Eva Knop
email: evaknop@gmx.ch

Leana Zoller

Remo Ryser

Christopher Gerpe

Maurin Hörler

Colin Fontaine

Artificial light at night as a new threat to pollination

Pollinators are declining worldwide 1 and this has raised concerns for a parallel decline in the essential pollination service they provide to both crops and wild plants 2,3 . Anthropogenic drivers linked to this decline include habitat changes, intensive agriculture, pesticides, invasive alien species, spread of pathogens and climate change 1 . Recently, the rapid global increase in artificial light at night 4 has been proposed to be a new threat to terrestrial ecosystems; the consequences of this increase for ecosystem function are mostly unknown 5,6 . Here we show that artificial light at night disrupts nocturnal pollination networks and has negative consequences for plant reproductive success. In artificially illuminated plantpollinator communities, nocturnal visits to plants were reduced by 62% compared to dark areas. Notably, this resulted in an overall 13% reduction in fruit set of a focal plant even though the plant also received numerous visits by diurnal pollinators. Furthermore, by merging diurnal and nocturnal pollination sub-networks, we show that the structure of these combined networks tends to facilitate the spread of the negative consequences of disrupted nocturnal pollination to daytime pollinator communities. Our findings demonstrate that artificial light at night is a threat to pollination and that the negative effects of artificial light at night on nocturnal pollination are predicted to propagate to the diurnal community, thereby aggravating the decline of the diurnal community. We provide perspectives on the functioning of plant-pollinator communities, showing that nocturnal pollinators are not redundant to diurnal communities and increasing our understanding of the human-induced decline in pollinators and their ecosystem service.

Animal-assisted pollination is a key ecosystem function for wild plant communities and 88% of all angiosperms depend on it to some extent 7 . Pollination by animals is also a crucial ecosystem service for global food supply 1,8 ; the estimated economic value of pollination was US$361 × 10 9 in 2009 (ref. 9). Thus, pollination by animals is fundamental to both the functioning of natural ecosystems and food security. Concerns have been raised that the pollination provided by insects may be at risk 2,3 due to a worldwide decline in wild and managed pollinators as a consequence of human activity 1 . The main factors causing this decline are considered to be habitat loss and degradation, conventional intensive agriculture including pesticide use, invasive alien species, pests and pathogens, and climate change 1 .

 and population dynamics 17 . Changes in species communities because of ALAN may in turn affect associated ecosystem functions and processes, as has recently been demonstrated for aquatic microbial communities 14 . For terrestrial systems, the effect(s) of ALAN on the functioning of communities, such as for pollinators and pollination, has yet to be demonstrated 5 .

The negative impact of ALAN might not be restricted to nocturnal pollinators and the plants they pollinate, but could propagate to Author Contributions E.K.

the diurnal pollinator community (Fig. 1), thereby increasing the overall environmental pressure on this community. Indeed, we know that plants and their pollinators are embedded in complex interaction networks [START_REF] Bascompte | The nested assembly of plant-animal mutualistic networks[END_REF] , where perturbations can cascade from species to species depending on network architecture [START_REF] Thébault | Stability of ecological communities and the architecture of mutualistic and trophic networks[END_REF] . These cascading effects have recently been shown for the effects of grazer presence on flower-visitation networks [START_REF] Vanbergen | Grazing alters insect visitation networks and plant mating systems[END_REF] . However, to our knowledge, the separate and combined responses of nocturnal and diurnal pollinator networks to environmental change have not been investigated (but see an example of a moth-pollen transport network [START_REF] Devoto | The 'night shift': nocturnal pollentransport networks in a boreal pine forest[END_REF] ). It is therefore unknown whether the architecture of pollination networks facilitates or impedes plant-mediated indirect interactions between the diurnal and nocturnal pollinator communities. Hence, we do not know whether the negative effects of ALAN potentially spread from the nocturnal to the diurnal pollinator community, with potential knock-on effects on pollination rates.

Here we show how the negative effects of ALAN on nocturnal pollinator communities translate into negative consequences for plant reproductive success, and we demonstrate that these effects have the potential to cascade from the nocturnal to the diurnal pollinator community (Fig. 1). On 7 out of 14 independent ruderal meadows, which had never been exposed to artificial lighting previously, we set up mobile street lamps, while leaving the other 7 as controls. On these meadows we sampled nocturnal interactions between plants and flower [START_REF] Potts | Safeguarding pollinators and their values to human well-being[END_REF] University of Bern, Institute of Ecology and Evolution, Baltzerstrasse 6, 3012 Bern, Switzerland. [START_REF] Biesmeijer | Parallel declines in pollinators and insect-pollinated plants in Britain and the Netherlands[END_REF] visitors and analysed how ALAN affects the structure of the interaction networks. In particular, we analysed the number of interactions and species, and two network metrics known to affect the stability of plant-pollinator communities: species generalism [START_REF] Waser | Generalization in pollination systems, and why it matters[END_REF] and network nestedness [START_REF] Bascompte | The nested assembly of plant-animal mutualistic networks[END_REF][START_REF] Thébault | Stability of ecological communities and the architecture of mutualistic and trophic networks[END_REF] . We found a significantly lower number of flower visits (z = -4.7, degrees of freedom (d.f.) = 10, P < 0.001, 62% fewer visitations on illuminated sites; Fig. 2a-c) and species of flower visitors (z = -4.4, d.f. = 11, P < 0.001; 29% fewer species on illuminated sites; Extended Data Table 1) on illuminated sites. The lower insect richness under the light treatment was mainly explained by the lower number of interactions sampled, suggesting that artificial light affects species equally (Extended Data Fig. 1). This negative effect of ALAN on insect visitation is most likely because of altered behaviour of nocturnal flower visitors, such as attraction to an artificial light source at night, as is known for many moth species [START_REF] Frank | Ecological Consequences of Artificial Night Lighting[END_REF] , and/or to a physiological reaction of flower visitors, although these have rarely been studied [START_REF] Van Geffen | Artificial light at night causes diapause inhibition and sex-specific life history changes in a moth[END_REF] . Alternatively, changes in plant physiology might have altered their attractiveness for flower visitors, but such mechanisms remain largely unexplored [START_REF] Bennie | Ecological effects of artificial light at night on wild plants[END_REF] . The number of plant species visited was similar between illuminated sites and dark control sites (subsequently referred to as dark sites) (z = -1.1, d.f. = 11, P = 0.271; Extended Data Table 1). However, the generalism of plant species, calculated as the weighted mean effective number of pollinator species per plant species [START_REF] Bersier | Quantitative descriptors of food-web matrices[END_REF] , was significantly lower for illuminated sites (t = -2.6, d.f. = 6, P = 0.043; Fig. 2d). As for flower-visitor richness, this reduction in plant generalism was explained by the lower number of interactions under the light treatment and did not reflect an actual change in plant generalism (Extended Data Fig. 2). Notably, flower-visitor generalism (that is, the weighted mean effective number of plant species per pollinator species [START_REF] Bersier | Quantitative descriptors of food-web matrices[END_REF] ) was unaffected by the light treatment (t = -0.2, d.f. = 6, P = 0.836; Fig. 2e and Extended Data Fig. 3), suggesting that the species visiting flowers with the light treatment were not more selective than those visiting flowers in the dark sites. Given the large differences in network size between control and illuminated sites, nestedness was standardized (z score of NODF (nestedness metric based on overlap and decreasing fill), see Methods). Nestedness was significantly higher in dark sites (t = -3.3, d.f. = 12, P < 0.001; Fig. 2f) compared to illuminated sites, suggesting that ALAN might have a destabilizing effect on nocturnal plant-pollinator communities. Because not all flower visitors are necessarily functional pollinators and not all specimens within the dataset were identified Data Table 5), suggesting that the disruption of the pollination function already occurs at low light intensities. Our results show that the pollination service provided by nocturnal flower visitors is disrupted in the vicinity of streetlamps, and that this leads to a reduced reproductive output of the plant, which cannot be compensated for by pollinators during the day. This further indicates that nocturnal and diurnal pollinators generate complementarity effects, which have previously been demonstrated for different functional groups of diurnal pollinators [START_REF] Fontaine | Functional diversity of plantpollinator interaction webs enhances the persistence of plant communities[END_REF] .

