Andrea Roskosch
email: roskosch@igb-berlin.de

Nicolas Hette
email: nicolas.hette@cemagref.fr

Michael Hupfer
email: hupfer@igb-berlin.de4lewe@igb-berlin.de

Jo ¨rg Lewandowski

Cemagref

Alteration of Chironomus plumosus ventilation activity and bioirrigation-mediated benthic fluxes by changes in temperature, oxygen concentration, and seasonal variations

Keywords: burrowing organisms, larvae, flow velocity, pumping rate, advective and diffusive water influx, endogenous clocks

Burrowing benthic organisms promote water and solute fluxes across the sediment-water interface. Water and solutes penetrate the burrow walls and are transported into/out of the sediment when organisms flush their burrows with overlying water. Few studies have been done to investigate bioirrigation under shifting environmental conditions. We experimentally quantified bioirrigation by Chironomus plumosus larvae in the laboratory at 3 ranges of O 2 saturation (low, medium, and high O 2 concentrations), 2 temperatures (10 and 20uC), and over different seasons. We measured ventilation activities with O 2 and flow-velocity microsensors, flow velocities during pumping periods with color tracers, pumping rates with conductivity exchange experiments, and rates of advective and diffusive water influx into the sediment by influx assays (NaCl was the tracer in both latter experiments). O 2 saturations ,12% extended pumping durations/h, whereas saturations ,3% decreased pumping durations to ,0. Flow velocities were 23 higher when O 2 saturation was .50% than when it was ,10%. Rising temperatures altered larval pumping (higher pumping frequency, lower pumping length) and increased flow velocity. Hence, pumping rate and rates of water influx were significantly higher at 20 than at 10uC. Seasonal variations in bioirrigation occurred despite constant laboratory conditions, i.e., the rate of water influx was significantly higher in spring/summer than in autumn. Our study shows that temporally varying environmental conditions should be considered when evaluating bioirrigation-mediated benthic fluxes across the sediment-water interface.

Burrowing benthic organisms enlarge the sediment surface area and promote active water transport across the sediment-water interface. Their ventilation activity, i.e., flushing their burrows with oxic water from the overlying water body (bioirrigation; [START_REF] Polerecky | High temporal resolution oxygen imaging in bioirrigated sediments[END_REF][START_REF] Gallon | Surviving in anoxic surroundings: how burrowing aquatic insects create on oxic microhabitat[END_REF]) transports solutes and particulate constituents into the sediment pore water or back into the overlying water [START_REF] Aller | Meiofauna and solute transport in marine muds[END_REF][START_REF] Aller | Transport and reactions in the bioirrigated zone[END_REF][START_REF] Koretsky | Quantifying bioirrigation using ecological parameters: a stochastic approach[END_REF]. Bioirrigation oxidizes the burrow walls, changes balances of redox reactions, and affects nutrient release or immobilization [START_REF] Aller | The effect of biogenic irrigation intensity and solute exchange on diagenetic reaction rates in marine sediments[END_REF][START_REF] Lewandowski | Effect of macrozoobenthos on two-dimensional small-scale heterogeneity of pore water phosphorus concentrations in lake sediments: a laboratory study[END_REF], Meysman et al. 2006a). These changes influence microbial community structure in the sediment [START_REF] Johnson | Interactions between Chironomus plumosus (L.) and the 2012] ALTERATION OF VENTILATION AND BIOIRRIGATION 279 microbial community in surficial sediments of a shallow, eutrophic lake[END_REF][START_REF] Stief | Microbial activities in the burrow environment of the potamal mayfly Ephoron virgo[END_REF][START_REF] Bertics | Biodiversity of benthic microbial communities in bioturbated coastal sediments is controlled by geochemical microniches[END_REF] and chemical diagenesis [START_REF] Andersson | The influence of animals on phosphorus cycling in lake ecosystems[END_REF][START_REF] Kristensen | Organic matter diagenesis at the oxic/ anoxic interface in coastal marine sediments, with emphasis on the role of burrowing animals[END_REF][START_REF] Stief | Probing the microenvironment of freshwater sediment macrofauna: implications of deposit-feeding and bioirrigation for nitrogen cycling[END_REF]. Thus, quantification of the benthic fluxes across the sediment-water interface induced by bioirrigating organisms is essential to understanding the microbiological and biogeochemical processes occurring in aquatic sediments [START_REF] Boudreau | A modelling study of discontinuous biological irrigation[END_REF][START_REF] Wang | A multicomponent reactive transport model of early diagenesis: application to redox cycling in coastal marine sediments[END_REF], Meysman et al. 2006b).

Quantification of bioirrigation-mediated benthic fluxes for a whole lake is difficult. First, the volumes of water pumped by diverse species differ greatly. Pumping rates between 1 and 16,000 mL h 21 individual 21 have been reported (Kristensen 1983a[START_REF] Leuchs | The ventilation activity of Chironomus larvae (Diptera) from shallow and deep lakes and the resulting water circulation in correlation to temperature and oxygen conditions[END_REF][START_REF] Osovitz | Burrow irrigation behaviour of Urechis caupo, a filter-feeding marine invertebrate, in its natural habitat[END_REF]. Population-wide pumping rates and, thus, quantification of bioirrigation-mediated fluxes depend on population densities. Moreover, fluxes are quite variable because pumping rates are susceptible to environmental biotic and abiotic conditions, such as food availability, presence of predators, and weather [START_REF] Seymour | Effects of temperature change on irrigation rate in Arenicola marina (L.)[END_REF][START_REF] Smethie | The use of radon-222 a tracer of sediment irrigation and mixing on the Washington continental shelf[END_REF][START_REF] Ho ¨lker | Adaptive behaviour of chironomid larvae (Chironomus riparius) in response to chemical stimuli from predators and resource density[END_REF]. The response of benthic organisms to changes in temperature and O 2 concentration and the consequences for bioirrigationmediated benthic fluxes, especially in freshwater sediments, have been investigated in few studies [START_REF] Walshe | The oxygen requirements and thermal resistance of chironomid larvae from flowing and from still waters[END_REF][START_REF] Riisga ˚rd | Filter-net structure and pumping activity in the polychaete Nereis diversicolor: effects of temperature and pump-modelling[END_REF][START_REF] Stief | Regulation of nitrous oxide emission associated with benthic invertebrates[END_REF]. Quantification of modified pumping rates under different environmental conditions is important because water temperatures and O 2 concentrations in freshwater lakes are expected to change in the future [START_REF] Gerten | Effects of climate warming, North Atlantic Oscillation and El Nin ˜o on thermal conditions and plankton dynamics in European and North American lakes[END_REF][START_REF] Wilhelm | Impact of summer warming on the thermal characteristics of a polymictic lake and consequences for oxygen, nutrients and phytoplankton[END_REF][START_REF] Kirillin | Modeling the impact of global warming on water temperature and seasonal mixing regimes in small temperate lakes[END_REF].

Season also has a strong effect on benthic organisms and their activity. Dipteran larvae [START_REF] Neumann | Photoperiodische Steuerung der 15tagigen lunaren Metamorphose-Periodik von Clunio Population (Diptera: Chironomidae)[END_REF], algae, fish, and mammals [START_REF] Lee | Female rats in a laboratory display seasonal variation in fecundity[END_REF][START_REF] Grey | Seasonal changes in the stable isotope values of lake-dwelling chironomid larvae in relation to feeding and life cycle variability[END_REF][START_REF] Van Dijk | Energy reserves during food deprivation and compensatory growth in juvenile roach: the importance of season and temperature[END_REF] show seasonal and annual cycles of activity, emergence, growth, or fecundity even when they are kept at constant conditions in the laboratory. Results of a few studies suggest seasonal variations of benthic fluxes across the sediment-water interface [START_REF] Hammond | Benthic fluxes in San Francisco Bay[END_REF][START_REF] Martin | Seasonal cycles of particle and solute transport processes in nearshore sediments: 222 Rn/ 226 Ra and 234 Th/ 238 U disequilibrium at a site in Buzzards Bay, MA[END_REF][START_REF] Schlu ¨ter | Seasonal variations of bioirrigation in coastal sediments: modelling of field data[END_REF]. However, ventilation activity and bioirrigation commonly are investigated under laboratory conditions without taking season into account. Consequently, alteration of benthic fluxes in response to the season is not completely understood and, especially for freshwater lakes, poorly investigated.

