Bert Cappelle

Ilse Depraetere

Mégane Lesuisse

The necessity modals have to, must, need to and should: using n-grams to help identify common and distinct semantic and pragmatic aspects 1

Keywords: necessity modals, n-grams, mutual information, lexically restricted saturation, shortcircuiting, illocutionary force, hierarchical clustering analysis, intersubjectivity

When an ambiguous lexical item appears within a familiar string of words, it can instantly receive an appropriate interpretation from this context, thus being saturated by it. Such a context may also short-circuit illocutionary and other pragmatic aspects of interpretation. We here extract from the British National Corpus over 500 internally highly collocating and high-frequency lexical n-grams up to 5 words containing have to, must, need to and/or should. These contexts-as-constructions go some way toward allowing us to group these four necessity modals into clusters with similar semantic and pragmatic properties and to determine which of them is semantico-pragmatically most unlike the others. It appears that have to and need to cluster most closely together thanks to their shared environments (e.g., you may have/need to…, expressing contingent, mitigated necessity), while should has the largest share of unique n-grams (e.g., rhetorical Why shouldn't I…?, used as a defiant self-exhortation).

Introduction: modal constructions, lexically restricted saturation and short-circuited meaning

Modal verbs, or modals for short, are notoriously ambiguous (see, among many others, [START_REF] Ruppenhofer | Yes we can!? Annotating English modal verbs[END_REF]Marasovic et al. 2016) and can be used in utterances with often specific pragmatic effects (see, for instance, [START_REF] Leech | The Pragmatics of Politeness[END_REF]. This paper aims to understand the role and nature of 'context' in interpreting a modal verb, here specifically one of the necessity modals have to, must, need to and should. In Cappelle and Depraetere (2016a), it was argued that a modal and the immediate context in which it occurs -one or more words to the left and/or to the right of it -may often be considered a construction. 'Construction' was used there in the sense, as is customary in Construction Grammar, of a form-function unit that is likely to be stored in a speaker's mental repository of words, idioms, recurrent sequences and semi-fixed patterns (cf. Goldberg 2006). This is also the sense of 'construction' adopted here. Our focus is on constructions towards the lexical pole of what is typically referred to as the syntax-lexicon continuum. We look at lexically specified strings, that is, contiguous sequences of actual words (not syntactic categories), one of which is a modal. However, most of these sequences do not constitute complete utterances. For instance, it should be noted that (one of the lexical constructions whose mental storage we posit) still needs to be followed by a clause to be used legitimately. In that sense, the vast majority of the constructions we deal with here are just lexical 'chunks' that are part of larger units.

We believe that a close investigation of these chunks may shed some light on how each of the four necessity modals can be characterized semantically as well as pragmatically. We do not pretend, though, that this focus on collocational preferences is the one and only way to reach such characterizations of the modals concerned. We do not claim either that speakers and hearers have access only to these very specific lexical chunks and not also to more schematic constructions with the necessity modals. A more general construction with must, for instance, probably specifies that, disregarding the concrete words this modal is used with, the necessity it expresses is speaker-driven, while a more general construction with have to specifies that the necessity is circumstantial, one with should that the necessity is of a relatively weak degree, and so on. What we do claim, however, is that, alongside these general constructions, many of the lexically more specific sequences definitely have their cognitive reality. It is even to be expected that in some cases, the function of a lexically specific string overrides the meaning inherited by the more general construction containing that modal. For instance, it must be noted that… is hardly any higher in strength or more obviously speaker-directed than it should be noted that….

Although the scarce attention to pragmatics in constructionist accounts of modals (for exceptions, see e.g. see e.g. [START_REF] Stefanowitsch | A construction-based approach to indirect speech acts[END_REF]Boogaert 2009;De Haan 2014;Boogaert and Fortuin 2016) might suggest that the integration of pragmatics in modal constructions is problematic, it is certainly possible to include pragmatic information in a modal construction's representation. After all, constructions can come with pragmatic specifications (for surveys, see [START_REF] Kay | Pragmatic aspect of grammatical constructions[END_REF][START_REF] Nikiforidou | Constructional analysis[END_REF]; the introduction to the papers in [START_REF] Bergs | Contexts and Constructions[END_REF][START_REF] Lee-Goldman | Context in Constructions[END_REF][START_REF] Cappelle | What's pragmatics doing outside constructions?[END_REF]; introduction to this issue). What is typical of the pragmatic value of a modal construction is that it can be 'shortcircuited', to use [START_REF] Morgan | Two types of convention in indirect speech acts[END_REF] imagery of how pragmatic aspects of our understanding of an utterance can be conventionally associated with specific expressions. For instance, Can you please… ? is immediately understood as a request, without the hearer having to compute this interpretation on the basis of a more basic ability meaning that can might have (see, e.g., [START_REF] Searle | Indirect Speech Acts[END_REF][START_REF] Aijmer | Conversational routines in English: convention and creativity[END_REF][START_REF] Leech | The Pragmatics of Politeness[END_REF][START_REF] Ruytenbeek | The comprehension of indirect requests: Previous work and future directions[END_REF]). Short-circuiting is in some respects similar to the process of conventionalisation that affects frequent invited inferences (cf. [START_REF] Traugott | The semantics-pragmatics of grammaticalization revisited[END_REF]. However, even if a short-circuited implicature is one that is conventionally associated with the form of a construction, short-circuited pragmatic aspects of meaning do not become part of a construction's semantics, as they can still (albeit with effort or facetiousness) be cancelled in context.

What is more, Cappelle and Depraetere (2016a) argue that modal verb constructions can also short-circuit 'non-pragmatic' aspects of interpretation. For instance, the modal in the common expression if I could just say... is standardly understood as expressing permission, which is why it sounds funny when this string of words ends up being used to express another kind of root possibility, such as ability (e.g. Homer Simpson: "If I could just say a few words… I'd be a better public speaker").2 So, while there is a semantic core of 'possibility' meaning (referred to as the 'context-independent semantic layer' in Depraetere 2014), in context, this meaning is made more specific and the hearer chooses between one of several limited options (a process which constitutes 'the context-dependent semantic layer' in [START_REF] Depraetere | Modal meaning and lexically-regulated saturation[END_REF]. That is, assigning to a modal a particular meaning such as 'have permission to' rather than 'have the ability to', 'have the opportunity to', etc. happens on the basis of contextual cues. Nevertheless, we adhere to the view that the output of disambiguation is a semantic level of interpretation [START_REF] Recanati | Literal Meaning[END_REF][START_REF] Depraetere | Some observations on the meaning of modals[END_REF]. Besides, there is little or no pragmatic reasoning involved when the disambiguating context is a well-entrenched sequence of words. When modals feature in a construction (a stored form-function unit), as in If I could just say…, the selection of the right meaning is 'short-circuited' by the familiar context around the modal.

Depraetere and Salkie (2017) consider this disambiguation to be a case of saturation [START_REF] Carston | The explicit/implicit distinction in pragmatics and the limits of explicit communication[END_REF]: 49, Recanati 2004: 7, Recanati 2010: 70), a pragmatic process which is obligatory: it is necessary in order to complete an incomplete proposition. As the search space for a contextually appropriate interpretation is constrained, Depraetere and Salkie (2017: 26) use the term lexically restricted saturation. In order to determine the propositional content of an utterance, one of several (unrelated or otherwise distinct) meanings has to be selected and the context gives clues as to what meaning is appropriate. For instance, There's a bat in the cupboard (cf. Clark 1991: 10) will be understood differently depending on whether it is reply to Why is she screaming? or to Let's go out and play a game. Specific contexts trigger interpretations but while there is a choice to be made, the choice for one rather than another meaning can be fairly immediate. To illustrate with a familiar textbook example, while selecting the meaning of 'financial institution' rather than 'shore of a river' for bank is a context-dependent process, arriving at the right semantic value for bank can hardly be called a pragmatic affair if we find this noun followed by the noun crisis. This is not to say that bank crisis can never be given another interpretation. For instance, a river's banks on the brink of collapsing due to erosion may be referred to by a journalist as a bank crisis. Yet, this will then have the flavour of a (poor) pun.

Just like bank crisis is stored as a lexical unit in which bank already comes with an appropriately disambiguated value, If I could just say… is a stored sequence that immediately helps the hearer to determine which semantic value of could is intended among a range of possible candidates. 3 An expression with a disambiguated modal meaning can still convey extra pragmatic information, that is, information at what [START_REF] Levinson | Presumptive Meanings[END_REF] and [START_REF] Recanati | Pragmatic enrichment[END_REF] call the post-semantic level (but this term's suggestion that the pragmatic information is added at a later stage than the disambiguation is not something we would subscribe to). In the case of If I could just say…, what is meant is more than just 'If I had permission to say…'; this sequence of words is a standard way of asking for permission (i.e., the expression functions as a request) and of expressing that the speaker assumes that this permission is in fact granted (i.e., the expression also functions as a floor-taker) (Cheng 2007: 206;Cappelle and Depraetere 2016a: 27). In [START_REF] Depraetere | Modal meaning and lexically-regulated saturation[END_REF] multi-layered approach to modal meaning, this layer is called the 'context-dependent, pragmatic layer'.

