Sajjad Sajjad
email: shsajjad@hotmail.com

Hussain

Sajjad Hussain Sajjad

Nadège Blond

Tanzina Mohsin

Khadija Shakrullah

Alain Clappier

TEMPERATURE VARIABILITY OVER URBAN, TOWN, AND RURAL AREAS: THE CASE OF PAKISTAN

Keywords: Urbanization, urban temperature, climate change, Pakistan

This study investigates the evolution of temperatures at several locations in Pakistan. Data on annual and seasonal minimum (Tn) and maximum (Tx) temperatures from 1950 to 2013 of urban (16 stations), town (11 stations) and rural (9 stations) areas are analyzed to establish the mean decadal rate of change in urban, town, and rural temperatures. The homogeneity of the data were assessed using HOMER 2.6. To measure the temporal intensity of change in temperature, the data were split into two different periods: 1950-1981 (P1, phase of less urbanization) and 1982-2013 (P2, phase of highly urbanized period) and were analyzed separately for both phases of 32 years each. The per decade changes of annual minimum and maximum temperatures (dTn a and dTx a , respectively) over most stations show an increase of temperatures. The trends of annual and seasonal dTn and dTx observed over urban, town, and rural stations during P2 are significantly higher than those observed during P1. The increase in minimum temperatures is more significant than that in maximum temperature and it is also more significant on urban stations than the town and rural stations. However, the maximum temperatures increase more at town stations than urban and rural stations. Overall, the tendencies in temperatures reflect less change in summer temperatures than other seasons of the year over the whole period.

Introduction

Human influence on the climate through the anthropogenic emissions of greenhouse gases (GHGs) is clear. These emissions are the highest in history while the impacts of climate change (CC) on human and natural systems are known to be important. The scientific community regularly alerts the population about these impacts and the need to reduce GHGs (IPCC, 2014). During the past 50 years , global annual-mean surface air temperature has increased at a per decade rate of 0.21°C -dec (Hansen et al., 2010). Populations must increasingly face heat waves due to this CC and urban populations are more exposed due to specific local air temperature conditions. Within urban areas, surface air temperatures, especially at night, are several degrees higher than the simultaneous temperatures of the surrounding rural areas [START_REF] Hung | Assessment with satellite data of the urban heat island effects in Asian mega cities[END_REF]. This temperature difference between urban and surrounding non-urban areas is known as an urban heat island (UHI). This is due to a combination of several factors [START_REF] Martilli | Is the Urban Heat Island intensity relevant for heat mitigation studies?[END_REF]: (1) highest absorption of the solar radiation by high buildings, enhanced by multiple reflections into street canyons. (2) Capture of infrared radiation emitted by warmed artificial materials into these street canyons. (3) Emissions of heat through heating or air conditioning systems, combustion in vehicles [START_REF] Li | Hidden Benefits of Electric Vehicles for Addressing Climate Change[END_REF] and industries [START_REF] Portela | Impact of urban and industrial features on land surface temperature: Evidences from satellite thermal indices[END_REF]. (4) Lower wind speeds that reduce heat dispersion. (5) Lack of evaporative cooling from vegetal areas [START_REF] Kohler | A city scale degree-day method to assess building space heating energy demands in Strasbourg Eurometropolis (France)[END_REF]. The UHI concept is often used to describe "excess" heat associated with urban areas, and is therefore considered a risk for urban populations in summer that requires mitigation. [START_REF] Martilli | Is the Urban Heat Island intensity relevant for heat mitigation studies?[END_REF] suggest that there are also positive effects of UHI within cities located in cold climates, as additional urban warmth reduces demand for residential heating. Cities in perennial hot climates would expect increased demand for air conditioning (AC), which transfers the indoor heat burden to the external local environment [START_REF] Roth | A historical review and assessment of urban heat island research in Singapore[END_REF].

According to World Urbanization Prospects of the United Nations, 55% of the world's population lives in urban areas, a proportion that is expected to increase to 68% by 2050 (United Nations, 2018a). Urbanization directly affects the development of UHI. When UHI is fully developed in urbanized areas, one major question arises: is it possible to mitigate the effect of UHI to contribute to reduce heat stress? This is the focus of several studies [START_REF] Viguie | Early adaptation to heat waves and future reduction of airconditioning energy use in Paris[END_REF][START_REF] Kim | Effects of Urban Heat Island mitigation in various climate zones in the United States[END_REF][START_REF] Wang | Comparing the effects of urban heat island mitigation strategies for Toronto, Canada[END_REF][START_REF] Salamanca | A numerical study of the Urban Heat Island over Madrid during the DESIREX (2008) campaign with WRF and an evaluation of simple mitigation strategies[END_REF]. [START_REF] Lemonsu | Evolution of the Parisian urban climate under a global changing climate[END_REF] proposed that in the present Parisian urban climate, UHIs vary between 0 and 6°C for Tn and between -2 and 4°C for Tx. Questions are raised for developing countries: could urbanization be controlled to avoid or minimize the development of UHI? Another upcoming question is what are the urbanization effects compared to global CC on increasing the local temperatures? [START_REF] Fujibe | Detection of urban warming in recent temperature trends in Japan[END_REF] concluded that the presence of warming trend over 0.3 °C/decade in Japan, even at non-urban stations, is largely attributable to background temperature increases on a large scale, rather than the development of UHI. However, the presence of significant urban anomalies is also observed where the anomalous trend for the urban areas with population density over 3000 km -2 or urban surface coverage over 50%, is about 0.1 °C/decade. For less urbanized sites with population density of ≈100 km -2 or urban surface coverage of ≈0.05, there is an anomalous trend of 0.03-0.05 °C/decade, namely 0.3-0.5 °C/century. Similarly, other studies revealed that at the global scale, combined urban and non-urban trends of maximum and minimum temperature are about 0.1°C per 100 years higher as compared to the trends of non-urban observing stations [START_REF] Easterling | Maximum and minimum temperature trends for the globe[END_REF].

As in many other developing countries, since the 1980s, Pakistan has experienced higher urbanization in different parts of the country. Major cities such as Karachi [START_REF] Sajjad | The preliminary study of urbanization, fossil fuels consumptions and CO2 emission in Karachi[END_REF], Lahore [START_REF] Sajjad | Urbanization effects on temperature trends of Lahore during 1950-2007[END_REF], and Faisalabad have expanded faster, and many old towns have now become prominent urban centers. Currently, almost 40% of the total population lives in cities (Fig. 1) and the rest in rural areas. According to an estimate by UNDESA (2011), almost 60% of the total population will be living in urban areas in 2050. Medium-and small sized cities, of which there are many, have grown at an even higher rate. Due to the rapid growth of cities (small, medium, and large), half of the total population is projected to live in urban areas by 2030 [START_REF] Arif | Urbanization, city growth and quality of life in Pakistan[END_REF]. The rapid urbanization has caused rapid changes in land use and increased environmental degradation.

Figure 1 The present study quantifies the increase in temperature at several locations in Pakistan, including stations located in different types of settlements such as mega cities (urban), smaller cities (town), and rural areas, and discusses the trends on minimum and maximum temperatures as a function of the size and locations of the cities. The major focus is on the estimation of local increase of temperature to highlight the local responsibilities compared to global effects. The results are discussed using the location of the stations and their physiographic features that may have introduced specific changes in the observed time series or anomalies for certain locations.

The data source, length, and data analysis method are presented in section 2. Results are discussed in section 3. Finally, concluding remarks are provided in section 4.

Data and methodology

This section describes the meteorological data collected in different parts of Pakistan, their homogenization, and statistical treatment.

Meteorological data

Based on data from 36 meteorological stations including urban ( 16), town (11), and rural (9) stations located in different parts of Pakistan (Fig. 2), temperature data of daily minimum (Tn) and maximum (Tx) averaged on a monthly basis from 1950 to 2013 were obtained from the Pakistan Meteorological Department (PMD). To measure the temporal intensity of change in temperature, the data was first analysed as a whole data set with no distinctions between stations. Breaks in trends were identified using the SeqMK method at several years depending on the station with few breaks noted around 1980 (see section 2.4 for methodology and 3.2 for results). First, to simplify the analysis, the data set was split into two different periods as per other regions of the world (e.g., [START_REF] Kalnay | Impact of urbanization and land-use change on climate[END_REF], [START_REF] Chung | Urbanization Effect on the Observed Change in Mean Monthly Temperatures between 1951-1980 and 1971-2000 in Korea[END_REF][START_REF] Cueto | Urbanization effects upon the air temperature in Mexicali, B. C[END_REF])): 1950-1981(P1) and 1982-2013 (P2) for all stations and was analyzed separately for both phases of 32 years each. The temperature data are split into two different periods to determine the per decade changes of annual minimum and maximum temperatures (dTn a and dTx a , respectively) during the low urbanization period (P1) and the high urbanization period (P2) in Pakistan.

The data were also averaged on seasonal and annual basis. The characteristics of all meteorological stations including international codes, complete name, latitude, longitude, elevation, total population density of population, and date range of available data are presented in Table 1. The data of different stations were classified into three categories: urban, town and rural stations. The distinction between the urban and the rural locations is not yet amenable to a single definition that would be applicable to all countries or even to the countries within a region. In many industrialized countries, the principal difference between urban and rural areas in terms of the circumstances of living tends to be related to the degree of concentration of population (United Nations, 2018b). But it is hard to follow a single definition even for population density to distinguish between different categories of stations. As for different countries, the population size and density are not the only rule to distinguish among different categories of settlements (UK Parliament, 2018;[START_REF] Jones | New Forms of Urbanization: Conceptualizing and Measuring Human Settlement in the Twenty-first Century[END_REF][START_REF] Pizzoli | How to Best Classify Rural and Urban[END_REF][START_REF] Bibby | Developing a new classification of urban and rural areas for policy purposes -the methodology[END_REF]. Other studies use night light observations via satellite to classify monitoring stations [START_REF] Imhoff | A technique for using composite DMSP/OLS "City Lights" satellite data to map urban area[END_REF][START_REF] Hansen | A closer look at United States and global surface temperature change[END_REF]. However, this type of method cannot be homogeneously used for classification of the stations into different categories as they are located in less developed areas of the world that are facing massive energy shortages.

