Monica Martinez-Haro

Andy J Green

Pelayo Acevedo

Rafael Mateo
email: rafael.mateo@uclm.es

Use of grit supplements by waterbirds: an experimental assessment of strategies to reduce lead poisoning

Keywords: Gastrolith, Hunting, Lead poisoning, Wildlife conservation, Wildlife management

The ingestion of spent Pb shot due to confusion with grit or inadvertently with food particles causes Pb poisoning in a large number of waterbirds, this being one of the main causes of mortality for some species. Lead ammunition for hunting is being progressively banned in more countries, while grit supplementation has been proposed as a management measure to reduce the ingestion of deposited Pb shot. Studies of grit selection with waterfowl in semicaptivity and in the wild were undertaken to evaluate preferences in the colour and geochemical composition of grit, whether it was available dry or in water, its position within the wetland and the relationship between grit ingestion and feeding behaviour. Grit ingestion was higher when food was included in the treatments. In the absence of food, red grit was taken in higher amounts than grey in semi-captivity but not in the wild. Siliceous grit was taken in a higher amount than calcareous when offered dry, but not in water. No differences in the amount of ingested grit were found among different positions within the wetland. The number of feeding attempts in plots supplemented with grit was higher than in those without grit, although the highest numbers of feeding birds were found in plots supplemented with food. Grit ingestion in waterfowl is intimately associated with feeding behaviour. To optimize the effectiveness of grit supplementation to reduce the risk of Pb poisoning in waterfowl, calcareous and siliceous grit may be combined and applied in feeding sites or mixed with bait to attract birds.

Introduction

Gastroliths, more commonly known as grit, are used by birds to grind food in the gizzard and in some species also as a dietary supplement [START_REF] Gionfriddo | Grit use by birds: a review[END_REF]. However, this implies a risk of exposure to toxic metals depending on the geochemical composition of grit [START_REF] Bendell-Young | Grit ingestion as a source of metal exposure in the spruce grouse, Dendragapus canadensis[END_REF]. Moreover, many species of aquatic and terrestrial birds ingest Pb shot pellets as grit or inadvertently with food particles and thus suffer from Pb poisoning [START_REF] Trost | Dynamics of grit selection and retention in captive mallards[END_REF]Mateo and Guitart 2000). Lead shot ingestion is therefore a threat for birds inhabiting intensively hunted environments with low availability of grit, e.g. coastal wetlands [START_REF] Pain | Lead shot ingestion by waterbirds in the Camargue, France: an investigation of levels and interspecific differences[END_REF][START_REF] Mateo | Lead shot pellets in the Ebro delta, Spain: densities in sediments and prevalence of exposure in waterfowl[END_REF], uplands or shooting ranges [START_REF] Ferrandis | Lead-shot exposure in red-legged partridge (Alectoris rufa) on a driven shooting estate[END_REF][START_REF] Thomas | Bone lead levels and lead isotope ratios in red grouse from Scottish and Yorkshire moors[END_REF].

Heavily hunted wetlands worldwide have accumulated spent Pb ammunition above 100 shot per square metre in the upper 20 cm of sediments [START_REF] Pain | Lead poisoning of waterfowl: a review[END_REF][START_REF] Mateo | Lead poisoning in wild birds in Europe and the regulations adopted by different countries[END_REF].

Annual mortality due to Pb poisoning in the USA before the ban of Pb shot use for waterfowl hunting was estimated as 2% to 3% (1.6-2.4 million birds) of the population of waterfowl [START_REF] Bellrose | Lead poisoning as a mortality factor in waterfowl populations[END_REF][START_REF] Friend | Field guide to wildlife diseases[END_REF]. In Europe, Pb poisoning is currently estimated to cause an annual mortality of 8.7% of the wintering populations of more than 11 million waterfowl from 17 species, i.e. a loss of almost one million waterfowl per winter [START_REF] Mateo | Lead poisoning in wild birds in Europe and the regulations adopted by different countries[END_REF]. In addition, an inverse correlation has been shown between Pb shot ingestion rates and population trends of waterfowl in Europe [START_REF] Mateo | Lead poisoning in wild birds in Europe and the regulations adopted by different countries[END_REF], and Pb poisoning is suggested as a major cause of mortality of several endangered species [START_REF] Fisher | A review of lead poisoning from ammunition sources in terrestrial birds[END_REF][START_REF] Taggart | Metal levels in the bones and livers of globally threatened marbled teal and white-headed duck from El Hondo, Spain[END_REF].

Despite efforts to reduce or eliminate the input of Pb shot in most wetlands [START_REF] Mateo | Lead poisoning in wild birds in Europe and the regulations adopted by different countries[END_REF], the problem often persists because spent Pb shot remains in soil for 100-300 years [START_REF] Rooney | Control of lead solubility in soil contaminated with lead shot: effect of soil pH[END_REF]). Thus, in addition to the ban of Pb ammunition, management practices such as land cultivation, regulation of water levels, and grit supplementation have been proposed to reduce the risk of Pb poisoning [START_REF] Godin | Test of grit types in alleviating lead poisoning in mallards[END_REF][START_REF] Sanderson | A review of the problem of lead poisoning in waterfowl[END_REF]Pain 1991;[START_REF] Thomas | Effects of tillage on lead shot distribution in wetland sediments[END_REF]. Grit supplementation has the advantages that it may be used in different types of habitats with low availability of grit, the environmental impact is relatively low, and it can be inexpensive in terms of material and labour cost. Grit supplementation has also been successfully applied for the control of parasitic infestations in red grouse (Lagopus lagopus scoticus; [START_REF] Newborn | Control of parasite burdens in wild red grouse Lagopus lagopus scoticus through the indirect application of anthelmintics[END_REF] and for a controlled gizzard release of contraceptives for the control of pest waterfowl [START_REF] Hurley | Poly(methyl methacrylate) synthetic grit formulations sustain the delivery of nicarbazin, a contraceptive agent, in pest waterfowl[END_REF].

Grit supplementation to reduce Pb poisoning in waterfowl was applied experimentally in 1958 in the Camargue (River Rhône delta, France; Hoffmann 1960). One year after the supplementation, a decrease in Pb shot ingestion from 39.4% to 8.3% was observed in tufted ducks (Aythya fuligula) but not in common teal (Anas crecca) or mallards (Anas platyrhynchos; Hoffmann 1960). More recently, grit supplementation helped reduce mortality by Pb poisoning due to the ingestion of Pb weights in mute swans (Cypus olor) at Cork Lough (Ireland;[START_REF] O'halloran | Trends in mute swan blood lead levels: evidence of grit reducing lead poisoning[END_REF][START_REF] Connell | Lead poisoning in mute swans (Cygnus olor) in Ireland: recent changes[END_REF]. Experiments with captive mallards showed that grit availability reduced both Pb shot ingestion (Mateo and Guitart 2000) and the effects of Pb poisoning [START_REF] Godin | Test of grit types in alleviating lead poisoning in mallards[END_REF][START_REF] Longcore | Toxicity of lead and proposed substitute shot to mallards. US Department of the[END_REF].

Several characteristics of grit may influence its use by birds, such as composition, particle size, colour or shape [START_REF] Gionfriddo | Grit use by birds: a review[END_REF]. Moreover, the chemical composition of grit may help reduce Pb absorption due to the presence of calcium or phosphates [START_REF] Heinz | Phosphorus amendment reduces bioavailability of lead to mallards ingesting contaminated sediments[END_REF][START_REF] Dauwe | Calcium availability influences lead accumulation in a passerine bird[END_REF][START_REF] Martinez-Haro | Avian digestive tract simulation to study the effect of grit geochemistry and food on Pb shot bioaccessibility[END_REF]. Grit composition has also been related to the retention time in the gizzard, calcareous grit (marble) having a shorter half-life than siliceous grit (quartzite ;[START_REF] Trost | Dynamics of grit selection and retention in captive mallards[END_REF]Mateo and Guitart 2000). In addition, the size of grit particles selected is an important characteristic for each species that is influenced by diet, being larger in granivorous species and more fine and abundant in herbivorous species [START_REF] Pain | Lead shot ingestion by waterbirds in the Camargue, France: an investigation of levels and interspecific differences[END_REF]Mateo et al. 2000a;Luttik and de Snoo 2004). However, grit size composition for a given species varies greatly between localities, whereas total grit quantity in the gizzard is a species attribute [START_REF] Figuerola | Grit selection in waterfowl and how it determines exposure to ingested lead shot in Mediterranean wetlands[END_REF].

Grit ingestion has been identified as independent of feeding behaviour for some species of geese (Anser spp.), which move toward certain areas, such as dunes or sandbars, to ingest grit (Mateo et al. 2000b;[START_REF] Lee | Grit-site selection of black particle size or calcium content?[END_REF]). However, there are few data about the gritting behaviour in most waterfowl species. In the present work, studies of grit selection with waterfowl in semi-captivity and the wild were undertaken to evaluate the relationship between grit ingestion and feeding behaviour, preferences in grit colour (red or grey), geochemical composition of grit (siliceous or calcareous), whether it is dry or in water, the position of grit within the wetland (shore, open water or on artificial platforms) and the use of areas supplemented with grit by different waterfowl species under field conditions. This information is essential to develop strategies of grit supplementation in wetlands contaminated with Pb shot.