The aim of our study was to use innovative measurement techniques to evaluate ventilation activity [START_REF] Roskosch | Measurement techniques for quantification of pumping activity of invertebrates in small burrows[END_REF]) and bioirrigation-mediated benthic fluxes under experimental conditions mimicking shifting environmental conditions (Fig. 1). We conducted experiments with Chironomus plumosus (Diptera: Chironomidae) larvae, which build U-shaped tubes in muddy sediment (burrow length = up to 40 cm, depth = up to 20 cm; [START_REF] Mclachlan | Sediment development and its influence on the distribution and tube structure of Chironomus plumosus L. (Chironomidae, Diptera) in a new impoundment[END_REF][START_REF] Roskosch | Bioirrigation by Chironomus plumosus: advective flow investigated by particle image velocimetry[END_REF][START_REF] Roskosch | The influence of macrozoobenthos in lake sediments on hydrodynamic transport processes and 280 A[END_REF] and periodically pump water through the tubes by undulating body movements [START_REF] Roskosch | Bioirrigation by Chironomus plumosus: advective flow investigated by particle image velocimetry[END_REF]. Chironomidae occur in high densities (100-1000 larvae/m 2 ) in the profundal area of freshwater lakes [START_REF] Mclachlan | Some effects of tube shape on the feeding of Chironomus plumosus L. (Diptera: Chironomidae)[END_REF][START_REF] Andersen | The influence of chironomids on decomposition of organic matter and nutrient exchange in a lake sediment[END_REF][START_REF] Kajak | Chironomus plumosus-what regulates its abundance in a shallow reservoir?[END_REF]. We hypothesized that changing O 2 concentrations and temperatures would alter ventilation activity (pumping frequency, length, and duration, and flow velocity) of C. plumosus larvae and result in significantly altered bioirrigation-mediated benthic fluxes (pumping rate and influx rate). We also hypothesized that seasonal variation caused by annual cycles would occur even if bioirrigation were measured under constant conditions in the laboratory.

Methods

Organisms, sediment, and water

We collected C. plumosus larvae from sediments at 6-m water depth at a fixed location (lat 52u44 9 N, long [Volume 31

13u65 9 E) from the shallow (mean water depth = 4.9 m), eutrophic, polymictic Lake Mu ¨ggelsee in Berlin, Germany, approximately every 2 mo over 3 y (November 2007 to November 2010). We used 4 thinstar (final-instar) larvae that were ,2 cm long and constructed burrows ,1.7 mm in diameter [START_REF] Roskosch | Measurement techniques for quantification of pumping activity of invertebrates in small burrows[END_REF]. We used sieved Lake Mu ¨ggelsee sediment (1-mm mesh size) topped with Lake Mu ¨ggelsee water for all experiments and for housing the larvae. Larvae began constructing burrows as soon as they were placed on sediments. Before the experiments, we kept larvae up to 1 wk in aerated tanks in darkness at 10uC without food.

Experiments

Experiment 1. Pumping frequency, length, and duration.-We measured the effects of O 2 concentrations and temperature on pumping frequency (number of periods of constant undulations/h), pumping length (time of constant undulations/period [s]), and pumping duration (total time spent ventilating/h [min/h]) with microsensors (1 st hypothesis). We filled U-shaped Perspex tubes (20-cm length, 7-mm inner diameter) with sediment and fixed them in water-filled plastic tanks (261 3160 3172 mm, 10 tubes each) as described in [START_REF] Roskosch | Bioirrigation by Chironomus plumosus: advective flow investigated by particle image velocimetry[END_REF][START_REF] Roskosch | Measurement techniques for quantification of pumping activity of invertebrates in small burrows[END_REF]. We placed larvae at one end of a tube to guide their digging of a burrow along the tube to the other end. In contrast to other studies in which water-filled tubes were used [START_REF] Walshe | The function of haemoglobin in Chironomus plumosus under natural conditions[END_REF][START_REF] Walshe | The function of haemoglobin in relation to filter feeding in leaf-mining chironomid larvae[END_REF], Kristensen 1983b, c), this arrangement allowed us to know burrow length while providing larvae with relatively natural conditions that did not alter their ventilation activity (Kristensen 1983a, Roskosch et al. 2011). We used 60 larvae (n = 60, 6 different tanks) per range of O 2 saturation and temperature. We acclimated larvae to the experimental conditions and allowed them to construct burrows for 2 d at the experimental temperature with constant aeration in darkness. Evaporation of water from the tanks was low, but if necessary, we replaced evaporated water with fresh lake water before the experiments were started.

We investigated the effects of low (0-3%), medium (.3-12%), and high (.12-100%) O 2 saturation on pumping frequency, pumping length, and pumping duration at 10 and 20uC. We chose these ranges of O 2 saturations to ensure that within-O 2 -group responses were similar (see Statistics below). We gassed the tanks constantly with a mixture of N 2 /CO 2 based on the partial pressure of the ambient air so that the pH was not affected by gassing. We covered the tanks with cling film to limit the redissolution of O 2 across the air-water interface. The different O 2 saturations were realized by altering the degree of gassing and covering the tanks. We checked O 2 concentration of the water continuously in the middle of the tank with O 2 microsensors (described below). Measurements were started 2 h after O 2 saturations were stable.

We investigated the effects of temperature at 10 and 20uC. We gassed the overlying water constantly with air (..50% O 2 saturation) because we did not want limited O 2 to alter ventilation activity. The experimental temperatures corresponded to differences between spring/autumn (10uC) and summer (20uC) above-ground temperatures in Lake Mu ¨ggelsee (R. Adrian, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, personal communication).

We measured pumping frequencies and lengths with Clark-type O 2 microsensors (OX-100, tip diameter = 50 or 100 mm; Unisense, Aarhus, Denmark) in burrow inlets and flow velocity microsensors (FS-25, tip diameter = 20 mm; Unisense Aarhus, Denmark; [START_REF] Revsbech | A novel microsensor for determination of apparent diffusivity in sediments[END_REF][START_REF] Brand | Microsensor for in-situ flow measurements in benthic boundary layers at sub-millimeter resolution with extremely slow flow[END_REF]) in burrow outlets as in [START_REF] Roskosch | Measurement techniques for quantification of pumping activity of invertebrates in small burrows[END_REF]. Sensor tips were 2 mm deep in the middle of a burrow opening, and the start of a pumping period was indicated by simultaneous increases in O 2 concentrations and flow velocities caused by pumping of the larva. When ventilation activity stopped, O 2 concentrations decreased gradually, whereas flow velocities decreased instantly. We have no indication that the presence of microsensors affected ventilation activity behavior. Chironomus plumosus is a filter-feeder that usually dwells deep in its burrow and is not a surface-deposit feeder [START_REF] Walshe | The function of haemoglobin in relation to filter feeding in leaf-mining chironomid larvae[END_REF][START_REF] Armitage | The Chironomidae: biology and ecology of non-biting midges[END_REF]. However, in few isolated instances, larvae detected the microsensors and built a new burrow tube beside the sensor tip. In such instances, only unaffected measurement periods were evaluated.

We calculated the arithmetic mean pumping frequency (f, /h) as the number of pumping periods divided by the length of the whole measurement time (average measurement time = 6 h). We estimated the arithmetic mean pumping length/period (l, /s) from all pumping periods during the whole measurement time. The mean pumping duration (D, min/h) was D=fl ½1

Experiment 2. Flow velocity.-We used color tracers to measure the effects of O 2 concentrations and temperatures on flow velocities (mm/s) within pumping periods (1 st hypothesis). We used the same sediment-filled U-shaped Perspex tubes, tanks, and larval collection/acclimation procedure described for experiment 1. We conducted the experiment analogously to experiment 1 except that temperature measurements were done at slightly different O 2 saturations (low = 3-12%, medium = .12-30%, high 2012]

ALTERATION OF VENTILATION AND BIOIRRIGATION = .30-100%) because pumping length was extremely short when O 2 saturation was ,3%, and reliable estimates of flow velocity were only possible for O 2 saturations .3%. Furthermore, the effect of O 2 concentration was investigated at 20uC only, whereas measurements in the range of high O 2 concentration (..50%) were done at 10 and 20uC. We used 40 larvae (n = 40, 4 tanks) per O 2 group and temperature. We chose treatments based on statistical criteria as described for experiment 1.

We measured flow velocity with color tracers as described by [START_REF] Roskosch | Measurement techniques for quantification of pumping activity of invertebrates in small burrows[END_REF]. We placed a dissolved color tracer (food color; Wusitta, Sitzendorf, Germany) with a pipette directly above the burrow inlet. When the larva started pumping, the tracer was sucked into the burrow. We immediately added a tracer of another color above the inlet. The time t (s) required for the 2 nd tracer to become visible at the burrow outlet was timed with a stopwatch.