If I could just say… is just one of very many recurrent sequences containing a modal. Cappelle and Depraetere (2016b) present a methodology to identify such 'lexical' constructions with modals, based on the mutual information of the modal and words occurring to the left and right of it, as well as of the words to the left and right of these context words, and so on. This methodology is a simple way of extracting from a corpus the lexical n-grams (i.e., continuous strings of 2, 3, or any number n of words) around a word such that each word entertains a strong collocational connection with its neighbouring words. Recurrent multi-word sequences have been referred to as 'lexical bundles' in the corpus-linguistic literature (cf. [START_REF] Biber | Lexical bundles in conversation and academic prose[END_REF], a term that we find potentially useful, even though we would like to stress that the n-grams retrieved with the methodology referred to above are not just high-frequency ones but especially also ones that are high in internal collocational strength, in terms of mutual information. 4Must served as a test case for Cappelle and Depraetere (2016b). The present paper applies the methodology described there to a larger range of necessity modals: have to, must, need to and should. We here aim to use this methodology as a tool to capture semantic and any pragmatic aspects that these modal verbs share or fail to share, by comparing the highly collocating lexical environments in which they occur. This study complements existing research into the semantics and pragmatics of modal verbs in at least two ways. First, as far as we know, a contrastive analysis of the lexical sequences with these modal verbs is new, at least for sequences of a size larger than two words. Our general approach has points in common with [START_REF] Hilpert | Change in modal meanings: Another look at the shifting collocates of may, Constructions and Frames[END_REF] and [START_REF] Flach | From big data to small data and back again: Using tokenbased semantic vector spaces for corpus-linguistic analyses[END_REF], but these studies of modal meanings focus on 2-grams. In the approach that is adopted here, there is potentially no limit on the size of the lexical sequences retrieved from the corpus. Secondly, previous studies paying attention to specific syntactic, semantic and pragmatic features only considered necessity modals in pairs (should and must, e.g. [START_REF] Rivière | Is should a weaker must?[END_REF][START_REF] Groefsema | Can, May, Must and Should: A Relevance Theoretic Account[END_REF]Keckses & Kirner-Ludwig (2017); have to and must, e.g. Larreya 1982; Depraetere and Verhulst 2008;should and ought to, e.g. Cappelle and De Sutter 2010). Our study fills this gap by taking into consideration four necessity modals at the same time.

In keeping with one of (Cognitive) Construction Grammar's basic tenets [START_REF] Goldberg | Constructions: A new theoretical approach to language[END_REF], we allow constructions to be not just highly schematic templates but also specific lexical strings that consist of words whose co-occurrence can be observed significantly more often than could be expected, based on their individual frequencies. Of course, we would not go as far as to say that, for instance, it should be noted that is really a full construction. As this n-gram is not a complete syntactic constituent and as speakers know it has to be followed by a clause, what speakers have stored as a unit in the mind is really [it should be noted that + clause]. Our methodology therefore does not yield lexical constructions necessarily -it typically allows us to retrieve lexical sequences that are part of constructions containing both fixed lexical material and at least one open slot yet to be filled in (cf. [START_REF] Cappelle | Towards and n-grammar of English[END_REF]. That constructions can be hybrid, combining lexical items and syntactic categories, is a common assumption in Construction Grammar and related frameworks (cf., e.g., [START_REF] Jackendoff | Twistin' the night away[END_REF]). Accordingly, multiple levels of abstraction should be considered when we chart speakers' knowledge of constructions (cf., e.g., [START_REF] Trousdale | A constructional approach to lexicalization processes in the history of English: Evidence from Possessive Constructions[END_REF][START_REF] Gyselinck | The role of expressivity and productivity in (re)shaping the constructional network[END_REF]. In view of this, we do not claim either that the modal constructions we here posit, by syntactically completing, where needed, the retrieved lexical sequences around a modal verb, are the only constructions with these modal verbs. Some modal verb constructions stored in the mind of the speaker will be considerably more abstract and general than the 'syntactically extended' patterns found here. They may, for instance, consist of just a modal verb preceded by a syntactic position to be filled by a subject NP and a syntactic position to be filled by a VP. Some other stored modal verb constructions may be lexically even more specific than the ones our methodology allowed us to retrieve. An example may be You should try this, which is possibly a stored exemplar of you should try {NP / to VP}.

The remainder of our paper is organized as follows. In Section 2, we describe the methodology used to identify (partial) modal constructions in the BNC (British National Corpus). In Section 3, we present the corpus findings and analyse the lexical n-grams, viewed as (partial) constructions, in an attempt to shed light on shared and different semantic and pragmatic aspects of the necessity modals studied. We provide a conclusion in Section 4.

Methodology

2.1.

Extracting highly collocating lexical n-grams The methodology adopted in Cappelle and Depraetere (2016b) is as follows. In Mark Davies's BYU-BNC interface [START_REF] Davies | Based on the British National Corpus from[END_REF] two-word strings (bigrams) with must were extracted that occur at least 50 times in the corpus and that have a mutual information (MI) score of at least 3. This latter cut-off point has been proposed by some researchers working on collocations (Hunston 2002: 71;Xiao and McEnery 2006: 105). The resulting strings were then used as search items in the next round of n-gram retrieval. The procedure remained the same: we again set 50 as the minimal frequency and 3 as the minimal MI score. The procedure was repeated until no more strings were found that occur at least 50 times in the corpus with an MI of 3. The recursive nature of the procedure would in principle allow the retrieval of n-grams to be carried out more or less automatically. For the present study, however, we did not write a script but used Davies's interface. N-grams were extracted, in the way just described, for must, should and the inflected forms and base form of have to and need to.

2.2.

Identifying shared and unique n-grams for the necessity modals In order to explore to what extent the four modals are found in the same or distinct n-grams, we created four sets (i.e., lists) of retrieved n-grams, one for each modal (with multiple inflectional forms of have to and need to collapsed into single lemmas5 ). In each set of n-grams, we replaced the modal verb in each n-gram by the generic label 'MOD'. With the help of an online tool for creating Venn diagrams, we then determined the size and members (if any) of the intersection(s) of these four sets of n-grams, as well as the n-grams that are unique to a particular set. 6We also performed hierarchical cluster analysis to find out which modals are most alike with respect to their lexical environments, as captured by the n-grams they share. For this, we used the function hclust in the R package pvclust, by which a dendrogram can be plotted. It is assumed here that similarity in collocational behaviour indicates similarity in function [START_REF] Harris | Distributional structure[END_REF][START_REF] Firth | A synopsis of linguistic theory 1930-1955[END_REF][START_REF] Mcdonald | Testing the distributional hypothesis: The influence of context on judgements of semantic similarity[END_REF][START_REF] Sahlgren | The Distributional Hypothesis[END_REF]).7

Results

Quantitative results

Out of the four necessity modals studied, should is the one to be found in the highest number of different n-grams (cf. Figure 1 Need to is the necessity modal with the lowest number of n-grams retrieved: 36 bigrams, 32 3-grams, 7 4-grams and no 5-grams. Except for need to, the number of extracted 3-grams containing a necessity modal is higher than the number of extracted bigrams with a necessity modal. This may be surprising, as one would expect there to be fewer collocational sequences as the number of words in the string increases -an expectation in fact supported by the observation that there are no sequences longer than 5 words to be obtained with our extraction method. The main reason why the 3-grams nonetheless outnumber the 2-grams is that for a single bigram such as should be, a large number of 3grams could be retrieved with a strongly collocating past participle or adjective (should be added, should be adopted, should be capable, should be careful, …).

Once we replace the actual modal by a generic label ('MOD'), we find that there are 517 different n-grams, some of which occur with multiple modals (see Appendix 1). Of these 517 n-grams, however, only 20 (3.9%) are shared by all four necessity modals (cf. Figure 2). 357 n-grams (69%) are unique to one particular modal. That more than half of the n-grams are specific to one modal or another suggests that the necessity modals have their own lexical profiles. This specificity is most pronounced for should, for which 171 out of the 286 n-grams (or 60%) are not shared by any other modal, and least so for need to, for which only 20 out of 75 n-grams (or 26.6%) are unique to this modal. We can make the following observations about must, have to and need to:

(i) must and have to share 9% (19 out of 211) and 11% (19 out of 170) of their n-grams, respectively, with one another; (ii) have to and need to share 11.7% (20 out of 170) and 27% (20 out of 75) of their ngrams, respectively, with one another; together with the previous observation, this suggests that have to has more n-grams in common with need to than with must; (iii) must shares only 2% (4 out of 211) of its n-grams with need to, and need to 5.3% (4 out of 75) of its n-grams with must. If we bring should into the picture, we find the following facts:

(iv) must shares as many as 30.8% (65 out of 211) of its n-grams with should and should 22.7% (65 out of 286) of its n-grams with must;

(v) have to shares 2.4% (4 out of 170) of its n-grams with should and should only 1.4% (4 out of 286) of its n-grams with have to; (vi) need to shares 4% (3 out of 75) of its n-grams with should and should only 1% (3 out of 286) of its n-grams with need to.

Table 1 summarizes these findings by showing for each necessity modal whether its unique and shared n-grams represent a surplus (in green) or shortage (in red), when observed values are compared to statistically expected values.

Table 1. Surplus and shortage (in percentage) of observed over expected n-grams

The mutual relatedness of the four modals is best brought out by the dendrogram shown in Figure 3, which is the output of our hierarchical cluster analysis. This statistical analysis measures the closeness between the modals in terms of their occurrence in n-grams. The degree of similarity is represented on the Y-axis. The dendrogram shows that have to is more closely related to need to than to must. Should is the least connected to the other modals, must being a somewhat closer 'relative' of should than of have to and need to. The figures in red and green correspond to p-values. The ones in red show the AU (approximately unbiased) p-value, while the ones in green show the BP ("bootstrap probability") p-value. The former of the two is more accurate. The p-values indicate how much support there is for distinct clusters at each split. Thus, that must forms a cluster of its own (leaving should behind on its own as well) is actually less strongly supported by the data than that have to and need to form a cluster together. Indeed, the cluster of need to and have to has maximum AU/BP values, i.e., 100%, which means that this cluster reliably follows from the data.