Material and methods

The study was conducted in semi-captivity and in the wild in 2007 and 2008. Semi-captive experiments were conducted in a Concerted Nature Reserve (CNR) for birds (Cañada de los Pájaros) in Andalusia near to Doñana Natural and National Park (south Spain; Fig. 1). This reserve contains a gravel pit with semi-natural management where food is supplied daily for captive waterfowl and where free-ranging waterfowl also come to feed from the surrounding wetlands. The most common species present at CNR were common coot (Fulica atra), common pochard (Aythya ferina), geese, mallard, red-knobbed coot (Fulica cristata) and shoveler (Anas clypeata).

Experiments in the wild were performed at Medina Lagoon (ML), a 120-ha Natural Reserve in Andalusia (Fig. 1). Owing to its importance for waterbirds [START_REF] Amat | Las poblaciones de aves acuáticas en las lagunas andaluzas: composición y diversidad durante un ciclo anual[END_REF][START_REF] Martí | La invernada de aves acuáticas en España. Dirección General de la Naturaleza-SEO/BirdLife, Organismo Autónomo Parques Nacionales[END_REF], it was protected under the Ramsar convention in 1989. It is particularly important for red-knobbed coot and the globally threatened white-headed duck (Oxyura leucocephala). Further, ML has the highest density of Pb shot pellets in sediments, resulting from hunting activity, recorded for any European natural wetland, with 399 shot per square metre in the upper 30 cm [START_REF] Mateo | Lead poisoning in wild birds from southern Spain: a comparative study of wetland areas and species affected, and trends over time[END_REF].

The experiments were conducted with grit of a fixed density 2.6 g/ml, siliceous grit (crushed quartzite), pur-chased from a pet shop (aquarium fine gravel) and calcareous grit (crushed marble) from a local floor tile factory. Before undertaking experiments, grit was thoroughly washed with tap water and dried. Finally, grit was sieved with mesh sizes of 1 and 4 mm to obtain particles within this range, in accordance with that most commonly found in gizzards of waterfowl species most affected by Pb poisoning [START_REF] Pain | Lead shot ingestion by waterbirds in the Camargue, France: an investigation of levels and interspecific differences[END_REF]Mateo et al. 2000a). Two different approaches were used to monitor grit ingestion in the experiments. The first one used in semi-captivity and the wild (September 2007 to March 2008) involved the monitoring of amounts of grit taken by birds from PVC watertight gutters (containers) of 50 cm long×20 cm wide× 12 cm high. After all the experiments carried out with this approach, remaining grit was sieved to separate and weigh the fraction of 1-4 mm. The second one, designed on the basis of results from the other experiments, was to record the number of waterbirds feeding in the wild in plots of 15×15 m seeded with different treatments (September 2008).

Experiments on grit ingestion

Experiment 1-September-October 2007 Grit colour is important for grit selection in several bird species [START_REF] Gionfriddo | Grit use by birds: a review[END_REF]. Two types of calcareous grit (red or grey) were offered in containers with or without wheat grain in a 1:4 ratio (grit/grain, v/v). The mass of grit taken from each one of the four treatments (three containers per treatment) was recorded and replaced daily during 7 days at CNR and every 4 days during 3 weeks at ML. Water was not added to the containers so as to facilitate the visual selection of grit by birds.

Experiment 2-February 2008 Differences in grit ingestion may depend on the flooding level and the presence of food at sites where waterfowl search for food and/or grit [START_REF] Lee | Grit-site selection of black particle size or calcium content?[END_REF]). To test these effects, red calcareous grit was offered in 12 containers located on the shore of the lake at CNR. Grit was offered alone or mixed with wheat in a 1:4 ratio (grit/grain, v/v) and with or without tap water at a depth of 5 cm. The mass of grit taken from each one of the four treatments (three containers per treatment) was recorded and replaced daily during 5 days at CNR. Three additional groups of two fenced containers, with henhouse mesh of 41 mm, were placed alongside experimental containers and used as controls to check for grit removal by non-waterfowl (e.g. passerines or rats (Rattus norvegicus)) during the experiments.

Experiment 3-March 2008

The geochemical composition of grit may influence the visual and tactile selection of grit by birds. Calcareous or siliceous grit was offered in containers with or without wheat grain in the same proportion as in the previous experiments. The mass of grit taken from each of the four treatments (three containers per treatment) was recorded and replaced daily during 5 days at CNR. As geochemical composition may affect tactile selection in water because of differences in the shape and surface texture of grit particles, this experiment was also repeated with containers with 5 cm depth of water. The calcareous grit used in our experiments was generally more flattened, and the siliceous grit was more rounded in shape. Since the shape of grit is largely affected by its geochemical nature, we cannot rule out an effect of shape on grit selection in our experiments. As in experiment 2, six additional fenced containers were used as controls.

Experiment 4-September 2008 Some waterfowl species show preferences for specific environments to pick up grit (e.g. greylag geese (Anser anser), Mateo et al. 2000b; purple gallinule (Porphyrio porphyrio), see Renner 2010). This experiment was performed at ML to study differences in grit ingestion from 18 containers located (1) on the shore of the lagoon, (2) on the water surface at 40 cm from the bottom and 1 m from the shore or (3) on wooden platforms located 4 to 5 m from the shore and 1 m from the bottom. A mixture of calcareous and siliceous grit (1:1, v/v) was offered with or without wheat at a ratio of 1:1 (grit/grain, v/v).

To limit the consumption of grain by rats, all the containers were filled with lagoon water. The mass of grit taken from each one of the six treatments (three containers per treatment) was recorded and replaced every 2 days for 2 weeks. Six additional fenced containers surrounded by henhouse mesh of 41 mm, two in each position, were used as controls.

Experiment 5-September 2008 Finally, the effect of grit and food supplementation on the feeding behaviour of waterbirds in the wild was studied at ML. In this experiment, the interaction between grit and food was studied, as in previous experiments, but was evaluated under more natural conditions as a pilot study of grit supplementation in the field. In this case, it was not possible to measure the exact amount of grit ingested, but we evaluated the use by birds of experimental plots with different supplies of grit and grain. Treatments were carried out in 5×5 m square plots in the lagoon, separated from each other by 15 m. Corners of plots were marked with four iron fence posts of 2 m height. The mean depth (SD) of the plots was 32 (±2)cm, within a range of 28-35 cm, so that the lagoon bottom was available for coots and ducks through diving, neck dipping and upending [START_REF] Thomas | Autumn and winter feeding ecology of waterfowl at the Ouse Washes, England[END_REF]Pöysä 1983a;[START_REF] Guillemain | Foraging methods can affect patch choice: an experimental study in mallard (Anas platyrhynchos)[END_REF]. Plots were located parallel to the shoreline in an area with a level bottom of soft sediment with macrophytes of 5 cm height above the bottom. Four different treatments with four replicates were tested with grey calcareous grit (1 kg/m 2 ) and grain (0.5 kg/m 2 ), which corresponds to a volumetric ratio of 1:1 (v/v). The treatments were either grit or grain, a mixture of the two, or neither.

Plots were installed 1 week before the treatments were started. Observations were made by telescope from a distance of 400 m from the experimental area. The observation period of 3 h started at 8.00 a.m. Every minute, one plot was observed, and the number of birds of each species eating in the plots was recorded, identifying three methods: neck dipping (for ducks only), upending or diving. Before plots were supplemented, birds were observed during 3 days to identify differences in the use of plots by birds. These results were used to balance the distribution of plots among the four different treatments according to their use before supplementation. Grit and food were supplemented, late in the afternoon on day 0, and then birds were monitored daily during 8 days. After each daily observation, plots were checked by touching the bottom manually because the water was too turbid to see the bottom, and grit (0-12 kg) or grain (0-6 kg) was added in order to maintain the initial proportions.

Statistical analyses

The mass of grit taken from containers was normalized with square root transformation, except for experiment 4 in which log (+0.1) was applied. Data were analysed using general linear models (GLM). The factors included in the models were grit colour and grain presence (experiment 1), water and grain presence (experiment 2), grit composition and grain presence with and without water (experiment 3) and location and grain presence (experiment 4). Replicate and day were included in the models as random effects to control for spatial and temporal variations. All models were calculated including the results of the fenced containers as controls (factor fenced/open). A backward stepwise procedure was used to select the final models, excluding the interactions when they had non-significant (p > 0.05) effects.

In experiment 5, species were analysed individually and also combined into three groups: dabbling ducks, diving ducks and coots. Statistical analyses were performed with data recorded after the lagoon plots were supplemented. The number of birds of each species feeding, or the number of attempts per bird feeding on the plots per minute, was used as dependent variables. In the same way, analyses were performed with the groups of dabbling ducks, diving ducks and coots. Generalized estimated equations (GEE) were used to fit a repeated measurements logistic regression with a negative binomial error structure, with a scale parameter calculated for each dependent variable as K=x 2 / (s 2 -x), with x and s 2 as the mean and variance of the distribution data [START_REF] Fowler | Practical statistics for field biology[END_REF]. A log-link function and the best assumption for a working correlation matrix (using the quasi-likelihood under independence model criterion goodness-of-fit coefficient, which helps to choose between two correlation structures) were selected for each model. The models were implemented using plot as subject variable and minute and day as within-subject variables. Models were constructed using presence of grit, grain and their interaction as factors. In those models in which the numbers of attempts per bird were considered as a dependent variable, the number of birds feeding was included in the model as a scale weight variable.