Assuming that the length l of the burrow (mm) built inside the Perspex tube was identical to the length of the Perspex tube, the flow velocity v (mm/s) of the color tracer during regular ventilation activity was

v= l t ½2
Experiment 3. Pumping rate.-We evaluated the effects of temperature (10 and 20uC) on pumping rates (mL/s) with conductivity exchange experiments (1 st hypothesis). We fixed a sediment-filled Perspex tube (22-cm length, 7-mm inner diameter) in a waterfilled plastic tank separated into 2 equal chambers (500 mL/chamber, 1 tube/tank, each end of the tube in a different chamber; see Roskosch et al. 2011 for details). We used 20 larvae and setups (n = 20, 20 tanks) per temperature, and allowed larvae to acclimate to the experimental conditions as described for experiments 1 and 2. We replaced evaporated water with fresh lake water before starting the experiments.

The measurements of the pumping rates were done as described in [START_REF] Roskosch | Measurement techniques for quantification of pumping activity of invertebrates in small burrows[END_REF]. We used color tracers to distinguish the inlet and outlet of a burrow. Repeated measurements confirmed that larvae were always oriented in the same direction during pumping periods. We added 50 mg/L NaCl to the chamber that housed the burrow inlet and ensured uniform mixing of the water by constant aeration of both chambers. The limit of salinity tolerance for C. plumosus (NaCl = 10,000 mg/L) is much higher than the concentration used in experiment 3 (50 mg/L NaCl corresponding to an increase in electrical conductivity of ,100 mS/cm). The natural salinity of Lake Mu ¨ggelsee is ,750 mS/cm, so it is very unlikely that the increased salinity affected ventilation activity [START_REF] Lauer | Osmotic regulation of Tanypus nubifer, Chironomus plumosus and Enallagma clausum in various concentrations of saline lake water[END_REF]. Equilibration of the tracer concentration in the inlet chamber was recorded continuously by conductivity meters (GMH3430; Greisinger Electronic, Regenstauf, Germany; accuracy = 0.5% but at least 62 mS/cm) for 2 d. We connected the inlet and outlet chambers with a water-filled tube (diameter = 1 mm) to ensure constant water levels in both chambers.

We calculated the pumping rate Q (mL/h) based on an equation for the exchange between 2 compartments [START_REF] Mutz | Effect of instream wood on vertical water flux in low-energy sand bed flume experiments[END_REF]). We estimated Q by minimizing the root mean squared error between the conductivity measured in the outlet chamber and the theoretical conductivity Lf 0 (mS/cm) at time t (h) based on the estimated Q. In the mass-balance equation 3, Lf Eq (mS/cm) is the conductivity in each chamber at equilibrium, Lf start (mS/cm) is the conductivity in each chamber at the start of the experiment, and V (mL) is the equal water volume in both chambers:

Lf 0 t ð Þ=exp {2t Q V Lf start t=0 ½ {Lf Eq zLf Eq ½3
Experiment 4a. Influx rate.-We evaluated the effect of temperature on the influx rates (= rate of advective and diffusive water influx of overlying water into the sediment [L/h]) (1 st hypothesis). We filled round plastic columns (diameter = 59 mm) with sediment and water and added different densities of larvae (1 larva/column = 360 larvae/m 2 , height (h) sediment = 10 cm, h water = 5 cm; 10 larvae/column = 3600 larvae/m 2 , h sediment = 15.5 cm, h water = 15.5 cm). We chose this setup to assure almost natural conditions. The larvae were able to build their burrows freely in the sediment. The number of larvae corresponded to in situ abundances that are within the range normally found in eutrophic lakes [START_REF] Svensson | Effects of bioturbation by tube-dwelling chironomid larvae on oxygen uptake and denitrification in eutrophic lake sediments[END_REF]Leonardson 1996, Kajak 1997).

We ran these experiments in batches (4 columns/ date and temperature) in summer and autumn 2008 to 2010 (between July and November) (1 larva, 10uC, n = 24 columns; 1 larva, 20uC, n = 10 columns; 10 larvae, 10uC, n = 34 columns; 10 larvae, 20uC, n = 26 columns). We ran all influx experiments immediately after larvae, sediment, and water were sampled from Lake Mu ¨ggelsee. We incubated columns for 2 d in darkness at 10uC so that larvae could build burrows and acclimate to the experimental conditions. On day 3, we raised temperatures to 20uC in ½ of the columns and allowed larvae to acclimate to the experimental temperatures for 1 d. We started measurements on day 4. We were focusing on the effect of temperature, but this effect could have been confounded by the effect of change in temperature. We aerated the

272

A. ROSKOSCH ET AL. [Volume 31

overlying water in all columns constantly. We replaced evaporated water with fresh lake water before the experiments were started.

To start, we removed ,10 mL of the overlying water and added NaCl dissolved in 10 mL of water (60 mg NaCl in experiments with h water = 5 cm; 250 mg NaCl in experiments with h water = 15.5 cm) to the laboratory column. The solution was mixed into the water column by aeration. We used the conductivity meters described above to record the decrease in NaCl in the overlying water caused by uptake of dissolved NaCl by the sediment for 7 d. We fitted the decrease in Lf 0 (mS/cm) and the influx rate Q ir (L/h) with the mass-balance Eq. 3 described above [START_REF] Mutz | Effect of instream wood on vertical water flux in low-energy sand bed flume experiments[END_REF]). However, in this experiment the volume of the overlying water V (mL) and the volume of the pore water V PW (mL) had to be taken into account separately:

Lf 0 = exp {t Q ir VzV PW ð Þ V V PW ð Þ Lf start {Lf Eq À Á h i zLf Eq 1000 ½4
Experiment 4b. Influx rate under seasonal variation.-We measured advective and diffusive influx rates (L/ h) with influx experiments conducted seasonally over a period of 3 y (2 nd hypothesis). We used methods identical to those described for experiment 4a (10 larvae; 3600 larvae/m 2 ). We did the experiments at 10 and 20uC (n = 4 columns, 40 larvae/temperature and date, done on the same day) approximately every 2 nd mo from November 2007 to November 2010 (18 dates for 10uC, 17 dates for 20uC). Experiment 4a for 10 larvae was a subset of experiment 4b (July to November).

Temperature coefficient.-We calculated the temperature coefficient (Q 10 ) from values measured at T 1 = 10uC and T 2 = 20uC as

Q 10 = R 2 R 1 10 T 2 {T 1

½5

The rate (R) represents any measurement at both temperatures, T 1 and T 2 . The Q 10 -value illustrates the temperature dependency of pumping frequency, pumping length, pumping duration, flow velocity, pumping rate, and water influx. Q 10 . 1 indicates that the variable increases, whereas Q 10 , 1 indicates that the variable decreases with rising temperature.

Statistics

We measured pumping frequency, pumping length, flow velocity, and pumping rate at least twice per larva and obtained measurements for every larva that built tubes and ventilated them. By using a threshold approach we established boundaries for low, medium, and high O 2 saturation. Therefore, observations between .0 and 100% were grouped so that the group variance was minimized (groups with low standard deviations were chosen).

We did all statistical analyses with the statistical software SPSS (version 14; IBM Corporation, Montauk, New York). We used Shapiro-Wilk tests to check for normality of all variables within O 2 groups. We compared pumping frequency, pumping length, flow velocity, and pumping rate among O 2 groups with 1-way analysis of variance (ANOVA) if data were normally distributed or the nonparametric Kruskal-Wallis H-test if they were not. We used post hoc (Least Significant Difference or Dunnett-T3) or repeated 2-sample tests to assess differences in the contributions between each pair of O 2 groups. To compare 2 groups of values (e.g., between temperatures), we used Student's t-tests if distributions were normal and Mann-Whitney U-tests if they were not normal. The test and the p-value are reported when results were statistically significant (p ƒ 0.05). If .2 measurement techniques were compared, we adjusted the p-value for significance with a Bonferroni correction.