Qualitative results

In this section we zoom in on a few qualitative generalizations that emerge from the sets of n-gramsas-constructions. We especially concentrate on pragmatic effects relating to illocutionary force.

Why should(n't)…: rhetorical questions

Should stands out among the other necessity modals in that it often occurs with why: there are 12 (partially overlapping) n-grams containing both why and should(n't). 9 These function as rhetorical questions denying the necessity to do something (as in (1a)) or not to do something (as in (1b)); as the negation here is situated at a higher level (negating the justification of the proposition), it is compatible with both should and shouldn't. Pragmatically, sequences containing Why shouldn't… often function as exhortations to do the opposite of what is expressed in the underlying proposition:

(1) a.

' Why should(n't)… questions may also be used in cases where should is epistemic. Here, too, the pragmatic function is that of denying that the underlying proposition is justified:

(2) She thought, looking dubious. 'It doesn't sound likely. Why should there be a conspiracy? (…)'

The illocutionary force is then something like 'Let's not assume/conclude too easily that…'.

Should with a passive infinitive: low intersubjectivity

Another striking finding is that should is particularly prone to combining with passive infinitives, as in this example:

(3) Any visual aid should be carefully selected and planned to add clarity to the presentation.

In 47 out of the 171 n-grams that should does not share with any other modals (27%), we find a passive infinitive (compared to 14 out of 78 unique n-grams with must (14%) and no passive infinitives in the unique n-grams with have to and need to). This suggests that should, more than any other of the necessity modals, occurs in contexts in which intersubjectivity [START_REF] Verhagen | Constructions of Intersubjectivity[END_REF]Davidse et al. 2010;[START_REF] Traugott | Inter)subjectivity and (inter)subjectification: A reassessment[END_REF][START_REF] Brems | English Text Construction, Special Issue[END_REF]) is low, in the sense that the utterance does not explicitly represent the addressee as the agent of the action. (Introducing the addressee as the agent in a by-PP would be very odd: ??Any visual aid should be carefully selected… by you). Given that must expresses strong necessity, we shouldn't be surprised to find that it collocates well with the adverb surely, which is harmonious with strong epistemic necessity:

(4) She must surely be the most beautiful in the world.

This adverb only occurs with must in the complete set of n-grams and suggests that must can be used for intersubjective modality in the sense of Nuyts (2012: 58), that is, the modal evaluation is "presented as being shared between the assessor and a wider group of people, possibly (but not necessarily) including the hearer". Should, which is generally seen as expressing a mitigated, weakened form of necessity, is nevertheless also used with with strong adverbs, like always and never. Never only occurs in combination with should in the complete set of n-grams.

(5) a.

You should always be very careful when dealing with persuasive equipment sales people. b.

Teaching strategies and classroom organization should never be viewed as ends in themselves. c.

The simple fact of the matter is that we should never have been there in the first place.

These combinations can be seen as cases where the relatively weak necessity expressed by should receives pragmatic (or at least contextual) strengthening.

On the other hand, should combines with think in hedging contexts involving negative raising, as in the following example, where it cannot be replaced by must (I don't think we should… has 62 occurrences in the BNC, as opposed to none for I don't think we must…):

(6)
It was kind of Anne to think of it and not condemn us, but -well, we've managed so far being discreet about it, and I don't think we should change things now.

This ties in with should being much more suitable than must for hedged expressions of root necessity. Note also that the mitigating adverbs maybe and perhaps are only found with should in our set of n-grams (maybe we should, perhaps you should, perhaps we should).

4.2.4. The modals with I/you/we: hedged performatives, positive politeness and speaker inclusion When it comes to the Subjects that each of the modals collocate with, the following patterns can be observed. First, I typically occurs in combination with performative verbs in so-called hedged performatives [START_REF] Fraser | Hedged Performatives[END_REF]). The other n-grams with the first person singular pronoun similarly occur in hedging contexts in combination with the verb think. Here, we find the modal should (think we should, I think we should, I don't think we should, I think they should, I think you should), as we mentioned in Section 3.2.2, but also the verb need (think we need to, I think we need to). Second, you MOD is at the intersection of the four sets of modal verb n-grams; in other words, the four modals are commonly used to issue a directive to the addressee. However, the communicative effect achieved by these constructions is quite different. Two verbs that collocate with must in particular are typically or often used as part of positive politeness markers (cf. [START_REF] Brown | Politeness: Some Universals in Language Usage[END_REF] aimed at bonding: (8) a.

You must come and have supper sometime. b.

But you must tell me all about it, I can't wait to hear.

In these cases, must, which is generally considered as the strongest modal, giving rise to very forceful orders, is not actually used with its unmarked, 'psychologically prototypical' [START_REF] Coates | The semantics of the modal auxiliaries[END_REF] force: it serves as a sign of comradery, whereby the speaker shows interest for the hearer. Third, we MOD is likewise an n-gram that is common to the four modals. Here, the selfexhorting character of the construction is likely to be part of a strategy that mitigates the effect of the directive by including the speaker into the set of addressees to whom the order is addressed. There is a clear semantico-pragmatic difference between you may have to wait and perhaps you should wait: the former expresses a weak degree of epistemic possibility that it will be necessary for you to wait due to external circumstances, while the latter is a way of suggesting politely that, as far as the speaker is concerned, it is advisable for the addressee to wait.

Conclusion

In this paper, we have laid out a particular approach to the pragmatic meaning of modal constructions: we have argued that there is a pragmatic layer that complements the semantic layer in the functional component of the constructions. Lexically restricted saturation results in the selection of one from a restricted set of meanings potentially communicated by the modal and the modal construction 'shortcircuits' the choice. Likewise, lexically specific modal constructions can give rise to short-circuited pragmatic effects.

We have also explained and applied a specific methodology to identify such lexically specific constructions (or at least lexically specific parts of constructions), in an attempt to pin down the semantico-pragmatic profiles of four necessity verbs. As argued in Cappelle and Depraetere (2016b) a constructional analysis of the type presented here is very useful from the perspective of second language acquisition in that it tells us what collocational patterns should be insisted upon. Section 2 shows that it is possible to make some precious generalisations about typical usage contexts of each of the modals on the basis of the range of lexical constructions of each of the modals. While this methodology has revealed interesting insights about lexical bundles and the relative kinship among the four modals (made explicit in the Venn diagram and in the dendrogram presented in Section 3), it does not in itself provide a comprehensive picture of the factors that lead the speaker to use say, have to, rather than need to, or should rather than need to. It follows from this that an additional feature analysis is needed of syntactic, semantic and pragmatic features that are significant if we want to find out what unites and sets apart the modal verbs.

It would also be worthwhile to use the n-grams retrieved here as predictors in a multinomial regression model to see how well they classify (i.e., predict the actual use in context of) the four modals in a different (but similar) corpus. In addition, we should compare or combine such n-grams as predictors with the predictive power provided by a model that implements a so-called Bag-of-Words approach (where one simply checks which words occur how often in a text, independently of these words' positions in the text -word order plays no role). In the Natural Language Processing community, such a Bag-of-Words model seems to be treated as a standard baseline for classification performance. Obviously, if we want to validate the insights from the present paper, a classifier that (also) incorporates lexical n-grams should perform better than a classifier that is (only) based on a Bagof-Words approach.

The present study has not presented a methodology to retrieve all the constructions that contain the modals concerned. There are, obviously also some more general modal verb constructions, which allow speakers to go beyond the set, large though it may be, of stored lexical sequences. For instance, Do you need to speak French to work at Disneyland Paris? is an utterance that is not a stored lexical sequence and thus can only have been formed on the basis of a more general need to VP construction, in combination with other smaller or larger constructions of a lexical or more syntactic nature (you, speak French, Disneyland Paris, work at NP, in order to VP, the inversion construction, etc.). If we want to adequately represent speakers' knowledge of how each of the four necessity modals discussed here are used, we should do more than just look at the lexical ngrams they are found in. One possible way of complementing our approach is to retrieve constructions towards the more syntactic pole of the syntax-lexicon continuum, for instance by finding out the preferred part-of-speech n-grams for each of the four necessity modals (cf. [START_REF] Cappelle | Towards and n-grammar of English[END_REF]. However, it will then still be necessary to perform a careful analysis of actual examples of these modals in context to get a complete picture of their semantic and pragmatic properties. Further research is therefore needed to meet this challenge.

Figure 1 .

 1 Figure 1. Number of high-collocation n-grams containing each of the four necessity modals should, must, have to and need to, extracted from the British National Corpus

Figure 2 .

 2 Figure 2. Venn diagram showing the number of n-grams that are common to two or more modals or that are unique to each modal.

Figure 3 .

 3 Figure 3. Cluster dendrogram with AU/BP values (%)

  / must} {say / tell (you)} I must admit, I must ask, must admit I I have to confess I should {imagine / think / think so} 10

  I've had enough of you!' she cried vehemently, not waiting for his response. 'Why should I care what you feel when all the time you behave like a monster?' she asked

	rhetorically. (= 'I see no reason why I should care…'; hence: 'I won't (allow myself to)
	care…')
	b.