Spearman rank correlations between the number of birds of each group feeding in the plots and the day of treatment were calculated to test for temporal trends due to learning of the locations of treated plots by birds. All tests were performed using SPSS 17.0, and the level of statistical significance used was p≤0.05.

Results

In experiments 1 to 4, the mass of grit taken from the fenced containers used as controls (Table S1) was lower than that taken from containers used for treatments (GLM, all p values <0.008), and the amount of grit spilled around treatment and control containers was negligible (<1%). Grain was completely consumed in all the non-fenced containers in experiments performed in semi-captivity and was almost completely consumed in those performed in the wild.

In experiment 1, the amount of grit taken was much higher in containers with grain in both the semi-captive and wild situations (Table 1). Red grit was consumed more than grey grit in semi-captivity when offered without grain, but this difference was not observed when grain was mixed with grit as shown by the interaction between grit colour and food (Table 1). No differences in colour selection were found in the wild (Table 1).

In experiment 2, the amount of grit taken by waterbirds was relatively low in containers filled with water and relatively high when grain was mixed with grit (Table 2).

In experiment 3 carried out without water in the containers, the amount of grit taken was much higher for siliceous than for calcareous grit (F 1, 82 =6.469, p=0.013; Fig. 2). However, no statistically significant differences were found in grit consumption related to its composition when the containers were filled with water (F 1, 84 =0.261, p= 0.611; Fig. 2). A significant interaction was found between grit composition and food in water (F 1, 84 =9.162, p=0.003), with a greater selection of siliceous grit when mixed with food and a greater selection of calcareous grit when offered alone (Fig. 2).

In experiment 4, the amount of grit taken was not affected (p=0.379) by the position of the containers in the lagoon, although a significant (p<0.001) interaction was found between location and presence of grain (Table 3). The presence of grain in the containers significantly (p< 0.001) increased the consumption of grit (Table 3).

The species observed in the experimental plots prior to starting experiment 5 were common coot, gadwall (Anas strepera), mallard and red-crested pochard (Netta rufina), and the list increased with common pochard and whiteheaded duck after supplementation. The numbers of waterbirds feeding in experimental plots supplemented with grain were higher than in non-supplemented plots for all avian species (GEE, all p values<0.03; not tested for red-crested pochard due to too few observations recorded). Similar results were found when grouping species into dabbling ducks (gadwall, mallard and unidentified ducks), diving ducks (common pochard, red-crested pochard and whiteheaded duck) and coots (χ 1 2 =19.841, p<0.001, χ 1 2 = 52.230, p<0.001; χ 1 2 =98.923, p< 0.001, respectively; Fig. 3a). The counts of feeding birds were not affected by supplementation with grit (GEE, all p values> 0.05). However, a positive effect of the presence of grit was found in the number of feeding attempts by foraging birds for mallards (χ 1 2 =244.189, p<0.001) and diving ducks (χ 1 2 =6.137, p=0.013; Fig. 3b).

The number of diving ducks and coots feeding in plots supplemented with grit and grain increased significantly over time (r s =0.976, n=8, p<0.001; r s =0.952, n=8, p< 0.001; respectively; Fig. 4), but this was not observed for dabbling ducks (r s =0.371, n=8, p=0.365). In the plots supplemented only with grain, the positive trend over time was only significant for diving ducks (r s =0.714, n=8, p= 0.047; Fig. 4b). No significant trends were found in plots supplemented only with grit or in controls (r s all p values> 0.05; Fig. 4).

Discussion

Grit ingestion by ducks and coots may be intimately associated with foraging behaviour. In all of our experiments, the amount of grit taken from containers or the number of birds attracted to supplemented plots was higher when grain was also supplied. However, some other waterbirds, such as greylag geese wintering in Doñana National Park, have been observed repeatedly visiting specific sites to get grit (Mateo et al. 2000b). Geese fly regularly from the Doñana marshes to neighbouring sand dunes, a predictable behaviour that was exploited historically by hunters to shoot geese early in the morning (Mateo et al. 2000b). Tide-dependent movement to sandbars to ingest grit has also been reported for black brant (Branta bernicla nigricans) in South Humboldt Bay, California [START_REF] Lee | Grit-site selection of black particle size or calcium content?[END_REF]). This preference for specific grit sites is not limited to geese. In the Ebro delta (northeastern Spain), purple gallinules were seen in groups taking grit from trails in places where food was not present [START_REF] Renner | Sick picnic-evaluating the legacy of lead ammunitions[END_REF]. Thus, some waterbird populations or species appear to have developed specific behaviours to search for grit.

The mechanisms of grit handling and ingestion vary among avian taxa. In Rallidae, grit must be picked up and directly swallowed, in a similar way as described for food items (Pöysä 1983b;[START_REF] Irwin | The wintering behaviour of coot Fulica atra L. at Cork Lough, south-west Ireland[END_REF]. But ducks and geese have different feeding mechanisms, including pumping through the lamellae to filter out particles of larger size than the interlamellar gap [START_REF] Kooloos | Comparative mechanics of filter feeding in Anas platyrhynchos, Anas clypeata and Aythya fuligula (Aves, Anseriformes)[END_REF][START_REF] Gurd | Filter-feeding dabbling ducks (Anas spp.) can actively select particles by size[END_REF]. In Anatidae, the presence of water at feeding or gritting sites may affect grit ingestion. In experiments 2 and 3, the amount of grit taken by the waterbirds from the containers was significantly lower in those with water. This result is probably explained because birds could wash the mixture of grit and grain in their mouths and selectively swallow the grain because of its lower density than grit. In fact, ducks feeding from the containers without water were seen going back and forth to the water, perhaps to facilitate the swallowing of grain and grit (authors, personal observation; this has been also described by [START_REF] Thomas | Autumn and winter feeding ecology of waterfowl at the Ouse Washes, England[END_REF] for wigeon). The presence of water allows waterfowl to eliminate undesired material such as soil or mud in food [START_REF] Mateo | Altered porphyrin excretion and histopathology of greylag geese (Anser anser) exposed to soil contaminated with lead and arsenic in the Guadalquivir Marshes, Southwestern Spain[END_REF]. In some situations, however, the presence of water may help birds take grit. [START_REF] Lee | Grit-site selection of black particle size or calcium content?[END_REF] found that grit-ingestion behaviour in black brant was more prevalent on sandbars that were still submerged, with water depth less than 0.5 m. There is a cost to ingesting too much grit as it reduces the intake rate of food, so birds may sometimes wish to ingest food without grit [START_REF] Amat | Grit ingestion and size-related consumption of tubers by graylag geese[END_REF].

Birds are able to distinguish food among other particles, such as grit, by sight and smell (Martin 2007;[START_REF] Steiger | Avian olfactory receptor gene repertoires: evidence for a well-developed sense of smell in birds?[END_REF]. During visual selection of grit, birds may be influenced by several grit characteristics such as size, colour, shape, surface texture and geochemical composition [START_REF] Gionfriddo | Grit use by birds: a review[END_REF]. Most of the grit found in waterfowl ranged from 0.5 to 3.0 mm in size, which was the size used in our experiments [START_REF] Thomas | Grit in waterfowl at the Ouse Washes, England[END_REF][START_REF] Pain | Lead shot ingestion by waterbirds in the Camargue, France: an investigation of levels and interspecific differences[END_REF]Mateo et al. 2000a). The size of grit selected by birds has been related to diet [START_REF] Thomas | Grit in waterfowl at the Ouse Washes, England[END_REF][START_REF] Pain | Lead shot ingestion by waterbirds in the Camargue, France: an investigation of levels and interspecific differences[END_REF]Mateo et al. 2000a;Luttik and de Snoo 2004) 2005). As for colour, we found that red grit was only consumed more than grey grit when it was not mixed with grain in the experiment in semi-captivity, although this difference was not observed in wild. There are no previous studies testing the effect of grit colour on grit selection by waterfowl, but several studies have described the colour of grit found in gizzards [START_REF] Thomas | Grit in waterfowl at the Ouse Washes, England[END_REF][START_REF] Gionfriddo | Grit use by birds: a review[END_REF][START_REF] Lee | Grit-site selection of black particle size or calcium content?[END_REF]Luttik and de Snoo 2004). Just as with grit size, the colour and composition of grit ingested by waterfowl depends partly on the availability in the environment [START_REF] Gionfriddo | Grit use by birds: a review[END_REF][START_REF] Lee | Grit-site selection of black particle size or calcium content?[END_REF]).