Results

Effect of O 2

Experiment 1.-Ventilation activities (pumping frequency, length, and duration) differed significantly among O 2 groups (0-3% [low], .3-12% [medium], .12-100% [high]) at both temperatures (10 and 20uC). At 10uC, pumping frequency, length, and duration were significantly lower at low than at medium or high O 2 and did not differ significantly between medium and high O 2 (Table 1, Fig. 2A-C). However, pumping length and duration peaked at medium O 2 , whereas pumping frequency did not differ appreciably between medium and high O 2 . At 20uC, pumping frequency was significantly lower at medium than at high O 2 and did not differ between medium and low or between low and high O 2 (Fig. 2A). Pumping length and duration were significantly longer at medium than at low or high O 2 and significantly longer at high than at low O 2 (Fig. 2B,C). At low O 2 , larvae conducted minor pumping (Fig. 3), but when O 2 saturation was ,,2%, larvae almost stopped pumping.

Experiment 2.-Flow velocities measured within pumping periods at 20uC differed significantly among O 2 groups (3-12% [low], .12-30% [medium], .30-

2012]

ALTERATION OF VENTILATION AND BIOIRRIGATION 273 100% [high]) and decreased with dropping O 2 concentrations (Table 1, Fig. 4).

Effect of temperature

Experiment 1.-Ventilation activity differed between temperatures. Pumping frequency was significantly higher at 20 than at 10uC (Fig. 5A), whereas pumping length was significantly lower at 20 than at 10uC (Fig. 5B). Pumping duration did not differ significantly between temperatures (Fig. 5C).

Experiment 2.-Flow velocity was significantly greater at 20 than at 10uC (Fig. 5D).

Experiment 3.-Constant pumping duration increased flow velocity at 20uC resulting in a significantly higher pumping rate at 20 than at 10uC (Fig. 5E). Q 10 -values for all variables except pumping length and duration were .1 (Table 2).

Rate of water influx affected by temperature and seasonal variation

Experiment 4a.-The rate of advective and diffusive influx of overlying water into the sediment was affected by temperature at both larval densities. Q 10values were 1.7 or 1.6, respectively, for experiments with 1 and 10 larvae (Table 2). We calculated per capita influx rate to provide a basis of comparison among experiments. Per capita influx rates were 1.73 (at 10uC) to 1.93 (at 20uC) higher at the lower than at the higher larval density (Fig. 6A,B). Originally, no comparison was planned between experiments, so experimental designs with 1 and 10 larvae differed slightly in terms of starting concentration of NaCl, sediment volume, and water volume, but sediment surface area was identical.

Experiment 4b.-Rates of water influx measured over 3 y under constant laboratory conditions were [Volume 31 significantly higher in spring/summer (April-July) than in autumn (September-October) (Fig. 7, Table 3). Across the entire period, the Q 10 -value was 1.7.

Discussion

Effects of O 2 concentration, temperature, and season

Our results supported hypothesis 1. Larvae modified their ventilation activity, and thus, bioirrigation, in response to the O 2 concentration in the overlying water column (experiments 1 and 2). Our results suggest that larvae are able to balance a decrease in O 2 by increasing their pumping duration. At very low O 2 saturation, normal ventilation activity is minimized (Fig. 2A-C). We assume that in this case, O 2 consumption, which is necessary to perform pumping, exceeds O 2 gained by pumping. Some authors have described a switch to anaerobic metabolism for chironomid larvae in such almost-anoxic conditions [START_REF] Augenfeld | Observations on the respiratory enzymes of various life-stages of Chironomus plumosus, Chironomus stageri, and Aedes aegypti[END_REF]Neess 1961, Nagell and[START_REF] Nagell | Resistance to anoxia of Chironomus plumosus and Chironomus anthracinus (Diptera) larvae[END_REF]. At extremely low O 2 saturation, larvae pump slowly, rarely, and briefly, but regularly (Fig. 3). Without occasional ventilation activity, larvae presumably would be unable to assess the O 2 concentration of the overlying water, and thus, would not register a renewal of O 2 , should it occur. In our experiments, ventilation activity stopped when the O 2 saturation in the overlying water decreased to ,,2%. In addition, the O 2 concentrations in the burrow adjacent to the larvae are probably even lower because O 2 saturation decreases further when the water from the surrounding anoxic sediments flows through the burrow. [START_REF] Walshe | The function of haemoglobin in Chironomus plumosus under natural conditions[END_REF] also reported that C. plumosus pumping length increased as O 2 concentrations decreased and that pumping length decreased drastically when O 2 was ,5% (cf. ,3% in our study). Chironomus thummi, another bioirrigating larva occurring in shallow lakes, increased pumping length and pumping duration as O 2 concentrations decreased [START_REF] Leuchs | The ventilation activity of Chironomus larvae (Diptera) from shallow and deep lakes and the resulting water circulation in correlation to temperature and oxygen conditions[END_REF]. In contrast to our study, the pumping frequency did not decrease with dropping O 2 concentrations in either of these studies. Both studies were done with larvae in water-filled tubes with a diameter wider than natural burrows. Such tubes are known to alter bioirrigation (Kristensen 1983a[START_REF] Roskosch | Bioirrigation by Chironomus plumosus: advective flow investigated by particle image velocimetry[END_REF]). In our study, tubes were filled with sediment. [START_REF] Roskosch | Bioirrigation by Chironomus plumosus: advective flow investigated by particle image velocimetry[END_REF][START_REF] Roskosch | Measurement techniques for quantification of pumping activity of invertebrates in small burrows[END_REF] showed that results from burrows built in sedimentfilled tubes were not significantly different results from burrows built directly in the sediment.

Our results confirmed that water temperature affects bioirrigation of C. plumosus significantly and temperature changes result in modified ventilation activity and pumping rates (Experiments 1, 2, and 3). Presumably this change in activity with temperature is explained by the fact that the larvae are poikilotherms

2012]

ALTERATION OF VENTILATION AND BIOIRRIGATION [START_REF] Platzer | Untersuchungen zur Temperaturadaption der tropischen Chironomidenart Chironomus strenskei Fittkau (Diptera)[END_REF][START_REF] Pinder | Biology of freshwater Chironomidae[END_REF]). Change in pumping frequency and length with temperature is consistent with results of studies on bioirrigating worms [START_REF] Seymour | Effects of temperature change on irrigation rate in Arenicola marina (L.)[END_REF], Kristensen 1983c) and larvae [START_REF] Walshe | The function of haemoglobin in Chironomus plumosus under natural conditions[END_REF][START_REF] Leuchs | The ventilation activity of Chironomus larvae (Diptera) from shallow and deep lakes and the resulting water circulation in correlation to temperature and oxygen conditions[END_REF]). However, [START_REF] Leuchs | The ventilation activity of Chironomus larvae (Diptera) from shallow and deep lakes and the resulting water circulation in correlation to temperature and oxygen conditions[END_REF] reported that the increased pumping rate of C. thummi with temperature was the result of a longer pumping duration, and not of increased flow velocity in the burrows as occurred in our study. The discrepancy may result from differences in the feeding behavior of the 2 species. Chironomus plumosus is a filter-feeder that catches particles from the water flowing through its burrow in a conical net [START_REF] Walshe | The function of haemoglobin in Chironomus plumosus under natural conditions[END_REF][START_REF] Armitage | The Chironomidae: biology and ecology of non-biting midges[END_REF], whereas C. thummi is a deposit-feeder. [START_REF] Roskosch | Bioirrigation by Chironomus plumosus: advective flow investigated by particle image velocimetry[END_REF] showed that filter feeding increases the pumping rate substantially because a larva pumps for respiration and to meet its food requirements. Therefore, depending on the available food quantity, filterfeeders must pump a considerable amount of water through their burrows [START_REF] Walshe | The function of haemoglobin in relation to filter feeding in leaf-mining chironomid larvae[END_REF][START_REF] Nielsen | Feeding, growth and respiration in the polychaetes Nereis diversicolor (facultative filter-feeder) and N. virens (omnivorous) -a comparative study[END_REF][START_REF] Riisga ˚rd | Water pumping and analysis of flow in burrowing zoobenthos: an overview[END_REF]. When larvae ventilate their burrows with a faster flow velocity, the net is more quickly packed with particles. Hence, we assume that rising temperatures might result in shorter pumping periods of C. plumosus.

The rate of advective and diffusive water influx into the sediment was significantly altered by rising temperatures (Experiments 4a, b). The Q 10 -values (1.6-1.7; Table 2) confirmed that larval activity was modified by temperature changes [START_REF] Naylor | Temperature relationships of the locomotor rhythm of Carcinus[END_REF]. Our results concur with those from a previous study indicating that bioirrigation affects the pore-water composition more strongly in warm summer (up to 22uC) than in cold winter (down to 5uC) [START_REF] Martin | Seasonal cycles of particle and solute transport processes in nearshore sediments: 222 Rn/ 226 Ra and 234 Th/ 238 U disequilibrium at a site in Buzzards Bay, MA[END_REF]. Seasonal variation of bioirrigationmediated benthic fluxes were assumed in a few in situ studies [START_REF] Hammond | Benthic fluxes in San Francisco Bay[END_REF][START_REF] Schlu ¨ter | Seasonal variations of bioirrigation in coastal sediments: modelling of field data[END_REF][START_REF] Smethie | The use of radon-222 a tracer of sediment irrigation and mixing on the Washington continental shelf[END_REF].