I felt so cross with myself, so stupid for wearing a short skirt. But on the other hand, I was furious; I thought: this is ridiculous, why shouldn't I go out wearing what I want without getting hassle? (= 'I see no reason why I shouldn't go out…'; hence: 'I will go out wearing…')

  3.2.5. Must and should with a perfect infinitive: epistemic necessity vs. reproach Must and should share many n-grams with a perfect infinitive. What these shared environments by themselves do not reveal, however, is that the meaning involved is very different. Must in this case usually expresses epistemic necessity, while should in these environments is used to make a reproach:

	(9)	a.	I'm sure Suzannah must have known him.
		b.	But I should have known better than to accept anyone recommended by you.
			contingency,
	not as a certainty:
	(10)	a.	Your financial situation can improve, but you may have to wait until next year before
			you are entirely out of the woods.
		b.	If you have serious drain or waste blockage problems, you may need to use specialist
			equipment or to call in a specialist drain clearing firm.

3.2.6. May have to and may need to: contingent necessity A syntactic property that distinguishes the semi-auxiliaries have to and need to on the one hand from must and should on the other is their combinability with auxiliaries (don't, doesn't, didn

't, will, 'll, shall, won't, would and may)

. Of particular interest is may, which in combination with you and have/need to helps to mitigate the addressee-directed necessity by presenting it as a

This paper was funded through a grant from the Agence Nationale de la Recherche (ANR-16-CE93-0009 REM).

If I could just say a few words usually also exemplifies insubordination (cf.[START_REF] Verstraete | Running in the family. Patterns of Complement Insubordination in Germanic[END_REF][START_REF] D'hertefelt | Insubordination in Germanic. A Typology of Complement and Conditional Constructions[END_REF]. Treating this sequence as an ordinary subordinate clause in a main clause, as does Homer Simpson in this example, is therefore already odd in itself (although one does very occasionally find sentences such as If I could say a few words, that'd be awesome).

We are aware that the use of the term 'disambiguated' is somewhat unfortunate here, as our view is that modals illustrate polysemy (related meanings) rather than ambiguity (unrelated meanings). For want of a less ambiguous verb, we will continue to use it, bearing in mind that in the case of modals, the choice is among one of several polysemes(Cruse 2011: 115).

As pointed out by a reviewer, our perspective aligns in a general way with[START_REF] Hunston | Pattern Grammar: A Corpus-driven Approach to the Lexical Grammar of English[END_REF] pattern grammar approach. Given the emphasis in our paper on phraseological units, we acknowledge that this may turn out to be the case, although we have at this stage not made a systematic comparison between our methodology and theoretical stances and Hunston and Francis's.

This lemmatization was done for ease of analysis but is also justified in that the n-grams we retrieved with the forms has and had and for needs and needed are also to be found in the list of n-grams with the forms have and need, respectively. It appeared, in other words, that the present tense third person singular use and the past tense use of the modals have to and need to do not exhibit lexical preferences that are specific to these inflectional forms only.

http://bioinformatics.psb.ugent.be/beg/tools/venn-diagrams, last accessed 6 March 2018.

A reviewer rightly comments that distributional similarity can also point to a paradigmatic opposition. For example, antonyms (e.g. increase and decrease) are known to have similar collocational profiles. However, as argued by[START_REF] Janda | Linguistic profiles and construction grammar[END_REF], "[a]ntonyms are synonyms that differ in just ONE value, they are the next closest thing to synonyms." Moreover, in the context of our study, there is little to no risk that any of the four necessity modals (have to, must, need to and should) is a paradigmatic opposite of any of the others.

Among the unique n-grams with should, there are also a small number of sequences in which should is not, in fact, a necessity modal but an alternative of would (e.g. I should imagine, I should think so, I should be grateful if). We have not discarded these few occurrences.

In fact, these cases do not, strictly speaking, involve should as a necessity modal but as an alternative to would.

Appendix 1. Overview of n-grams extracted from the BNC 1. Intersection have to -must -need to -should: 20 n-grams MOD be able, MOD be considered, MOD be done, MOD be made, MOD be taken, MOD ask, MOD be, MOD consider, MOD go, MOD make, MOD take, MOD write, MOD learn, you MOD go, you MOD make, you MOD, we MOD look, we MOD, we MOD make, we MOD get, 2. Intersection have to -must -should: 17 n-grams MOD accept, MOD bear, MOD be careful, MOD be carried, MOD be seen, MOD be kept, MOD be paid, MOD carry, MOD choose, MOD decide, MOD give, MOD leave, MOD pay, MOD prove, MOD stay, MOD stop, we MOD go 3. Intersection must -need to -should: 6 n-grams MOD be aware, MOD be given, we MOD consider, MOD develop, MOD ensure, MOD ensure that 4. also, MOD also be, MOD always, MOD always be, MOD apply, MOD avoid, MOD be added, MOD be addressed, MOD be based, MOD be allowed, MOD be borne, MOD be borne in, MOD be borne in mind, MOD be capable, MOD be established, MOD be exercised, MOD be included, MOD be interpreted, MOD be maintained, MOD be noted, MOD be obtained, MOD be placed, MOD be provided, MOD be recognized, MOD be regarded, MOD be remembered, MOD be sent, MOD be stressed, MOD be prepared, MOD be treated, MOD begin, MOD contain, MOD continue, MOD continue to, MOD expect, MOD follow, MOD have, MOD have been, MOD have done, MOD have gone, MOD have known, MOD have seen, MOD have taken, MOD have thought, MOD include, MOD involve, MOD provide, MOD recognize, MOD remain, MOD seek, MOD take place, MOD therefore, MOD try, MOD try to, applicants MOD, candidates MOD, care MOD be, care MOD be taken, user MOD, they MOD, we MOD also, we MOD take, we MOD try 9. Intersection must -need to: 4 n-grams you MOD know, MOD understand, MOD remember, MOD look 10. Intersection need to -should: 3 n-grams think we MOD, I think we MOD, MOD check 11. should: 171 n-grams attention MOD be, authorities MOD, client MOD, consideration MOD, consideration MOD be, consideration MOD be given, courts MOD, don't think we MOD, everyone MOD, gentleman MOD, hon gentleman MOD, how they MOD, I don't think we MOD, I MOD be grateful if, I MOD imagine, I MOD think, I MOD think so, I think they MOD, I think you MOD, it MOD be noted that, local authorities MOD, managers MOD, maybe we MOD, MOD adopt, MOD advise, MOD aim, MOD aim to, MOD attend, MOD be adopted, MOD be applied, MOD be assessed, MOD be available, MOD be avoided, MOD be banned, MOD be brought, MOD be carefully, MOD be checked, MOD be completed, MOD be consulted, MOD be dealt, MOD be directed, MOD be discussed, MOD be doing, MOD be drawn, MOD be emphasised, MOD be encouraged, MOD be entered, MOD be entitled, MOD be examined, MOD be excluded, MOD be extended, MOD be fitted, MOD be granted, MOD be grateful, MOD be grateful if, MOD be informed, MOD be introduced, MOD be judged, MOD be noted that, MOD be permitted, MOD be pointed, MOD be ready, MOD be reduced, MOD be referred, MOD be removed, MOD be replaced, MOD be restricted, MOD be retained, MOD be returned, MOD be sought, MOD be spent, MOD be sufficient, MOD be taught, MOD be undertaken, MOD be used, MOD be viewed, MOD bear in, MOD bear in mind, MOD concentrate, MOD concentrate on, MOD consult, MOD contact, MOD cover, MOD die, MOD discuss, MOD draw, MOD enable, MOD encourage, MOD enjoy, MOD enter, MOD focus, MOD happen, MOD have known better, MOD have told, MOD have won, MOD ideally, MOD imagine, MOD inform, MOD n't, MOD never, MOD never be, MOD never have, MOD never have been, MOD normally, MOD not, MOD not be, MOD not expect, MOD note, MOD note that, MOD occur, MOD pay for, MOD proceed, MOD receive, MOD reduce, MOD refer, MOD reflect, MOD seek to, MOD serve, MOD specify, MOD stick, MOD therefore be, MOD think, MOD treat, MOD write to, -n't think we MOD, no reason why you MOD, nor MOD, patient MOD, perhaps we MOD, perhaps you MOD, procedures MOD, pupils MOD, pupils MOD be, purchaser MOD, reader MOD, readers MOD, reason why they MOD, reason why you MOD, reference MOD, reference MOD be, so why MOD, teachers MOD, tenant MOD, that they MOD, that they MOD be, the drafter MOD, think they MOD, think we MOD be, think you MOD, we MOD be, we MOD be able, we MOD be able to, we MOD expect, we MOD have, we MOD not, whether they MOD, whether we MOD, why MOD, why MOD I, why MOD n't, why MOD n't I, why MOD we, why they MOD, why we MOD, why you MOD, you MOD always, you MOD ask, you MOD n't, you MOD n't have, you MOD never, you MOD try 12. Must: 78 n-grams care MOD, I MOD admit, I MOD ask, I MOD go, if you MOD know, it MOD be admitted, it MOD be remembered, it MOD be remembered that, it MOD be stressed, it MOD be stressed that, MOD address, MOD admit I, MOD also have, MOD assume, MOD be accepted, MOD be accompanied, MOD be accompanied by, MOD be admitted, MOD be based on, MOD be capable of, MOD be joking, MOD be mad, MOD be prepared to, MOD be regarded as, MOD be remembered that, MOD be satisfied, MOD be seen as, MOD be stressed that, MOD be understood, MOD come, MOD comply, MOD comply with, MOD decide whether, MOD depend, MOD exist, MOD have felt, MOD have heard, MOD have looked, MOD have seemed, MOD inevitably, MOD learn to, MOD lie, MOD make a, MOD meet, MOD necessarily, MOD realize, MOD satisfy, MOD speak, MOD surely, MOD surely be, MOD surely have, MOD take into, plaintiff MOD, somebody MOD, someone MOD have, there MOD, there MOD be, there MOD be some, there MOD be something, there MOD have, there MOD have been, they MOD also, they MOD be, they MOD have, they MOD have been, we MOD ask, we MOD ensure, we MOD ensure that, we MOD keep, we MOD remember, we MOD remember that, you MOD be, you MOD be joking, you MOD