The geochemical composition of grit is important because it affects the hardness, shape and surface texture of the particle, its ability to grind food and the presence of essential elements such as calcium [START_REF] Gionfriddo | Grit use by birds: a review[END_REF]. We observed a higher selection of siliceous than calcareous grit when it was offered to waterbirds mixed with grain, especially when it was not in water. Siliceous grit has been commonly found in gizzards, and this may be related to its abundance in some environments and to its hardness and thus resilience in the gizzard [START_REF] Gionfriddo | Grit use by birds: a review[END_REF]. Silicon dioxide is one of the most abundant components of the continental crust (66%), whereas calcium oxide only represents 4.2% [START_REF] Rudnick | Nature and composition of the continental crust: a lower crustal perspective[END_REF]. The half-life of grit in mallard gizzards was 3.1 days for siliceous grit (calculated according to the results and equations given by Trost 1981 in his experiment #6) and 1.4 days for calcareous grit (Mateo and Guitart 2000). The higher ingestion of siliceous grit observed in the present study may also be due to its more rounded shape than calcareous grit, so the effect of shape in grit ingestion by waterbirds should be studied in the future. Differences in the type of grit ingested were related to sex in experimental studies with captive mallards [START_REF] Trost | Dynamics of grit selection and retention in captive mallards[END_REF]. When offered a choice among commercial calcium, granite, oyster shell, quartzite and sandstone, female mallards selected oyster shell and sandstone whereas males selected sandstone [START_REF] Trost | Dynamics of grit selection and retention in captive mallards[END_REF], although the author suggested that the basis for grit selection was not purely mineral composition. However, calcium supplements for females during the egglaying period play an important role. This has been well documented in poultry production where limestone and oyster shell are commonly used to improve yolk pigmentation and increase resistance of eggshells and tibia bones [START_REF] Koreleski | Calcium from limestone meal and grit in laying hen diets-effect on performance, eggshell and bone quality[END_REF][START_REF] Lichovnikova | The effect of dietary calcium source, concentration and particle size on calcium retention, eggshell quality and overall calcium requirement in laying hens[END_REF][START_REF] Safaa | Productive performance and egg quality of Brown egg-laying hens in the late phase of production as influenced by level and source of calcium in the diet[END_REF]. Furthermore, several studies have addressed calcium requirements and the influence of this on the distribution and breeding success of pheasants and passerines [START_REF] Mccann | A study of the grit requirements of certain gallinaceous game birds[END_REF][START_REF] Mccann | Grit as an ecological factor[END_REF][START_REF] Harper | The influence of calcium on the distribution of the pheasant in North America[END_REF][START_REF] Graveland | Calcium availability limits breeding success of passerines on poor soils[END_REF][START_REF] Reynolds | Calcium supplementation of breeding birds: directions for future research[END_REF][START_REF] Wilkin | Calcium effects on life-history traits in a wild population of the great tit (Parus major): analysis of long-term data at several spatial scales[END_REF][START_REF] Jones | Effects of calcium-loading on egg production in ring-necked pheasants[END_REF]. Despite this, little attention has been paid to the study of calcium use and requirements for waterfowl. [START_REF] Flint | Observations of geese foraging for clam shells during spring on the Yukon-Kuskokwim Delta, Alaska[END_REF] found that, in spring on the Yukon-Kuskokwim Delta (Alaska), geese of several species, especially females, were foraging on empty clam shells, supposedly to meet calcium requirements.

Very little information exists about the sites selected by waterbirds to seek for grit, but preferences have been recorded in a few species (Mateo et al. 2000b;[START_REF] Lee | Grit-site selection of black particle size or calcium content?[END_REF]. In our experiment 4, we could not detect significant differences in grit consumption from containers located at different positions in a lagoon (shore, bottom, platform), but in all of these locations, grit consumption was enhanced by the inclusion of grain.

Grit per se seemed to be little attractive for waterbirds in the containers in the wild, probably because it was presented in an artificial context that permitted us to quantify the amounts consumed by birds. However, grit supplementation in a more natural way, as in experiment 5, showed again that the attraction of large numbers of birds to specific sites was only accomplished with grain supplementation. Up to 50 of the estimated 7,700 coots at ML were observed feeding at the same time in the 200-m 2 area that we supplemented with grain. Food supplementation in the experimental plots of the present study (500 g/m 2 ) supplied a higher grain density than commonly observed in natural wetlands (50-70 g/m 2 ; [START_REF] Baldassarre | Field-feeding ecology of waterfowl wintering on the Southern high plains of Texas[END_REF][START_REF] Arzel | Experimental functional response and interindividual variation in foraging rate of teal (Anas crecca)[END_REF]). The greater attractiveness of grain compared with grit was not an unexpected result because grit, despite being necessary for birds, is required in smaller quantities than food. Grit is also retained in the gizzard for relatively long periods. However, the possible usefulness of supplementing grit alone is supported by the observation in experiment 5 that, for mallards and diving ducks, a higher number of feeding attempts were seen in plots supplemented with grit than in control plots.

Management implications

Grit ingestion is mostly associated with foraging behaviour in ducks and coots. Therefore, the first recommendation for grit supplementation strategies to reduce Pb poisoning would be to distribute the grit at natural feeding sites in the wetlands. The artificial supply of grain may not be desirable because of the risk of eutrophication and the proliferation of rats. As these natural feeding sites are usually flooded and thus visibility is reduced, the colour of grit may be of little importance in grit selection. Our recommendation would be to use a mixture of both calcareous and siliceous grit for several reasons: (1) calcareous grit may decrease the solubility of Pb in the digestive tract of birds because it tends to increase gizzard pH and helps to precipitate insoluble Pb carbonate [START_REF] Martinez-Haro | Avian digestive tract simulation to study the effect of grit geochemistry and food on Pb shot bioaccessibility[END_REF]); (2) calcareous grit produces a higher calcium concentration in the intestine than siliceous grit [START_REF] Martinez-Haro | Avian digestive tract simulation to study the effect of grit geochemistry and food on Pb shot bioaccessibility[END_REF]. Calcium is a natural antagonist of Pb absorption in the intestine and thus alleviates the toxic effect of Pb [START_REF] Longcore | Toxicity of lead and proposed substitute shot to mallards. US Department of the[END_REF][START_REF] Mykkanen | Gastrointestinal absorption of lead (203Pb) in chicks: influence of lead, calcium, and age[END_REF][START_REF] Scheuhammer | Influence of reduced dietary calcium on the accumulation and effects of lead, cadmium, and aluminum in birds[END_REF]; and (3) siliceous grit is harder and therefore has a longer halflife in the gizzard (Mateo and Guitart 2000). Thus, siliceous grit may reduce the need to replace grit in the gizzard and thus the probability to ingest Pb shot. Few differences in grit consumption were observed among those locations tested in the present study. However, we propose grit supplementation in shallow waters, close to the shore. The ideal depth range is up to 30-45 cm, to allow both diving and dabbling ducks to reach the sediments. Some species, such as white-headed duck, are very reluctant to leave the water and are unlikely to use grit placed on the shore.

Until now, only two applications of grit supplementation to reduce the ingestion of Pb shot or Pb fishing weights have been attempted under field conditions [START_REF] Hoffmann | Le saturnisme fleau de la sauvagine en Camargue[END_REF][START_REF] O'halloran | Trends in mute swan blood lead levels: evidence of grit reducing lead poisoning[END_REF]. The first effort was in 1958, when three tonnes of grit were supplemented in a heavily hunted 12-ha area of the Camargue, where grit was absent. One year after supplementation, Pb shot prevalence was reduced significantly in tufted ducks but not in mallards or common teal [START_REF] Hoffmann | Le saturnisme fleau de la sauvagine en Camargue[END_REF]. The second application was carried out at Cork Lough, an urban site of 6 ha and 1.5 m maximum water depth where coarse fishing was popular. In this case, 40 tonnes of sandstone <10 mm was first spread in 1986, and 50 tonnes was added annually from 1997 to 2000 [START_REF] O'halloran | Trends in mute swan blood lead levels: evidence of grit reducing lead poisoning[END_REF]. Later in the same area, 150 tonnes of grit was supplemented from 2001 to 2005 (O' Connell et al. 2009). After grit supplementation, declines in the median blood lead level and in the proportion of swans suffering from acute lead poisoning were observed [START_REF] O'halloran | Trends in mute swan blood lead levels: evidence of grit reducing lead poisoning[END_REF][START_REF] Connell | Lead poisoning in mute swans (Cygnus olor) in Ireland: recent changes[END_REF].

In Spain, calcareous grit can be purchased at 21 €/tonne and siliceous grit at 100 €/tonne. The transportation cost from local quarries may be 4 €/tonne. So we estimate that the cost of incorporating calcareous and siliceous grit (1:1, w/w, with 65 particles per gram of mix) to achieve a grit density in sediments ten times higher than 400 Pb shot per square metre would be about 40 €/ha. Depending on the input rate of additional spent shot, burial rates and rates of grit ingestion and retention by waterbirds, grit should be supplemented regularly to maintain a higher proportion of grit than shot pellets in sediments.