Because ALAN affects nocturnal pollinators such that this causes a lower fruit set of the plants they pollinate, this negative impact could further affect diurnal pollinators, given that these plants represent an important food source for them. Even in the case of C. oleraceum, which can also reproduce clonally, reduced sexual reproductive output is still likely to have negative effects on fitness over the long term [START_REF] Vallejo-Marin | The ecological and evolutionary consequences of clonality for plant mating[END_REF] and hence on the food resource of the pollinators using that plant. To investigate whether the structure of intertwined nocturnal and diurnal networks tend to propagate or buffer such effects, we quantified the potential for indirect effects from nocturnal to diurnal flower visitors (Fig. 4). We then compared the observed potential for indirect effects to the effect of networks in which plants connect the nocturnal and diurnal sub-networks in a randomized manner (see Methods). The observed potential for indirect effects was significantly higher than the effect of randomly inter-connected 3. to a high taxonomic resolution, all analyses were repeated with two different subsets in which specimens were identified to a high taxonomic resolution and more strictly assigned to pollinators on the basis of expert knowledge and whether they carried pollen (see Methods). These analyses gave consistent results (Extended Data Table 1).

To test whether the negative impact of ALAN on night-time pollinators translates into a lower pollination function, we conducted a fruit set experiment using the model plant Cirsium oleraceum. This plant was common to all our sites and was one of the most frequently visited species. In the year before the light treatment, this plant species received the highest number of flower visits during the day and during the night (11% of all diurnal visits, 56% of all nocturnal visits; Fig. 3), and the mean number of visits was similar during day and night (Extended Data Table 2). This was consistent for the two subsets of the flower visitors (Extended Data Table 2).

The impact of ALAN on pollination function was assessed by analysing the reproductive output of a total of 100 experimental plants of C. oleraceum, distributed equally across 5 illuminated and 5 dark sites. On each site the 10 plants were paired and one plant within a pair was bagged to exclude flower visitors. The number of developed fruits of plants exposed to flower visitors was significantly reduced on the illuminated sites compared to the dark sites (z = -2.5, d.f. = 140, P = 0.014; Fig. 4a). This effect was most likely not driven by potential bottom-up effects of altered plant growth [START_REF] Bennie | Ecological effects of artificial light at night on wild plants[END_REF] , as plant biomass did not differ between illuminated and dark sites (t = -1, d.f. = 8.1, P = 0.261, mean ± s.e.m. = 17.5 ± 1.0 for dark sites, mean ± s.e.m. = 15.0 ± 0.8 for illuminated sites), and the number of developed fruits of selfpollinated plants (that is, bagged plants) was similar between the two treatments (z = 1.47, d.f. = 140, P = 0.140, Fig. 4a). Similar results were found for the percentage of developed fruits per flower head (Fig. 4b and Extended Data Table 3). A small number of flower heads (4%) were infested with herbivores, which negatively affected the number of developed fruits (Extended Data Table 3). The effect of herbivores on the number of developed fruits did not differ between illuminated and dark sites. No difference was found in the percentage of developed fruits per flower head between flower heads infested by herbivores and herbivore-free flower heads (Extended Data Table 3). Our network data show that the number of flower visits to C. oleraceum was significantly reduced for illuminated sites, and this was consistent for the two subsets of flower visitors analysed (Extended Data Table 4). There was no relationship between light intensity measured on the flower head of the plants and number and percentage of developed fruits (Extended species and the frequency of visitations (day and night) are reflected by the width of the rectangles. Cirsium oleraceum was the most frequently visited plant species during day and night (framed in black). A list of all plant and insect species is given in Supplementary Tables 2,3. networks (P = 0.008; Extended Data Table 6), indicating that plants connect nocturnal and diurnal flower visitors in a way that favours the spread of indirect interactions between them. This might arise from correlated plant generalism in both sub-networks, a pattern that favours the spread of indirect interactions [START_REF] Fontaine | The ecological and evolutionary implications of merging different types of networks[END_REF] . Similar results were obtained for analyses of the two subsets of flower visitors (Extended Data Table 6).

The worldwide decline in pollinators has received much attention in recent years, due to the essential pollination service they provide to both crops and wild plants [START_REF] Potts | Safeguarding pollinators and their values to human well-being[END_REF] . Here, we demonstrate for the first time that ALAN is a threat to pollination that is rapidly spreading globally [START_REF] Hölker | The dark side of light: a transdisciplinary research agenda for light pollution policy[END_REF] . Because nocturnal and diurnal pollinators act as complementary functional groups and are linked by plant-mediated indirect interactions, ALAN has the potential to further aggravate the decline of diurnal pollinators through plant-mediated indirect effects from the nocturnal pollinator community, with further knock-on effects on plant pollination. We provide novel perspectives for the understanding of the functional complementarities among pollinators, and show how plant-pollinator interactions are threatened by a hitherto little acknowledged driver of global change.

Letter reSeArCH

MethODS

Data reporting.

No statistical methods were used to predetermine sample size.

Where possible experiments were randomized, for example, plants were selected randomly for treatment out of all plants on the study site. Insect sampling on the site was not done in a blinded manner, but the experts who identified the insect species were blinded to which site the insects were obtained from. Experimental set-up. In 2014, a total of eight independent ruderal meadows were selected in the Prealps of Switzerland (nearest ground distance between two adjacent sites (dist): mean ± s.e.m. = 0.9 ± 0.3 km, median = 0.6 km). In 2015, nine additional meadows were selected and five of the sites of 2014 were used, resulting in a total of 14 sites (dist: mean ± s.e.m. = 1.0 ± 0.2 km, median = 0.6 km; see Supplementary Table 1). This region still has low levels of light emission with a radiance of less than 0.25 × 10 -9 W sr -1 cm -2 (data from http://www.lightpollutionmap.info). All sites were comparable in vegetation, C. oleraceum was present on all sites and a varying subset of the following plant species was common to all sites: Eupatorium cannabinum, Valeriana officinalis, Epilobium angustifolium and Silene vulgaris. In 2015, on 7 out of the 14 sites LED street lamps (Schréder GmbH, type: AMPERA MIDI 48 LED, colour temperature: neutral white (4,000 K), nominal LED flux: 6,800 lm) were installed (subsequently referred to as illuminated sites). The lamps were installed on poles at a height of 4 m and were equipped with a twilight switch, which turned the lamps on when the surrounding brightness fell below 5 lx. Varying with the topography of the meadow, this resulted in a mean ± s.e.m. = 52.0 ± 4.2 lx within a 5-m radius circle around the streetlamp, 25.7 ± 1.1 lx 5-10 m away and < 3.9 ± 0.4 lx more than 10 m away. Each of the seven illuminated sites was paired with a dark site (Supplementary Table 1). All dark sites were situated at least 100 m from any permanent light source (that is, street lamp) and had no major light source, such as an illuminated sports ground, in the surrounding 500 m. Pairing was done based on spatial proximity. In addition, due to high nightly variance in nocturnal insect activity, the sites within one pair were sampled at the same time during the same night. Assessment of interactions between plants and flower visitors. Two datasets were collected, one in 2014 comprising paired diurnal and nocturnal interactions between plants and flower visitors (subsequently referred to as the daynight dataset), and one in 2015, comprising nocturnal observations only, but on illuminated and dark sites (subsequently referred to as the ALAN dataset). The two datasets were collected according to the same method, but with a slightly different sampling effort. Subsequently, the sampling procedure for the day-night dataset is described, and deviations from it in the ALAN dataset are given in brackets. The plant-flower visitor interactions were sampled by collecting all flower visitors actively touching the reproductive organs of the flowers within the area of 1 m on both sides of a transect using a hand net and walking at a steady speed [START_REF] Gibson | Sampling method influences the structure of plant-pollinator networks[END_REF] . Each flower visitor was individually caught and immediately transferred to a separate vial to avoid contamination by contact with other collected insects. For each collected visitor, the plant species was recorded. The flower visitors were frozen, pinned and if possible determined to species level (day-night dataset: 77%, ALAN dataset: 82%) otherwise to morphospecies (day-night dataset: 6%, ALAN-dataset: 8%), genus (day-night dataset: 12%, ALAN dataset: 7%) or family level (day-night dataset: 4%, ALAN dataset: 3%). For simplicity, subsequently, they are all referred to as species. However, to ensure that the specimens identified to a low taxonomic resolution and flower visitors that were not functional pollinators did not influence the results, all analyses were repeated using two subsets of the two datasets. The first two subsets (further referred to as day-night_p and ALAN_p) included only specimens that were identified to the level of genus, species, or morphospecies. Furthermore, all specimens of insect orders generally regarded as pollinators, namely Hymenoptera (except specimens of the family Formicidae) and Diptera [START_REF] Orford | The forgotten flies: the importance of non-syrphid Diptera as pollinators[END_REF][START_REF] Rader | Non-bee insects are important contributors to global crop pollination[END_REF] , were included, whereas specimens of taxa groups generally not regarded as pollinators were excluded, namely specimens of Carabidae, Neuroptera, Araneae and Blattodea. Finally, all specimens that were found to carry more than 5 pollen grains at least once were assigned as pollinators (assignment of specimens identified at the genus level only, if for all the specimens this was the highest taxonomic resolution). The second two subsets (further referred to as day-night_p.strict and ALAN_p. strict) were obtained following the same approach as for the first two subsets, but with a more strict exclusion of taxa generally not regarded as pollinators: all orders were excluded except for Lepidoptera and Coleoptera (except for Carabidae, which were also excluded), Diptera and Hymenoptera. For more details regarding the datasets see Supplementary Table 3.