Betty Chaumet

Nicolas Mazzella

Julie Neury-Ormanni

Soizic Morin

Light and temperature influence on diuron bioaccumulation and toxicity in biofilms

Keywords: Freshwater periphyton, herbicide, microbial ecotoxicology, seasons, toxicokinetics-toxicodynamics

Variations of temperature and photoperiod throughout different seasons can affect aquatic communities such as biofilms. Biofilms, generally present at the base of trophic chains in freshwaters, are also subject to organic contamination, and are especially affected by herbicides.

Many studies have investigated the effect and interactions of herbicides and environmental factors on biofilms, but never with a toxicokinetic point of view.

The objective of this study was to assess structural and functional changes in biofilms exposed to diuron, and to link them with contaminant accumulation, under the influence of temperature and light variations. To this aim, biofilms were exposed to all possible combinations of three concentrations (0, 5 and 50 µg.L -1 ) of diuron, two temperatures (10 and 26°C), and two light/dark photoperiods (16/8, 10/14), for durations of 0, 1 and 3 days. Diuron accumulation in biofilms was quantified and structural descriptors (protein and polysaccharide contents, dry weight) and functional endpoints (photosynthetic and enzymatic activities) were analysed.

The results obtained mainly highlighted the influence of temperature on diuron bioaccumulation and the associated toxic impact on biofilms. Bioaccumulation in biofilms exposed during three days at 10°C, at the highest diuron concentration, was in average 1.4 times higher than bioaccumulation on biofilms exposed to 26°C. Accordingly, the photosynthetic yield was more inhibited at lower than at higher temperatures. Temperature was also the highest impacting factor for metabolism regulation;

for example, at 26°C after three days of exposure, polysaccharide production was boosted under both photoperiods tested.

Introduction

It is well known that temperature and incident light are two of the main environmental factors able to modulate primary production [START_REF] Dodds | The relationship of floods, drying, flow and light to primary production and producer biomass in a prairie stream[END_REF][START_REF] Gomes | Temperature and Light Modulation of Herbicide Toxicity on Algal and Cyanobacterial Physiology[END_REF][START_REF] Staehr | Seasonal changes in temperature and nutrient control of photosynthesis, respiration and growth of natural phytoplankton communities[END_REF]. In temperate regions, both factors change seasonally, subjecting primary production to seasonal variations. Temperature and photoperiod influence organisms at the base of trophic chains in aquatic environments such as biofilms [START_REF] Flemming | The EPS matrix: The "House of Biofilm Cells[END_REF] which are composed of microalgae, fungi and bacteria embedded in an EPS matrix (Aguilera et al., 2008;[START_REF] Flemming | The biofilm matrix[END_REF].

The EPS matrix contains polysaccharides, which make-up around 97 % of its total constituents (Aguilera et al., 2008), proteins, humic acids and DNA. Additionally, biofilm integrates and reacts quickly to contamination, making it a good bioindicator [START_REF] Edwards | Applications of biofilms in bioremediation and biotransformation of persistent organic pollutants, pharmaceuticals/personal care products, and heavy metals[END_REF].

Biofilms are increasingly constrained by environmental stresses, coupled with anthropogenic pressures such as organic pollution [START_REF] Delorenzo | Toxicity of pesticides to aquatic microorganisms: a review[END_REF][START_REF] Guasch | The use of biofilms to assess the effects of chemicals on freshwater ecosystems[END_REF][START_REF] Kim Tiam | Improving toxicity assessment of pesticide mixtures: The use of polar passive sampling devices extracts in microalgae toxicity tests[END_REF]. In fact, watercourses are among the final receptacles of pollution, including pesticide contamination brought about by runoff or leaching processes [START_REF] Kohusova | A long-term survey of heavy metals and specific organic compounds in biofilms, sediments, and surface water in a heavily affected river in the Czech Republic[END_REF]. Due to their massive use, pesticides are widely monitored in aquatic environments. This is especially true for diuron, a photosynthesis inhibitor of photosystem II herbicide [START_REF] Yebra | Antifouling technology -Past, present and future steps towards efficient and environmentally friendly antifouling coatings[END_REF], considered as a priority substance in the European Water Framework Directive (Directive 2000/06/EC). Herbicides, and diuron in particular, have been shown to drastically reduce photosynthetic activity [START_REF] Kim Tiam | Herbicide toxicity on river biofilms assessed by pulse amplitude modulated (PAM) fluorometry[END_REF][START_REF] Pesce | Effects of Organic Herbicides on Phototrophic Microbial Communities in Freshwater Ecosystems[END_REF][START_REF] Tlili | Responses of chronically contaminated biofilms to short pulses of diuron. An experimental study simulating flooding events in a small river[END_REF].

Many studies have already tackled the biological impacts of temperature, light and herbicide contamination on biofilm (e.g: [START_REF] Bérard | Tolerance of Oscillatoria limnetica Lemmermann to atrazine in natural phytoplankton populations and in pure culture: Influence of season and temperature[END_REF][START_REF] Larras | The effect of temperature and a herbicide mixture on freshwater periphytic algae[END_REF][START_REF] Villeneuve | Influence of slight differences in environmental conditions (light , hydrodynamics) on the structure and function of periphyton[END_REF]. The effects of these parameters on the growth and photosynthetic efficiency of autotrophic organisms are well documented. In fact, it has already been shown that an increase in temperature, or in the length of the photoperiod can increase biofilm resistance to contaminants [START_REF] Staehr | Seasonal changes in temperature and nutrient control of photosynthesis, respiration and growth of natural phytoplankton communities[END_REF][START_REF] Herzig | Photoacclimation, photosynthesis, and growth in phytoplankton[END_REF]. Moreover, [START_REF] Duong | Experimental toxicity and bioaccumulation of cadmium in freshwater periphytic diatoms in relation with biofilm maturity[END_REF] and [START_REF] Villeneuve | In situ interactions between the effects of season, current velocity and pollution on a river biofilm[END_REF] showed that biofilm community composition could be modified according to season. Also, [START_REF] Wolfstein | Production of extracellular polymeric substances (EPS) by benthic diatoms: Effect of irradiance and temperature[END_REF] highlighted a variation in EPS content associated with different temperatures and degrees of irradiance exposure. Also, [START_REF] Chalifour | Combined effect of temperature and bleaching herbicides on photosynthesis, pigment and fatty acid composition of Chlamydomonas reinhardtii[END_REF] showed that temperature can affect the sensitivity of biofilms to herbicides.

According to the literature, a biofilm exposed to high temperatures, greater irradiance (such as during summer) and herbicides would be less sensitive to toxic effects than a winter biofilm [START_REF] Chalifour | Combined effect of temperature and bleaching herbicides on photosynthesis, pigment and fatty acid composition of Chlamydomonas reinhardtii[END_REF]. Variations of temperature and light intensity target processes involved in photosynthesis and metabolism [START_REF] Guasch | The use of biofilms to assess the effects of chemicals on freshwater ecosystems[END_REF]. As these environmental factors, pesticides such as herbicides can interact on photosynthesis and metabolism process due to their mode of action [START_REF] Draber | Herbicides in Photosynthesis Research[END_REF]. These combined effects may be synergistic, additional or antagonistic, but remain little known.

As herbicides, light and temperature co-occur in natural environments, we can hypothesise the interaction between all these factors may disturb the normal functioning of biofilm, and potentially more during the winter period. Consequently, it is crucial to study the fate and effect of herbicides in biofilms, and their impact, under environmental factors variations.

To answer this question, the aim of this study was to use a toxicokinetic-toxicodynamic approach to gain a better understanding of the different steps related to the effect of diuron under contrasting seasonal conditions. Thanks to this approach, more details would be provided about the time needed for metabolism processes and variation among herbicide contamination.

The objectives of the study were to investigate i) bioaccumulation by microorganisms in the biofilm, ii) subsequent modification of photosynthetic yield, and finally, iii) overall impact on metabolism via the determination of biochemical contents, and the measurement of complementary heterotrophic enzymatic activities.

Materials and methods

To reach the objectives arising from the problematic of this study, an experimental design was set up in which a mature biofilm was exposed to a combination of two different temperatures (10 and 26°C) and photoperiods (light/dark: 16/8 and 10/14), and to three diuron concentrations (0, 5 and 50 µg.L - 1 ).

Experimental design

During January-February 2017, bare glass slides (L: 26 cm, l: 6 cm) were placed in Gazinet-Cestas pond (South-West France, geographical coordinates: 44°46'30.1"N, 0°41'44.3"W). They were collected after four weeks of immersion, the time needed for biofilm to colonize the slides. During the colonization period, water temperature was 8.6 ± 0.5 °C (n= 9), and daily hours of sunlight numbered 10h09 ± 6min (n= 50).