Figure 2 For the current study, the classification of stations into urban, town, and rural was made by following the traditional method based on population density and size, surface characteristics (paved or natural land cover) and position of the station (within populated area, outside of characteristic urban area, or in rural type landscape even located nearby cities or small towns).

The stations where there is a population density of more than 4000 persons/km 2 are considered as urban, and those where the population density is between 2000 to 4000 persons/km 2 and less than 2000 persons/km 2 are considered as town and rural, respectively.

Test for homogeneity of data

Several non-climatic factors such as station relocation, changes in instruments, screen of the instrument, observation timings, surrounding areas, observational procedure, calculation procedures and instrumental inaccuracies, observer capability, attentiveness, and observing regulations cause the inhomogeneities in time series data. Such non-climatic factors hide the real signal of CC during one specific period [START_REF] Aguilar | Guidelines on Climate Metadata and Homogenization, World Climate Programme Data and Monitoring[END_REF]. While they may have provided valuable information on the immediate surroundings of stations (typically metadata within tens of meters help to better understand the local effect), the detailed metadata of the stations could not be obtained from the data source. To control the quality and remove inhomogeneities from the time series data, HOMER version 2.6, which includes tools for quality control, homogenization (detection and adjustment), and visualization of monthly climate network, is used. Based on relative techniques, it combines different detection approaches: pairwise detection (based on multiple-comparisons of station pairs), joint detection (based on simultaneous statistical modeling of a station network), and traditional weighted references. HOMER applies an ANOVA model for simultaneous adjustments of detected and validated breaks [START_REF] Osadchyi | Homogenization of Ukrainian air temperature data[END_REF][START_REF] Freitas | Homogeneity of monthly air temperature in Portugal with HOMER and MASH[END_REF][START_REF] Mestre | HOMER: a homogenization softwaremethods and applications[END_REF]. HOMER has been constructed exploiting the best characteristics of some other state-of-the-art homogenization methods, that is, PRODIGE, ACMANT, and CLIMATOL [START_REF] Mestre | HOMER: a homogenization softwaremethods and applications[END_REF].

Table 1 To avoid any bias of urban area's temperatures on non-urban stations over homogenized data series, the homogeneity of the data was checked for urban and non-urban stations separately. We used HOMER without any development, modification, addition, or deletion. The procedure follows the preparation of files for network numbers, selection of parameter (temperature, precipitation), unit of parameter (°C), selection of correlation for interactive option with Pearson's-r value of 0.99 with correlation of eight neighboring stations for comparison. Annual and seasonal pairwise detection with visualization of series was performed for the homogenization process.

Figure 3 For the fast quality control, for the detection of any major errors in the data, the fast CLIMATOL command was used. Data were then quality controlled followed by the detection and removal of outliers within time series data in comparison with other neighboring stations. For homogenization, pairwise and joint detection were performed and data were then corrected using the "correction" option to obtain the homogenized data series for further analysis. For the statistical procedure and HOMER's source code, see (2013), and the HOMER manual on http://www.homogenisation.org/homer.2.6.R, which explains the installation, sourcing of HOMER, preparation of the computer, and communication with the software.

Figure 3 highlights the trends of Tn a of Lahore (Fig. 3a) and Quetta (Fig. 3b) with and without homogeneity check. It shows that after detecting and removing the outliers and inhomogeneities within the Tn time series data in comparison with other neighboring stations, the data obtained for further analysis were without biases. The adjustment value of temperature data using HOMER remained different for each station, such as 0 to 1.1 °C for Lahore and 0 to 1.9 °C for Quetta. See [START_REF] Mestre | HOMER: a homogenization softwaremethods and applications[END_REF] for details of the statistical procedure and functioning of HOMER.

Temperature trends

To assess the temperature trends using the homogenized data series, anomalies dT of annual and seasonal mean of minimum (Tn) and maximum (Tx) temperatures were computed.

The linear least squares regression technique was applied to extract the following:

o Trends of annual mean of anomalies of minimum (dTn a ) and maximum temperatures (dTx a ), that is, the slopes of the linear variations.

o Trends of seasonal mean of dTn s and dTx s for all four seasons in Pakistan with s an index to indicate different seasons: autumn (s=Au), winter (s=Wi), spring (s=Sp), and summer (s=Su).

o Trend differences of dTn p and dTx p between P2 (1982-2013) and P1 (1950-1981), respectively.

o Trend of dTn u-r and dTx u-r , which represent annual and seasonal difference in anomalies of minimum and maximum temperature between urban and rural stations, respectively (Table 2).

o Assessment of trends of dTn t-r and dTx t-r , which represents annual and seasonal difference in anomalies of minimum and maximum temperature between town and rural stations, respectively.

Mann-Kendall test

The Mann-Kendall [START_REF] Khambhammettu | Mann-Kendall analysis[END_REF] test is used to determine whether the observations in the data tend to increase or decrease over the study period (and not only for 2 periods of time). 6. Here, u(ti) is a standardized variable that has zero mean and unit standard deviation. Therefore, its sequential behavior fluctuates around zero.

7. The values of u'(ti) can be computed backward in a similar manner as the forward series but starting from the end of the series.

8. u(ti) and u'(ti) can be plotted to detect the systematic change in climate data [START_REF] Sneyers | On the Statistical Analysis of Series of Observations[END_REF]).

The "systematic change" refers to any change in the time series data that represents sustained changes for the area under investigation. In the case of a significant trend, the graphical representation of u(t) and u'(t) curves identifies (at the intersection) the start of the systematic change in the time series. In the absence of any statistically significant trend, the curves usually overlap several times toward the end of the time series.

The normalized values of the test statistic, u(ti), are also used to test the null hypothesis in favor of the existence of a trend through the p-value. When the p-value is lower than 0.01, there is a strong significance in favor of a trend. When the p-value is greater than 0.01 but below 0.05, there is a significance in favor of a trend, but when it is greater than 0.05, the potential trend is not significant.

More information on this procedure can be obtained from the WMO paper by [START_REF] Sneyers | On the Statistical Analysis of Series of Observations[END_REF].

Results

Temperature trends

The summary of the results reported in Table 2 highlights the dTn and dTx during P1 and P2 on an annual and a seasonal basis at urban, town, and rural stations. To assess the long-term UHI, Table 2 also highlights the long-term temperature differences between urban and rural stations (dTn u-r and dTx u-r ) and between town and rural stations (dTn t-r and dTx t-r ) calculated by taking the average of annual and seasonal dTn and dTx of all urban, town, and rural stations separately. General assessment of the results reveals:

-During P1, there were increasing trends in Tn a at urban, town, and rural stations, but there are decreasing trends in Tx a only at urban and town stations. -During P2, there were increasing trends in Tn a and Tx a at all types of settlements, that is, urban, town, and rural stations. -During P2, dTn a and dTn s were observed significantly higher at urban stations than town and rural stations. -During P1, the per decade trends of dTn a at urban, town, and rural stations were 0.04°C, 0.13°C, and 0.20°C, respectively. -During P2, the per decade trends of dTn a at urban, town and rural stations were 0.37°C, 0.10°C, and 0.03°C, respectively. -The trend of dTn a during P2 at urban stations was significantly higher than the dTn a during P1 (0.37°C/decade vs 0.04°C/decade, respectively). -Among all the seasons, spring is highly affected by changes in which minimum and maximum temperatures at urban, town, and rural stations increased more than any other season.

-Long-term annual net change in trend of dTn u-r is 0.50°C/decade, which is the highest in spring (0.62°C/decade). -Long-term annual net change in trend of dTx u-r is only 0.03°C/decade, which is the highest in spring (0.42°C/decade). The results indicate that not all the stations display positive trends; a number of stations also show negative temperature trends. However, maximum and minimum temperatures have increased at most stations, especially in P2 (Fig. 4a and4b). Figure 4a reveals that during P1, there is significant variation in trends of dTn a and dTx a at individual urban, town, and rural stations. Panjgur is one of the town stations, which showed an irregular trend for dTn a compared to its neighboring stations and other stations of the same category. Faisalabad and Gilgit are two urban stations, but the both show negative dTn a during P1 with -0.46°C -dec and -0.30°C -dec , respectively.

Figure 4(b) indicates that most of the urban stations show increasing dTn a and dTx a during P2, with the minimum temperature showing higher increasing tendency than the maximum temperature. The average dTn a trend of all urban stations is also much higher than at town and rural stations, while the average dTx a trends at rural, urban, and town stations are similar. The temperature difference, especially at urban stations, (Figure 4(c)) highlights the effect of rapid urbanization in recent decades. Most of the urban stations except Hyderabad show the higher increasing shift in dTn a and dTx a where it is larger for dTn a .

Figures 5(a) and 5(b) highlight the comparison of changing order of stations based on dTn a for P1 and P2. Overall analysis reveals that the variation in temperature depends not only on rate of urbanization; there may be many other factors that can affect the temperature trends of a particular place. This fact is evident as among all the data series, dTn a at Panjgur (a town station) during P1 increased more rapidly than any other station. The figure highlights that there were only two out of a total 36 stations where dTn a is more than 0.50°C -dec during P1. During P2, the number of stations where dTn a is more than 0.50°C -dec reached to nine stations (Table 3). It is therefore evident that temperature at urban stations increased faster in recent decades. Figures 6(a) and 6(b) explain that dTx a is higher at more stations during P2 than P1. There are some urban stations where dTn a increased, however dTx a is higher mostly at rural and town stations.

Figure 7 illustrates the geographical distributions of stations' linear changes of dTx a (top) and dTn a (bottom) for P1 and P2 over the entire period (32 years for each period). Night warming during P2 is higher at number of stations mainly situated in Indus valley area where several major cities are located near to one another such as Sialkot, Jhelum, Lahore, Faisalabad, Sargodha, Multan, and Bahawalpur. Most of these cities are located on flat land on the Indus plain. The maps highlight that day warming is substantial mostly at stations in northern mountain areas, which are a major source of fresh water for large parts of the country. The rise in maximum temperature at stations in northern areas indicates an alert which may affect the precipitation pattern and may also accelerate the retreat of glaciers in the northern highlands, which are considered as the lifeline of Pakistan. Other studies have also found that mountain regions are warming faster than the global or hemispheric average [START_REF] Beniston | Climatic change at high elevation sites: an overview[END_REF][START_REF] Liu | Climatic warming in the Tibetan Plateau during recent decades[END_REF][START_REF] Ceppi | Revisiting Swiss temperature trends 1959-2008[END_REF].