Our measurements of influx rates support the hypothesis that seasonal variation (circumannual cycling) occurs even if bioirrigation is measured when laboratory conditions, such as O 2 supply or temperature, are constant (experiment 4b). We kept experimental conditions constant during the 3-y study, but rates of water influx into the sediment (spring/ summer vs autumn) clearly differed between seasons.

Advanced environmental implications

A quantitative evaluation of bioirrigation-mediated benthic fluxes and their alterations is essential to understanding the biogeochemical processes in aquatic sediments and for applying dynamic diagenetic models [START_REF] Boudreau | A modelling study of discontinuous biological irrigation[END_REF]Marinelli 1994, Wang and[START_REF] Wang | A multicomponent reactive transport model of early diagenesis: application to redox cycling in coastal marine sediments[END_REF]. Predictive modelling is becoming increasingly important because environmental conditions, such as water temperature and O 2 concentrations, are shifting rapidly with global climate change. For example, long-term measurements of the water temperature of Lake Mu ¨ggelsee show that its mean annual temperature has been increasing by ,0.3uC per decade [START_REF] Kirillin | Modeling the impact of global warming on water temperature and seasonal mixing regimes in small temperate lakes[END_REF]. Moreover, [START_REF] Kirillin | Modeling the impact of global warming on water temperature and seasonal mixing regimes in small temperate lakes[END_REF] predicted that mixing patterns in the shallow lake eventually will change from polymictic (mixed water column) to dimictic (stratification periods in summer and winter) and, finally, to warm monomictic (no winter stratification, strong stratification in summer). Consequently, the hypolimnetic O 2 concentration will decrease because warmer temperatures and earlier diatom spring blooms will lead to a higher mineralization rate, especially at the lake bottom [START_REF] Wilhelm | Impact of summer warming on the thermal characteristics of a polymictic lake and consequences for oxygen, nutrients and phytoplankton[END_REF]. In Lake Mu ¨ggelsee, O 2 saturation in the hypolimnion (measured directly above the sediment, 5-m water depth) usually does not decrease below 25% (R. Adrian, personal communication). However, during short-term stratification periods in summer, low O 2 saturation (,2 mg/L) may occur [START_REF] Wilhelm | Impact of summer warming on the thermal characteristics of a polymictic lake and consequences for oxygen, nutrients and phytoplankton[END_REF]. In the future, stratification periods are expected to become longer [START_REF] Kirillin | Modeling the impact of global warming on water temperature and seasonal mixing regimes in small temperate lakes[END_REF]. Our results indicate that under such low O 2 conditions, ventilation activity will be affected and pumping rate of C. plumosus larvae in Lake Mu ¨ggelsee will decrease significantly.

Pumping rates and, thus, bioirrigation-mediated benthic fluxes are affected by population density. Mean larval density over 3 y in Lake Mu ¨ggelsee was 514 6 299 (n = 20) 4 th -instar C. plumosus larvae/m 2 sediment [START_REF] Roskosch | The influence of macrozoobenthos in lake sediments on hydrodynamic transport processes and 280 A[END_REF]. Assuming this abundance remains constant all year and that C. plumosus is the only bioirrigating species in the lake, a change in temperature from 10 to 20uC would yield a 40% increase (Q 10 = 1.6-1.7; Table 2) in the populationwide pumping rate (from 430 to 600 L m 22 d 21 , based on rates of 34.7 and 48.3 mL h 21 individual 21 , respectively). Given the volume of Lake Mu ¨ggelsee (3.65 310 9 L), this rate implies that the equivalent of the volume of the entire lake is pumped through the sediment in 10.4 d at 20uC instead of 14.4 d at 10uC [START_REF] Roskosch | Bioirrigation by Chironomus plumosus: advective flow investigated by particle image velocimetry[END_REF]). This extrapolation is only an approximation of the in situ pumping rate, but the values confirm the strong effects of bioirrigation on the aquatic environment.

Water influx into the sediment per individual decreased with increasing abundance of larvae (experiment 4a with 360 and 3600 larvae/m 2 sediment). The efficiency per larva decreased with increased abundance by a factor of 0.58 in our experiments. [START_REF] Mclachlan | Some effects of tube shape on the feeding of Chironomus plumosus L. (Diptera: Chironomidae)[END_REF] reported that C. A. ROSKOSCH ET AL.

[Volume 31

plumosus kept at very high densities build shorter tubes. Consequently, water influx into the sediment depends not only on the pumping rate, but also on variables, such as community composition, burrow shape (I-, U-, Y-or branched tubes), and burrow dimension (effective exchange surface) [START_REF] Graneli | The influence of Chironomus plumosus larvae on the oxygen-uptake of sediment[END_REF][START_REF] Koretsky | Quantifying bioirrigation using ecological parameters: a stochastic approach[END_REF]. Hence, the influx rate per individual might be lower at densities .3600 larvae/ m 2 . Bioirrigation-mediated fluxes at the sedimentwater interface are highly sensitive to environmental conditions, population density, tube dimensions, and sediment physics (permeability and porosity, muddy or sandy sediment) (Meysman et al. 2006a, b). Bioirrigation-mediated benthic fluxes should be quantified under different environmental conditions and for diverse species and sediments. O 2 concentration, temperature, and season alter ventilation activity and advective and diffusive water influx into the sediment. As O 2 concentration dropped, pumping duration increased and subsequently decreased, whereas flow velocity constantly decreased. As temperature rose, pumping duration remained the same, whereas flow velocity increased. Thus, changing conditions led to changes in pumping rates. Moreover, the rate of water influx was always higher in spring/summer than in autumn. This result shows that the season should be considered even when test organisms are brought into the laboratory and bioirrigation measurements are done under laboratory conditions. For meaningful quantification of bioirrigation-mediated benthic fluxes, measurements must be made carefully and under precise experimental conditions, especially when the data are being interpreted or extrapolated to population-scale rates.

FIG. 2 .

 2 FIG. 2. Box-and-whisker plots for pumping frequency (A), pumping length (B), and pumping duration (C) of Chironomus plumosus larvae at 0 to 3% (low), .3 to 12% (medium), .12 to 100% (high) O 2 saturations at 10 and 20uC. Dotted lines indicate means, solid lines indicate medians, box ends are quartiles, whiskers show 2 standard deviations, and * indicates outliers. p-values indicate probabilities associated with 1-way analysis of variance done at each temperature (10 and 20uC) with O 2 group as the main effect (see Table1for post hoc comparisons of means and results of tests for differences between temperatures). Bars with the same letters are not significantly different.

  10uC l , m: p = 0.012 a l , h: p = 0.011 a l , m: p , 0.001 a l , h: p = 0.009 a l , m: p , 0.001 a l , h: p = 0.001 a 20uC m , h: p = 0.009 a l , m: p = 0.002 a l , h: p = 0.001 a m . h: p = 0.012 a l , m: p , 0.001 a l , h: p , 0.001 a m . h: p , 0.001 a l , m: p = 0.013 b l , h: p , 0.001 c m , h: p , 0.001 c Low (l) 20 . 10: p = 0.007 b Medium (m) 20 . 10: p = 0.006 b High (h) 20 . 10: p = 0.001 b 20 , 10: p = 0.010 b 274 A. ROSKOSCH ET AL.

FIG. 4 .

 4 FIG. 4. Box-and-whisker plot for flow velocity of Chironomus plumosus larvae at 3 to 12% (low), .12 to 30% (medium), and .30 to 100% (high) O 2 saturations measured at 20uC in experiment 2. Dotted lines indicate means, solid lines indicate medians, box ends are quartiles, whiskers show 2 standard deviations, and * indicates outliers. Data were analyzed with a Kruskal-Wallis H-test (seeTable 1 for p-values). Bars with the same lower case letter are not significantly different.

FIG. 5 .