Observations during the night started 30 min after astronomical sunset. A 50-m transect (ALAN dataset: 100-m transect) was sampled every 30 min for a period of 3 h (ALAN dataset: 4 h), resulting in six sampling rounds per night and site (ALAN dataset: eight sampling rounds). Between May and September 2014 (ALAN dataset: June and September 2015) sampling was repeated six times (ALAN dataset: five times, except for one illuminated and one dark site, which were only sampled four times). As visual aids, night-vision goggles (BIG25-CV, Vectronix) and LED head torches (873155 Intertronic, Interdiscount) were used in a randomized, alternating pattern (that is, for each hour, either LED head torches or night-vision goggles were randomly used first). Observations during the day started at 13:00 and they were paired with the night sampling, that is, on the same day or the day before or after, except for two times, when there were 3 and 10 days in between two sampling events owing to bad weather conditions. Sampling was only conducted without strong wind and rain, and during the day only in sunny conditions. Estimation of pollination service. In 2015, on each of 5 dark and 5 illuminated sites (Supplementary Table 1), 5 pairs of 2 individuals of C. oleraceum were selected, resulting in 10 plants per site. The plants within a pair were similar in size and were at maximum 1 m apart from each other. Before the onset of flowering, one plant within each pair was randomly selected and its flower heads were bagged with a white nylon mesh (mesh size: 0.5 mm) in order to exclude flower visitors. This resulted in a total of 100 observed plants subjected to four treatments (the 25 plants for each treatment were distributed equally among the sites). Between the end of July and the beginning of September, flower heads were collected as soon as they started to wither and to produce a feathery pappus. All fruits of two flower heads per plant were counted, and a visual distinction between the developed white fruits and the aborted brown ovules was made (Extended Data Fig. 4). Two measurements of reproductive output were calculated: the number of developed fruits and the percentage of developed fruits per flower head. Lux intensities for each position of a plant pair were determined by a Photocurrent Amplifier (Ph-Amp MB7 Version 2.0, Czibula and Grundmann GmbH). The withered plants were harvested (above ground) and dried for three consecutive days in a drying cabinet (70 °C) to assess the dry biomass using a Mettler Toledo scale (NewClassic MF; ML203E). A few flower heads were infested with small herbivores and thus categorized according to whether they were infested or not (herbivores: yes versus no). Pollen analysis. Pollen grains on insects were assessed using a 1 × 1-mm 3 cube of glycerin gel (1.04094.1000, Merck KGaA) stained with Fuchsin (3256.1, Roth). The insects were swabbed on the entire body, except for wings and parts that are specialized for pollen-carrying, that is, the corbiculum and scopa of bees, as the pollen there is not available for pollination. The gel was then placed on a microscope slide and gently melted. Each microscope slide (that is, one per insect specimen caught) was then scanned for pollen grains using a light microscope, and up to five grains were counted. Indirect effects from nocturnal to diurnal pollinators. The potential for indirect effects from nocturnal to diurnal pollinators via shared plant species was calculated following the approach developed by Müller et al. [START_REF] Müller | The structure of an aphid-parasitoid community[END_REF] , hereafter referred to as Müller's index. Müller's index is well-suited for assessing the potential for any indirect influence between species (that is, apparent competition or facilitation) and is coded in the PAC function of the R package bipartite. For each pair of nocturnal and diurnal flower visitors within each of the eight networks, we computed the potential for plant-mediated indirect effects from the nocturnal to the diurnal flower visitor. For each of the eight sites, we then computed the mean potential for indirect effects from nocturnal to diurnal flower visitors, hereafter referred to as the observed mean. The observed means were then compared to means derived from a null model similar to the one that was used previously [START_REF] Sauve | How plants connect pollination and herbivory networks and their contribution to community stability[END_REF] . This null model keeps the structure of the diurnal and nocturnal plant-pollinator sub-networks identical to the observed ones, but randomizes how plants connect these two subnetworks. In other words, while the network structure of the plant-flower visitor sub-network remained identical, plant names were randomly assigned before merging networks, resulting in a random interconnection of the two sub-networks. For each of the eight sites, we performed 1,000 randomizations and computed for each randomization the mean potential for indirect interactions, hereafter referred to as randomized means. For each site we then calculated the percentage of randomized means that were below the observed mean. A quantile test (sign test) using the quantile function in R was performed to test whether the observed means were above the median of the randomized means across the eight sites. Network parameters. For the ALAN dataset, six network parameters were calculated: the number of species visiting the flower heads; the number of flower visits; the number of plant species visited; the generalism of plants, that is, the weighted mean effective number of species of flower visitors per plant species [START_REF] Bersier | Quantitative descriptors of food-web matrices[END_REF] ; the generalism of flower visitors, that is, the weighted mean effective number of plant species per pollinator species [START_REF] Bersier | Quantitative descriptors of food-web matrices[END_REF] ; and nestedness (NODF), which reflects the tendency for specialist species to interact with generalists. The last three parameters are known to affect the stability of plant-pollinator communities [START_REF] Thébault | Stability of ecological communities and the architecture of mutualistic and trophic networks[END_REF][START_REF] Waser | Generalization in pollination systems, and why it matters[END_REF] and were calculated using the function networklevel of the R package bipartite. Data analysis. Nocturnal plant-flower visitor networks. The network parameters were taken as a response variable and analysed using either linear mixed-effects (LME) models with the lmer function in the lme4 package or generalized linear mixed-effects (GLMM) models with a Poisson distribution (see Extended Data Table 1). Treatment (two levels, dark versus illuminated) was included as a fixed factor, and site (seven paired sites of which one was the dark and the other the illuminated site) as a random factor. All GLMM models were checked for overdispersion by including an observation-level random factor (as many levels as observations) into the model and comparing it to a model without this additional variance parameter. When the observation-level random factor significantly improved the model, it was retained in the model. All LME models were visually checked for a normal distribution of residuals and homoscedasticity, and, if necessary, the dependent variable was transformed to fulfil model assumptions (see Extended Data Table 1). To account for different numbers of interactions sampled between dark and illuminated sites, we generated accumulation curves for species richness and generalism of insects and plants. For each site and treatment, 500 accumulation curves were performed by re-sampling the observed interactions, see for example ref. 20. To account for differences in network size between dark and illuminated sites when estimating nestedness, we calculated z scores by comparing the observed NODF values to 1,000 NODF values generated using a null model that keeps network size, connectance and heterogeneity in generalism constant [START_REF] Vázquez | Evaluating multiple determinants of the structure of plant-animal mutualistic networks[END_REF] .

Flower visitations to C. oleraceum. The effect of artificial lighting on nocturnal flower visitations to C. oleraceum was analysed with GLMM models that use the number of species of flower visitors and the number of visits of the ALAN dataset as response variables and include the same model structure as described for the network parameters. Similarly, the models were checked for overdispersion and, if necessary, an observation-level random factor was included (see Extended Data Table 4 for specifications). The difference between the flower visitations to C. oleraceum between day and night was analysed using the day-night dataset. A GLMM model was run, with the number of flower visitors as the response variable, time of sampling (two levels, day versus night) as a fixed factor, site (eight sites, that is, eight levels) as a random factor, and, if necessary, including an additional observation-level random factor.

Fruit set of C. oleraceum. The effect of artificial lighting on the number of developed fruits was analysed with a GLMM model assuming a Poisson distribution and including plant pair (5 pairs per site, 5 levels) nested within sites (10 sites, 10 levels) nested within site pair (5 pairs, 5 levels) as a random factor. Treatment (two levels, dark versus illuminated), bagging of the flower head (two levels, bagged versus open) and herbivores (two levels, yes versus no) were included as fixed factors, the dry biomass as a co-variable, and treatment-bagging, treatment-biomass and treatment-herbivores as interactions. If necessary, an additional observation-level random factor was included to correct for overdispersion. The minimal adequate model was determined by Akaike information criterion (AIC)-based stepwise deletion of the interactions first and then single predictors of the full model using likelihood ratio tests. Similarly, the percentage of developed fruits was analysed with a GLMM model and the same model structure was used, except for the assumption of a binomial distribution instead of a Poisson distribution. To test whether the reduction of fruit set varies according to the lux intensity on the flower head, GLMM models were run for the number of developed fruits assuming a Poisson distribution and the percentage of developed fruits assuming a binomial distribution. For these models only data from the unbagged flower heads and illuminated sites were used. In all models plant biomass and lux intensity were included as explanatory variables, and sites (5 sites, 5 levels) as a random factor. The fixed factor, herbivores (yes versus no), was not included as no herbivores were found in the subset. If necessary, the GLMM models were corrected for overdispersion (see Extended Data Table 5 for specifications).