Table 2 Figure 4 Figure 5 Figure 6 Figure 7 The percentage of stations out of the total number of stations where annual and seasonal minimum and maximum temperature showed positive trends/increased is reported in Table 4. It reflects that during P2, at almost 94% of urban stations, dTn a and dTn s were positive while this was only 51% during P1. Further details for town and rural stations are also presented in Table 4.

Table 3 Table 4 Figure 8 Figure 8 describes the magnitude of urban warming in comparison of urban station to its surrounding rural station or smaller town. Figure 8a highlights the trends of dTn a over Quetta, an urban station, and Kalat, a nearby rural station. Over a period of 64 years, long-term annual increase in trends of dTn a over Quetta city are found up to 0.25 °C-dec more than that of Kalat. Figure 8b highlights the trends of dTn a over Karachi, the largest city in Pakistan, and Lasbella, a nearby town station. The long-term annual increase in trends of dTn a for Karachi are up to 0.011 °C-dec more than the nearby smaller town. These two comparisons reveal that there is substantial urban impact on rising temperature trends. These types of trends are similar for many other stations in other parts of the world [START_REF] Brunetti | Urban heat island bias in Italian air temperature series[END_REF][START_REF] Liu | Temporal characteristics of the Beijing urban heat island[END_REF] as well as in Pakistan when urban and rural and urban and town stations are compared.

Results from SeqMK test

The SeqMK test is used to detail the changes in time series data of maximum and minimum temperature (not only for 2 periods in section 3.1, but also over the entire period), station by station to identify specific features. It provides new information compared to the HOMER method, which only allows us to detect and correct changes mainly concerning the station itself (change of instruments or localization). Here, we evaluate the possibility to automatically detect significant changes in the trends due to CC, urbanization, or other local land use change. For all stations, two types of information are presented:

o The years for which a break of trend is observed. Indeed, in cases where the forward and the backward u(ti) and u'(ti) series intersect, we note the associated years as breaks in the trends. If they intersect several times in a short period, we do not consider these changes as significant. Figures 9, 10 and 11illustrate specific examples for urban, town, and rural stations, respectively. Islamabad is an example for which both maximum and minimum temperatures show statistically significant abrupt change in trend, whereas Karachi-AP shows no well-defined commencement of such abrupt changes for maximum and minimum temperatures. Sialkot has a strong increase of minimum temperatures while these results are not considered significant. This result indicates a strong time variability around the trend.

o The significance of the observed trends using the p-value. Considering the results from the previous section on trend analysis, we expect to see systematic changes mostly in the minimum temperature due to the expansion of urbanization in urban areas compared to the town and rural areas. Table 5 shows that for all urban stations except Peshawar, Bahawalpur, D.I. Kahn, and Faisalabad, the SeqMk methodology detects statistically significant trends (p-value<0.05) for either the maximum or the minimum temperatures, or both. It is noted that half of breaks detected in Tn trends are between 1975 and 1985, while breaks in Tx appear at any time, but mostly late 1960s/early 1970s, and late 1990s/early 2000s. The SeqMk method suggests progressive trends of Tn temperatures at most urban stations over the entire period, while most of these stations showed quite high increases in Tn temperatures between P1 (before 1981) and P2 (after 1981) (see section 3.1). One can cite the example of Lahore where no abrupt changes of trend in Tn are observed by the SeqMk, but the trend for dTn a during P1 (urban) is 0.27 °C/decade and 0.73 °C/decade for P2. Karachi and Hyderabad are two urban areas situated near the sea with green surrounding areas and show significant abrupt changes in maximum temperature trends in the late 1970s/early 1980s. Islamabad shows a break in trends for Tn and Tx in the late 1990s.

Table 5 Table 5 also shows the SeqMk methodology detects statistically significant trends (p-value<0.05) for either the maximum or the minimum temperatures, or both for all town stations except Sibbi. Only five out of 11 town stations showed statistically significant trends in Tn. However, eight town stations show statistically significant trends (MK-test) in Tx, with several breaks detected between 1965-1975, the 1980s and late 1990s. The results of SeqMK test for the rural stations should be analyzed differently, since the abrupt changes cannot be due to urbanization (i.e. the stations are far away from urban areas). The changes in the rural location can only be explained by changes in climate or the surface characteristics. We note that for all rural stations, except Kalat, the detected trends are statistically significant (p-value<0.05) for either the maximum or the minimum temperatures, or both. We notice a slight increase of breaks since the 1980s.

Discussion and Conclusions

Both local and global warming make cities more vulnerable in terms of local extremes such as heat waves, which, in extreme conditions, aggravate the mortality rate mainly among children and the elderly. This study explored the variation in temperature over major cities, town areas, and at rural stations in Pakistan. Using the data of 36 meteorological stations with different physical characteristics (urban, town, and rural), the results of each type of station were not uniform and there were significant variations in temperature trends even among stations of the same category, that is, urban or rural stations.

It was observed that, on average, minimum and maximum temperature increased at all types of stations, but not at all stations. We show that the effect of urbanization is more pronounced on the minimum temperature than the maximum temperature and more at urban stations than town and rural stations. This is evident from the trends of averaged annual Tn of all urban stations, which are higher than the average annual Tn of rural stations (0.37°C -dec vs 0.03 °C-dec , respectively). The strength of warming over urban areas signifies alarming changes in the climate of urban areas.

Thus, the present study confirms the general notion that the larger the city, the greater its impact on local warming [START_REF] Hung | Assessment with satellite data of the urban heat island effects in Asian mega cities[END_REF]. However, based on our findings, it can also be asserted that larger cities' minimum or maximum temperatures do not necessarily increase more than those of smaller cities. It was found that even in the recent period (P2), dTn or dTx at different urban stations were not ranked according to the size of the cities (Figure 5b and Table 1). This may be influenced by other factors present in or around the individual urban stations, such as very local effects (impact of vegetation or specific urban structures), elevation, relief, or latitude; this needs to be evaluated by developing different scenarios to find this type of cause of change in temperature.

As the topography of Pakistan is not uniform, it is unsurprising that there are not uniform characteristics of dTn and dTx at all sets of stations (urban, town, and rural sets) or even a single set of stations. The observed dTn or dTx of nearby stations may also differ in ways that are unexpected in view of the classifications of the sites. For example, Hyderabad is an urban station that is larger than Chhor, a nearby town station. Both stations are located in the same type of environment; however, Tn at Hyderabad has decreased whereas it has increased at Chhor. Similarly, Gilgit (urban station) and Bunji (rural station) are located in the same kind of environmental conditions, at almost the same altitude and have the same type of topography (mountainous). However, this study revealed that during recent decades, the rise in Tn at Gilgit is substantially lower than Bunji even though the former is an urban station and larger in size than the latter. On the other hand, Tx at Gilgit increased more than at Bunji. Although a majority of studies suggest that warming is more rapid at higher elevation which is substantial in dTn a of P1 and dTx a of P2 of the present study, a number of studies show either no relationship or a more complex situation. A majority of studies also suggest that minimum temperatures show a stronger tendency toward elevation-dependent warming (EDW) than maximum temperatures [START_REF] Pepin | Elevation-dependent warming in mountain regions of the world[END_REF]. In certain cases, it may be true but not in others. It is also important that rising temperatures at stations located in northern parts of Pakistan, containing major glaciers, may definitely lead to retreat of glaciers at higher elevations as the overall climate system is warming. As a result of local/regional warming such as in the Tibetan/Himalaya region, the snow cover season has shortened and more precipitation is now falling as rain [START_REF] Rikiishi | Height dependence of the tendency for reduction in seasonal snow cover in the Himalaya and the Tibetan Plateau region, 1966-2001[END_REF][START_REF] Archer | Spatial and temporal variations in precipitation in the Upper Indus Basin, global teleconnections and hydrological implications[END_REF][START_REF] Bhutiyani | Climate change and the precipitation variations in the northwestern Himalaya: 1866-2006[END_REF].

Thus, it can be concluded that the trends of dTn and dTx are strongly influenced by the local conditions where a particular station is located. Local characteristics of different stations, even with similar terrain, population density, elevation, and mountain valley shape where a station is located, greatly affect the dTn and dTx and should be detailed as metadata. The orographic climatic conditions, nearness to sea, and flat areas also affect the local area temperature. Differing influences on dTn and dTx are evident from other regional cities of India where at Mumbai's annual dTn shows decreasing trends while dTx shows increasing tendencies. Similarly, annual dTn at Kolkata shows lower trends than annual dTx [START_REF] Dhorde | Long-term temperature trends at four largest cities of India during the twentieth century[END_REF]. 1950-1981 1982-2013 1950-1981 1982-2013 1950-1981 1982

Figures

  To identify the significant change of trends in temperature data of various stations of Pakistan, the sequential Mann-Kendall (SeqMK; Mohsin and Gough, 2010) test was used. The application of sequential Mann-Kendall test has the following steps: 1. The N values of the original data of a data series (say) x are replaced by their ranks in a new data series y, arranged in ascending order 2. The magnitudes of yi (i = 1,...,N) are compared with yj (j = 1,...,i-1). At each comparison, the number of cases yi>yj need to be counted and denoted by ni 3. A statistic ti can be defined as follows: ti = 4. The distribution of the test statistic has an expectation mean E(ti) and a variance as E(ti) = i(i -1)/4 and var(ti) = i(i -1)(2i + 5)/72 5. The sequential values of the statistic u(ti) can then be computed as u(ti) = [ti -E(ti)] /SQRT(var(ti))

Figure 4

 4 Figure 4 illustrates the details of the results, showing the annual net change in trends of dTx a as a function of the annual net change in trends of dTn a on all the 16 urban, 11 towns, and 9 rural stations for both periods.