In some wetlands where hunters regularly bait with grain around the hunting blinds, grit could be mixed with the grain with a minor labour cost. Grit supplementation as defined here could also be applied in uplands or shooting ranges that have a shortage of grit where Pb shot ingestion by wild birds is documented [START_REF] Ferrandis | Lead-shot exposure in red-legged partridge (Alectoris rufa) on a driven shooting estate[END_REF][START_REF] Thomas | Bone lead levels and lead isotope ratios in red grouse from Scottish and Yorkshire moors[END_REF]. In conclusion, the present paper provides new information to understand grit selection and ingestion by waterbirds as well as the economic cost per hectare for grit supplementation by wetland managers.

Fig. 1

 1 Fig. 1 Geographic location of the study areas in Andalusia (southern Spain). Study areas, Concerted Nature Reserve (CNR) of Cañada de los Pájaros and Medina Lagoon (ML), are represented by asterisks. Rivers (grey lines) and the perimeter of Doñana National Park (grey area) are also displayed

Fig. 2

 2 Fig. 2 Mean mass of siliceous (Si) and calcareous (Ca) grit taken (± SE) by semi-captive waterfowl in the Concerted Nature Reserve of Cañada de los Pájaros when the effect of grit composition and presence of food were tested in dry versus underwater contexts (experiment 3)

Fig. 3

 3 Fig.3Estimated marginal means (± SE) obtained in GEE models of a the number of wild birds feeding and b the number of attempts per minute, adjusted for the number of wild birds feeding (experiment 5) in Medina Lagoon. Means for dabbling ducks, diving ducks and coots sharing a number, capital letter or lowercase letter, respectively, did not differ significantly (p>0.05)

Fig. 4

 4 Fig. 4 Temporal evolution of the number of wild birds feeding per minute on plots with different treatments after supplementation in experiment 5 for a dabbling ducks, b diving ducks and c coots in Medina Lagoon

  and also to

	Table 2 Mass of grit taken when
	the effect of the presence of water	Experiment 2	Treatment	Number	Grit taken (g)	Statistics
	and food were tested in semi-
	captive waterfowl in the Concert-				Mean	SE
	ed Nature Reserve of Cañada de
	los Pájaros (Andalusia, southern	Semi-captive	Grit-water	15	1.3	0.5	Water: F 1, 85 =95.540, p<0.001
	Spain)		Grit-dry	15	20.1	3.3	Food: F 1, 85 =26.318, p<0.001
			Grit-water-food	15	5.9	1.0
			Grit-dry-food	15	49.0	4.1

Eur J Wildl Res (2011) 57:475-484

Acknowledgements We are grateful to P. Rodríguez and M. Adrián for their permission for and assistance with the work in semi-captivity. H. Rodríguez-Pérez helped in the design of the experimental containers. We wish to thank E. Márquez, L. Monsalve, G. Taberner, P. Delgado and many others for helping both during the laboratory and field work. This work was supported by a project funded by the Consejería de Medio Ambiente, Junta de Andalucía. P. Acevedo is funded by a Juan de la Cierva research contract awarded by the Ministerio de Ciencia e Innovación-Fondo Social Europeo. This study was also funded by MICINN (under CGL2007-62797). We also thank two anonymous referees for their comments that improved the manuscript.


MPH, MSc, PhD Maribel Almonte
email: m.almonte@qmul.ac.uk

Isabel Dos

Santos Silva

Akua Asare

Clare Gilham

Alexandra Sargent

Andrew Bailey

Andrew Turner

Mina Desai

Henry C Kitchener

Julian Peto

Sexual behaviour and HPV infection in British women, by postal questionnaires and telephone interviews

Keywords: HPV, cervical cancer, sexual behaviour, HPV acquisition, postal questionnaires, telephone interviews HPV Cervical cancer Sexual behaviour HPV acquisition

Sexually transmitted human papillomaviruses (HPVs), most frequently HPV 16, are the primary cause of cervical carcinogenesis. The aim of this study was to evaluate the relationship between sexual behaviour and prevalence and acquisition of HPV infection among British women attending regular screening who responded to postal questionnaires and/or telephone interviews. A total of 1880 women who had been tested for HPV in the ARTISTIC trial were randomised to three methods of data collection: group 1 (questionnaire only), group 2 (questionnaire excluding sexual history, short interview) and group 3 (questionnaire, longer interview). Sexual history collected included age at first sexual intercourse, sexually transmitted diseases, lifetime (total and regular) sexual partners and partners in the last five years (total and new). Demographics, reproductive, screening and smoking history were also collected in questionnaires. The overall participation rate was 35%.

There was good agreement (87.4%-95.5%) on sexual behaviour answers in questionnaires and interviews in women in group 3 and no significant differences between data obtained by questionnaire or interview. Odds ratios (OR) for both HPV prevalence and acquisition increased consistently with increasing numbers of lifetime sexual partners, regular partners and new partners in the last five years. No significant association was found for other characteristics investigated. The effect of recent partners on HPV acquisition (OR for 2+ new partners: 4.4, 95%CI: 1.7-11.2) was stronger than that of earlier partners (OR for 2+ earlier partners: 2.2, 95%CI: 0.7-6.7) suggesting that most incident HPV infections are newly acquired rather than recurrent.

Introduction

Cervical cancer is the second most common cancer among women worldwide [START_REF] Parkin | International variation[END_REF], and is more frequent in many socio-economically deprived populations [START_REF] Almonte | Risk factors for human papillomavirus exposure and co-factors for cervical cancer in Latin America and the Caribbean[END_REF]. Human papillomaviruses (HPVs), most frequently HPV 16, are the primary cause of cervical carcinogenesis [International Agency for Research on Cancer, 1995]. Over 100 HPV types have now been described, including 13 'high-risk' types that are definitely associated with cervical cancer and several more that are probably carcinogenic [International Agency for Research on Cancer, 2007]. The overall prevalence of HPV in cervical cancers worldwide is over 99%, implying the highest attributable fraction ever identified for a specific cause of a major human cancer [START_REF] Walboomers | Human papillomavirus is a necessary cause of invasive cervical cancer worldwide[END_REF].

HPV is one of the most common sexually transmitted infections [START_REF] Bosch | Epidemiology and natural history of human papillomavirus infections and type-specific implications in cervical neoplasia[END_REF]. Most women become infected during their lives, although the majority of the infections are transient and clear within two years [START_REF] Plummer | A 2-year prospective study of human papillomavirus persistence among women with a cytological diagnosis of atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion[END_REF]. HPV genital acquisition is associated with sexual behaviour patterns such as early age at first sexual intercourse, number of sexual partners and sexual behaviour of the partner; and certain co-factors (high parity, long-term use of hormonal contraceptives and cigarette smoking) help in the progression from infection to cancer [START_REF] Almonte | Risk factors for human papillomavirus exposure and co-factors for cervical cancer in Latin America and the Caribbean[END_REF][START_REF] Vaccarella | Sexual behavior, condom use, and human papillomavirus: pooled analysis of the IARC human papillomavirus prevalence surveys[END_REF][START_REF] Vaccarella | Smoking and human papillomavirus infection: pooled analysis of the International Agency for Research on Cancer HPV Prevalence Surveys[END_REF].

This study describes sexual behaviour patterns in relation to HPV prevalence and acquisition rates among British women who participated in the ARTISTIC trial (A Randomised Trial In Screening To Improve Cytology), which was conducted in Greater Manchester [START_REF] Kitchener | HPV testing in routine cervical screening: cross sectional data from the ARTISTIC trial[END_REF]. In order to compare responses and participation rates, the method of ascertaining sexual behaviour was randomised between postal questionnaire, telephone interview, and both.

Materials and Methods

The ARTISTIC trial

In the ARTISTIC (A Randomised Trial In Screening To Improve Cytology) trial, 24,510 women aged 20-64 who were attending for routine cervical cytology in four health authorities (Manchester, Salford &Trafford, Wigan andStockport) in 2001-2003 were screened with liquid-based cytology (LBC) and HPV testing [START_REF] Kitchener | HPV testing in routine cervical screening: cross sectional data from the ARTISTIC trial[END_REF]. LBC samples were collected and processed using ThinPrep (Cytic). Hybrid Capture 2 (hc2, Qiagen) was used to test for high-risk HPV DNA. This nucleic acid microplate chemiluminescent detection assay detects 13 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). Results were read and calculated on the Digene Microplate Luminometer 2000 (DML 2000; Qiagen) using the hc2 software at the recommended relative light units (RLU) to control ratio of greater than or equal to 1.0. HPV results were reported independently of cytology.

At entry, women were randomly allocated in a 1:3 ratio to have their HPV results concealed or revealed.

Cytologically normal women in the revealed arm who were HPV positive (HPV+) were recalled a year later, and those with persistent HPV were offered colposcopy. Women were invited to a second screening round between 26 and 54 months after entry into the study.

Study population

A random sample of 1880 ARTISTIC participants who attended the second screening round (26 to 54 months after entry) and who had an adequate cytology in the second round was selected. All women had a satisfactory HPV test at entry, and the majority (87%) had adequate HPV tests at the next screening round.