 5 FIG. 5. Box-and-whisker plots for pumping frequency (A), pumping length (B), pumping duration (C), flow velocity (D), and pumping rate (E) of Chironomus plumosus larvae at 10 and 20uC under high O 2 concentrations (..50%) in experiments 1 to 3. Dotted lines indicate means, solid lines indicate medians, box ends are quartiles, whiskers show 2 standard deviations, and * indicates outliers. Data were analyzed with t-tests. Bars with the same lower case letter are not significantly different.

  276

FIG. 6 .

 6 FIG. 6. Box-and-whisker plots for rate of advective and diffusive water influx into the sediment per individual caused by pumping of Chironomus plumosus larvae at the individual (1 larva = 360 larvae/m 2 ; experiment 4a) (A) and population level (10 larvae = 3600 larvae/m 2 ; experiment 4b) (B) at 10 and 20uC. Experiment 4a was done in summer and autumn 2008, whereas experiment 4b was done in summer and autumn 2008, 2009, and 2010 (July to November each year). Dotted lines indicate means, solid lines indicate medians, box ends are quartiles, whiskers show 2 standard deviations, and * indicates outliers. Data were analyzed with t-tests. Bars with the same lower case letter are not significantly different.

FIG. 7 .

 7 FIG. 7. Mean (61 SD) rate of advective and diffusive water influx into the sediment caused by the pumping of a Chironomus plumosus population (10 larvae = 3600 larvae/ m 2 ) at 10 and 20uC during the 3-y study (experiment 4b). Experiments in grey boxes were conducted in summer and autumn 2008, 2009, and 2010 and were used to compare the rate of water influx into the sediment caused by pumping of C. plumosus larvae at 10 and 20uC.

  278

  FIG.1. Schematic diagram of the measurement techniques used for the 4 variables (experiment 1: pumping frequency, length, and duration; experiment 2: flow velocity; experiment 3: pumping rate; and experiment 4a, b: influx rate), and environmental variables that were manipulated in each experiment.

270

A. ROSKOSCH ET AL.

TABLE 1 .

 1 p-values for pairwise comparisons of pumping frequency, pumping length, and pumping duration among low, medium, and high O 2 saturation groups at 10 and 20uC (experiment 1) and for flow velocity among low, medium, and high O 2 saturation groups at 20uC (experiment 2). Superscripts indicate statistical test used to compare groups ( a indicates analysis of variance, b indicates t-test, c indicates Mann-Whitney U-test). Bonferroni-corrected p-value for significance in all tests comparing a pair of groups was p ƒ 0.0167.

	Experimental		Experiment 1		Experiment 2
	conditions	Pumping frequency	Pumping length	Pumping duration	Flow velocity

  Table 1 for p-values). Bars with the same lower case letter are not significantly different. FIG. 3. Normal pumping at high O 2 saturation (.20%) and minor pumping at low O 2 saturation (,2%) measured by an O 2 microsensor in a burrow inlet (2 mm deep) in experiment 1. Data were taken from separate experimental trials. Time is coded as days, hours, and minutes.

TABLE 2 .

 2 The temperature coefficient (Q 10 ) for pumping frequency, pumping length, pumping duration, flow velocity, pumping rate, and rate of water influx at

	2012]			ALTERATION OF VENTILATION AND BIOIRRIGATION
			Experiment 4b	1.7
	Rate of water influx	Experiment 4a	(10 larvae)	1.6
		Experiment 4a	(1 larva)	1.7
	Experiment 3		Pumping rate	1.4
	Experiment 2		Flow velocity	1.9
		Pumping	duration	1.0
	Experiment 1		Pumping length	0.5
		Pumping	frequency	2.3
	10 and 20uC.		10:20uC	Q 10

TABLE 3 .

 3 Mean (61 SD; median in parentheses) spring/summer (April-July) and autumn (September-October) advective and diffusive water influx (mL/h) into the sediment measured for 10 larvae at 10 and 20uC.

	Season	10uC	2 0 uC
	Spring/summer	17.0 6 4.

2 (16.0); n = 24 31.9 6 9.0 (31.1); n = 24 Autumn 12.7 6 3.6 (12.0); n = 12 22.3 6 7.8 (21.7); n = 10 Spring/summer . autumn t-test: p = 0.020; df = 34 t-test: p = 0.006; df = 32

Acknowledgements

The Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB) and the German Research Foundation (DFG, LE 1356/3-1) are acknowledged for financial support. The authors thank S. Jordan, R. Ho ¨lzel, and B. Schu ¨tze (IGB) for data sampling and technical support, K. Pohlmann (IGB) for statistical advice, and A. Arthur-Goettig (bioXpress) for language editing. We thank T. Mehner and the participants in the course on scientific writing at IGB for helpful discussion on an earlier draft of this article.


Ben Khaoula

Juliette Messaoud

Jean Guibert

Elise Bouyer

De La

Khaoula Ben Messaoud
email: khaoula.ben.messaoud@gmail.com

Juliette Guibert

Jean Bouyer

Elise De

La Rochebrochard

Messaoud

Strong social disparities in access to IVF/ICSI despite free cost of treatment: a French population-based nationwide cohort study

Keywords: Assisted reproduction technologies, Health services accessibility, Socioeconomic disparities, Infertility, Nationwide study, IVF/ ICSI, Clomiphene citrate, Gonadotropins

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Background

Access to assisted reproduction technologies (ART) may be prevented by their high cost [START_REF] Collins | An international survey of the health economics of IVF and ICSI[END_REF][START_REF] Chambers | The economic impact of assisted reproductive technology: a review of selected developed countries[END_REF][START_REF] Domar | Burden of care is the primary reason why insured women terminate in vitro fertilization treatment[END_REF][START_REF] Maeda | Out-of-pocket payment and patients' treatment choice for assisted reproductive technology by household income: a conjoint analysis using an online social research panel in Japan[END_REF]. Some studies carried out in Canada, the United States and Australia have shown the importance of economic barriers in access to in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI) by demonstrating that use of IVF/ICSI increased when health insurance coverage or low-cost programmes were offered [START_REF] Mccarthy-Keith | Will decreasing assisted reproduction technology costs improve utilization and outcomes among minority women?[END_REF][START_REF] Chambers | Socioeconomic disparities in access to ART treatment and the differential impact of a policy that increased consumer costs[END_REF][START_REF] Vélez | Universal coverage of IVF pays off[END_REF][START_REF] Bissonnette | Impact of government health coverage for ART: The results of a 5-year experience in Quebec[END_REF][START_REF] Lee | Association of state insurance coverage mandates with assisted reproductive technology care discontinuation[END_REF]. During the first year that universal coverage of IVF/ICSI was introduced in Quebec, the number of procedures increased by 192% [START_REF] Vélez | Universal coverage of IVF pays off[END_REF].

Access to ART is probably greatly facilitated in countries such as France, where national health insurance fully covers ART for all women, including those below the poverty line, until their 43rd birthday. However, even in such a favourable national context, barriers to ART access may still exist. Studies have shown an increased risk of nonaccess among less socially advantaged populations and among populations living in remote areas far from fertility centres [START_REF] Chambers | Socioeconomic disparities in access to ART treatment and the differential impact of a policy that increased consumer costs[END_REF][START_REF] Feinberg | Economics may not explain Hispanic underutilization of assisted reproductive technology services[END_REF][START_REF] Harris | Socio-economic disparities in access to assisted reproductive technologies in Australia[END_REF].

Infertile couples are rarely directly treated by IVF/ICSI in France, like in the USA [START_REF] Chandra | Infertility service use in the United States: data from the National Survey of Family Growth, 1982-2010[END_REF][START_REF] Belgherbi | Can men be trusted in populationbased surveys to report couples' medical care for infertility?[END_REF]. Nine in 10 infertile couples first receive a much less invasive and complex treatment based on hormonal drugs, clomiphene citrate (CC) and/or gonadotropins alone, known as ovulation induction [START_REF] Belgherbi | Can men be trusted in populationbased surveys to report couples' medical care for infertility?[END_REF][START_REF] Carson | Diagnosis and management of infertility: a review[END_REF]. In infertility treatment, IVF/ICSI is often a second-line treatment after ovulation induction by CC and/or gonadotropins has failed.

Our aim was to measure non-access to ART and to identify associated factors among women unsuccessfully treated by CC and/or gonadotropins.

Methods

Data source and inclusion criteria

In France, national health insurance covers 98% of the resident population. Its databases exhaustively record all individual drug reimbursements, medical devices, laboratory tests, medical procedures, private and public hospital stays and healthcare. They also provide some information on the patient: sex, age, commune of residence and registration with health insurance for lowincome patients. This data source has been described in detail elsewhere [START_REF] Tuppin | Value of a national administrative database to guide public decisions: From the système national d'information interrégimes de l' Assurance Maladie (SNIIRAM) to the système national des données de santé (SNDS) in France[END_REF]. Access to French health insurance databases is subject to strict national legislation and is granted only by a personal time-limited authorisation for a specific research project.