Extended Data Figure 1 | Accumulation curves for the species richness of flower visitors on illuminated and dark sites. For each site 500 randomizations were performed and 95% confidence intervals are given (illuminated sites in white, dark sites in grey). Numbers above the plots correspond to the number of the pair of sites (see Supplementary Table 1).

Extended Data Figure 2 | Accumulation curves for generalism of plant species on illuminated and dark sites. For each site 500 randomizations were performed and 95% confidence intervals are given (illuminated sites in white, dark sites in grey). Numbers above the plots correspond to the number of the pair of sites (see Supplementary Table

  Centre d'Ecologie et des Sciences de la Conservation, Muséum National d'Histoire Naturelle, UMR 7204 CNRS-MNHN, 61 Rue Buffon, 75005 Paris, France.

Figure 1 |

 1 Figure 1 | Interaction web showing the pathway by which artificial light at night affects plant reproduction and diurnal pollinator communities. Solid arrows indicate direct interactions; dashed arrows denote indirect interactions. The sign (+ or -) refers to the expected direction of the direct or indirect effect (see text). The figures and table that present data supporting each of the predicted effects are indicated.

Figure 2 |

 2 Figure 2 | Effects of artificial lighting on parameters of nocturnal plant-flower visitor networks. a, b, Overall quantified plant-flower visitor network of seven dark sites (a) and seven experimentally illuminated sites (b). The rectangles represent insect species (top) and plant species (bottom), and the connecting lines represent interactions among species. Species codes for the plants and a list of insect species are given in

Figure 4 |

 4 Figure 4 | Effects of artificial lighting on fruit set. a, b, Number of developed fruits (a) and percentage of developed fruits (b) for flower heads with insect pollination (open) and without insect pollination (bagged) on dark sites (light blue bars) and experimentally illuminated sites (white bars). Fifty flower heads were observed per treatment group (n = 50 per treatment group), and mean ± s.e.m. are shown. Results from statistical models are given in Extended Data Table3.

Figure 3 |

 3 Figure 3 | Merged overall quantified diurnal and nocturnal plantflower visitor network. The top rectangles represent species of diurnal visitors, the bottom rectangles are species of nocturnal visitors, and the middle rectangles show plant species, respectively. The abundance of all

1

 1

). Extended Data Figure 4 |

 4 Fruits and aborted ovules of C. oleraceum. Letter reSeArCH extended Data table 1 | Comparison of network parameters of dark and illuminated sites Results of linear mixed-effects (LME) models comparing the number of pollinator species (visitor richness), number of visits, number of plant species visited (plant richness), generalism of plants, generalism of flower visitors and z score of NODF (nestedness) of dark sites (dark, n = 7) and sites that were experimentally illuminated (illuminated, n = 7). Test statistics (test stat.): t value for lmer, z value for glmer. n, models without overdispersion or transformation of response variable; o, models that included an observation-level random factor; s, square-root transformation of the response variable. extended Data table 2 | the number of flower visits to C. oleraceum during day and night Results of generalized linear mixed effects (GLMM) models analysing the difference between the number of flower visits to C. oleraceum during the day (n = 8) compared to during the night (n = 8). Analyses were run with the full dataset of all flower visitors (day-night) and two subsets (day-night_p and day-night_p.strict; for specifications regarding subsets, see Methods). All models were overdispersed and therefore included an observation-level random factor. extended Data table 3 | effects of artificial lighting on fruit set Results of the minimal adequate models showing the effect of artificial lighting on the number and percentage of developed fruits per flower head of C. oleraceum. treat, light treatment (Lamp (illuminated sites) versus Dark (dark sites)); bagging, exclusion treatment of flower visitors (Bagged (bagged flowers) versus Open (open flowers)); presence of herbivores (Yes versus No). EST, estimate; SE, standard error. Both models were overdispersed and therefore included an observation-level random factor. extended Data table 4 | effects of artificial lighting on the diversity of insects visiting C. oleraceum Results of GLMM models comparing the diversity of species visiting C. oleraceum on dark sites (dark, n = 7) and experimentally illuminated sites (illuminated, n = 7). Analyses were run with the full dataset of all flower visitors (ALAN) and two subsets (ALAN_p and ALAN_p.strict; for specifications regarding subsets see Methods. n, models without overdispersion; o, models which included an observation-level random factor. extended Data table 5 | effect of lux intensities on fruit set Results of GLMM models analysing the effect of different lux intensities and plant dry biomass on the number and percentage of developed fruits of C. oleraceum. EST, estimate; SE, standard error. Both models were overdispersed and therefore included an observation-level random factor.

Online Content Methods, along with any additional Extended Data display items and Source Data, are available in the online version of the paper; references unique to these sections appear only in the online paper.

Acknowledgements

We thank all those who assisted with fieldwork, E. Thébault for discussions, M. Visser and M. Menz for comments that helped to improve this manuscript and D. Sanders for contributing to early ideas on the project. Furthermore, we are grateful to all experts who helped with identification of species: H.-P. Wymann (Lepidoptera), E. Obrecht (Diptera), S. Oertli (Hymenoptera) and C. Germann (Coleoptera). This study was supported by the Swiss National Sciences Foundation.

Code availability. The computer code used for this study is available from the corresponding author upon request. Data availability. The datasets analysed during the study are available from the corresponding author upon request.

Supplementary Information is available in the online version of the paper.

extended Data table 6 | Potential for indirect plant-mediated effects from nocturnal to diurnal flower visitors

Results of the analyses quantifying the potential for indirect plant-mediated effects from nocturnal to diurnal flower visitors. The observed mean potential for such effects was compared to the effect for networks in which plants connect the nocturnal and diurnal sub-networks in a randomized manner. A quantile test was performed to test whether the observed value across sites was significantly higher than for randomly inter-connected networks. Analyses were run with the full dataset of all flower visitors (day-night) and the two subsets (day-night_p and day-night_p.strict).


Christian Mougin

Albert Kollmann

Jacqueline Dubroca

Paul Henri Ducrot

Roger Alvinerie

Pierre Galtier

Michel Alvinerie

Fate of the veterinary medicine ivermectin in soil

Keywords: Avermectin, Ivermectin, Soil, Transformation, Bioavailability

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

Introduction

Avermectins are a class of macrocyclic lactone drugs with insecticidal and anthelmintic properties that have been developed for the protection of animals [START_REF] Campbell | Ivermectin today: an update[END_REF], humans [START_REF] Dadzie | Ocular findings in a double-blind study of ivermectin versus diethylcarbamazine versus placebo in the treatment of onchocerciasis[END_REF]) and crops (Putter et al. 1981). Examples of avermectins used for cattle protection include ivermectin, doramectin, eprinomectin and moxidectin. A recent formulation, the intraruminalsustained release bolus, is considered as an efficient means to control endoparasitic nematodes in ruminants because of the long-term protection of animals. 80-90% of the drug dose administered by the bolus is excreted without transformation in faeces during a 4-month period [START_REF] Alvinerie | Persistence of ivermectin in plasma and faeces following administration of a sustained-release bolus to cattle[END_REF]. Faecal excretion of ivermectin induces toxic effects on non-target organisms, including key dung-colonising families of insects. The drug has also been shown to negatively influence the decomposition of dung organic matter in soil [START_REF] Sommer | The influence of veterinary medicines on the decomposition of dung organic matter in soil[END_REF], but data are lacking concerning its impact on soil inhabiting (micro)-organisms.

The ecotoxicological risk assessment of bolus on pasture-land ecosystems has not been performed in detail in the past. Our aim is to provide data concerning possible side effects of high levels of residues deposited in the soil. In this paper, we report the results concerning the fate of ivermectin in soils, as a basis for exposure assessment. These data will make it possible to design impact studies of ivermectin on soil fungi. The impact studies will be presented in a further paper.