Data collection

These women were randomised to three methods of obtaining the sexual behaviour data: postal questionnaire only (group 1), postal questionnaire excluding sexual history and a short interview including sexual behaviour (group 2) and both postal questionnaire and longer interview containing sexual behaviour including a calendar of sexual partners (group3). All women received an invitation package containing a description of the study, a consent form and a postal questionnaire that included age, current weight and height, marital status, education, F o r P e e r R e v i e w 7 smoking, reproductive history (menarche, menopause, parity and use of hormonal contraceptives), and age at first smear . The questionnaire for groups 1 and 3, which was similar to that used in a previous study in the Manchester area [START_REF] Deacon | Sexual behaviour and smoking as determinants of cervical HPV infection and of CIN3 among those infected: a case-control study nested within the Manchester cohort[END_REF], had an additional section with questions on sexual behaviour. These included age at first sexual intercourse, ever having a sexual transmitted disease (STD), and numbers of regular (more than 3 months) and shorter relationships with male sexual partners overall and in the previous five years.

The interview for groups 2 and 3 included the same sexual behaviour questions, so questionnaire and interview responses could be compared for women in group 3. The interview for group 3 also included further details on each relationship (age, duration and contraceptive method). Additionally, questionnaires and both short and long interviews included three common questions: current weight, current height, and age at first cervical screening so these variables could also be compared in questionnaires and interviews in groups 2 and 3. Short interviews lasted eight minutes and long ones 16 minutes on average and were conducted by a trained research nurse with expertise in conducting telephone interviews for different cancer related studies.

Women were informed in the invitation letter whether they would also be interviewed or would only need to return the questionnaire. All women were asked to give a blood sample for future studies and signed consent for their screening records to be reviewed by the investigators. Non-responders were sent a reminder letter 3-10 months later.

Ethical approval was provided by the North West Multicentre Research Ethics Committee (MREC).

Statistical Analysis

To ensure the return of 200 questionnaires in each group, a minimal response rate of 32% was assumed, so invitation packages were sent to 1880 women.

Non-response and refusal rates were based on all women who were sent a questionnaire. Further analyses were restricted to women who returned both the consent form and questionnaire. Current BMI was calculated in kg/m 2 and was categorised in four groups (<18.5, 18.5-24.9, 25-29.9, and, 30 or more kg/m 2 ). Kappa statistics were used to compare questionnaire and interview responses of women in group 2 (BMI and age at first smear) and group 3 (BMI, age at first smear, and, sexual behaviour variables). Analyses of HPV status at the second 8 screening round were restricted to women who had an HPV test result both at entry and at the second screening round 26 to 54 months later. Age-adjusted odds ratios (AORs) with corresponding 95% confidence intervals (95%CI) for HPV prevalence at the second round (HPV detection at the second round irrespectively of HPV test result in the first round) and for HPV acquisition (HPV detection at round 2 in women who were HPV negative in round 1) were calculated by unconditional logistic regression for single variables, and for the combined effects of new partners in the last five years and earlier partners. Two-sided significance levels were calculated using Fisher's exact test or chi-square tests for trend or heterogeneity. All analyses were carried out using Stata 10.

Results

Of the 1880 women selected, 661 (34% in group 1, 37% in group 2 and 34% in group 3) agreed to participate (512 at first invitation), 182 (10%) refused and 1037 (55%) did not reply to the invitation or reminder. Among 1037 non-respondents, two women died, three withdrew from the ARTISTIC trial and 163 (16%) changed address during the invitation process (information from the trial coordination office or by returned mail). There were no significant differences between randomisation groups (group 1: questionnaire; group 2: questionnaire excluding sexual history, short interview; group 3: questionnaire, longer interview) in response, refusal and non-response rates. Nine women in group 2 and 14 in group 3 returned the questionnaire but were not interviewed. Table I shows overall participation rates in this study and numbers in each group by age, allocated screening protocol (HPV result revealed or concealed), cytology at entry, and HPV status at entry and at the second screening round in the ARTISTIC trial. Participation rates ranged from 29% to 39%, being lower among women aged under 35 (P = 0.03) and those who had been randomised in the ARTISTIC trial to have their HPV results concealed (P = 0.01). Among women who responded there were no significant between-group differences for any of the screening results shown in Table I.

Similarly, there were no statistically significant between-group differences for any variables not related to sexual behaviour collected in questionnaires (Supplementary Table I) , therefore these data was pooled and analyse for all women (three groups). Age at second screening round was associated with both HPV acquisition and HPV prevalence, while marital status, ever been pregnant, menopausal status and ever used hormonal contraceptives smear were associated with HPV prevalence at second round (Table II). But no associations remained significant after adjusting for age at second round (Age-adjusted odds ratios (AORs): 0.6 (95%CI: 0.3-1.1) for being married or cohabiting, 0.9 (95%CI: 0.4-1.8) for ever been pregnant, 1.4 (95%CI: 0.4-4.6) for being menopausal, and 3.9 (95%CI: 0.9-17.5) for ever use of hormonal contraceptives).

Table III shows responses to questions on sexual behaviour from questionnaires for groups 1 and 3, from interviews for groups 2 and 3, and for all women (questionnaire data in groups 1 and 3, interview data in group 2). The only statistically significant difference was in the proportion of women reporting first intercourse at age IV shows percentage agreement (exact and within one category) on questionnaire and interview responses to these questions by women in group 3. There was good agreement (87.4%-95.9% exact, 99.5%-100% within one category) for all questions. A similar analysis for BMI and age at first smear also showed good agreement between responses in questionnaires and interviews (agreement for current BMI: 92.2% in group 2 and 90.4% in group 3 and for age at first smear: 85.3% in group 2 and 81.3% in group 3, Supplementary Table II).

Table IV shows age-specific rates for HPV prevalence at the next screening round and for HPV acquisition (HPV positive at round 2 in women who were HPV negative at entry) for all women with an HPV test at the second screening round. The lower part of Table IV shows age-adjusted HPV prevalence and acquisition ORs for each sexual history question, and for a multiple regression analysis including age, number of sexual partners within the last five years, and number of earlier partners. ORs for both prevalence and acquisition increased consistently with increasing numbers of lifetime sexual partners and lifetime regular partners. For HPV acquisition the effect of new partners in the last five years (OR for 2+ new partners: 4.4, 95%CI 1.7-11.2) appeared to be stronger than that of earlier partners (OR for 2+ earlier partners: 2.2, 95%CI 0.7-6.7).

Table V tabulates the age at first sexual intercourse by number of sexual partners over life and in the last five years according to year of birth. There were marked changes in sexual behaviour between women born before 1940 and those born in 1970-1979. The percentage with more than one lifetime sexual partner increased from 50% (10/20) to 94% (125/133) (P for trend < 0.001) and the percentage reporting a sexual relationship before age 17 increased from zero (0/20) to 44% (58/133) (P for trend < 0.001). Similar cohort trends were observed for number of sexual partners in the last five years.

Table VI shows lifetime numbers of sexual partners by age, reported by women participating in a large national survey of sexual attitudes and lifestyles (NATSAL) conducted in 2000 [START_REF] Johnson | Sexual behaviour in Britain: partnerships, practices, and HIV risk behaviours[END_REF] and2006). The distributions of the number of lifetime sexual partners in women aged 25 or older were similar (average number of sexual partners 7.1 in this study and 6.9 in NATSAL 2000, respectively), but below age 25 a substantially larger proportion of respondents reported five or more partners (63%) than in NATSAL (36%), presumably reflecting a tendency for women with several partners to attend for screening earlier.

Discussion

Cervical cancer is the second most common cancer among women worldwide [START_REF] Parkin | International variation[END_REF], with the highest attributable fraction ever identified for a specific cause of a major human cancer [START_REF] Walboomers | Human papillomavirus is a necessary cause of invasive cervical cancer worldwide[END_REF].

HPV 16 and 18 are the most frequent types associated with cervical cancer and two prophylactic vaccines against these high-risk types are available and being used to vaccinate young women (mostly adolescents) in several countries. Most HPV infections are no longer detectable two years later [START_REF] Plummer | A 2-year prospective study of human papillomavirus persistence among women with a cytological diagnosis of atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion[END_REF][START_REF] Rodriguez | Rapid clearance of human papillomavirus and implications for clinical focus on persistent infections[END_REF]], but it is not known whether HPV in a woman who was HPV negative at a previous test is usually newly acquired or a recurrence of a latent infection. HPV testing may soon replace cytology as the primary cervical screening test, at least in older women, and the significance of transient and persistent infections is an important aspect of the natural history of HPV infection and cervical neoplasia in relation to screening policy and clinical management. Viral latency is also relevant to the effectiveness of HPV vaccination in older women, many of whom have cleared a previous HPV infection. This study describes the sexual behaviour patterns in relation to HPV prevalence and HPV acquisition rates in a sample of British women attending regular cervical screening.

