after extracting samples, white bars represents analyses on hermetically closed extracts stored 3-5 months in dark and cold (A-G) or at room temperature (H-J). 150x194mm (150 x 150 DPI) Page 28 of 27 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

Table 1 .

 1 Contents of BP and 4-MBP in 46 paperboard samples.

	Sample number	Sample type	Supplier	BP b (mg dm -2 )	4-MBP (mg dm -2 )
	1 a	cake	Supermarket	2.56	n.d.
	2 a	cake	Supermarket	0.07	1.47
	3	cereals	Supermarket	n.d.	n.d.
	4	cereals	Supermarket	n.d.	n.d.
	5	cookies	Supermarket	n.d.	n.d.
	6	dried fruits	Supermarket	n.d.	n.d.
	7	dried fruits	Supermarket	n.d.	n.d.
	8	cookies	Supermarket	n.d.	n.d.
	9	cake	Supermarket	2.10	n.d.
	10	cake	Supermarket	0.06	2.24
	11	cereals	Supermarket	0.05	n.d.
	12	crêpes	Supermarket	3.38	n.d.
	13	cake	Industry	0.04	1.69
	14	cake	Industry	0.10	4.41
	15	cake	Industry	0.22	n.d.
	16	cake	Industry	n.d.	n.d.
	17	cake	Industry	0.06	n.d.
	18	coffee	Industry	3.99	0.05
	19	cake	Industry	0.10	4.33
	20	cookies	Industry	1.57	n.d.
	21	cookies	Industry	0.02	n.d.
	22	cake	Industry	0.09	0.98
	23	cake	Industry	0.21	1.10
	24	cake	Industry	2.23	n.d.
	25	cake	Industry	n.d.	n.d.
	26	cake	Industry	3.21	n.d.
	27	cake	Industry	1.43	n.d.
	28	cake	Industry	3.02	n.d.
	29	cake	Industry	2.97	n.d.
	30	cake	Supermarket	1.66	n.d.
	31	cake	Supermarket	3.84	0.05
	32	cake	Supermarket	n.d.	n.d.
	33	chocolate	Supermarket	1.07	n.d.
	34	cake	Industry	0.02	1.00
	35	cake	Supermarket	0.35	1.04
	36	cake	Industry	2.98	n.d.
	37	cake	Supermarket	n.d.	n.d.
	38	chocolate	Industry	n.d.	n.d.
	39	chocolate	Industry	n.d.	1.48
	40	chocolate	Industry	n.d.	n.d.
	41	chocolate	Industry	n.d.	0.03
	42	chocolate	Industry	n.d.	1.47
	43	nuts	Supermarket	n.d.	n.d.
	44	pastry	Supermarket	n.d.	n.d.
	45	pastry	Supermarket	n.d.	n.d.
	46	cake	Supermarket	n.d.	n.d.

a The food was analysed b n.d. = not detectable

Table 2 .

 2 Validation parameters for the method to measure benzophenone and other derivatives in food packaging paperboard.

	Food Additives and Contaminants		Page 22 of 27
		Intercept	Slope	r 2	Range [mg L -1 ]	LOD [mg L -1 ]	LOQ [mg L -1 ]
	BENZOPHENONE
	wl = 256 nm, rt = 8.03 min	154.42	265.88	0.9996	0.46-118.4	0.046	0.14
	4-METHYL BENZOPHENONE
	wl = 260 nm, rt = 9.1 min	290.41	237.56	0.9991	0.46-118.3	0.046	0.14
	BENZOPHENONE ACRYLATE
	wl = 256 nm, rt = 10.8-12 min	151.3	74.36	0.9996	0.45-253.05	1.22	3.67
	4-BENZOYLBIPHENYL
	wl = 290 nm, rt = 11.15 min	844.42	201.01	0.9916	0.45-118.0	0.045	0.14

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Table 3 .

 3 Levels of three photo-initiators on printed paperboard samples after different additional drying trials.

	Page 23 of 27
		BP	4-MBP BPacr
			(mg dm -2 )
	Basic procedure	0.10	4.41	1.52
	Additional treatments (oven drying)
	I: 200 ºC for 10 min	0.12	4.38	1.20
	II: 200 ºC for 2 x 10 min	0.13	4.64	1.26
	III: 200 ºC for 20 min	0.14	2.86	3.25
	IV: 200 ºC for 2 x 20 min	0.12	4.49	1.19

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Acknowledgements 9

The authors are grateful to Mrs. Giorgia Beldì, Mr. Sandro Valzacchi and Gruppo 10 FINCARTA Spa for their technical assistance. 11 12

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