Experimental

Chemicals

High-purity ivermectin (22,23-dihydroavermectin B1) and chemical reagents were purchased from Sigma-Aldrich. 8,9-and 10,11-Z isomers of ivermectin were obtained by photo-transformation of a drug solution layered on glass plates under sunlight, then identified using mass spectrometry, 1 H and 13 C NMR, according to [START_REF] Mrozik | Photoisomers of avermectins[END_REF].

Soil characteristics

The soil used in this study was a silt loam collected in the 10-20 cm layer of a meadow in Versailles, France. It contains 25.5% sand, 55.0% silt and 19.5% clay, and its organic matter content was 1.65%. The soil pH was 8.1 and the cationic exchange capacity was 10.2 meq 100 g -1 .

The soil was 2 mm sieved then used immediately.

Degradation experiments in soils

Incubations were performed on 25 g dry weight soil samples spiked with 25 µg ivermectin (1 mg kg -1 ). The soil was kept at 80% of its moisture-holding capacity by adding water. Soil samples were incubated either in sterile or in non-sterile conditions in the dark at 25 °C, or under sunlight at ambient temperature of June, with a mean value of 20 °C).

Bioavailability of ivermectin

The amount of ivermectin bioavailable for soil fungi was determined using the protocol described by [START_REF] Gaillardon | Influence of soil moisture on short-term adsorption of diuron and isoproturon by soil[END_REF]. Soil samples (10 g equivalent dry matter) were placed in 5-cm diameter Petri dishes to give a 3-4 mm thick layer. Acetonic solutions of ivermectin were applied to the surface of the soil to obtain a final concentration of 1 mg kg -1 . The solvent was allowed to evaporate for 30 min and water was added to ensure that 80% of the moisture-holding capacity of the soil was maintained. Concentrations of ivermectin in the soil solution were determined 24 h after treatment. Two superposed 42.5-mm-diameter glass microfibre filters GF/A (Whatman) were laid on the soil surface and slight pressure was applied for 10 s to favour wetting of the filters. The upper filter was then recovered, weighed to determine the volume of soil solution, and dried overnight at 40 °C.

Degradation experiments in fungal liquid cultures

The ability of two strains of filamentous fungi, Trametes versicolor (ATCC 32745) and Fusarium solani (isolated from the soil) was evaluated in liquid cultures. They were grown on a culture medium (10 mL in 150-mL Erlen-meyer flasks, [START_REF] Lesage-Meesen | A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus[END_REF] with maltose and ammonium tartrate as carbon and nitrogen sources, inoculated with a mycelial mat on agar plug (10-mm diameter). Cultivation was carried out statically in the dark at 25 °C for 2 days. Then, the medium was supplemented with 1 mg L -1 ivermectin. Drug transformation was then checked after 8 days of growth.

Analytical methods

During the soil degradation experiments, residual ivermectin and possible transformation products were extracted every week by shaking the soil (25 g dry weight) with 50 mL acetonitrile for 1 h.

The organic extract was then centrifuged (5 min, 2,000 g), and aliquots of 6 mL were mixed with 9 mL distilled water; 10 mL of the solution was concentrated on an MCH-10 C18 guard column (30x4.6 mm; Varian) with an isocratic high performance liquid chromatography (HPLC) pump at a flow rate of 1 mL min -1 . The guard column was fixed to a ten-way valve. The extracted compounds were then eluted on a C18 Nova-Pak analytical column (150 x 4.6 mm; Waters) with an HPLC pump delivering a solvent system composed of H3PO4 (0.1% in water), acetonitrile and methanol (20:57:23, v/v/v). The column eluate was monitored at 245 nm using a variable wavelength UV-Vis detector. The detection limit was 80 ng ivermectin.

To analyse bioavailability assays, dry filters were extracted with 10 mL acetonitrile. The eluate was then evaporated to dryness and the dry residue was subjected to the derivation procedure already published [START_REF] Alvinerie | Persistence of ivermectin in plasma and faeces following administration of a sustained-release bolus to cattle[END_REF]. Briefly, the dry residue was dissolved in 100 µL of an Nmethylimidazole solution in acetonitrile. To initiate the derivatisation, 150 µL trifluoroacetic anhydride solution in acetonitrile was added. After derivatisation, 100 µL of the solution was analysed hy HPLC. A mobile phase of H3PO4 (0.1% in water), acetonitrile and methanol (4:66:30, v/v/v) was used. Fluorescence was detected at an excitation wavelength level of 377 nm and an emission wavelength of 427 nm. The detection limit was 1 ng derivatised ivermectin.

Ivermectin in fungal cultures was analysed using the following procedure: Fungal biomass (700-800 mg dry weight) from an Erlenmeyer flask was separated from the medium by filtration on a glass fiber filter after 8 days. The biomass was then heated in the presence of MeOH (20 mL per Erlenmeyer flask) at 70 °C for 10 min. After cooling, each extract was concentrated under vacuum and dissolved in 20 mL diethyl ether. The organic extract was then pooled with the corresponding culture medium and shaken for 2 min in a separatory funnel. Finally, the organic fraction was recovered and dried on MgSO4. These steps were performed in triplicate. The resulting organic extracts were pooled, evaporated to dryness and dissolved in 1 mL MeOH for HPLC analysis without derivatisation. Each experiment was done in triplicate. Results are expressed as means. The standard deviation was less than 10% of the mean.

Results and discussion

Transformation of ivermectin in the soil

The fate of ivermectin in soil was studied under various incubation conditions (Fig. 1). The initial amount of the ivermectin added (1 mg kg -1 ) is commonly found in the faeces of calves treated with the bolus [START_REF] Alvinerie | Persistence of ivermectin in plasma and faeces following administration of a sustained-release bolus to cattle[END_REF], and may represent the maximal amount of ivermectin in the subsurface of the soil.

The results show that under sterile conditions in the dark, the drug ivermectin was not degraded (Fig. 1). Further, under non-sterile conditions, only a slight decrease of ivermectin concentrations was observed, as a possible biological effect of endogenous microflora. A half-life of the chemical of around 240 days was calculated under these conditions. By sharp contrast, exposing the soil samples to sunlight induced in a rapid decrease of ivermecin amounts extracted from the soil, which was stimulated by the mixing of the soil. The corresponding half-life of the chemical was 21 days. These results are consistent with the data previously published [START_REF] Halley | Environmental aspects of ivermectin usage in livestock: general considerations[END_REF].

Two minor peaks from extracts obtained from soils incubated under sunlight were detected on the HPLC chromatograms. They co-eluted with standards of 8,9-and 10,11-Z isomers of the drug formed through photo-transformation (Fig. 2), but their low amounts prevented any structural determination. Formation of these com-pounds has also been reported previously [START_REF] Mrozik | Photoisomers of avermectins[END_REF]. No other transformation products could be identified in organic extracts.

Bioavailability of ivermectin in soil

We measured the amounts of ivermectin in the soil solution after a 24-h period of sorption of the chemical onto soil samples. The ivermectin measured by HPLC amounted to 23.8±13.8 ng (mean ± SD of three replicates) per filter, and corresponded to a concentration of 6 µg L -1 soil solution. This value fits with the aqueous solubility of the highly hydrophobic ivermectin, which could be approximated to 10 -8 M for the design of toxicity experiments on soil micro-organisms.

The soil used in this study exhibited a moderate organic carbon content, responsible for the adsorption of ivermectin, a neutral compound. As a consequence, the bioavailability of the drug appears essentially governed by the organic carbon content of the soil rather than its other properties.

Biotransformation of ivermectin in fungal liquid cultures

We studied the biotransformation of the drug in liquid cultures of T. versicolor and F. solani (Table 1). The ability of these strains to transform xenobiotics has been extensively studied in the laboratory.

After 8 days, ivermectin concentration in the medium decreased to 70.4% of the initial amount added in controls flasks non-inoculated with either strain. This decrease was a result of adsorption of the chemical on glass surfaces. Ivermectin amounts found after the same period in the Erlenmeyers flasks inoculated with of T. versicolor or F. solani were slightly higher, evidencing the lack of transformation of the drug by the fungal strains.

T. versicolor is a white-rot basidiomycete producing exocellular oxidases, namely laccases, involved in the transformation of natural or xenobiotic compounds. Spiking the cultures with 20 mM xylidine, a well-known inducer of laccase production [START_REF] Mougin | Enhanced production of laccase in the fungus Trametes versicolor by the addition of xenobiotics[END_REF], did not enhance ivermectin transformation.

In order to increase the uptake of ivermectin by F. solani, a fungal strain transforming xenobiotics using intracellular enzymes, several cultures were treated with neutral surfactants, namely Triton X100, Tween 20 and Brij 35, each at two-fold its critical micellar concentration. As the concentrations of ivermectin globally increased in the treated cultures, we concluded that the presence of the surfactants did not have an enhancing effect on ivermectin transformation by the fungus.