  Figure 9 Figure 10 Figure 11

Figure 1 :

 1 Figure 1: Total population (million) and total urban population (%) of Pakistan for the period 1955 -2020.

Figure 2 :

 2 Figure 2: Location map of network of meteorological stations of Pakistan. Large circles represent the stations located at higher altitude and small circles the stations at lower altitude..

Figure 3 :

 3 Figure 3: Comparison of trends of Tn a with and without the homogeneity check for Lahore (a) and Quetta (b).

Figure 4 :Figure 5 :Figure 6 :

 456 Figure 4: Per decade change in trends of dTn a (x-axis) as a function of trends of dTx a (y-axis) for urban (red filled circles), town (blue filled circles) and rural (green filled circles) stations for P1 (a); P2 (b) and dTP2-P1 (c). The hollow red, blue and green circles in a (P1) and b (P2) represent the trends of dTn a as a function of trends of dTx a drawn by taking the mean of all urban, town and rural stations, individually, respectively.

Figure 7 :

 7 Figure 7: Graphical presentation of linear changes in dTx a (top) and dTn a (bottom) of all stations for each of P1 and P2 (32 years for each period).

Figure 8 :

 8 Figure 8: Magnitude of urban warming in comparison of (a) Quetta (urban) vs Kalat (rural) and (b) Karachi (urban) vs Lasbella (town).

Figure 9 :

 9 Figure 9: Sample plots showing the changes for urban stations associated with u(t) and u'(t), that is, the forward and backward time series, respectively obtained from the SeqMK test.

Figure 10 :

 10 Figure 10: Sample plots showing the changes for town stations associated with u(t) and u'(t), that is, the forward and backward time series, respectively obtained from the SeqMK test.

Figure 11 :

 11 Figure 11: Sample plots showing the changes for rural stations associated with u(t) and u'(t), that is, the forward and backward time series, respectively obtained from the SeqMK test.

dT n (°C/decade) dT x (°C/decade) dT n (°C/decade) dT x (°C/decade) dT n (°C/decade) (P2 -P1) dT x (°C/decade) (P2 -P1)

	dT u	0.04	-0.03	0.37	0.21	0.33	0.25
	dT t	0.13	-0.07	0.10	0.16	-0.03	0.23
	dT r	0.20	0.04	0.03	0.26	-0.18	0.22
	dT u -dT r	-0.17	-0.07	0.34	-0.04	0.50	0.03
	dT t -dT r	-0.07	-0.11	0.07	-0.10	0.15	0.02
	dT u	-0.07	-0.11	0.29	-0.02	0.36	0.09
	dT t	0.04	-0.17	0.21	0.22	0.16	0.39
	dT r	0.06	-0.06	0.14	0.36	0.08	0.42
	dT u -dT r	-0.13	-0.05	0.14	-0.38	0.27	-0.33
	dT t -dT r	-0.02	-0.11	0.06	-0.14	0.08	-0.03
	dT u	0.04	0.10	0.71	0.90	0.67	0.79
	dT t	0.26	0.05	0.27	0.73	0.02	0.67
	dT r	0.32	0.26	0.37	0.63	0.05	0.38
	dT u -dT r	-0.28	-0.16	0.34	0.26	0.62	0.42
	dT t -dT r	-0.06	-0.20	-0.10	0.09	-0.03	0.30
	dT u	-0.18	-0.23	0.22	-0.09	0.39	0.14
	dT t	0.07	-0.12	0.08	-0.07	0.01	0.05
	dT r	0.19	-0.02	-0.02	-0.13	-0.20	-0.11
	dT u -dT r	-0.36	-0.21	0.23	0.04	0.60	0.25
	dT t -dT r	-0.11	-0.11	0.10	0.05	0.21	0.16
	dT u	-0.12	0.02	0.44	0.04	0.56	0.01
	dT t	0.11	-0.11	0.16	0.09	0.06	0.19
	dT r	0.08	-0.10	0.14	0.26	0.07	0.36
	dT u -dT r	-0.19	0.12	0.30	-0.23	0.49	-0.35
	dT t -dT r	0.03	-0.01	0.02	-0.18	-0.01	-0.17

Summer Autumn Net change from P1 to P2 dT u , dT t , dT r Period P1 (1950-1981) P2 (1982-2013) Annual Winter Spring

Table 3 :

 3 Number of stations in four ranges of dTn a and dTx a for P1 and P2.

				dT (°C -dec )
	dT	Period					Total stations
			< 0	0 -0.30	0.30 -0.50	> 0.50
	dTn a	P1 (1950-1981) P2 (1982-2013)	14 11	15 9	5 7	2 9	36 36
	dTx a	P1 (1950-1981) P2 (1982-2013)	15 7	20 15	1 6	0 8	36 36

Table 4 :

 4 Percentage of stations showing increasing trends in annual and seasonal Tn and Tx for each type of stations for the period1950-1981 and 1982-2013.

	dTn and dTx	Urban	Town	Rural

Table 5 :

 5 Summary of the Mann-Kendall test comparing maximum and minimum temperature for urban stations. Significant values (p value<0.05) highlighted in gray.

	Stations

Years of Breaks in Tn trend MK test-Tn (p-value) Years of Breaks in Tx trend MK test-Tx (p-value)

			Urban stations
	Gilgit	none	6.10 -5	none	3.10 -4
	Quetta	none	2.10 -7	none	1.10 -4
	Lahore_ur	none	2.10 -6	none	8.10 -3
	Karachi	none	3.10 -6	1968	1.10 -5
	Hyderabad	none	2.10 -3	1967, 1984	0.02
	Islamabad	1999	1.10 -3	1997	0.01
	Multan	none	1.10 -6	none	0.45
	Sargodha	none	2.10 -5	1971, 1999	0.11
	Jhelum	none	1.10 -6	1996, 2001, 2010	0.81
	Kakul	none	1.10 -5	1993, 2002	0.27
	Sialkot	1975,	0.11	none	1.10 -4
	Muzaffarabad	1987, 1990	0.48	1997	2.10 -5
	Peshawar	1998	0.058	1968, 1971	0.07
	Bahawalpur	1969,1982	0.37	1969, 1982	0.05
	D.I.Khan	1977, 1983	0.17	1974, 2003, 2011	0.37
	Faisalabad	1984, 2002	0.12	1969, 2002	0.63
	Panjgur	none	Town stations 2.10 -7	none	1.10 -5
	Badin	none	2.10 -6	1971,1977	3.10 -5
	Jacobabad	none	9.10 -4	1969,1975	0.207
	Padidan	none	7.10 -4	1985, 1994	0.561
	Kohat	1964,1987,1994	0.025	1985	7.10 -4
	Risalpur	1969,1982,2012	0.54	1965,1999	0.039
	Lasbella	1969, 1994, 2000	0.50	1967, 1984	3.10 -6
	Nawabshah	1994, 2000	0.98	1996, 2002	2.10 -3
	Chhor	1996, 2003	0.14	none	4.10 -5
	Skardu	1971, 1987, 2003	0.15	1971,1987,2003	2.10 -7
	Sibbi	1970, 1988, 2002	0.68	1987,2005	0.74
	Dalbandin	none	Rural stations 1.10 -7	none	2.10 -5
	Astore	none	0.025	none	0.035
	Cherat	1997	5.10 -3	none	6.10 -4
	Bunji	1988	9.10 -4	1961, 1988	0.13
	Chilas	none	1.10 -3	1967, 1999	0.85
	Gupis	1969 1993, 2007	0.58	1969,1996, 2007	2.10 -3
	Jiwani	1979, 2002	0.78	1976	2.10 -3
	Pasni	1960, 2002	0.13	none	2.10 -4
	Kalat	1989,2005	0.86	1993,2005	0.59

Sajjad Hussain SAJJAD a * , Nadège BLOND a , Tanzina MOHSIN b , Khadija

Acknowledgment

The principal author thanks College de France, Université de Strasbourg and Laboratoire Image Ville Environment (LIVE) for providing funding for research work under PAUSE project (BPC 2018 / ARC number 28503 and BPC 2019 / ARC number 33684 NTYPOP 1) and Nadège Blond (second author). We also thank the Pakistan Meteorological Department (PMD) for providing meteorological data for this study, Oleg Skrynyk from the Ukrainian Hydrometeorological Institute for his help regarding the use of HOMER 2.6 to check the homogeneity of the data, Nasir Ashraf, GIS Expert JEC, Pakistan and Seema Saadi, University of Tabriz, Iran for their help to finalize the maps.

This study focused on temperature trends in urban, town and rural stations of Pakistan. It was observed that minimum and maximum temperature increased on average at all kinds of stations, though not at every station. The effect of urbanization is more pronounced on the minimum temperature rather than the maximum temperature and more at urban stations than the town and rural stations. This is evident from the obtained trends of averaged annual Tn of all urban stations which are many times higher than the average annual Tn of rural stations (0.37°C -dec vs 0.03°C -dec , respectively in 1982-2013). The predominance of warming over urban areas signifies alarming changes in urban climate. The study also revealed that the rate of increase in minimum temperature based on data analysis from 1982-2013 at urban stations is significantly higher than in the smaller towns." Minimum temperature is especially increasing in the big cities in the Indus valley where more than half of the population of Pakistan lives.

TEMPERATURE VARIABILITY OVER URBAN, TOWN AND RURAL AREAS: THE CASE OF PAKISTAN

Sajjad Hussain SAJJAD a * , Nadège BLOND a , Tanzina MOHSIN b , Khadija SHAKRULLAH c , Alain CLAPPIER a Tables Table 1: Geographical description of meteorological stations located in different parts of Pakistan.

Station description

Table 2: Observed trends of dTn a, s , dTx a, s ; dTn u-r , dTx u-r ; dTn t-r and dTx t-r (°C -dec ) for P1 and P2, respectively.