The collected biofilm was immediately re-immersed in artificial channels (L: 50 cm, l: 9.2 cm , H: 5 cm) containing recirculating dauta medium [START_REF] Dauta | Conditions de développement du phytoplancton. Etude comparative du comportement de huit espèces en culture. II. Rôle des nutriments : assimilation et stockage intracellulaire[END_REF] at a pH of 7.5-8, with a flow velocity of 2 cm.s -1 , in a thermoregulated room. Two temperatures were tested, with room temperature set at 26°C and 10°C, and for each temperature, two light/dark photoperiods were applied: 16/8 and 10/14, to mimic four contrasting "seasonal conditions" (named "Seasons" 1 to 4) as presented in Figure 1. The four experiments were performed independently, starting from Season 1 (S1) on February 13 th , to Season 4 (S4), ending on March 9 th , with one experiment per week during this period. A diuron (CAS: 330-54-1, purity: 99 %, Dr Ehrenstorfer GmbH) stock solution was prepared in acetonitrile (CAS: 75-05-8, purity: 99.97 %, Biosolve) to reach a final concentration of 200 mg.L -1 . The toxicity of acetonitrile was tested on biofilms prior to the experiment. The NOEC was established at 1%, based on the measurement of photosynthesis inhibition (Neury-Ormanni, unpublished data). All exposure conditions were prepared to ensure that the final concentration of acetonitrile reached 0.25%, which is below the established NOEC. For each condition, three nominal concentrations of diuron were selected: 0, 5 and 50 µg.L -1 (respectively named Ctrl, 5D and 50D, prepared from stock solution in ultrapure water), and the biofilms were exposed to these conditions during one and three days, to ensure that the equilibrium between diuron concentrations in water and in biofilms was reached [START_REF] Chaumet | Flow conditions influence diuron toxicokinetics and toxicodynamics in freshwater biofilms[END_REF]. Moreover, the 3-day exposure was expected to be enough to highlight likely effects of photoperiod (16/8 photoperiod condition has 37 % more hours of illumination than 10/14 photoperiod condition).

Biofilm preparation

At each collection time (T 0 , one day and three days) for each condition (S1, S2, S3 and S4 for all the diuron exposure: Ctrl, 5D and 50D), four replicates from the same channel were sampled and prepared as described below. Biofilms were scrapped with a razor blade and introduced into a beaker previously filled with 40 mL of dauta medium. Biofilm directly collected after colonisation in the pond (i.e. without exposure in the channels) was also analysed in the same way as those after one and three days of exposure and was named T 0 . All samples were homogenized by agitation at room temperature for 30 minutes at 150 rpm (Variomag multi-station agitator, Thermo-Scientific).

Several analyses were then performed on biofilms in suspension: determination of dry weight (2.3.2), polysaccharide (2.4.1) and protein content (2.4.2), enzymatic (2.4.3) and photosynthetic activities (2.4.4), and pesticide quantification (2.3.4).

Sample processing and physicochemical analyses

Experimental conditions

Three mL of water from each channel were collected at each sampling time (T 0 , one day and three days) for all conditions (S1, S2, S3 and S4) and exposure conditions (the control and the two contaminated); and filtered on syringe-filters (pore: 0.45 µm, hydrophilic regenerated cellulose membrane, Whatman), in order to quantify pesticide content in the medium. A volume of 10 µL of internal standards was added to 990 µL of the filtrate prior to analysis (see 2.3.4). The solution of internal standards contained Diuron D6 (CAS: 1007536-67-5 -purity: 98 %, provided by Dr.

Ehrenstorfer GmbH) prepared at 10 mg.L -1 .

The temperature in the channels and in the thermoregulated room was measured by data loggers (Tinytag, Radiospare) every ten minutes for each condition. A light meter (LI-COR Li-250, USA) was used to control incident light intensity reaching the slides. The data are listed in Table 1.

Dry weight

For dry weight measurement, 8.8 mL of the 40 mL of suspended biofilm were filtered on a Buchner with previously-weighed ashed GF/F filters (Ø 90 mm, pore size: 0.7 µm, Whatman). Then, filters were frozen for 12 hours, lyophilised, and re-weighed. Dry weight data were used to standardize bioaccumulation data, as described in 2.3.4.

Biofilm sample preparation for diuron analysis

Diuron was extracted from biofilm matrices using Accelerated Solvent Extraction (ASE 2000, Dionex, Samples were purified by Solid Phase Extraction (SPE) on HR-X Chromabond cartridges (Macherey-Nagel, Germany) previously conditioned with 5 mL of methanol (CAS: 67-56-1, purity: 99.95 %, Biosolve) and 5 mL of ultrapure water. Samples were percolated through the cartridge, then the cartridge was rinsed with 20 % -80 % methanol (CAS: 67-56-1, purity: 99.95 %) -ultrapure water buffer, and dried in a vacuum with a Visiprep TM for 30 minutes. The cartridges were eluted with 5 mL of acetonitrile (CAS: 75-05-8, purity: 99.97 %), and 10 µL of internal standard were added to the eluate collected. The mixture was evaporated until dry in a SpeedVac (Thermo Fisher Scientific, France), and the extracted diuron was dissolved into 1 mL of ultrapure water before analysis (2.3.4).

Diuron quantification in water and biofilm

Diuron and its main metabolites (DCPMU and DCPU) in biofilms and water samples were quantified by UPLC-ToF (Ultra-Performance Liquid Chromatography -Time of Flight Mass Spectrometry, Xevo G2-S ToF, Waters) as proceeded in a previous study (see [START_REF] Chaumet | Flow conditions influence diuron toxicokinetics and toxicodynamics in freshwater biofilms[END_REF]). An ACQUITY BEH C18 column 50 × 2.1 mm, 1.7 μm (Waters) was used for chromatographic separation at 45°C with a binary gradient of ultrapure water (at pH= 5) and methanol (both solutions with ammonium acetate to a concentration of 10 mM, CAS: 631-61-8, ULC quality Biosolve), and 20 µL of samples were injected.

The gradient was performed with the following range: 98 % of ultra-pure water solution (0-3.6 min), 2 % of methanol solution (3.6-6 min) at a flow rate of 0.45 mL.min -1 .

Data were processed with MassLynx V4.1 software, and diuron concentrations were inferred via a calibration curve from 0 to 100 µg.L -1 , comprising 10 points. Every 10 samples, 3 quality controls were carried out at 0, 1 and 10 µg.L -1 on the calibration curve. For biofilm samples, data were normalized by dry weight and then expressed in µg.g -1 . Limits of quantification were established at 5 µg.L -1 for water samples and at 0.05 µg.mg -1 for biofilm samples.

Biofilm characterization

Polysaccharide analysis

Polysaccharide content was analysed according to the method outlined by [START_REF] Dubois | Colorimetric Method for Determination of Sugars and Related Substances[END_REF].Aliquots of 500 µL of samples were prepared by adding 12.5 µL of phenol (CAS: 108-95-2, purity: 99 %, Scharlab) and 2.5 mL of sulfuric acid (CAS: 7664-93-9, purity: 95 %, VWR). Samples were incubated at room temperature for 10 minutes, and then 20 minutes in a water bath at 30°C.

Absorbance was read with a microplate reader (Synergy TM HT, Biotek, USA) at 485 nm. Absorbance data were converted into µg glucose-equivalent per cm² using a standard curve of D(+) -glucose anhydrous solution (CAS: 50-99-7, ACS Reagent Grade, Scharlab) from 0 to 750 µg.L -1 . Concentrations were expressed in percentage of T 0 values for each condition, to account for variability of the inoculums used.

Protein dosage

A protocol adapted to low concentrations of proteins was developed based on the methodology presented by [START_REF] Bradford | A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding[END_REF]. An aliquot of 500 µL of the suspended biofilm was collected and 4.5 mL of methanol were added, then the mixture was sonicated for 20 minutes. Samples were then evaporated close to dryness with a SpeedVac (Thermo Fisher Scientific, France) and filled again with 500 µL of ultrapure water and 2.5 mL of Bradford reagent, prepared using 3.3 mg of Brilliant blue G (CAS: 6104-58-1, Sigma), 33.3 mL of methanol, 66.67 mL of phosphoric acid (CAS: 7664-38-2, purity: 85 %, Chem-Lab) and 900 mL of ultra-pure water. Samples were gently agitated to homogenize the color thus formed. Absorbance was measured using a microplate reader (Synergy TM HT, Biotek, USA) at 595 nm. Protein content was determined using a standard curve from 0 to 250 µg.L -1 performed with Bovine Serum Albumine (BSA, CAS: 9048-46-8, Sigma). Data obtained in µg of BSA-equivalent per cm² were then expressed in percentage of T 0 values for each condition.