Conclusion

Our data show that that ivermectin can be stored for long periods in the soil in dark conditions. We were unable to detect any biotransformation of ivermectin by filamentous fungi, including a powerful white-rot strain. Bioavailability of the drug for soil inhabiting organisms was extremely low in the soil solution, but it may be increased according to the exposure routes. For example, ivermectin associated with the surface of particulate matter could penetrate the gastrointestinal tract of animals. Ivermectin could also represent a risk for organisms living in aquatic ecosystems that can be contaminated by the drug strongly adsorbed onto soil particles or suspended materials.

We know the concentration of the drug in the soil solution and its main chemical form, the parent com-pound. It is therefore now possible to design impact studies of ivermectin on soil fungi.

Fig. 1 .

 1 Fig. 1. Transformation of ivermectin in soil under various incubation conditions

Table 1 .

 1 Ivermectin extracted from 8-day liquid cultures submitted to various incubation conditions 9

Acknowledgements

This work was supported by the French program PNETOX. We thank Christine Young (INRA, Jouy en Josas) for proofreading the manuscript.


Alexa Latapy
email: alexa.latapy@gmail.com

Arnaud Héquette

Amandine Nicolle

Nicolas Pouvreau

Assessing the Influence of Changes in Shoreface Morphology since the 19th Century on Nearshore Hydrodynamics and Shoreline Evolution in Northern France: A Modeling Approach

Keywords: Sand banks, northern France, hydrodynamic circulation, residual tidal currents, Calais

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

INTRODUCTION

Sand banks are large sedimentary features that occur in many coastal and shelf seas where there is a large amount of sediment and strong enough currents to transport it [START_REF] Caston | Linaer sand banks in the Southern North Sea[END_REF][START_REF] Dyer | The origin, classification and modelling of sand banks and ridges[END_REF][START_REF] Kenyon | Offshore Tidal Sand-Banks as Indicators of Net Sand Transport and as Potential Deposits[END_REF][START_REF] Pan | Modelling the hydrodynamics of offshore sandbanks[END_REF]. The southern North Sea is characterized by multiple tidal sand banks that have been extensively studied [START_REF] Berné | Architecture and long term evolution of a tidal sandbank: The Middelkerke Bank (southern North Sea)[END_REF][START_REF] Caston | Linaer sand banks in the Southern North Sea[END_REF][START_REF] Deleu | Morphodynamic evolution of the kink of an offshore tidal sandbank: the Westhinder Bank (Southern North Sea)[END_REF][START_REF] Houbolt | Recent sediments in the Southern Bight of the North Sea[END_REF][START_REF] Lanckneus | Environmental setting, morphology and volumetric evolution of the Middelkerke Bank (southern North Sea)[END_REF][START_REF] Tessier | Internal Structure of Shoreface Banks Revealed by High-Resolution Seismic Reflection in a Macrotidal Environment (Dunkerque Area, Northern France)[END_REF]. Sand banks are particularly widespread off the coast of northern France where they form linear shoreparallel or slightly oblique sand bodies about 10-30 km long and 1-3 km wide (Figure 1, inset map). They belong to the Flemish Banks [START_REF] Houbolt | Recent sediments in the Southern Bight of the North Sea[END_REF]) and generally occur as groups of banks from shallow coastal areas to depths of several tens of meters (Anthony, 2002;[START_REF] Vicaire | Dynamique hydro-sédimentaire en mer du Nord mériodionale (du Cap Blanc-Nez à la frontière Belge)[END_REF]. Near the coast, sand bank mobility can be responsible for significant changes in bathymetry and seabed morphology (Aernouts and Héquette, 2006;[START_REF] De Moor | Evaluation of sea-floor sediment dynamics in the Flemish Banks (southern North Sea) between 1985 and 1995[END_REF][START_REF] Héquette | The influence of nearshore sand bank dynamics on shoreline evolution in a macrotidal coastal environment, Calais, Northern France[END_REF][START_REF] Tessier | Internal Structure of Shoreface Banks Revealed by High-Resolution Seismic Reflection in a Macrotidal Environment (Dunkerque Area, Northern France)[END_REF]. Previous studies carried out in different coastal regions, including the coast of northern France, suggested strong relationships between shoreline evolution and nearshore sand bank mobility that would have induced changes in cross-shore and longshore sediment transfer [START_REF] Carter | Sub-tidal ebbshoal control of shoreline erosion via wave refraction, Magilligan foreland, Northern Ireland[END_REF][START_REF] Héquette | The influence of nearshore sand banks on coastal hydrodynamics and sediment transport, northern coast of France[END_REF][START_REF] Héquette | The influence of nearshore sand bank dynamics on shoreline evolution in a macrotidal coastal environment, Calais, Northern France[END_REF][START_REF] Shaw | Role of a Submarine Bank in the Long-Term Evolution of the Northeast Coast of Prince Edward Island, Canada[END_REF][START_REF] Thomas | A multi-century record of linked nearshore and coastal change[END_REF]. However, the modes of sediment transfer between nearshore sand banks and adjacent coastline are still unclear.

Historical bathymetries were used in several studies to modeling hydrodynamic circulation evolution to investigate interactions between coastal morphology and hydrodynamics (e.g., [START_REF] Bertin | Contribution of simulations on historical bathymetries to the understanding of evolutions of estuarine sand banks[END_REF][START_REF] Cooper | Natural bathymetric change as a control on century-scale shoreline behavior[END_REF][START_REF] Héquette | The influence of nearshore sand bank dynamics on shoreline evolution in a macrotidal coastal environment, Calais, Northern France[END_REF]. For example, Horrillo-Caraballo and Reeve (2008) showed that the 150 year-long changes in In northern France, topographic and hydrographic surveys of coastal and nearshore morphology were carried out by the French Hydrographic Service (Shom) since the beginning of the 19th century, enabling to describe the long-term characteristics of the seabed evolution. To assess changes in nearshore circulation, these historical bathymetries were used to simulate tidal currents at different dates. In this paper, we present the results obtained near Calais (Figure 1) where two prominent sand banks developed near the coast during historical time [START_REF] Ruz | Development of an incipient foredune field along a prograding macrotidal shoreline, northern France. Géomorphologie : relief, processus[END_REF]: the Riden de la Rade, which extends over 13 km alongshore, is connected to the Riden de Calais (11.5 km long) at its western end, forming a V-shaped sediment body with an angle of 20°(Figure 1). Strong tidal currents are channelized between the sand banks, with a flood-dominated asymmetry (Figure 1) that is responsible for a net sediment transport to the ENE [START_REF] Héquette | Sediment transport under wave and current combined flows on a tide-dominated shoreface, northern coast of France[END_REF] that can explain the longshore migration of the nearshore sand banks in the same direction [START_REF] Latapy | Mesoscale Morphological Changes of Nearshore Sand Banks since the Early 19th Century, and their Influence on Coastal Northern France[END_REF].

METHODS

Bathymetry Changes

Hydrographic field sheets from the Shom, spanning from the mid-19th century to the beginning of the 21st century (Table 1) were used to study the morphological evolution of the sand banks and of the adjoining seafloor. These documents were scanned, digitized and then interpolated using the krigging method to produce Digital Elevation Models (DEMs). The coordinate system is in WGS84 and elevations are in meters below chart datum. To quantify bathymetry changes, differential DEMs, alongshore and cross-shore transects and sediment volume calculation were made (more details on interpolation and computing methods can be found in [START_REF] Latapy | Mesoscale Morphological Changes of Nearshore Sand Banks since the Early 19th Century, and their Influence on Coastal Northern France[END_REF]).

Numerical Model

To investigate the influence of seabed morphological changes on coastal hydrodynamics, a numerical approach was developed using TELEMAC-2D. Model domains extend from the Dover Strait to the Belgium border and integrate historical bathymetry (Table 1 in bold). The offshore open boundary is forced with specified elevations for 143 tidal harmonics from the tidal model cstFRANCE developed and used by the Shom (Le [START_REF] Roy | Réalisation et validation d'un modèle de marée en Manche et dans le Golfe de Gascogne (application à la réalisation d'un nouveau programme de réduction des sondages bathymétriques[END_REF]. ) with sea-level elevations over the whole domain at 10 min intervals. Astronomical spring tides enabled to simulate the strongest and most intense currents in each mesh in order to identify high energy areas and those that have potentially undergone the most significant changes. Residual tidal current were computed over the complete simulated period and the generated maps were used to identify possible transport pathways. Tidal friction velocities were also computed to assess the variations in bed shear stress that could led to shoreface morphology change.