Jinane Farah

Marie Choël

Patricia De Nadaï

Joanne Balsamelli

Sylvie Gosselin

Nicolas Visez
email: nicolas.visez@univ-lille.fr

ORGANIC AND AQUEOUS EXTRACTION OF LIPIDS FROM BIRCH POLLEN GRAINS EXPOSED TO GASEOUS POLLUTANTS

Keywords: Ozone, Nitrogen dioxide, Bioaccessibility, Pollen Cytoplasmic Granules, Pollen Rupture, Asthma

The lipid fraction of birch pollen grains (BPGs) is not yet fully described, although pollen lipid molecules may play a role in the allergic immune response. The mechanisms by which atmospheric pollutants modify allergenic pollen grains (PGs) are also far from being elucidated despite high potential effects on allergic sensitization. This work is a contribution to a better description of the lipid profile (both external and cytoplasmic) of BPGs and of alterations induced by gaseous air pollutants. Several lipid extractions were performed using organic and aqueous solvents on BPGs following exposure to ozone and/or nitrogen dioxide and under conditions favoring the release of internal lipids. Ozone reacted with alkenes to produce aldehydes and saturated fatty acids, while nitrogen dioxide was shown to be unreactive with lipids. NO 2 exhibited a protective effect against the reactivity of alkenes with ozone, probably by competition for adsorption sites. The decreased reactivity of ozone during simultaneous exposure to NO 2 /O 3 raised the possibility of a Langmuir-Hinshelwood mechanism. Oxidation reactions induced by exposure of BPGs to ozone did not substantially modify the extraction of lipids by aqueous solvent, suggesting that the bioaccessibility of lipids was not modified by oxidation. On the contrary, the rupture of PGs appeared to be a key factor in enhancing the bioaccessibility of bioactive lipid mediators (linoleic and α-linolenic acids) in aqueous solution. The internal lipid fraction of BPGs has specific characteristics compared to external lipids, with more abundant hexadecanoic acid, tricosanol and particularly unsaturated fatty acids (linoleic and α-linolenic acids). Several mechanisms of action of gaseous pollutants on allergenic pollen were identified in this study: gaseous air pollutants can (i) modify the external lipid fraction by reactivity of alkenes, (ii) adsorb on the surface of PGs and be a source of oxidative stress after inhalation of PGs, and (iii) promote the release of cytoplasmic bioactive lipids by facilitating pollen rupture.

Introduction

Birch Pollen Grain (BPG) is one of the most abundant tree pollen in Europe and a major cause of allergenic rhinitis and asthma attacks [START_REF] Biedermann | Birch Pollen Allergy in Europe[END_REF]. Seasonal allergic asthma has been linked to pollen aeroallergens even though the majority of pollen grains (PGs) are too large in size (>20 µm in diameter) to reach the lower respiratory airways where asthmatic reactions occur (Schäppi et al. 1997a;[START_REF] Taylor | Birch Pollen Rupture and the Release of Aerosols of Respirable Allergens[END_REF][START_REF] Amato | Thunderstorm-Asthma and Pollen Allergy[END_REF][START_REF] Taylor | Links Between Pollen, Atopy and the Asthma Epidemic[END_REF]Abou Chakra et al. 2011a;[START_REF] Caillaud | Effects of Airborne Birch Pollen Levels on Clinical Symptoms of Seasonal Allergic Rhinoconjunctivitis[END_REF][START_REF] Amato | Thunderstorm-Related Asthma: What Happens and Why[END_REF]. Birch pollen allergens have been detected in inhalable aerosol fractions (particulate matter of less than 10 µm in diameter), particularly on days of light rainfall during the birch pollen season [START_REF] Schäppi | Direct Determination of Allergens in Ambient Aerosols: Methodological Aspects[END_REF](Schäppi et al. , 1997b;;[START_REF] Grote | Abortive Pollen Germination: A Mechanism of Allergen Release in Birch, Alder, and Hazel Revealed by Immunogold Electron Microscopy[END_REF][START_REF] Taylor | Birch Pollen Rupture and the Release of Aerosols of Respirable Allergens[END_REF]). Pollen cytoplasmic granules (PCGs) are microscopic particles such as starch granules, polysaccharide particles and other cytoplasmic pollen fragments; PCGs released from the pollen cytoplasm are capable of triggering allergic asthmatic reactions (Abou Chakra et al. 2011a, b, 2012). Several mechanisms are proposed in literature for the release of PCGs which could be the result of pollen grain bursting due to sporoderm breakage, to an abortive germination (growth and burst of the pollen tube) or to the ejection of PCGs from the grain aperture after pollen immersion in an aqueous solution [START_REF] Suphioglu | Mechanism of Grass-Pollen-Induced Asthma[END_REF][START_REF] Grote | Release of Allergen-Bearing Cytoplasm from Hydrated Pollen: a Mechanism Common to a Variety of Grass (Poaceae) Species Revealed by Electron Microscopy[END_REF][START_REF] Taylor | Release of Allergens as Respirable Aerosols: a Link Between Grass Pollen and Asthma[END_REF][START_REF] Motta | Traffic-Related Air Pollutants Induce the Release of Allergen-Containing Cytoplasmic Granules from Grass Pollen[END_REF][START_REF] Rogerieux | Modifications of Phleum pratense Grass Pollen Allergens following Artificial Exposure to Gaseous Air Pollutants (O 3 , NO 2 , SO 2 )[END_REF][START_REF] Ghiani | Ragweed Pollen Collected Along High Traffic Roads Shows a Higher Allergenicity Than Pollen Sampled in Vegetated Areas[END_REF][START_REF] Smiljanic | Sub-Pollen Particles Are Rich Carriers of Major Short Ragweed Allergens and NADH Dehydrogenases: Quantitative Proteomic and Allergomic Study[END_REF][START_REF] Smiljanic | In-Depth Quantitative Profiling of Post-Translational Modifications of Timothy Grass Pollen Allergome in Relation to Environmental Oxidative Stress[END_REF].

PCGs from allergenic PGs are a source of allergenic proteins and NAD(P)H oxidases in the respirable fine fraction of atmospheric aerosols, which can cause allergic airway inflammation [START_REF] Bacsi | Effect of Pollen-Mediated Oxidative Stress on Immediate Hypersensitivity Reactions and Late-Phase Inflammation in Allergic Conjunctivitis[END_REF][START_REF] Bacsi | Subpollen Particles: Carriers of Allergenic Proteins and Oxidases[END_REF][START_REF] Boldogh | Ros Generated by Pollen NADPH Oxidase Provide a Signal That Augments Antigen-Induced Allergic Airway Inflammation[END_REF]. Immunological studies have highlighted the potential immunological role of pollen lipid molecules in allergic immune response regulation (Behrendt et al. 2001;[START_REF] Traidl-Hoffmann | Impact of pollen on Human Health: more than Allergen Carriers?[END_REF][START_REF] Gilles | The Pollen Enigma: Modulation of the Allergic Immune Response by Non-Allergenic, Pollen-Derived Compounds[END_REF][START_REF] Bublin | Do Lipids Influence the Allergic Sensitization Process?[END_REF][START_REF] Dahl | Pollen Lipids Can Play a Role in Allergic Airway Inflammation[END_REF][START_REF] Aglas | Context Matters: Th2 Polarization Resulting from Pollen Composition and not from Protein-Intrinsic Allergenicity[END_REF]. However, lipid molecules in allergenic airborne pollen fragments of respirable size are still under-studied (Farah et al. 2020a). The lipid fraction of mature pollen is distributedbetween the external and internal lipids constituting respectively the extracellular pollen wall and the cytoplasm [START_REF] Evans | A Study of Phospholipids and Galactolipids in Pollen of Two Lines of Brassica napus L. (Rapeseed) with Different Ratios of Linoleic to Linolenic Acid[END_REF][START_REF] Evans | Quantitative Analysis of Lipids and Protein from the Pollen of Brassica napus L[END_REF][START_REF] Piffanelli | Intra-and Extracellular Lipid Composition and Associated Gene Expression Patterns During Pollen Development in Brassica napus[END_REF][START_REF] Piffanelli | Biogenesis and Function of the Lipidic Structures of Pollen Grains[END_REF][START_REF] Pacini | Pollenkitt -Its Composition, Forms and Functions[END_REF]. The sporderm of PGs act as barriers for the extraction of cytoplasmic lipids and PGs must therefore be fractured in order to release their internal lipids [START_REF] Evans | Quantitative Analysis of Lipids and Protein from the Pollen of Brassica napus L[END_REF]).

Among external lipids extracted from allergenic BPGs, the following chemical families have been identified: alkanes, alkenes, alcohols, aldehydes, fatty acids (saturated and unsaturated), aldehydes, ethers, amino-alcohols and phenolic compounds [START_REF] Henricsson | Chemical Characterisation of Extractable Compounds Found in the Coating of Birch (Betula) Pollen[END_REF][START_REF] Traidl-Hoffmann | Lipid Mediators from Pollen Act as Chemoattractants and Activators of Polymorphonuclear Granulocytes[END_REF][START_REF] Bashir | Pollen Lipidomics: Lipid Profiling Exposes a Notable Diversity in 22 Allergenic Pollen and Potential Biomarkers of the Allergic Immune Response[END_REF][START_REF] Mueller | A Metabolomic, Geographic, and Seasonal Analysis of the Contribution of Pollen-Derived Adenosine to Allergic Sensitization[END_REF][START_REF] Zhu | Uptake of Ozone and Modification of Lipids in Betula Pendula Pollen[END_REF]. To the best of our knowledge, the internal lipid composition of birch pollen has not been previously described in literature. In Phleum pratense PGs, large differences were detected between external and internal lipid fractions, in particular for unsaturated fatty acids (Farah et al. 2020a).