Criteria of inclusion in the cohort study were being a woman living in mainland France, aged 18-43 years, having started CC and/or gonadotropin treatment between 1 January and 31 August 2016, and whose treatment was unsuccessful (i.e. no birth or pregnancy leading to ultrasound monitoring). All women were followed up for 24 months after the start of ovulation induction.

Outcomes and factors

The outcome of interest was non-access to IVF and ICSI, measured as absence of IVF and ICSI during the 24-month follow-up.

The woman's age was the age reached by the woman during the year that ovulation induction was started. Social disadvantage (yes/no) was assessed by registration with the health insurance for low-income patients. This indicates that the annual household income is below the poverty line (i.e. < 50% of median annual household income). A complementary social indicator was used measuring the level of deprivation in the patient's commune of residence, or residence deprivation index [START_REF] Rey | Ecological association between a deprivation index and mortality in France over the period 1997-2001: variations with spatial scale, degree of urbanicity, age, gender and cause of death[END_REF]. This validated index combines four indicators measured at the level of the commune (local administrative unit) based on data from the 2013 French census (percentage of unemployed individuals, percentage of workers in the active population, percentage of individuals aged 15 and over with high-school graduation and median income per household).

Driving time by road between the commune of residence and the nearest fertility centre was calculated using the tool developed by the French Biomedicine Agency which monitors ART and by the French Institute for Research and Information in Health Economics. It is based on the French road dataset (French National Geographic Institute BD TOPO ® ) and is weighted by population density and land use.

Statistical analysis

The risk of non-access to IVF/ICSI was analysed using bivariate and multivariable mixed effect logistic regressions to take into account some clustering in the data for the women's commune of residence [START_REF] Goldstein | Multilevel modelling of medical data[END_REF][START_REF] Austin | Intermediate and advanced topics in multilevel logistic regression analysis[END_REF]. We reported non-adjusted and adjusted odds ratios (OR), 95% confidence intervals (CI) and P values. All statistical analyses were performed using SAS Enterprise Guide software (version 4.3, SAS Institute Inc., Cary, NC, USA). The GLIMMIX procedure was used to estimate OR and 95% CI.

Results

Our study cohort included 20,240 French women aged 18-43 years unsuccessfully treated by ovulation induction. The median age of the women in this cohort was 34 years and 46.1% were aged 35 years or older. Fourteen percent were socially disadvantaged and 11.6% lived at least 60 min drive from the nearest fertility centre. The global proportion of women who did not access IVF/ICSI treatment within 24 months after the start of ovulation induction was 65.4%, i.e. only 34.6% accessed IVF/ICSI. More than four out of five women did not access IVF/ ICSI among socially disadvantaged women (87.2%) and among women aged 40-43 (82.7%) (Table 1).

Factors associated with non-access to IVF/ICSI were similar in univariate and multivariate mixed effect logistic regression (Table 2). The woman's age was associated with non-access to ART according to a U-shaped curve: risk of non-access was lowest among women aged 30-34 years and higher among younger and older women. The adjusted odds ratio of non-access to IVF/ICSI was (OR = 2.17, 95% CI: 1.85-2.54) for women aged 18-24, (OR = 1.23, 95% CI: 1.13-1.33) for ages 25-29, (OR = 1.29, 95% CI: 1.19-1.39) for ages 35-39 and (OR = 3.60, 95% CI: 3.25-3.98) for ages 40-43 compared with women aged 30-34 years (P < 0.001). Non-access to ART was higher among socially disadvantaged women with an adjusted odds ratio of (OR = 3.76, 95% CI: 3.34-4.23) compared with non-disadvantaged women. Non-access to IVF/ICSI showed a significant upward trend with increasing deprivation of place of residence, with adjusted odds ratios of non-access to IVF/ICSI increasing from (OR = 1.15, 95% CI: 1.03-1.27) in the 2 nd richest fifth, (OR = 1.26, 95% CI: 1.13-1.41) in the 3 rd fifth, (OR = 1.33, 95% CI: 1.19-1.49) in the 4 th fifth to (OR = 1.60, 95% CI: 1.43-1.78) in the poorest fifth compared with the richest fifth (P < 0.001). Driving time to the nearest fertility centre was not significantly associated with IVF/ICSI non-access with an adjusted odds ratio of (OR = 0.99, 95% CI: 0.92-1.07) for driving time to the nearest fertility centre of 30 to 60 min and (OR = 1.04, 95% CI: 0.93-1.16) for driving time of 60 min or more compared with less than 30 min (P = 0.70).

Discussion

After unsuccessful treatment by ovulation induction, only one out of three women (34.6%) accessed IVF/ICSI. This demonstrates that non-access to IVF/ICSI is very frequent, even in a context of full health insurance coverage as in France.

Previously, a Dutch study explored the infertility treatment care of women with WHO-II anovulation, i.e. normogonadotropic normoestrogenic anovulation. Of 104 Dutch women unsuccessfully treated with ovulation induction, 53% accessed IVF/ICSI and 47% did not in the following 36-month period [START_REF] Braam | Treatment strategies and cumulative live birth rates in WHO-II ovulation disorders[END_REF]. This percentage is well below the 65.4% level of non-access observed in our study, but it remains extremely high considering the longer follow-up (36 versus 24 months). More importantly, these women were in a particularly favourable context to access IVF/ICSI since they had already had ovulation induction in a fertility centre and so did not need to seek another centre to pursue their treatment. We have not identified any literature on this particular point, and it would be interesting to examine whether similar results have been observed in other countries.

Other studies have investigated other stages of infertility treatment care and have also found high levels of discontinuation. As many as 25% to 50% of women do not pursue IVF/ICSI after one or two unsuccessful IVF/ICSI cycles [START_REF] Troude | Medical factors associated with early IVF discontinuation[END_REF][START_REF] Kreuzer | Possible Reasons for Discontinuation of Therapy: an Analysis of 571 071 Treatment Cycles From the German IVF Registry[END_REF], probably in part because of the psychological burden of these treatments [START_REF] Swift | Comparing infertility-related stress, coping, and quality of life among assisted reproductive technology and non-assisted reproductive technology treatments[END_REF]. Even before ART, 2 out of 5 infertile couples do not access infertility care in France and in the United States [START_REF] Duron | Cumulative incidence rate of medical consultation for fecundity problems-analysis of a prevalent cohort using competing risks[END_REF][START_REF] Kelley | Disparities in accessing infertility care in the United States: results from the National Health and Nutrition Examination Survey, 2013-16[END_REF]. Our study therefore complements a coherent body of knowledge showing a surprisingly high level of non-access and drop-out at each stage of infertility treatment care. As infertility treatments are fully reimbursed in France, non-access to IVF/ICSI could be even higher in populations with less favourable insurance coverage.

Women aged over 35 years, who made up about half of our cohort (46.1%), faced a higher risk of non-access to IVF/ICSI, and women over 40 years in particular. Since it is very well established that IVF/ICSI success rates decline after the age of 35 years and even more sharply after 40 years [START_REF] Malizia | Cumulative live-birth rates after in vitro fertilization[END_REF][START_REF] Van Loendersloot | Predictive factors in in vitro fertilization (IVF): a systematic review and meta-analysis[END_REF][START_REF] Mclernon | Predicting the chances of a live birth after one or more complete cycles of in vitro fertilisation: population based study of linked cycle data from 113 873 women[END_REF], it can be hypothesized that gynaecologists and ART specialists may tend to discourage or refuse to treat older women, especially if infertility checkup revealed a low ovarian reserve [START_REF] Maheshwari | A survey of clinicians' views on age and access to IVF and the use of tests of ovarian reserve prior to IVF in the United Kingdom[END_REF][START_REF] Gizzo | Ovarian reserve test: an impartial means to resolve the mismatch between chronological and biological age in the assessment of female reproductive chances[END_REF]. At the opposite end of the age scale, the few young women in our study (5.4% aged 18-24 years) also had a higher risk of non-access to IVF/ICSI. It would be useful to investigate the characteristics of these young women and the rationale of their medical care.