Enzymatic activity measurement

Two extracellular enzymatic activities related to the heterotrophic component of the biofilm were measured: the β-glucosaminidase (EC 3.2.1.30) and the β-glucosidase (EC 3.2.1.21) activities, carriedout according to the methodology outlined in [START_REF] Romaní | Relevance of polymeric matrix enzymes during biofilm formation[END_REF]. The two substrates, 4methylumbelliferyl N-acetyl-β-D-glucosaminide (CAS: 37067-30-04, Sigma) and 4-methylumbelliferylβ-D-glucopyranoside (CAS: 18997-57-4, Sigma), respectively associated to β-glucosaminidase and βglucosidase, were prepared at a final concentration of 0.3 mmol.L -1 (corresponding to the saturation concentration of the substrate [START_REF] Romanı | Romani et al 2004-The influence of substratum type and nutrient supply on biofilm organic matter utilization in streams-L and O.pdf[END_REF]

Data analysis

Data obtained were not normal and different conditions tested were independent, consequently, non-parametric tests were applied. The aim of the statistical analysis was to highlight if they were significant differences between conditions tested like between the two temperatures, or the two photoperiods, or the three exposure concentrations, or time exposure. A Kruskal-Wallis test followed

Results

Seasonal condition effects on control samples

The dry weight of the biofilm samples measured immediately after collection from the pond (T 0 ) for each season showed high variations. Indeed, biomass regularly increased between S1 and S4, with values 6-fold higher at S4 than S1 (DW S1 = 33 ± 5 µg.cm -2 and DW S4 = 203 ± 27 µg.cm -2 , Table 2)

highlighting that biofilm colonisation depends on the period of the year (meteorological parameters) in temperate settings. It was observed that the biomass in the inoculum can vary greatly from one sample to another. Therefore, the results obtained for the structural and functional descriptors were standardized by the associated T 0 , thus avoiding biomass bias. The samples could then be compared with each other at the level of absolute variations observed after exposure. Variations between dates and diuron exposure treatments were expressed as percentages of T 0 , to highlight only the effect of temperature, photoperiod and diuron. Biofilm dry weight generally increased over the three days of channel exposure, and the 3-day increase in biomass was little more pronounced at 26°C. In fact, for season 1 and 2, the growth rate was of 0.68 and 0.69 µg.cm -2 .h -1 respectively (2.5 times increase in 3 days for S1 and 1.9 times greater for S2, Table 2) and of 0.4 and -0.4 for season 3 and 4 respectively (1.4 times increase in 3 days for S3, Table 2). Even, a slight decrease in dry weight was visible after three days of exposure for S4. On the other hand, in terms of photosynthetic yield of the control samples, there were no marked effects of temperature nor of photoperiod, whatever the exposure time.

Protein dosage and β-glucosidase activity measurement showed an increase in percentage at three days compared to T 0 (Table SI 1 andFigure 2). The same trend was visible for polysaccharide results and β-glucosaminidase activity measurements, but only for biofilms exposed to 26°C. For these descriptors, samples exposed to 10°C did not highlight any particular variation (around 100 % T 0 ). Generally, for all descriptors, results highlighted an evolution between T 0 , one day and three days of exposure, with results slightly more marked after three days. For data collected after three days of exposure, the temperature effect was noticeable for all descriptors (Table SI 1 andFigure 2).

Nevertheless, data were not statistically different but they allow to highlight a trend because all showed an increase of percentage in comparison to T 0 for samples exposed to 26°C. There were almost no differences between conditions for β-glucosaminidase activity among the different photoperiods. However, β-glucosidase activity was slightly affected by the most extended photoperiod independently of the temperature after three days of exposure. Indeed, we observed an increase of this enzymatic activity with the longer photoperiod (e.g. S1 was 3.8 times higher than S2 and S3 1.6 greater than S4, Figure 2, Table SI1). The same trend was visible for polysaccharide percentage, but more moderately, also for protein percentage but only between S3 and S4. About biofilm characterization, temperature and photoperiod had an influence on structural biofilm parameters. For biofilms exposed to the highest temperature (26°C), polysaccharide content increased between day 1 and day 3 (Figure 2). This effect was considerably less marked in biofilms exposed at a temperature of 10°C than at 26°C. In general, the polysaccharide percentage from control treatments increased in the following order: S4 < S3 < S2 < S1. This result resembles that observed for other descriptors, like protein content and enzymatic activities. At equal temperature, polysaccharide concentrations were higher with a longer photoperiod (S2 < S1, S4 < S3).

From bioaccumulation to toxic impact

Diuron concentration in the water channels was quantified at 5.1 ± 0.2 (n= 4) and 43.9 ± 0.4 µg.L -1 (n= 4) of diuron, respectively, for 5D and 50D treatments which is extremely close to nominal concentrations. The results from control water channels and metabolite concentrations in exposed channels (for water and biofilms samples) were below the limit of quantification. No statistical difference in diuron exposure for each treatment was found between conditions or dates of sampling. The concentrations of the main metabolites of diuron were below the limits of quantification A small amount of diuron was quantified in biofilm samples from control conditions (mean= 88.4 ± 19.9 µg.g -1 of diuron) which could originate from the environment during colonisation. The total amount of diuron measured within biofilms exposed to 10°C was globally higher (but not significantly) than within those exposed to 26°C (Figure 3). This observation was more noticeable for 50D treatments. For 50D, at a given temperature of exposure, bioaccumulation tended to be higher when light exposure time increased.

In general, the results presented in Figure 2 did not show very marked trends in terms of the effect of diuron on functional and structural descriptors. However, although there was no significant difference to confirm these observations, it would appear that for all descriptors, after 3 days of exposure, there was an increase with exposure concentration for biofilms exposed to 10°C, and a slight decrease (proteins, polysaccharides, β-glucosidase activity), or a stability (β-glucosaminidase activity) for biofilms exposed to 26°C. The size of the points is proportional to the dose of exposure (small = Ctrl, middle = 5D, large = 50D).

The effects of temperature on toxicity were not as clear as on bioaccumulation. Indeed, the photosynthetic yield was lower at 10°C for the 50D treatment and at 26°C for the 5D treatment (Figure 3, also see Table SI2 in the Supplementary Material). However, at 10°C the photosynthetic yield tended to decrease with the luminosity amplitude (S4 < S3), and at 26°C, the contrary was observable, but the photosynthetic yields were quite close between S1 and S2. Photosynthetic activity was negatively correlated with diuron bioaccumulation in the biofilm (R²= 0.684, p-value < 0.001; Figure 3), correlation coefficients calculated for each condition separately increased as follow:

S1 (R²= 0.48, p-value< 0.001) <S2 (R²= 0.52, p-value< 0.001) <S3 (R²= 0.87, p-value< 0.001) <S4 (R²= 0.94, p-value< 0.001) (see Figure SI 1).

Influence of environmental variation on metabolism

The Kruskalmc test performed on data from all analyses did not show significant differences according to temperature, photoperiod or diuron effect. It was subsequently performed with data from each descriptor, and there were no significant trends with regard to the different environmental exposure conditions and contaminations. The linear discriminant analysis carried out with structural descriptors (proteins, polysaccharides) and enzymatic activities, after three days of exposure, all treatments combined (as diuron had no effect on these descriptors), discriminated the different conditions.

Seasons 1 and 2 were separated along axis 1 (Figure 4), which was mostly explained by an increasing relative activity of β-glucosidase ("BetaGlu"; mean of three days for all treatments S1= 706 % and S2= 215 %). In fact, conditions were discriminated along this axis in relation to the β-glucosidase activity as follows: S2 < S4 < S3 < S1. Thus, axis 1 highlighted photoperiod influence on biological responses, mainly related to the amplitude of variation in β-glucosidase activity over three days. Season 2 was also characterized by a slightly higher relative protein content and β-glucosaminidase activity, and Season 1 by slightly higher polysaccharide content and β-glucosidase activity. In contrast to the conditions presenting higher temperatures, Seasons 3 and 4 were anticorrelated with Seasons 1 and 2 along axis 2, due to their slightly lower polysaccharide content. Axis 2 discriminated seasons according to temperature as follows: S2 < S1 < S3 < S4.

Additionally, the fact that conditions sharing a seasonal characteristic (equal temperature or photoperiod) were not fully identical suggests an interaction between the influence of temperature and photoperiod. These results highlight the influence of photoperiod and temperature on biofilm metabolism and illustrate the previous conclusions that these factors influenced the variation over three days in polysaccharide and protein content, as well as in β-glucosaminidase activity over three days (section 3.1).

Discussion

Diuron impact on PSII

The results show that diuron bioaccumulation in biofilm was higher at 10°C than at 26°C (e.g. 27 % more at 10°C) except for S1 with 5D treatment, and consequently, the photosynthetic activity was more inhibited at 10°C for associated samples, at equal photoperiods (Figure 3). Toxic effects observed in this study are in accordance with many others carried out on similar microorganisms (green algae, diatoms) with pesticides. For example, [START_REF] Tasmin | Elevated water temperature reduces the acute toxicity of the widely used herbicide diuron to a green alga, Pseudokirchneriella subcapitata[END_REF] found a 12-17 % increase of photosynthesis inhibition on microalgae with a decrease in temperature from 25 to 10°C for a contamination at 32 µg.L -1 of diuron during three days. These observations are in accordance with our results: an increase of 16 % of photosynthesis inhibition with a decrease in temperature.

Moreover, [START_REF] Chalifour | Combined effect of temperature and bleaching herbicides on photosynthesis, pigment and fatty acid composition of Chlamydomonas reinhardtii[END_REF] measured a photosynthetic yield decrease from 57 to 98 % between 25 and 15°C in green algae exposed to 2.5 µM of norflurazon (a herbicide). Although the literature dealing with combined herbicide and temperature impacts on photosynthetic yield is abundant, there is very little information on bioaccumulation aspects. This work complements the existing knowledge by showing that higher PSII inhibition at lower temperatures is likely explained by higher amounts of accumulated diuron.

