Danilo Rheinheimer

José Augusto Santos

Monteiro

Castro Lima

Jocelina Paranhos
email: jocelinaparanhos@mail.ufsm.br

Rosa De Vargas

Marilia Camotti Bastos

Maria Alice Santanna

Leslie Santos

Jérôme Mondamert

Labanowski

Pesticide bioaccumulation in epilithic biofilms as a biomarker of agricultural activities in a representative watershed

Keywords: contamination, soil use management, soil erosion, runoff, riparian forest

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

Introduction

As elsewhere in the world, the model of Brazilian agriculture is based on the production and export of major crops such as soybean, corn, cotton, citrus, and coffee [START_REF] Graminha | Enzyme production by solid-state fermentation: Application to animal nutrition[END_REF]. The availability of genetically modified seeds, advanced agricultural machinery, intensive usage of fertilizers and pesticides, and management of no-till farming led to the consolidation of this agricultural model. A problem affecting most of the producing countries is the increase in pesticide use, resulting in an increase in the augmentation of pesticide use per area of cropland from 1.5 kg ha -1 in 1990 to 2.7 kg ha -1 in 2016 (FAO, 2018). Based on these values, it is assumed that a significant part of the pesticides applied to the cultures (active compounds and metabolites) are not achieving their primary targets or are not being metabolized by plants.

Nowadays, environmental contamination with pesticides has received great scientific and societal attention due to the major risk posed to the natural environment and human health [START_REF] Özkara | Environmental Health Risk -Hazardous Factors to Living Species[END_REF]Tang et al. 2018). The occurrence of various contaminants in the surface water is correlated with agricultural activities, exhibiting the conflict between productivity and contamination [START_REF] Bastos | Presence of Anthropogenic Markers in Water: A Case Study of the Guaporé River Watershed, Brazil[END_REF][START_REF] Fernandes | Indiscriminate use of glyphosate impregnates river epilithic biofilms in southern Brazil[END_REF][START_REF] Kaiser | High Nitrogen Fertilization of Tobacco Crop in Headwater Watershed Contaminates Subsurface and Well Waters with Nitrate[END_REF][START_REF] Zafar | Phosphorus seasonal sorption-desorption kinetics in suspended sediment in response to land use and management in the Guaporé catchment, Southern Brazil[END_REF]. Pesticide residues can be transferred to water sources by soil erosion, surface runoff, and percolation. These losses are related to low-quality no-tillage management (Tiecher et al. 2017a), the withdrawal of the terraces, and a deficient supply of straw, leading to high loss rates of water, soil [START_REF] Deuschle | Erosion and hydrological response in no-tillage subjected to crop rotation intensification in southern Brazil[END_REF], and nutrients [START_REF] Zafar | Phosphorus seasonal sorption-desorption kinetics in suspended sediment in response to land use and management in the Guaporé catchment, Southern Brazil[END_REF].

The transfer of pesticides to aquatic ecosystems occurs mainly in diffuse form, that is, most of it is originated by the transport system, in this case, mainly rainwater transport carrying contaminated soil particles; therefore, pesticide residues are divided between the aqueous phase and adsorbent matrix eroded from the soil (Vryzas et al. 2018), depending on their physicochemical properties (aqueous solubility S W , octanol-water partition coefficient K OW , vapor pressure P, Henry's law constant H, octanol-air partition coefficient K OA , octanol solubility S O (Shen and Wania 2005). In water, these compounds are present in low concentrations (µg L -1 to ng L -1 ), making it difficult for the monitoring process to perform active sampling. Once in the aquatic ecosystem, the relations between the pesticides found in soluble form and those adsorbed to a given matrix can change in the face of new geochemical and biological conditions in the environment. An example are the epilithic biofilms, which can interact with pesticides by adsorbing soluble compounds or incorporating contaminated sediments in its extracellular polymeric matrix [START_REF] Battin | The ecology and biogeochemistry of stream biofilms[END_REF][START_REF] Lawrence | Sorption and metabolism of selected herbicides in river biofilm communities[END_REF].

The epilithic biofilms are composed of an inorganic matrix (weathering products with sediment attached), a diversity of autotrophic and heterotrophic microorganisms (bacteria, algae, fungi, and microfauna), and detritus exogenous or produced in situ [START_REF] Flemming | The biofilm matrix[END_REF]. Biofilms are of fundamental ecological importance in the aquatic ecosystem because their water/substrate interface is an autotrophic primary producer, an organic matter decomposer, and an adsorption/desorption reactor [START_REF] Feckler | Impacts of Contaminants on the Ecological Role of Lotic Biofilms[END_REF]. Biofilms have a high physicochemical reactivity due to the great diversity of surface functional groups, such as amine, amide, carboxyl, and phenolic in organic matrixes provided by organic constituents, as well as a permanent charge and surface hydroxyl sites in clay minerals and oxides in the inorganic colloid matrix provided by the inorganic constituents [START_REF] Essington | Soil and water chemistry: an integrative approach[END_REF][START_REF] Fernandes | Indiscriminate use of glyphosate impregnates river epilithic biofilms in southern Brazil[END_REF]. [START_REF] Proia | Response of biofilm bacterial communities to antibiotic pollutants in a Mediterranean river[END_REF] have shown that microorganisms in biofilms have a short life cycle and there is a strong interaction between the microbiota. Due to these characteristics, biofilms can be used as a powerful ecological indicator [START_REF] Edwards | Applications of biofilms in bioremediation and biotransformation of persistent organic pollutants, pharmaceuticals/personal care products, and heavy metals[END_REF][START_REF] Sabater | Monitoring the effect of chemicals on biological communities. The biofilm as an interface[END_REF]) because the availability of diverse sorption sites and their likely saturation contribute to multiple kinetics of bioaccumulation, being consecutive or stationary [START_REF] Zhang | Inhibitory effects of extracellular polymeric substances on ofloxacin sorption by natural biofilms[END_REF].