The interactions of atmospheric pollutants with pollen allergens can worsen respiratory allergy and asthma [START_REF] Sénéchal | A Review of the Effects of Major Atmospheric Pollutants on Pollen Grains, Pollen Content and Allergenicity[END_REF][START_REF] Frank | Effects of NO 2 and Ozone on Pollen Allergenicity[END_REF]. For example, in Denmark, peaks in ozone concentrations were observed in spring concomitantly with peaks in BPGs concentrations (Ørby et al. 2015). Ozone has been associated with a worsening of allergic symptoms during birch pollen season [START_REF] Berger | Impact of Air Pollution on Symptom Severity During the Birch, Grass and Ragweed Pollen Period in Vienna, Austria: Importance of O 3 in 2010-2018[END_REF] but also with increased Bet v 1 allergen content in BPGs [START_REF] Beck | High Environmental Ozone Levels Lead to Enhanced Allergenicity of Birch Pollen[END_REF]. Moreover, Pollen Associated Lipid Mediators (PALMs) were extracted in higher quantities in BPGs from areas with higher ozone exposure [START_REF] Beck | High Environmental Ozone Levels Lead to Enhanced Allergenicity of Birch Pollen[END_REF]. Chemical modifications of external lipids have been highlighted for BPGs exposed to ozone in laboratory conditions [START_REF] Zhu | Uptake of Ozone and Modification of Lipids in Betula Pendula Pollen[END_REF]. Moreover, Bet v 1 nitration by exposure to polluted air induced an increased potency to trigger and enhance an immune allergic reaction [START_REF] Franze | Protein Nitration by Polluted Air[END_REF][START_REF] Reinmuth-Selzle | Nitration of the Birch Pollen Allergen Bet v 1.0101: Efficiency and Site-Selectivity of Liquid and Gaseous Nitrating Agents[END_REF]. The co-exposure of birch pollen extracts to both NO 2 and O 3 gases highlighted a synergistic effect between those pollutants [START_REF] Franze | Enzyme Immunoassays for the Investigation of Protein Nitration by Air Pollutants[END_REF]. In addition, the exposure to atmospheric pollutants is a facilitating factor for pollen rupture in the atmosphere [START_REF] Motta | Traffic-Related Air Pollutants Induce the Release of Allergen-Containing Cytoplasmic Granules from Grass Pollen[END_REF][START_REF] Sénéchal | A Review of the Effects of Major Atmospheric Pollutants on Pollen Grains, Pollen Content and Allergenicity[END_REF][START_REF] Puc | Birch Pollen Grains Without Cytoplasmic Content in the Air of Szczecin and Bialystok[END_REF]. The molecular mechanisms behind gaseous pollutant (NO 2 or O 3 ) effects on PGs are largely unknown [START_REF] Frank | Effects of NO 2 and Ozone on Pollen Allergenicity[END_REF]. Moreover, tropospheric ozone concentrations are predicted to rise in polluted areas during the 21 st century as a result of climate change [START_REF] Lu | Meteorology and Climate Influences on Tropospheric Ozone: a Review of Natural Sources, Chemistry, and Transport Patterns[END_REF]). In addition, the concentrations of BPGs have been observed to rise in various locations in recent decades, notably because of the increase in atmospheric concentrations of carbon dioxide [START_REF] Frei | Climate Change and Its Impact on Birch Pollen Quantities and the Start of the Pollen Season an Example from Switzerland for the Period 1969-2006[END_REF][START_REF] Hoebeke | Thirty-Four Years of Pollen Monitoring: An Evaluation of the Temporal Variation of Pollen Seasons in Belgium[END_REF][START_REF] Besancenot | Pollen et changement climatique. Bouleau et graminées en France métropolitaine[END_REF]. Thus, individual exposure to the ozone-BPG cocktail is already affecting the health of allergic patients, and this harmful co-exposure is expected to increase further in the coming decades [START_REF] Berger | Impact of Air Pollution on Symptom Severity During the Birch, Grass and Ragweed Pollen Period in Vienna, Austria: Importance of O 3 in 2010-2018[END_REF].

Lipidic fractions (both external and internal) of allergenic PGs are still under-studied. In this work, BPGs were exposed in laboratory to O 3 , NO 2 and a mixture of both gases. Lipid extractions were performed with both organic and aqueous solvents in order to discuss the bioaccessbility of lipids and their potential modification by oxidation reactions. As the pollen sporoderm is an efficient barrier against solvent extraction, BPGs were also artificially ruptured in order to gain insights about the internal lipids. This work contributes to a better description of the lipid profile of BPGs and its alteration by gaseous urban air pollutants in the perspective of an increase of birch pollen allergy in the 21 st century.

Materials and methods

Sampling of birch pollen grains

Male catkins were harvested during birch (Betula pendula) pollination period in March 2017 on the campus of the University of Lille (France). Fresh PGs were sieved at 40 μm in laboratory, placed in a closed container under a dry atmosphere for 24 h and then stored hermetically in sealed vials at 4ºC until used. Microscopic observations of birch pollen after this drying step shows that PGs were all intact (no broken PG was observed). Broken pollen grains were artificially produced in laboratory by grinding pollen grains with a magnetic stirrer for 4 hours at 1000 rpm (Farah et al. 2020a).

The water content of stored BPGs was estimated to less than 5% by weighing BPGs at different temperatures up to 150°C. All experiments were therefore carried out with dry pollen. In the text below, 'intact PGs' refers to dried pollen grains without any further treatment and 'broken PGs' refers to dried pollen grains artificially broken (crushed by grinding) in laboratory.2.2 Exposure of pollen grains to ozone and nitrogen dioxide PGs were deposited between two sheets of cellulose filter paper (GE Healthcare, Life Sciences Whatman TM , diameter 55 mm) and exposed to gaseous pollutant flow into a stainless-steel fluidized bed reactor. Exposures were performed for 16 hours under dry conditions and laboratory temperature (20±1 °C). PGs were exposed separately to 130±5 ppb of O 3 and to 100±5 ppb of NO 2 . PGs were also exposed to a mixture of NO 2 and O 3 at concentrations of 100±5 pbb for each gas. Ozone was generated from synthetic air by a UV-generator at a flow rate of 1.35 L.min -1 . O 3 concentration was measured at the outlet of the filter holder every 10 s with a detection limit of 1 ppb using an ozone analyzer (Thermo Fisher Scientific, model Megatec 49i). Nitrogen dioxide was generated from a flow (2.5 L.min -1 ) of nitric oxide using a NO x generator (Ansyco, SYCOS KT-GPTM). NO x concentration was measured at the reactor outlet by a nitrogen oxide analyzer (Thermo Fisher Scientific, Model Megatec 42i) performing a measurement every 60 s with a detection limit of 0.4 ppb.

Organic extraction of external and internal lipids

The extraction procedure for external lipids without disrupting pollen cell-wall integrity was described elsewhere [START_REF] Zhu | Uptake of Ozone and Modification of Lipids in Betula Pendula Pollen[END_REF]Farah et al. 2020b, a). In brief, external molecular lipids were extracted from 50 mg of either intact or broken PGs with 5 mL of methylene chloride (≥99.8%; Sigma-Aldrich). The organic extract was then dried under a stream of nitrogen. Finally, the residue was solubilized in 50 μL of methylene chloride and derivatized by reconstituting with 50 μL of 99 % of BSTFA -1% TMCS (N, O-bis(trimethylsilyl) trifluoroacetamide, trimethylchlorosilane; Sigma-Aldrich).

Chemical analyses were carried out with a gas chromatograph coupled to a mass spectrometer (GC/MS) detector for identification and with a flame ionization detector (GC/FID) for quantification. Chromatographic parameters were as follows: injection of 1 µL, helium column flow of 1 mL•min -1 , split ratio of 1:10, injector temperature of 250°C, oven temperature initially at 50°C and raised at 4ºC.min -1 to reach 310ºC (hold for 19 min). The MS ionization was done by electronic impact at 70 eV. Lipid molecular species were quantified using carbon equivalent number as previously described [START_REF] Scanlon | Calculation of Flame Ionization Detector Relative Response Factors Using the Effective Carbon Number Concept[END_REF][START_REF] Zhu | Uptake of Ozone and Modification of Lipids in Betula Pendula Pollen[END_REF]) excepted for unsaturated fatty acids (linoleic acid and α-linolenic acid) which were directly quantified using their FID response factor determined by injection of standard solutions. For each lipid extraction condition, three separate extractions and separate GC analyses were performed. Every single sample was treated as an independent variable and the error bars for mass concentrations were expressed as the maximum deviation for those three analyses around the mean value. The mass of lipids A i in internal fraction was calculated from the mass of A i calculated from extracts of broken and intact PGs following equation 1:

m(A i ) internal = m(A i ) extraction of broken PGs -m(A i ) extraction of intact PGs Equation 1
Negatives values calculated for m(A i ) internal and values inferior to the maximum deviation were set to zero.

Total dry extractible mass

In order to estimate the total mass of internal extractable matter by organic solvent, 1 gram of broken and intact PGs was extracted with 5 mL of methylene chloride. The total amount of extractable lipids was then weighed with a precision balance (±0.1 mg) after complete evaporation of the solvent.

Aqueous lipid extraction

PGs were extracted with Phosphate Buffered Saline (PBS) solution (Thermo fisher Scientific) to be close to physiological conditions. The aqueous pollen extraction steps have been described elsewhere (Farah et al. 2020b). In brief, 50 mg of PGs were extracted for 2 hours by suspension in 2 mL of PBS solution under different conditions:

intact PGs with no agitation or with magnetic stirring agitation (1000 rpm), laboratory-ruptured PGs deposited in PBS without agitation and laboratory-ruptured PGs with gentle agitation. In order to identify the lipid molecular species released in aqueous phase from broken pollen, PGs were fractured during 4 hours using mechanical agitation with a magnetic stirrer (Farah et al. 2020a) followed by extraction with 2 mL of PBS in two ways: suspension without agitation or gentle agitation by rotation of extraction tubes.

Pollen lipids extracted by PBS were then transferred to organic solvent by the following steps: centrifugation at 1800 g for 2 min after PBS extraction, filtration of the supernatant, liquid-liquid extraction (PBS-methylene chloride) by adding 2 mL of organic solvent, hand-shake for 2 min, centrifugation at 1800 g for 2 min, recovery of the organic phase (repeated three times) followed by evaporation to dryness of solvent. The dry residue was finally solubilized in 50 μL of methylene chloride and 50 μL of BSTFA-TMCS.

Scanning electron microscopy (SEM)

BPGs were observed by SEM to determine the extent of physical damage due to thorough mechanical stirring. BPGs were spread over a conductive carbon tape and gold-coated prior to SEM imaging using a field-emission gun scanning electron microscope (Hitachi S4700 FEG-SEM) operated at 3 kV.