RESULTS

Calais Nearshore Evolution since the 19 th Century

The Calais seabed morphology changed considerably since 1861 (Figure 2c), mainly due to significant variations in the morphology and position of one particular nearshore sand bank: the Riden de la Rade. Since 1861, a longshore growth of this sediment body was clearly visible on bathymetric maps (not shown). The lengthening of the bank was accompanied by an elevation of the crest of about 10 m (Figure 2c). This sand bank has evolved in 3 distinct phases:

 From 1861 to 1911, the Riden de la Rade was barely developed (Figure 2a-b). The increase of both width and height at the end of the 19 th century, visible on cross-shore profiles, reveals a recent development.

 Between 1911 and 1983, the western part of the Riden de la Rade rose (+ 5 m) and gradually migrated onshore, with a maximum displacement of up to 800 m (Profile A) while the easternmost part experienced exclusively a vertical growth (Profile B). In the western part of the Calais harbor, the strong lowering of the foreshore (Figure 2c) can be attributed to the shoreward movement of the bank which induced the onshore of the channel between the coast and the bank (Profile A).

 From 1983 to 2009, the landward migration of the Riden de la Rade led to a deepening of the channel and its migration towards the coast (Profile B). The profile A reveals a widening of the Riden de la Rade as well as a shift from a seaward asymmetry to a symmetrical profile.

Evolution of Tidal Circulation

Figure 3 shows the computed tidally induced residual currents in the Calais nearshore zone for each bathymetry configuration between 1878 and 1975. Residual currents are essentially directed to the NE which corresponds to the flood direction. In the interbank area (between the Riden de la Rade and the Riden de Calais) currents are weaker (< 0.04 m.s -1 ) and mainly directed towards NE, except in the 1975 configuration where the flow is towards WSW. Our results suggest significant changes in the hydrodynamic circulation with an overall increase in velocities of residual currents related to the formation and growth of the Riden de la Rade. In 1878, the strongest currents were close to the shore and did not exceed 0.20 m.s -1 (Figure 3a). In 1910, the zone of highest current velocities expanded alongshore, accompanied by an increase in current velocities in some areas (Figure 3b). In 1930, while the channel continued to deepen, current speeds continued to increase, reaching 0.30 m.s -1 particularly in the central part of the channel (Figure 3c). Finally in 1975, residual currents above 0.15 m.s -1 in the channel and in the junction between the shoreface and the Riden de la Rade represent an area 3 times greater than that obtained in 1878 and 4 times greater for residual currents higher than 0.2 m.s -1 (Figure 3d).

On the foreshore, east of the Calais port, residual currents are gradually decreasing through time. Currents directions are mostly directed to the ENE in the 1878 and 1910 configurations. However, with the 1930 and 1975 set-ups, clockwise gyre flows are detected (Figure 3f). Figure 3e highlights a decrease and an increase of the friction velocity on the top of the Riden de la Rade and in the foreshore respectively. These changes could have favored sediment accumulation by reducing shear stress on the sand bank. On the foreshore, clockwise gyre flows combined with an increase in friction velocity may have enhanced sediment redistribution. West of Calais, there is an increase in velocities between 1878 and 1975, not only on the foreshore but also in the nearshore (up to the -10m isobaths).

DISCUSSION

Using historical bathymetry surveys, this study revealed significant morphological changes since the middle of the 19 th century. The Riden de la Rade experienced sediment accumulation, with a maximum gain of +17.9 × 10 6 m 3 between 1930 and 1975 [START_REF] Latapy | Mesoscale Morphological Changes of Nearshore Sand Banks since the Early 19th Century, and their Influence on Coastal Northern France[END_REF].

Channelization appears to be an important process within the study area. Since the early 1910s, the coastal channel increased in both width and depth (Figure 2a-c). This process, induced by the growth and the landward migration of the Riden de la Rade, resulted in higher flow velocities in the channel, potentially leading to erosion in the western part of the nearshore zone of Calais. The ebb-dominated current in the interbank, wellidentified in the 1975 model, is consistent with observations made in this area [START_REF] Augris | Le domaine marin du Nord-Pas de Calais[END_REF] and may be a result of the Riden de la Rade formation. Likewise, gyres at the eastern end of the bank in the 1930 and 1975 models may be a direct consequence of tidal current deflection by the welding of the Riden de la Rade with the shoreface, similarly to what was observed by [START_REF] Horrillo-Caraballo | Morphodynamic behaviour of a nearshore sandbank system: The Great Yarmouth Sandbanks[END_REF] in the eastern UK coastal zone. Shoreline changes since the 1950s revealed that the western area has been rather stable even if some erosion was locally detected [START_REF] Chaverot | Changes in storminess and shoreline evolution along the northern coast of france during the second half of the 20 century[END_REF]. However, shoreline progradation occurred eastward reaching about 60 m from 1949 to 2000 [START_REF] Crapoulet | Evolution du trait de côte, bilan sédimentaire et évaluation des zones à risques sur le littoral du Nord-Pas-de-Calais : analyse multi-échelles par LiDAR aéroporté[END_REF][START_REF] Héquette | The influence of nearshore sand bank dynamics on shoreline evolution in a macrotidal coastal environment, Calais, Northern France[END_REF].

Simulation of wave propagation using historical bathymetry from 1910 onwards made by [START_REF] Héquette | The influence of nearshore sand bank dynamics on shoreline evolution in a macrotidal coastal environment, Calais, Northern France[END_REF] suggested that the onshore movement of the Riden de la Rade resulted in a decrease of wave energy in the nearshore, leading to more dissipative conditions that could enhance sediment supply. The present investigation proposes additional hydrodynamic processes. The increasing flows since 1878 on the foreshore and in the coastal channel, east of Calais, likely enhanced sediment remobilization and transport. Potentially, the coastal erosion observed in the mid-20th century may have also been favored by these strongest currents. On the other hand, the velocity decrease of clockwise gyres currents in the eastern section could have led to sediment deposition which potentially contributed to shoreline progradation.

Even if tidal-induced sediment transport processes are not the only mechanisms that drive sand bank dynamic, the combination of numerical model output and historical measurements suggests that tidal residual currents have a significant role in governing the maintenance and evolution of this sandbank. However, because waves may intensify or diminish tidal currents and play an important role in coastal sediment transport (notably through longshore drift), a tidal-wave coupling model would bring new insights in the coastal hydrodynamic evolution and thus in the sandbanks dynamics in this area.

CONCLUSIONS

Morphological changes on the Calais shoreface and associated sand banks have been investigated from comparison of historical charts as well as with results from a tidal model applied to different bathymetries (1878, 1910, 1930 and 1975) to calculate residual currents.

The Riden de la Rade underwent major movements in almost 150 years with vertical accretion, landward migration and alongshore displacements. This led to the deepening of the coastal channel between the shore and the sand bank and the welding of the Riden de la Rade with the foreshore east of Calais.

These significant morphological changes induced variations in nearshore hydrodynamics with a strengthening of residual tidal currents in the channel. Since residual currents can be indicative of sediment transport patterns, the increasing longshore flow velocities in the channel could have favored longshore sediment dispersal rather that cross-shore transport, which may have limited onshore sediment transport to the beach. In the same way, the sediments transported in the nearshore were probably more easily deposited in the eastern part of the study area when numerical modelling reveals a decrease in the longshore current speeds. Gyres flows may have also favored sediment deposition leading to the shoreline progradation observed in the eastern part of the study area.

ACKNOWLEDGMENTS

The first author (A.L) was funded by the Université du Littoral Côte d'Opale through a PhD fellowship. Hydrographic field sheets are stored at Shom's archives and used in this study, thanks to the hosting agreement Shom-ULCO (n° 133/2016).

LITERATURE CITED

Aernouts, D., Héquette, A., 2006

  sand bank morphology in the UK coastal zone can be mostly explained by variations in the pattern of tidal residual currents.

Figure 1 .

 1 Figure 1. Location map of Calais with position of A and B bathymetry profiles presented in Fig. 2 and directional distribution of the mean currents recorded in 5 m water depths from 25/04/1975 to 13/05/1975

  for 2009, made by the port of Calais). MHE: Mean horizontal error; MVE: Maximum vertical error calculated using the method developed by Wheaton et al. (2010). In bold, bathymetry survey used in the numerical model. prediction, water elevation observations and tidal current records were used to adjust and validate each model. The time step was set to 10 seconds and the model was run for a period of 30 days during the highest astronomical tides of 2015 (03/01/2015 to 03/30/2015

Figure 2 .