Even though in France infertility treatments are fully reimbursed, women who were socially disadvantaged or living in a deprived area had a higher risk of non-access to IVF/ICSI than more advantaged women. This result is in line with a US study of women with low annual incomes who benefited from complete coverage of IVF/ ICSI treatments [START_REF] Jain | Socioeconomic and racial disparities among infertility patients seeking care[END_REF]. Another US study pointed out that the social gradient may partly reflect difficulties in finding a doctor with whom disadvantaged women felt comfortable, as well as difficulties in taking time off for the multiple appointments needed during infertility treatment [START_REF] Missmer | Cultural factors contributing to health care disparities among patients with infertility in Midwestern United States[END_REF]. More broadly, studies have suggested potential social barriers such as difficulties in communicating with medical providers, the indirect cost of time-consuming infertility treatment, the complexity and bureaucracy of the ART process, and the stigma attached to infertility that may be stronger depending on cultural background [START_REF] Jain | Disparities in access to infertility services in a state with mandated insurance coverage[END_REF][START_REF] Brandes | When and why do subfertile couples discontinue their fertility care? A longitudinal cohort study in a secondary care subfertility population[END_REF][START_REF] Nachtigall | The challenge of providing infertility services to a low-income immigrant Latino population[END_REF][START_REF] Quinn | Racial and ethnic disparities in assisted reproductive technology access and outcomes[END_REF].

Our finding that driving time to the nearest fertility center was not significantly associated with non-access to IVF/ICSI is in line with a US study [START_REF] Chin | Differences in women's use of medical help for becoming pregnant by the level of urbanization of county of residence in Georgia[END_REF]. However, an Australian study concluded on an increased risk of nonaccess to IVF/ICSI in remote areas compared with major cities [START_REF] Harris | Socio-economic disparities in access to assisted reproductive technologies in Australia[END_REF]. This latter result may be explained by a very low population density in Australia and much longer distances to remote areas.

Using French health insurance databases, access to assisted reproduction techniques was investigated for the first time in the general population by considering all national fertility centres. Unlike our study, previous work has investigated access to or pursuit of IVF/ICSI based only on patients recruited in specific fertility centres [START_REF] Brandes | When and why do subfertile couples discontinue their fertility care? A longitudinal cohort study in a secondary care subfertility population[END_REF][START_REF] Farr | Predictors of pregnancy and discontinuation of infertility services among women who received medical help to become pregnant, National Survey of Family Growth[END_REF][START_REF] Bedrick | Factors associated with early in vitro fertilization treatment discontinuation[END_REF]. Moreover, these earlier studies were not able to distinguish between non-access and treatment in another fertility centre [START_REF] Ghorbani | Dropout of infertility treatments and related factors among infertile couples[END_REF].

This study was limited by the lack of information on causes of infertility, duration of infertility or parity. It was also limited by the lack of the indication on the reasons for non-access to IVF/ICSI that could be due to barriers to access but also to medical reasons, the decision to access cross-border reproductive care or to choose adoption, or to abandon the project of having a child [START_REF] Gomez | Cross-border reproductive care among French patients: experiences in Greece Spain and Belgium[END_REF]. Finally, only infertile women treated by ovulation induction (CC and/or gonadotropins) were included in this study. Nevertheless, this represents 9 of 10 infertile women in France [START_REF] Belgherbi | Can men be trusted in populationbased surveys to report couples' medical care for infertility?[END_REF].

Conclusions

Our findings suggest that several mechanisms are involved in the very high level of non-access to IVF/ ICSI even when care is fully covered by health insurance. Firstly, an expected low probability of successful IVF/ICSI may play a part in older women, either through a medical decision or a mutual patient-doctor decision not to treat. This decision process deserves further investigation. Secondly, we observed that a strong social gradient affected access to IVF/ICSI. Crucially, we need to understand the underlying social mechanisms. Possibly, IVF/ICSI may be less accepted among socially disadvantaged patients and/or may be more difficult to reconcile with their daily work constraints. Moreover, socially disadvantaged patients may find it more difficult to interact with ART specialists, either because these specialists do not sufficiently adapt to these patients or because they (unconsciously) discriminate against them [START_REF] James | The strangest of all encounters: racial and ethnic discrimination in US health care[END_REF]. To unravel the mechanisms of social barriers to IVF/ICSI access would be of primary importance in developing an efficient and equitable health policy regarding infertility care.

Table 1

 1 Characteristics of the cohort study (n = 20,240)

	Characteristics	Distribution %	N
	Age (years)
	18-24	5.35	1,083
	25-29	19.49	3,945
	30-34	29.10	5,889
	35-39	27.71	5,609
	40-43	18.35	3,714
	Below the poverty line
	No	85.98	17,403
	Yes	14.02	2,837
	Residence deprivation index
	Richest fifth	22.80	4,614
	2nd quintile	19.94	4,036
	3rd quintile	18.70	3,785
	4th quintile	17.94	3,632
	Poorest fifth	20.62	4,173
	Driving time to the nearest fertility centre
	< 30 min	58.19	11,777
	[30;60[ min	30.21	6,114
	≥ 60 min	11.61	2,349

Table 2

 2 Non-access to IVF/ ICSI during the 24-month period after the start of ovulation induction by CC and/or gonadotropins among included in the cohort (n=20,240); univariate and multivariable analysis a

	Characteristics	Non-access to	Univariable analysis a		Multivariable analysis a
		ART	OR	95% CI	P-value	OR	95% CI	P-value
	Age (years)				< 0.001			< 0.001
	18-24	77.75	2.58	[2.21;	3.01]	2.17	[1.85;	2.54]
	25-29	61.85	1.23	[1.13;	1.33]	1.17	[1.07;	1.27]
	30-34	56.83	1			1
	35-39	63.06	1.29	[1.19;	1.39]	1.30	[1.20;	1.40]
	40-43	82.66	3.57	[3.23;	3.95]	3.60	[3.25;	3.98]
	Below the poverty line				< 0.001			< 0.001
	No	61.84	1			1
	Yes	87.20	4.06	[3.61;	4.56]	3.76	[3.34;	4.23]
	Residence deprivation index				< 0.001			< 0.001
	Richest fifth	59.93	1			1
	2nd quintile	62.49	1.11	[1.00;	1.24]	1.15	[1.03;	1.27]
	3rd quintile	65.42	1.24	[1.11;	1.38]	1.26	[1.13;	1.41]
	4th quintile	66.82	1.34	[1.20;	1.49]	1.33	[1.19;	1.49]
	Poorest fifth	72.99	1.79	[1.61;	2.00]	1.60	[1.43;	1.78]
	Driving time to the nearest fertil-				0.45			0.70
	ity centre
	< 30 min	65.68	1			1
	[30;60[ min	64.83	1.01	[0.93;	1.09]	0.99	[0.92;	1.07]
	≥ 60 min	65.43	1.07	[0.96;	1.19]	1.04	[0.93	1.16]

a From univariate and multivariable mixed effect logistic regression models

Acknowledgements

We thank Pierre-Louis Bithorel (Ined) for his invaluable assistance in managing data from the complex French health insurance databases. We thank the StimHo research team for help and discussions through this work, especially Elisabeth Morand, Arnaud Bringé, Elodie Baril, Marie-Laure Trudel. We thank Florian Bayer from the Agence de Biomédecine for sharing with us data on driving time from the commune of residence to the nearest ART centre, for his help in updating these data and his rapid feedback that made our work easier. We thank Nina Crowte for professional assistance in language editing.

Funding

This work was supported by the ANR StimHo project, grant ANR-17-CE36-0011-01 from the French Agence Nationale de la Recherche.

Availability of data and materials

To request access to French health insurance database, please contact the French health Data Hub (website: https:// www. health-data-hub. fr/) in accordance with French law (National Decree no. 2021-848, 29 June 2021). The SAS code underlying this article will be shared on reasonable request to the corresponding author.

Abbreviations

IVF

In vitro fertilisation ICSI Intracytoplasmic sperm injection CC Clomiphene citrate ART Assisted reproduction technologies Authors' contributions KBM, ELR and JB were involved in the conception and design of the study. KBM and ELR have access to the data. KBM managed the data and carried out statistical analysis with the help of ELR, JB and JG. All authors interpreted the data, drafted the manuscript, read and approved the final version.

Declarations Ethics approval and consent to participate

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

• fast, convenient online submission • thorough peer review by experienced researchers in your field

• rapid publication on acceptance

• support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year

•

At BMC, research is always in progress.

Learn more biomedcentral.com/submissions

Ready to submit your research Ready to submit your research ? Choose BMC and benefit from:

? Choose BMC and benefit from:

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.