Results

Description of external and internal lipid composition of birch pollen grains

A total dry mass of 27±1 mg.g -1 of extractable lipids from BPGs collected in 2017 was obtained; such mass is identical to the total mass extracted from BPGs collected in 2016 on the same site [START_REF] Zhu | Uptake of Ozone and Modification of Lipids in Betula Pendula Pollen[END_REF]. Identified and quantified lipids from BPGs collected in 2017 (this work) summed up for 3591 ng.mg -1 (i.e. about 13% of the total dry extract). Extractable lipids can be classified in 6 chemical families (table 1 and fig. 1): alkenes (1655±167 ng.mg -1 ), alcohols (1052±123 ng.mg -1 ), fatty acids (671±81 ng.mg -1 ), alkanes (164±19 ng.mg -1 ), aldehydes (30±5 ng.mg -1 ) and unsaturated fatty acids (19±3 ng.mg -1 ). Total masses per family were very similar between organic extracts of BPGs from the 2016 and 2017 pollination seasons [START_REF] Zhu | Uptake of Ozone and Modification of Lipids in Betula Pendula Pollen[END_REF]. Unsaturated fatty acids were not identified and quantified in the work of [START_REF] Zhu | Uptake of Ozone and Modification of Lipids in Betula Pendula Pollen[END_REF].

The total dry residue of the extract from crushed BPGs weighed 33 mg.g -1 corresponding to the sum of extractable organic matter from both internal and external parts of PGs. The total mass of identified lipids was almost equally distributed between internal (4181 ng.mg -1 ) and external fractions (3591 ng.mg -1 ). The same lipids were identified between both external and internal regions, only their mass fractions differed. Mass fractions of unsaturated fatty acids were most different between external and internal lipids of BPGs: more than 99% of unsaturated fatty acids were recovered from broken BPGs (i.e. internal lipids). Hexadecanoic acid and tricosanol were also more abundant in internal lipids (respectively 97% and 98%). Internal lipid fraction of BPGs was described for the first time in this work and it has great similarities with that of Phleum Pratense PGs (Farah et al. 2020a). Aldehydes and saturated fatty acids are produced by the reactions between ozone and alkenes from the external lipid fraction of BPGs [START_REF] Zhu | Uptake of Ozone and Modification of Lipids in Betula Pendula Pollen[END_REF]. This reactivity induces a decrease in alkenes mass fractions (from 1655 to 542 ng.mg -1 ) and an increase from 671 to 960 ng.mg -1 for saturated fatty acids and from 30 to 413 ng.mg -1 for aldehydes (table 2). Mass fractions of alkenes are similar between BPGs samples from pollination period 2017 and 2016

(respectively 1655 and 1651 ng.mg -1 ). The consumption of alkenes by ozone is however much more important for BPGs from 2017 (decrease of 70%) compared to 2016 (decrease of 30%) despite exposure of pollen samples to very close doses of ozone (2017: 16 hours at 130±5 ppb; 2016: 16 hours at 115±5 ppb) [START_REF] Zhu | Uptake of Ozone and Modification of Lipids in Betula Pendula Pollen[END_REF].

Exposure of BPGs to NO 2 alone (100 ppb, 16 hours) had no observable effects on the lipid molecular species identified in this work (table 2). New compounds were not identified consecutively to NO 2 exposure. Alkenes consumption was less marked when BPGs were exposed simultaneously to NO 2 and O 3 : observation of a decrease of 70% for alkenes after exposure to ozone alone and of 30% after exposure to both NO 2 and O 3 (table 2).

Discussion and conclusion

The internal lipid fraction of BPGs has specific characteristics compared to external lipids with more abundant hexadecanoic acid, tricosanol and particularly unsaturated fatty acids (linoleic and α-linolenic acids). The most abundant lipids extracted from BPGs indeed originated from the internal fraction: linoleic acid 2.26 µg.mg -1 and αlinolenic acid 1.75 µg.mg -1 . Rupturing of the membrane of BPGs is a crucial mechanism to account for the dispersion of allergens in the fine fraction of the atmospheric aerosol (Schäppi et al. 1997a;[START_REF] Grote | Abortive Pollen Germination: A Mechanism of Allergen Release in Birch, Alder, and Hazel Revealed by Immunogold Electron Microscopy[END_REF][START_REF] Taylor | Birch Pollen Rupture and the Release of Aerosols of Respirable Allergens[END_REF][START_REF] Visez | Wind-Induced Mechanical Rupture of Birch Pollen: Potential Implications for Allergen Dispersal[END_REF]. Inhalation of broken BPGs and/or their PCGs therefore exposes the immune system simultaneously to internal lipids and to the protein allergens.

Understanding of the bioaccessibility of biochemical compounds carried by PGs in physiological conditions needs to be improved. Pollen rupture seems indeed a crucial mechanism for the release of potentially bioactive lipids. PBS used as an aqueous solvent proved to be a very poor solvent for lipid extraction from BPGs, including the most hydrophilic lipids (saturated and unsaturated fatty acids), except under conditions of extraction with strong stirring.

To be effective, the extraction must be carried out with mechanically ruptured BPGs. Pollen rupture seems to be a prerequisite to enhance the bioaccessibility of certain lipids in aqueous solution, especially for unsaturated fatty acids mostly found in the cytoplasm of the BPGs. Inhalation of broken BPGs may be common, at least on specific meteorological conditions, as the fraction of BPGs without their cytoplasm has been estimated to up to 9% in atmospheric environment [START_REF] Puc | Birch Pollen Grains Without Cytoplasmic Content in the Air of Szczecin and Bialystok[END_REF]. Investigations of immune response reactions should take into account this cytoplasmic-specific lipid fraction in order to determine whether inhalation of broken pollens grains fragments (or PCGs) may play a role in allergic sensitization or in exacerbation of allergic symptoms. Moreover, the biochemical composition of BPGs is not completely elucidated, which limits our knowledge of the biochemical microenvironment surrounding a PG deposited on the nasal epithelium or a PCG deposited on the lower airways.

Chemical alterations following BPG exposure to ozone do not seem to be able to modify lipid bioaccessibility in aqueous phase, at least for the lipids identified in this study. Unsaturated fatty acids are less extracted in aqueous phase after pollen exposure to ozone: extraction for intact pollen was 4010 ng.mg -1 as compared to 3359 ng.mg -1 for pollen exposed to ozone. The release of these compounds may be impaired after exposure to ozone as it was previously observed for some proteins after exposure to pollutants [START_REF] Behrendt | Localization, Release and Bioavailability of Pollen Allergens: The Influence of Environmental Factors[END_REF]. Unsaturated fatty acids may have also been degraded by ozone although the expected products during ozonolysis of α-linolenic acid (nonanedioic acid for example) were not detected. The degradation of lipidic compounds by ozone in the pollen cytoplasm raises the question of ozone diffusion through the sporoderm, while chemical reactions on solid particles are essentially expected at their surface (Shiraiwa et al. 2011a). It has been shown that NO can diffuse into the BPG cytoplasm but in experimental conditions with excessively high doses of pollutant exposure (48 hours at 1 to 5% of NO) [START_REF] Lhuissier | Secondary Ion Mass Spectrometry Imaging of the Fixation of 15 N-Labelled NO in Pollen Grains[END_REF]. Conversely, nitrogen dioxide only reacted on the surface of BPGs and the chemical alterations did not pass through the pollen wall for exposures ranging from 1 to 400 ppm.day (Goschnick andSchuricht 1995, 1996). Following ozone exposures, SDS/PAGE analyses of water-soluble BPGs proteins showed no changes in the polypeptide profiles suggesting protection of proteins within the PG cytoplasm [START_REF] Cuinica | Exposure of Betula pendula Roth pollen to atmospheric pollutants CO, O 3 and SO 2[END_REF][START_REF] Cuinica | Effect of Air Pollutant NO 2 on Betula pendula, Ostrya carpinifolia and Carpinus betulus Pollen Fertility and Human Allergenicity[END_REF]. Proteins extracted and then exposed outside the BPGs underwent for their part post-translational modifications [START_REF] Franze | Enzyme Immunoassays for the Investigation of Protein Nitration by Air Pollutants[END_REF][START_REF] Franze | Protein Nitration by Polluted Air[END_REF]. More work is clearly needed to unravel the diffusion of gaseous pollutants into pollen cytoplasm.

Alkenes are strongly consumed by ozone during laboratory exposure of timothy and birch PGs [START_REF] Zhu | Uptake of Ozone and Modification of Lipids in Betula Pendula Pollen[END_REF]Farah et al. 2020b). It is not possible to easily extrapolate this reactivity to atmospheric conditions because the distribution in residence time of the PGs in the atmosphere is not known at all. The residence time of BPGs in the atmosphere can be long enough to allow them to travel considerable distances [START_REF] Sofiev | Towards Numerical Forecasting of Long-Range Air Transport of Birch Pollen: Theoretical Considerations and a Feasibility Study[END_REF][START_REF] Skjoth | The Long-Range Transport of Birch (Betula) Pollen from Poland and Germany Causes Significant Pre-Season Concentrations in Denmark[END_REF][START_REF] Sofiev | On Impact of Transport Conditions on Variability of the Seasonal Pollen Index[END_REF]). However, most tree pollen probably has much shorter travel distances but potentially with periods of deposition and resuspension that are poorly described in the literature [START_REF] Proctor | The Natural History of Pollination, Updated, Subsequent[END_REF]. PGs can also be exposed to pollutants directly on the plant. A dose of 130 ppb of ozone for 16 hours probably corresponds to a high pollen exposure value in comparison with real atmospheric conditions. There is a lack of analysis of PGs collected in the atmosphere and analyzed at the individual grain level for molecular tracers of exposure to pollutants. Aldehydes are, for example, excellent tracer molecules for the exposure of birch and timothy grass PGs to ozone [START_REF] Zhu | Uptake of Ozone and Modification of Lipids in Betula Pendula Pollen[END_REF]Farah et al. 2020b). These chemical modifications, or their absence, could provide information on the degree of PGs exposure to air pollution during airborne lifetime of pollen. Synergistic effects have been described between ozone and nitrogen dioxide resulting in an enhanced nitration of bet v 1 allergen after O 3 and NO 2 simultaneous exposure compared to NO 2 exposure alone [START_REF] Franze | Enzyme Immunoassays for the Investigation of Protein Nitration by Air Pollutants[END_REF]). On the contrary, NO 2 has in this work a protective effect towards alkenes reactivity with ozone. Ozone reactivity may proceed via two reaction pathways, either ozone first adsorbs before reacting or either gas-phase ozone reacts directly with adsorbed alkenes (mechanisms 1 and 2) [START_REF] Baxter | Insight into why the Langmuir-Hinshelwood mechanism is generally preferred[END_REF]. The decreasing reactivity of ozone during simultaneous NO 2 /O 3 exposure suggests a reactivity following Langmuir-Hinshelwood mechanism. Indeed, competition for adsorption on the PG surface between NO 2 and O 3 could result in a decreased surface concentration of adsorbed ozone resulting in a lower probability of reaction with alkenes. Direct measurements of ozone adsorption have already been performed and an estimate of 29 ng of adsorbed ozone per mg of BPG has been proposed [START_REF] Cvitas | Ozone Destruction on Solid Particles[END_REF][START_REF] Zhu | Uptake of Ozone and Modification of Lipids in Betula Pendula Pollen[END_REF]. Washing of BPGs with organic solvent has also resulted in a decreased of ozone adsorption which was tentatively attributed to the loss of ozone adsorption on lipids [START_REF] Zhu | Uptake of Ozone and Modification of Lipids in Betula Pendula Pollen[END_REF]). The washing with organic solvent could also change adsorbing properties of the surface resulting in a lowered ozone adsorption.