 2 Figure 2. (a) -(b) Bathymetry profiles evolution from 1861 to 2009; A and B are cross-shore profiles (location on Figure 1), (c) bathymetric differential map between 1879 and 2009, red-orange patterns indicate a seabed lowering, conversely blue-green areas correspond to vertical accretion, areas in grey correspond to the error margin and are considered as stable over the study period.

Figure 3 .

 3 Figure 3. Simulations of changes in tidal residual currents over (a) 1878, (b) 1910, (c) 1930 and (d) 1975 bathymetries; (e) profiles of the friction velocity obtained along the transect C during the high water springs of March 2015 (location on (b)) for each simulation; (f) represents a zoom of the 1975 map showing clockwise gyre flows.

Table 1 .

 1 Bathymetric surveys used in this study (source: Shom, except

  . L'évolution du rivage et des petits-fonds en baie de Wissant pendant le XXe siècle (Pas-de-Calais, France). Géomorphologie: Relief, Processus, Environnement, 12, 49-64. Anthony, E.J., 2002. Long-term marine bedload segregation, and sandy versus gravelly Holocene shorelines in the eastern English Channel. Marine Geology, 187, 221-234.


Leila Ben Khelifa

Modern diatom in Tunisia as indicators of water chemistry and their use for hydrologic and climatic reconstruction

MODERN DIATOM IN TUNISIA AS INDICATORS OF WATER CHEMISTRY AND THEIR USE FOR HYDROLOGIC AND CLIMATIC RECONSTRUCTION

  d'Histoire Naturelle, UMR 7245 MNHN-CNRS MCAM Eq. « Cyanobactéries, cyanotoxines et environnement », 12 rue Buffon, 75231 Paris Cedex 05, France Email : benkhelifa.leila@bbox.fr Thanks to Conclusions In Cerastoderma lens, no thalassic species are observed in the chott area whereas in Akarit, the occurrence of few marine species suggest a tidal influence. However, more Cerastoderma lens' samples from the chott regions need to be studied and dated for a definitive response. The purpose of this work is to update taxonomic identification of Diatom from Tunisia described in Ben Khelifa,L., 1989, for a better understanding of quaternary diatom occurrence and its implication. In particular, have quaternary Cerastoderma lens a marine or a continental origine? II. Modern Sites location with in particular, photos of the diverses biotopes in Wadi El Akarit The euryhalobous specie Terpsinoe musica from Akarit Cerastoderma lens I. Quaternary Sites location : II. Diatom assemblages, stenotypic species, autoecological results and paleoecological interpretations Akarit Cerastoderma lens Les Chotts Cerastoderma lens Modern Diatom Assemblages in Modern samples of TUNISIA Diatom distribution in Cerastoderma lens of Wadi El Akarit 1.limon, 2. silt, 3.sand,4.gravel&pebble,5.concretion,6.gastropod,7.cardium,8.peat MEB photo for major species occuring in both fossil and modern biotopes Hydrothermal sources Eusaline? Marine origin? ; in Akarit Cerastoderma lens The most frequent specie in holocene sediment Denticula elegans var. africana Hustedt is abondant in modern sample EH90 Metasaline Assemblage from modern (TN62: C=-(65600µ/cm, pH=9)and fossil samples of the Chott region. Rhopalodia musculus occur in very big size and the two Nitzschia do not correspond exactly to Nitzschia graciloides Hust. And (kütz.)Smith thus more taxonomic work is needed to make sure wether these species are euryhalobous or stenotypic metasaline. samples from the chott area with only two or three species dominant Eusaline Synedra crystallina? In the matrix, Surirella striatula (Turpin) and Chaetoceros kystes are abondant III. When Diatom allow a detailed paleorecontruction : in Akarit Cerastoderma lens, we can note an evolution from a lagunar environment to a marine one, in Villafranchien time Achnanthes brevipes? is very big in fossil sediment


L Ben Khelifa

& S Hamlaoui

DIATOM FROM

THANKS TO

1. Cyanobacteria (trichome) and microalgae were isolated using a tapered pipette and observed with a ZEISS LM (S.Hamlaoui).

In Majerda River we have observed Phormidium sp. and Planktothrix sp. (Cyanphyceae), (Cyanphyceae) , Vaucheria sp.

(Xanthophyceae), Ceratium hirundinella (O.F.Müller) Dujardin (Dinophyceae), Closterium aciculare T. West (Zygnematophyceae). Some species of Phormidium and Planktothrix might be toxic (Hamlaoui, perso.comm.). Toxicity tests must be conducted to affirm that the decrease in the stock of Alevins of Mugil Cephalus, Liza Ramada and Carp (Mili S. et al., 2016), in the aquaculture Basin of the Majerda River, is due to the proliferation of these cyanobacteria. Thurty three years ago, In Majerda River, Diatom assemblage was rich with Cyclotella sp. ocellata Pant, probably corresponding to Cyclotella choctawatcheeana Prasad, (in Carvalho et al., 1995), in all the major ten sites of the river. In 2019, it is less distributed and often replaced by Nitzschia acicularis (Kütz.) W.Smith) which suggest an eutrophic freshwater habitat cf. L. Bertalot et al., 2017. In particular, in the four sites of Kalaât El Andalous, it is absent where eutrophic taxa, in particular Thalassiosira aff. Weisslogii, Gyrosigma and Halamphora are abundant in several sites and mostly near the Kalaät El Andalous 'station de Pompage' and the bay of Tunis. 2. In Tunis Lake 36°49'03 ''N, 10°14'33''E, around 40 marine species are observed (Ben Khelifa, L.,1989) and more than ten species from the Majerda River are observed with in particular, Gyrosigma, Cocconeis placentula and Halamphora species. Subsurface sediment of two cores (in North lake), show a rapid diagenese of diatom frustules because of the high eutrophication, however other micro-organismes are observed. Some Desmidiaes type Pleurotaenium Nägeli (Hamlaoui, perso.comm.), suggest hot humid period in the Holocene recent whereas, one enigmatic specie observed may be nowdays near the edge of the Majerda River, suggest an aridification events and /or eustatic change whithin the quaternary section.

In 1984 and 1986, in all sites of the Majerda River, Cyclotella sp. ocellata Pant. was often abundant and in diverse size and spread in different part of the river but never rich the Tunis lagoon. Today, in 2019, this specie is less abundant, and seems more homogene in size and is observed frequently only in Sidi Salem site. In sites from Kalaât el Andalous, this taxa is absent.

Nowdays, in all the other sites of Kalaât El Andalous, near the bay, Eutrophication is important and Cyclotella sp ocellata is replaced by Thalassiosira abundant with Gyrosigma, Entomoeneis and Navicula. They are observed even in the Tunis lagoon. Nitzschia species are present but not the Nitzschia acicularis observed in Sidi Salem

Andalous sites near the bay and in Tunis lagoon

In quaternary wet land of Tunisia this enigmatic form is often observed and some holocene layers are really rich (Tunis Lagoon, sebkha Kelbia, Gabes Gulf, etc.., here in Kalaât El Andalous (Site1 Pont Mobile), in subaerian sample near the edge of the river, a modern analogue, for me, is obvious and so we have an indication of quaternary paleoenvironment when Diatom are absent , Liza Ramada and Carp (Mili S. et al., 2016) in the aquaculture Basin of the Majerda River. 2. To distinguish taxa linking with eutrophic polluted area by anthropic effect, and thus to contribute to water management of Majerda River which provides the drinking water for all the big cities of Tunisia; Tunis, Cap-Bon, Sahel et Sfax and the water for the principal agricultural area as Grombalia, Soliman, Cap-Bon, Mornag, Ras Jebel, Ousja and Kalaat El Andalous. 3. To contribute in the study of recent sedimentation in the Gulf of Tunis to prevent urbanisation effect and to understand the paleoenvironmental and paleoclimatical change during the quaternary alternance of glacial-interglacial input from the Majerda river. 4. A modern analogue for the Cyclotella spp. earlier referred as Cyclotella caspia in fossil african assemblages ' yet it is absent from the modern flora of this region (in Carvallo, L.R. et al., 1995)', is observed. A MEB identification on the frequent small specimens of Cyclotella sp.ocellata Pant. would give a taxonomic answer. 5. Two rare Nitzschia specie, one marine and one continental are of interest, if similitude in between, we might have an information about eustatic event in that area which would have affected the whole region of the SW Mediterranean Sea.

METHODOLOGY AND PRELIMINARY RESULTS

  Diatom, Cyanophyceae and other micro flora and fauna to contribute to explain the diminution of the stock of Alevins of Mugil Cephalus