The multiple regression results suggest that most incident infections are newly acquired, as HPV acquisition was determined mainly by the number of new sexual partners in the last five years (OR for 2+: 4.4, 95%CI 1.7-11.2). The smaller and non-significant effect for earlier partners (OR for 2+: 2.2, 95%CI 0.7-6.7) is presumably due at least partly to residual confounding, as the partner of a woman who has had multiple partners is more likely to have had several partners himself. A weak association of lifetime number of sexual partners with HPV acquisition once the effect of recent partners was taken into account has been seen in a number of studies [START_REF] Moscicki | Risks for incident human papillomavirus infection and low-grade squamous intraepithelial lesion development in young females[END_REF][START_REF] Velicer | Prevalence and incidence of HPV genital infection in women[END_REF]. Several studies have shown a high risk of new HPV infection in young women following a new sexual relationship [START_REF] Giuliano | Incidence, prevalence, and clearance of type-specific human papillomavirus infections: The Young Women's Health Study[END_REF][START_REF] Ho | Natural history of cervicovaginal papillomavirus infection in young women[END_REF][START_REF] Moscicki | Risks for incident human papillomavirus infection and low-grade squamous intraepithelial lesion development in young females[END_REF][START_REF] Nielsen | Acquisition of high-risk human papillomavirus infection in a population-based cohort of Danish women[END_REF][START_REF] Wiley | Acquisition of new infection and clearance of typespecific human papillomavirus infections in female students in Busan, South Korea: a follow-up study[END_REF][START_REF] Velicer | Prevalence and incidence of HPV genital infection in women[END_REF][START_REF] Winer | Condom use and the risk of genital human papillomavirus infection in young women[END_REF]. A study in young women in the US [START_REF] Moscicki | Risks for incident human papillomavirus infection and low-grade squamous intraepithelial lesion development in young females[END_REF] reported that 55% of uninfected women aged under 22 years attending family planning clinics acquired an HPV infection within three years, the risk increasing with numbers of new partners. 13 Data on HPV acquisition in older women are sparse, and the reason for the high HPV prevalence seen in older women in many developing countries [START_REF] Ferreccio | Population-based prevalence and age distribution of human papillomavirus among women in Santiago, Chile[END_REF][START_REF] Herrero | Population-based study of human papillomavirus infection and cervical neoplasia in rural Costa Rica[END_REF][START_REF] Lazcano-Ponce | Epidemiology of HPV infection among Mexican women with normal cervical cytology[END_REF][START_REF] Molano | Prevalence and determinants of HPV infection among Colombian women with normal cytology[END_REF] is not known. Twenty-seven (10.0%) of the women aged over 45 in this study reported one or more new partners in the last five years and 11.1% (3/27) of these had a new HPV infection at the second round, compared with 3.7% (9/243) of those with no new partner.

The results of this study are limited because it was not powered to evaluate the association between sexual activity and HPV status; instead the sample size was fixed to a minimum of 200 women targeted by each method of data collection assuming a minimal response rate of 30%. However, these limited data clearly suggest that HPV is often recently acquired rather than persistent or recurrent even in older women.

Postal questionnaires are less expensive and perhaps less intrusive than personal interviews, but they may provide less reliable information on sensitive sexual behaviour data. The comparison of responses on sexual behaviour showed no substantial differences between British women returning postal questionnaires and those interviewed by telephone, or between questionnaire and interview responses by women who provided both.

Among women aged 25 or older the distribution of sexual partners observed in this study was similar to the results of Natsal 2000 [START_REF] Johnson | Sexual behaviour in Britain: partnerships, practices, and HIV risk behaviours[END_REF], which was based on a national random sample of women interviewed at home (Table VI). Similarly, the patterns of sexual behaviour reported by South Korean students returning a postal questionnaire [START_REF] Shin | Prevalence and determinants of genital infection with papillomavirus, in female and male university students in Busan, South Korea[END_REF] were similar to those obtained by a household interview in The National Health and Nutrition South Korea Survey [START_REF] Wiley | Acquisition of new infection and clearance of typespecific human papillomavirus infections in female students in Busan, South Korea: a follow-up study[END_REF]. A recent study conducted in the US suggests that reporting of potentially sensitive items such as very early sexual intercourse in women and late first intercourse in men is increased when telephone interviews are automated rather than conducted by an interviewer [START_REF] Turner | Improving epidemiological surveys of sexual behaviour conducted by telephone[END_REF].

For both scientific and practical reasons it thus appears that personal interviews, at least in this field, have no advantages over more impersonal and less expensive alternatives. Postal questionnaires or automated telephone interviews are easier and less expensive to administer and code consistently than personal interviews. This is an important finding, as large samples will need to be surveyed to monitor future changes in sexual behaviour

  later, which was higher in group 2 (short interview) than in group 3 (extended interview) (29.2% v 19.1%: P = 0.02). The right-hand part of Table

  and in women in the present study who were aged 17-45 in 2000. (Lifetime number of partners up to 2000 was estimated as the number of partners more than five years before the survey, as 89% of questionnaires/interviews were filled

  are now being vaccinated against HPV and will not reach the age of 25 years when cervical screening begins for more than a decade.

Table I . Study participants: characteristics at entry and second round screening in the ARTISTIC trial

 I

		Group 1	Group 2	Group 3	Participants	All women	Participation
		No. %	No. %	No. %	No. %	invited	rate (%)
	Total	215 100	233 100	213 100	661 100	1880	35.2
	Age at entry in the ARTISTIC Trial
	20-24	23 10.7	16 6.9	18 8.5	57 8.6	191	29.8
	25-34	42 19.5	57 24.5	41 19.3	140 21.2	459	30.5
	35-44	67 31.2	77 33.1	71 33.3	215 32.5	562	38.3
	45-54	47 21.9	52 22.3	51 23.9	150 22.7	407	36.9
	55-64	36 16.7	31 13.3	32 15.0	99 15.0	261	37.9
						P = 0.03 (4 df)
	Allocated screening protocol in ARTISTIC Trial
	HPV Revealed	181 84.2	174 74.7	160 75.1	515 77.9	1 399	36.8
	HPV Concealed	34 15.8	59 25.3	53 24.9	146 22.1	481	30.4
	Cytology at entry in the ARTISTIC Trial Negative 185 86.1 206 88.4 F o	181 85.0	572 86.5	P = 0.01 (1 df) 1 598 35.8
	Borderline/Mild	22 10.2 r 20 8.6	21 9.9	63 9.5	216	29.2
	Moderate/worse HPV testing in the ARTISTIC Trial 8 3.7 At entry Negative 181 84.2 Positive 34 15.8	7 3.0 P 195 83.7 38 16.3 e e 11 5.2 178 83.6 35 16.4 r	26 3.9 554 83.8 107 16.2	66 P = 0.12 (2 df) 39.4 1 567 35.4 313 34.2 P = 0.69 (1df)
	At second round Negative Positive Not done At entry/second round Neg/Neg Neg/Pos Pos/Neg Pos/Pos	167 77.7 17 7.9 31 14.4 147 68.4 11 5.1 20 9.3 6 2.8	189 81.1 18 7.7 26 11.2 165 70.8 11 4.7 24 10.3 7 3.0	162 76.1 19 8.9 32 15.0 145 68.1 10 4.7 17 8.0 9 4.2 R e v 518 78.4 54 8.2 89 13.5 457 69.1 32 4.8 61 9.2 22 3.3 i e w	1 419 151 310 P = 0.03 (2 df) 36.5 35.8 28.7 1 257 36.4 82 39.0 162 37.7 69 31.9
	Neg/no test	23 10.7	19 8.2	23 10.8	65 9.8	228	28.5
	Pos/no test	8 3.7	7 3.0	9 4.2	24 3.6	82	29.3
						P = 0.17 (5 df)
	Group 1: Questionnaire (included sexual behaviour).
	Group 2: Questionnaire (without sexual behaviour) and short interview (with sexual behaviour).
	Group 3: Questionnaire and long interview (both included sexual behaviour).
				21

Table II . Characteristics of participants collected in questionnaires by HPV prevalence and HPV acquisition at second round HPV prevalence at second round 1 HPV acquisition at second round 2

 II

		HPV neg	HPV pos		HPV neg	HPV pos
		No.	%	No.	%	P (df)	No.	%	No.	%	P (df)
	Age at second screening round
	20-24	10	62.5	6	37.5 < 0.001	4	57.1	3	42.9 < 0.001
	25-34	85	80.2	21	19.8	(5)	66	85.7	11	14.3	(5)
	35-44	165 93.8	11	6.3		145 96.0	6	4.0
	45-54 55-64 65+ Being married/cohabiting Yes No Age when education completed <18 18 Menarche <12 12+ Ever pregnant Yes No No. alive births 0	145 95.4 98 95.2 15 79.0 396 92.3 121 85.2 271 90.6 244 90.7 81 85.3 433 91.5 425 91.8 83 84.7 25 86.2 F o r P e e r R e v i e w 7 4.6 135 97.1 4 2.9 5 4.9 93 94.9 5 5.1 4 21.1 14 82.4 3 17.7 33 7.7 0.01 359 94.5 21 5.5 21 14.8 (1) 97 89.8 11 10.2 28 9.4 0.98 238 92.6 19 7.4 25 9.3 (1) 218 94.8 12 5.2 14 14.7 0.06 71 92.2 6 7.8 40 8.5 (1) 382 93.6 26 6.4 38 8.2 0.03 382 93.9 25 6.2 15 15.3 (1) 65 91.6 6 8.5 4 13.8 0.18 22 91.7 2 8.3	0.08 (1) 0.33 (1) 0.65 (1) 0.47 (1) 0.33
	1-2	299 93.4	21	6.6	(2)	272 95.1	14	4.9	(2)
	3+	107 89.2	13	10.8		94	91.3	9	8.7
	Menopausal status
	Menopausal	200 93.9	13	6.1	0.04	183 95.3	9	4.7	0.18
	Pre-menopausal	317 88.6	41	11.5	(1)	273 92.2	23	7.8	(1)
	Ever used hormonal contraceptives
	Yes	448 89.6	52	10.4	0.04	393 92.9	30	7.1	0.22
	No	70	97.2	2	2.8	(1)	64	97.0	2	3.0	(1)
	Age at first smear
	<20	127 87.0	19	13.0	0.14	105 91.3	10	8.7	0.38
	20+	288 91.4	27	8.6	(1)	257 93.8	17	6.2	(1)
	Ever smoker
	Yes	238 90.2	26	9.9	0.76	207 92.8 22	16	7.2	0.61
	No	280 90.9	28	9.1	(1)	250 94.0	16	6.0	(1)