Metabolism variations with temperature and light

Environmental modulation effect

Heterotrophic organisms were not accurately identified, however, the choice of the dosage of the two enzymatic activities allowed to provide information on the activity of the heterotrophic component of the biofilm. Indeed, β-glucosaminidase plays a role in the degradation of peptidoglycans (a component of the bacterial wall) and chitin (a polysaccharide derived from fungal walls), and β-glucosidase is involved in carbohydrate degradation. In our study, the linear discriminant analysis highlighted that β-glucosidase activity was boosted by longer periods of light, and the contrary for β-glucosaminidase activity, both at higher temperatures. This means that the metabolism of organisms such as bacteria and fungi were intensively activated at 26°C, indicating a favourable environment for them [START_REF] Lurling | Comparison of cyanobacterial and green algal growth rates at different temperatures[END_REF].

For autotrophic organisms, photosynthetic activity is directly related to metabolism, itself dependent on temperature [START_REF] Brown | Toward a metabolic theory of ecology[END_REF]. In this study, polysaccharide quantities produced after three days of exposure were greater at 26°C than at 10°C, suggesting that the biofilm was less productive at colder temperatures. These results are in accordance with those of [START_REF] Wolfstein | Production of extracellular polymeric substances (EPS) by benthic diatoms: Effect of irradiance and temperature[END_REF] who found a slight increase (around 1.25 -1.3 times higher) in EPS content (composed of polysaccharides and proteins) after three and nine days of exposure between biofilm exposed from 10°C to 25°C, with a photoperiod of 12/12.

Interaction of environmental factors effects with the impact of diuron

The effects of environmental factors interacting with diuron were not clearly visible and not significant on heterotrophic organisms. In fact, as diuron is a photosynthetic inhibitor herbicide, it should have a greater influence on autotrophic compartment. Results for β-glucosaminidase activity showed a slight decrease at 10°C and a stability at 26°C after three days of exposure between controls and the most contaminated condition. In contrary β-glucosidase activity was marked by a small increase. Indeed, diuron is a photosynthetic inhibitor herbicide, so it does not directly target bacteria and fungi over such short periods of exposure as strongly as microalgae and their photosynthetic capacity. Because of the very short duration of biofilm exposure (three days), the toxic effect of these factors may not have reached the whole community through cascading effects [START_REF] Proia | The Use of Attached Microbial Communities to Assess Ecological Risks of Pollutants in River Ecosystems: The Role of Heterotrophs[END_REF]. The same phenomenon was found by [START_REF] Proia | Resistance and recovery of river biofilms receiving short pulses of Triclosan and Diuron[END_REF], who observed that after two days of exposure to diuron at 15 µg.L -1 , the bacteria showed no structural nor functional changes. However, following this exposure and during one week of recovery, they observed indirect effects such as a transient increase in bacterial mortality.

However, this study, combining toxicokinetic and toxicodynamic approaches, showed the evolution of the effects of environmental factors and diuron on biofilm metabolism over time. Although the data showed little difference over a three-day period exposure, they still highlight an evolution and a dynamic system, suggesting a greater impact on longer-term exposure in the natural environment.

Although our results showed no significant differences between conditions, the effect of diuron on algae seemed to be modulated by temperature and light to variable extents, depending on the concentration of exposure. Nevertheless, the results showed that among all the descriptors analysed, the only part of the metabolism significantly impacted by diuron was photosynthetic activity.

Contrastingly, polysaccharide and protein production and enzymatic activities were mainly affected by the interaction between temperature and photoperiod (Figure 2).

Overall, biofilm appeared to be less impacted and more productive at 26°C than at 10°C. This can be explained by the fact that the biofilm originated from conditions close to Season 4 (10°C, light/dark cycle of 10/14). A priori, the biofilms having experienced the highest degree of disturbance should be the ones from S1, with a drastic change in environmental conditions (+6h of light/day and +16°C). As conditions in S1 seem to be favourable to the activation of metabolism, this could explain the high polysaccharide and protein production under the conditions of this treatment. That means that even if the change of environmental conditions can affect the biofilms (e.g. from around 10°C in the pond to 26°C in the channels), it appears that this case the increase of temperature and photoperiod duration is not the most impacting external stress like cold temperature and dark/light cycle reduced.

Indeed, in the case of stressful conditions, organisms can slow their metabolism in order to reduce their energy needs [START_REF] Brown | Toward a metabolic theory of ecology[END_REF]. The results of [START_REF] Chalifour | Combined effect of temperature and bleaching herbicides on photosynthesis, pigment and fatty acid composition of Chlamydomonas reinhardtii[END_REF] support this fact by

showing that algae use their energy reserves to maintain a growth rate similar to controls in the presence of herbicide (2.5 µM norflurazon) and over 4 days, they observed a strong decrease in photosynthetic efficiency. Our results present the same kind of phenomenon for contaminated conditions: at 26°C polysaccharide production was boosted, while at 10 °C, production remained stable. Also, photosynthetic efficiency was greater under 26°C exposure. All this shows that biofilm exposed to low temperatures and a shorter photoperiod is forced to restrict the energy allocated for its growth, hence the lower metabolic activity of the biofilm during Season 4 and its greater sensitivity to diuron.

All the variations observed in this study on biofilm show only trends and not significant differences, which begs the question of what the major changes would be that would take place over longer exposure times, and at the level of community composition.

Conclusion

This study focused on the influence of interactions between diuron exposure, temperature and photoperiod on pesticide bioaccumulation and toxic impact on biofilm. Our results suggest some influence of temperature on bioaccumulation, and consequently on photosynthetic activity. They imply that Winter biofilms in temperate regions are more sensitive to diuron exposure than Summer ones.

Our results agree with the existing literature dealing with the joint influence of temperature and herbicides on biofilm vulnerability. However, this study also demonstrates the influence of photoperiod duration on several biofilm endpoints, not only related to the autotrophic component of the community. Moreover, this work goes further by coupling toxicokinetic and toxicodynamic aspects, allowing to link the bioaccumulation of pesticides in biofilms with their metabolism.

Figure 1 :

 1 Figure 1: Graphical representation of experimental design and abbreviations associated with each

  France). A solution of surrogates was prepared with Monuron d6 (CAS: 217488-65-8) at a final concentration of 10 mg.L -1 , Prometryn d6 (CAS: 1705649-52-0) at 1 mg.L -1 and Simazine d5 (CAS: 220621-41-0) at 20 mg.L -1 , all provided by Dr. Ehrenstorfer GmbH. The ASE cells were filled as follows: the GF/F filter containing the lyophilized biofilm, Fontainebleau sand, and 10 µL of surrogates (to control the percentage of recovery) were introduced into the ASE device. The extraction was performed at 70 bars and 100°C, with acetone (CAS: 67-64-1, purity: 99.5 %, Chem-Lab) as the solvent. Once the extraction complete, acetone samples were evaporated under nitrogen flux with a Visiprep TM (Supelco) and the extracted pesticides were dissolved in 10 mL of ultrapure water (Synergy UV, Millipore).

  Photosynthetic activity (effective photosystem II quantum yield, Φ PSII ) of all samples was assessed by

  by a posthoc kruskalmc test [Package pgirmess version 3.6.1] were performed on data from all the analyses (in percentage of T 0 ), and then on each biological descriptor individually. A p-value of 0.05 was considered significant. A linear discriminant analysis was applied on the same dataset (in percentage of T 0 ) with R software [Package ade4 version 1.6-2], with the aim of individualising data into similarly-composed groups.

Figure 2 :

 2 Figure 2: Polysaccharide and protein contents, and β-Glucosaminidase and β-Glucosidase activities

Figure 3 :

 3 Figure3: Mean photosynthetic quantum yield (±sd, n=4) as a function of diuron bioaccumulation in the biofilm in µg.g -1 (mean±sd), for biofilms exposed in channels during one and three days. The

Figure 4 :

 4 Figure 4: Linear discriminant analysis of the different conditions after three days of exposure (left

Table 1 :

 1

	Mean (± standard error) temperatures (n= 475) and light intensity (n=8) during the
	experiments
	Conditions Water temperature (°C) Room temperature (°C) Light intensity (µmol.s -1 .m -2 )
	Season 1	na*	na*
	Season 2	22.70 ± 0.59	25.16 ± 0.01
			7.47 ± 0.42
	Season 3	11.60 ± 0.18	11.23 ± 0.00
	Season 4	11.65 ± 0.15	11.17 ± 0.00

*: data not available. A technical problem was encountered for the recording of Season 1 temperature data, but the temperature settings of the thermoregulated room were identical between Season 1 and Season 2.

Table 2 :

 2 Mean (±sd) dry weight in µg.cm -2 and photosynthetic yield in Ctrl biofilms. * indicates

	significant differences within the conditions (Kruscalmc test)
	Conditions	Exposure time
			Mean dry weight (µg.cm -2 ) Photosynthetic yield
	(temperature, photoperiod)	(day)
		0	33±5	0.41±0.02
	Season 1
		1	35±6	0.48±0.02
	(26°C, 16/8)
		3	83±10	0.49±0.02
		0	57±10	0.44±0.02
	Season 2
		1	65±11	0.51±0.03
	(26°C, 10/14)
		3	106±18	0.51±0.01

Acknowledgments

This study was carried out with financial support from the French National Research Agency (ANR) in the framework of the Investments for the future Programme, within the Cluster of Excellence COTE (ANR-10-LABX-45).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.