Another aspect in controlling pollutants adsorption and reactivity is the potential competition with adsorbed water.

In this work, pollutant exposures were performed in dry atmosphere with desiccated BPGs. With Langmuir-Hinshelwood mechanism, gaseous and adsorbed water indeed contribute to competitive adsorption with reactive species [START_REF] Kolb | An Overview of Current Issues in the Uptake of Atmospheric Trace Gases by Aerosols and Clouds[END_REF][START_REF] Chu | Effect of Ozone Concentration and Relative Humidity on the Heterogeneous Oxidation of Linoleic Acid Particles by Ozone: An Insight into the Interchangeability of Ozone Concentration and Time[END_REF]). The strong observed consumption of alkenes with ozone in dry laboratory conditions may be a limit case illustrating maximum reactivity not limited by competitive adsorption.

Involvement of water molecules in oxidative reactivity is probably much more complex as reactivity occurs between H 2 O and NO 2 to produce acids (HNO 2 , HNO 3 ) on the pollen surface [START_REF] Okuyama | Adsorption of Air Pollutants on the Grain Surface of Japanese Cedar Pollen[END_REF][START_REF] Chassard | Kinetic of NO 2 Uptake by Phleum Pratense Pollen: Chemical and Allergenic Implications[END_REF].

Whereas two BPGs samples with the same mass fractions of alkenes were exposed to the same amount of ozone, an important increase in reactivity was observed for BPGs harvested in 2017 compared to those harvested in 2016.

Different properties of pollen surfaces regarding ozone adsorption may tentatively explain this differential reactivity.

Reactivity of pollutants on the pollen surface is known to be species-specific, dose-dependent and this specific example shows that reactivity could also be different for PGs coming from different batches collected on different years at the same location.

Besides surface reactivity, adsorption of pollutants on the PG surface may have a role in allergic symptom exacerbations. Adsorbed pollutants could be transferred to nasal and ocular mucous membranes and become a source of pollen-associated oxidative stress with subsequent inflammatory processes [START_REF] Okuyama | Adsorption of Air Pollutants on the Grain Surface of Japanese Cedar Pollen[END_REF]Shiraiwa et al. 2011b[START_REF] Shiraiwa | Hazardous Components and Health Effects of Atmospheric Aerosol Particles: Reactive Oxygen Species, Soot, Polycyclic Aromatic Compounds and Allergenic Proteins[END_REF]. Transfer of adsorbed pollutants to the immune system via inhaled PGs is to our opinion understudied especially since PGs have been demonstrated to possess high adsorption capabilities [START_REF] Thio | Magnetic Pollen Grains as Sorbents for Facile Removal of Organic Pollutants in Aqueous Media[END_REF][START_REF] Zhang | A Novel Phenanthrene Sorption Mechanism by Two Pollens and Their Fractions[END_REF]. Ragweed PGs are for example adsorbent as strong as activated carbon for removing hydrophobic organic compounds from aqueous solutions [START_REF] Thio | Magnetic Pollen Grains as Sorbents for Facile Removal of Organic Pollutants in Aqueous Media[END_REF]. Adsorption of acid and basic pollutants (HNO 2 , HNO 3 , HCl and NH 3 ) were quantified in concentrations of up to 1 µg.mg -1 in cedar PGs [START_REF] Okuyama | Adsorption of Air Pollutants on the Grain Surface of Japanese Cedar Pollen[END_REF]. Cedar pollen has adsorption capability for these pollutants higher than other tested particles of biogenic origins (humic acid and yellow sand) [START_REF] Okuyama | Adsorption of Air Pollutants on the Grain Surface of Japanese Cedar Pollen[END_REF].

Conclusion

Knowledge of the lipid fraction of birch pollen grains is still incomplete. Only a fraction of the material extractable by organic or aqueous solvents has been identified and quantified. The bioaccessibility of pollen lipids under physiological conditions is largely unknown and constrains our understanding of their potential role in the allergic reaction. Moreover, pollen rupture is a key factor in the bioaccessibility of allergens and lipids. Pollen lipid fraction can be modified by air pollution at least during its airborne journey. Some lipids are indeed modified in laboratory conditions by ozone, but it is difficult to extrapolate to atmospheric conditions because competition for surface adsorption between several gaseous species and ozone could greatly limit the reactivity. It is also not clear whether gaseous pollutants can pass through the sporoderm of intact pollen grains to reach the cytoplasm and react with internal lipids and allergens. The adsorption of gaseous pollutants (NO 2 or O 3 ) on the pollen surface allows their transfer to the mucous membranes upon inhalation where they can produce oxidative stress or modify the acidity of the environment. The role of these gaseous pollutants adsorbed on the pollen surface in the allergic reaction is largely unknown for both health effects and germination-related processes.

Figure 1 .

 1 Figure 1. Mass fractions of the main chemical families extracted from birch pollen grains with organic solvent (methylene chloride) and aqueous solvent (PBS: phosphate buffer solution).

Figure 2 .

 2 Figure 2. Birch Pollen Grain (BPG) observed after thorough magnetic stirring under dry conditions with shredded sporoderms letting out micrometer-sized pollen cytoplasmic granules (PCGs). Internal lipids are no longer enclosed in BPGs. Yellow arrows point to PCGs. Green arrows mark BPG apertures. Pink arrows indicate fragments of pollen wall.

Figure 3 .

 3 Figure 3. Extraction of unsaturated fatty acids with phosphate buffer solution (PBS) under different conditions: grey = gentle agitation of Birch Pollen Grains (BPGs); white = broken BPGs without agitation; white dashed = broken BPGs with gentle agitation; grey dashed = broken BPGs with magnetic stirring.

Mechanism 1 :

 1 Langmuir-Hinshelwood type O 3 (g) = O 3 (adsorbed) O 3 (adsorbed) + alkene (adsorbed) = fatty acid (adsorbed) + aldehyde (adsorbed) Mechanism 2: Eley-Rideal type O 3 (g) + alkene (adsorbed) = fatty acid (adsorbed) + aldehyde (adsorbed)

Table 1 .

 1 Mass fractions of lipids extracted from birch pollen grains either with organic (methylene chloride) or with aqueous

	176
	177	solvent (PBS: phosphate buffer solution).

Table 2 .

 2 Mass fractions of external lipids extracted from birch pollen grains with organic solvent after exposure to gaseous

	194
	195	pollutants (NO 2 and/or O 3 ) for 16 hours. Mass fractions of total lipids extracted from birch pollen grains with aqueous solvent
	196	after exposure to O 3 for 16 hours.

Acknowledgements

Not applicable

Funding

This research was funded by the Université de Lille, the Centre National de la Recherche Scientifique (CNRS) and Institut de Recherches Pluridisciplinaires en Sciences de l'Environnement (IREPSE Fed 4129), the French National Research Agency (ANR) through the PIA (Programme d'Investissement d'Avenir) under contract ANR-11-LABX-005-01 (Chemical and Physical Properties of the Atmosphere) and the research Project CLIMIBIO (the French Ministère de l'Enseignement Supérieur et de la Recherche, the Hauts de France Region and the European Funds for Regional Economic Development).

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Lipid extraction from BPGs with organic solvent does not allow to estimate the bioaccessibility of pollen lipids under physiological conditions. Hence, several lipid extraction methods were used with phosphate buffer solution (PBS) as the aqueous solvent. Different levels of mechanical stress were applied on PGs in order to break the sporoderm of pollen and enable internal (cytoplasmic) lipid extraction. PGs were severely damaged by 4 hours of magnetic stirring (fig. 2); after 4 hours of mechanical grinding, no intact pollen grain could be observed by scanning electron microscopy. Aqueous extraction of unsaturated fatty acids was enhanced by a factor of almost a hundred (fig. 3) when lipids were extracted from broken BPGs with thorough magnetic stirring (1000 rpm) compared to lipid extracted from intact BPGs with gentle agitation (linoleic acid: compared 2260±211 to 18±4 µg.mg -1 ; α-linolenic acid: 1750±172 compared to 15±4 µg.mg -1 ). Aqueous extraction of unsaturated fatty acids from broken PGs with magnetic stirring was even more efficient than that with organic solvent (4010±383 compared to 1704±135 µg.mg - 1 ). However, the use of PBS as the extraction solvent resulted in less alkanes, alcohols and alkenes extracted than using methylene chloride (table 1).

Oxidation reactions induced by ozone exposure of BPGs did not substantially modify the extraction by aqueous solvent (table 2). The total mass of extractable lipids was not significantly different between non-exposed (5576±662 ng.mg -1 ) and O 3 -exposed BPGs (4948±417 ng.mg -1 ) (table 2). Only unsaturated fatty acids were less extracted with PBS for O 3 -exposed BPGs (3359±157 ng.mg -1 ) compared to non-exposed ones (4010±383 ng.mg -1 ).

DECLARATIONS Ethics approval and consent to participate

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Consent for publication

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Competing interests

The authors declare that they have no competing interests