Table III . Agreement on reported sexual behaviour variables between groups, and between questionnaires and interviews within group 3

 III

		Between	Group 1	Group 2	Group 3	All	Group 3	Group 3: questionnaires v interviews
		group	questionnaires interviews questionnaires	women	interviews Exact agreement	± one category
		P (4 df)	No. (%)	No. (%)	No. (%)	No. (%)	No. (%)	%	kappa	%	kappa
	Total		215 (100)	224 (100)	213 (100)	652 (100) 199 (100)
	Age at first sexual intercourse	0.04
	<17		74 (34.6)	60 (26.9	74 (35.1)	208 (32.1) 71 (35.7)	87.4	0.80	100	1.0
	17-19		98 (45.8)	98 (44.0)	99 (46.9)	295 (45.5) 90 (45.2)
	20+		42 (19.6)	65 (29.2)	38 (18.0)	145 (22.4) 38 (19.1)
	Not answered		1	1	2	4	-
	Ever having STDs	0.41
	None		151 (75.9)	161 (81.7)	151 (77.4)	463 (71.1) 157 (80.1)	88.8	0.68
	Any specified 1		30 (15.1)	25 (12.7)	33 (16.9)	88 (13.5) 30 (15.3)
	Other unspecified 1		18 (9.1)	11 (5.6)	11 (5.6)	40 (15.4)	9 (4.6)
	Not answered		16	27	18	61	3
	Lifetime sexual partners	0.55
	0-1		54 (25.1)	54 (24.4)	41 (19.3)	149 (23.0) 41 (20.8)	94.4	0.91	100	1.0
	2-4		71 (33.0)	70 (31.7)	68 (32.1)	209 (32.3) 63 (32.0)
	5+		90 (41.9)	97 (43.9)	103 (48.6)	290 (44.8) 93 (47.2)
	Not answered		0	3	1	4	2
	Lifetime regular sexual partners 2	0.56
	0-1		61 (29.2)	64 (28.8)	51 (25.1)	176 (27.8) 52 (26.3)	89.0	0.83	99.5	0.97
	2-4		95 (45.5)	89 (40.1)	92 (45.3)	276 (43.5) 89 (45.0)
	5+		53 (25.4)	69 (31.1)	60 (29.6)	182 (28.7) 57 (28.8)
	Not answered		6	2	10	18	1
	Sexual partners in the last five years	0.74
	0-1		170 (80.6)	184 (82.5)	170 (81.3)	524 (81.5) 162 (81.8)	95.9	0.87	99.5	0.94
	2-4		31 (14.7)	32 (14.4)	27 (12.9)	90 (14.0) 26 (13.1)
	5+		10 (4.7)	7 (3.1)	12 (5.7)	29 (4.5)	10 (5.1)
	Not answered		4	1	4	9	1
	New partners in the last five years	0.45
	0-1		176 (85.9)	198 (88.8)	171 (84.7)	545 (86.5) 171 (86.4)	94.7	0.79	99.5%	0.94
	2-4		20 (9.8)	21 (9.4)	21 (10.4)	62 (9.8)	18 (9.1)
	5+		9 (4.4)	4 (1.8)	10 (5.0)	23 (3.7)	9 (4.6)
	Not answered		10	1	11	22	1

Page 22 of 26 John Wiley & Sons Journal of Medical Virology

Table IV . HPV prevalence at second screening round and HPV acquisition in women who were HPV negative at round 1. Age-adjusted ORs for each sexual variable, and multivariate analysis fitting age, new partners in last five years and partners over five years ago

 IV ORs compared two or more sexual partners with less than two partners.HPV prevalence at second round HPV acquisition at second round (women HPV neg at entry)

		All	No. (%)	Age-adjusted OR	Multivariate OR	HPVneg	No. (%)	Age-adjusted OR	Multivariate OR
	women	HPV pos		(95%CI)		(95%CI)	at entry	HPV+		(95%CI)		(95%CI)
	Age at second screening round
	20-24	15	5 (33.3)			1.4	(0.4-4.9)	7	3 (42.9)			3.2	(0.6-17.9)
	25-34	104	20 (19.2)			1.0		77	11 (14.3)			1.0
	35-44	174	11 (6.3)			0.4	(0.2-0.9)	150	6 (4.0)			0.4	(0.1-1.1)
	45-54	151	7 (4.6)			0.3	(0.1-0.8)	138	4 (2.9)			0.3	(0.09-1.2)
	55-64	103	5 (4.9)			0.4	(0.1-1.3)	98	5 (5.1)			0.8	(0.2-2.8)
	65+	19	4 (21.1)			2.1	(0.6-8.1)	17	3 (17.7)			3.2	(0.6-15.4)
	Age at first sexual intercourse
	≤16	175	22 (12.6)	1.0				148	14 (9.5)	1.0
	17-19	259	19 (7.3)	0.7	(0.4-1.5)			213	9 (4.2)	0.6	(0.2-1.6)
	20+	128	11 (8.6)	0.9	(0.4-2.2)			122	9 (7.4)	1.0	(0.3-2.9)
	Ever having STDs
	None	400	34 (8.5)	1.0				354	21 (5.9)	1.0
	Specified 1	77	14 (18.2)	2.0	(1.0-4.2)			59	9 (15.3)	2.5	(1.0-6.1)
	Unspecified 1	88	4 (4.6)	0.5	(0.2-1.6)			73	2 (2.7)	0.5	(0.1-2.2)
	Lifetime sexual partners
	0-1	141	6 (4.3)	1.0				137	4 (2.9)	1.0
	2-4	178	11 (6.2)	1.5	(0.5-4.2)			155	7 (4.5)	1.8	(0.5-6.8)
	5+	246	35 (14.2)	3.4	(1.2-9.6)			194	21 (10.8)	5.0	(1.4-18.7)
	Lifetime regular partners
	0-1	164	6 (3.7)	1.0				154	3 (2.0)	1.0
	2-4	231	22 (9.5)	2.5	(0.9-6.7)			194	12 (6.2)	3.8	(0.9-14.9)
	5+	155	22 (14.2)	3.6	(1.3-10.3)			124	15 (12.1)	8.1	(1.9-34.2)
	Sexual partners > 5 years ago
	0-1	150	8 (5.3)	1.0		1.0		143	5 (3.5)	1.0		1.0
	2-4	183	13 (7.1)	1.4	(0.5-3.6)	1.7	(0.7-4.2) 2	157	8 (5.1)	1.7	(0.5-5.8)	2.2	(0.7-6.7) 2
	5+	227	31 (13.7)	2.4	(0.9-6.3)			182	19 (10.4)	3.6	(1.1-12.1)
	New partners in last 5 years
	0-1	492	33 (6.7)	1.0		1.0		435	20 (4.6)	1.0		1.0
	2-4	52	12 (23.1)	2.6	(1.1-5.9)	3.1	(1.5-6.5) 2	37	7 (18.9)	3.5	(1.2-10.1)	4.4	(1.7-11.2) 2
	5+	16	7 (43.8)	6.0	(1.9-19.3)			10	5 (50.0)	12.0	(2.6-55.8)

1 Specified STDs: trichomonas, genital warts, herpes, gonorrhea, chlamydia. Unspecified STDs: any other infection excluding thrush.

2

Journal of Medical Virology

John Wiley & SonsJournal of Medical Virology

HPV prevalence at second round: HPV detection at round

irrespectively of HPV test result at round 1. 2 HPV acquisition at second round: HPV detection at round 2 in women HPV negative at round 1.

Specified STDs: trichomonas, genital warts, herpes, gonhorrea and chlamydia. Unspecified STDs: any other infection excluding thrush.

Regular sexual partners defined as relationships lasting three or more months.

Acknowledgements

The current study was funded by Cancer Research UK. The ARTISTIC trial was funded by the UK Health Technology Assessment Programme.

The authors have no conflict of interest to disclose.

Sexual partners in the last five years (n = 618