In areas of intense agriculture, aquatic ecosystems and food webs are exposed to pesticides that alter species abundance and diversity, causing negative effects even at low concentrations [START_REF] Beketov | Pesticides reduce regional biodiversity of stream invertebrates[END_REF].

When exposed to this type of contamination, the photosynthetic activity of biofilms can decrease, and inhibition of growth and changes in community structure can be observed [START_REF] Kim | Improving Toxicity Assessment of Pesticide Mixtures: The Use of Polar Passive Sampling Devices Extracts in Microalgae Toxicity Tests[END_REF][START_REF] Pesce | Effects of Organic Herbicides on Phototrophic Microbial Communities in Freshwater Ecosystems[END_REF]. For example, [START_REF] Bricheux | Pyrosequencing assessment of prokaryotic and eukaryotic diversity in biofilm communities from a French river[END_REF] showed that by exposing biofilms to glyphosate and diuron, the growth of the autotrophic community was inhibited, and the bacterial communities adapted by changing their composition. In addition, areas with water collection for drinking water distribution may be contaminated, presenting a risk to the population if the treatment is inefficient for the removal of these organic compounds from the water [START_REF] Beketov | Pesticides reduce regional biodiversity of stream invertebrates[END_REF]. Previous studies in the Guaporé catchment showed that bioaccumulation in epilithic biofilms could be used as biomarkers of anthropic contamination by glyphosate and its metabolite AMPA [START_REF] Fernandes | Indiscriminate use of glyphosate impregnates river epilithic biofilms in southern Brazil[END_REF] and by carbamazepine and sucralose [START_REF] Bastos | Presence of Anthropogenic Markers in Water: A Case Study of the Guaporé River Watershed, Brazil[END_REF]). This study aimed at identifying and quantifying pesticides present in epilithic biofilms in order to evaluate the effectiveness of this matrix as a bioindicator capable of discriminating areas and periods with different inputs of pesticides.

Materials and Methods

Catchment description

Located in the state of Rio Grande do Sul, southern Brazil, the Guaporé River catchment drains an area of 2,490 km² and is 153 km long [START_REF] Lima | Modern agriculture" transfers many pesticides to watercourses: a case study of a representative rural catchment of southern Brazil[END_REF]. Classified as Cfa in the Köppen system, the climate is characterized by an average annual temperature of 17.9 °C and an average annual minimum and maximum temperatures of 12.6 and 24.7 °C, respectively. In this catchment, previous studies revealed that rain events have high erosion potential (8,800 MJ mm ha -1 h -1 year -1 ) [START_REF] Didoné | Impact of no-tillage agricultural systems on sediment yield in two large catchments in Southern Brazil[END_REF] with greater occurrence from July through November [START_REF] Inmet | Multi-residue method for trace level determination of pharmaceuticals in solid samples using pressurized liquid extraction followed by liquid chromatography/quadrupole-linear ion trap mass spectrometry[END_REF]. Sediment production in 2012 amounted to 390.2 Mg km -2 year -1 , and the average was estimated at 140 mg km -2 year -1 from 2000 to 2010, with a runoff coefficient of up to 31% [START_REF] Didoné | Impact of no-tillage agricultural systems on sediment yield in two large catchments in Southern Brazil[END_REF]. The land use in this area is dominated by cropland (60.0%), natural forest (30.1%), pastureland (5.6%), forestry (3.1%), urban areas (0.6%), and water bodies (0.4%) (Fig. 1) characteristic of rural areas with high production in southern Brazil. In the upper third of the catchment, the relief is nearly level to gently sloping and predominated by Ferralsols; the no-tillage system has been adopted and has predominated for two decades. Besides soybean (Glycine max L. Merr.) cultivation, livestock, swine, and poultry are also produced in the catchment in spring/summer. The animals reside in marginal noncultivated areas, generally along the watercourses. The pesticides used are characterized by high doses of herbicides and fungicides applied to soybean production, including 2,4-D for plants resistant to glyphosate. The annual production density of this region is 161.6 mg year -1 km -2 of soybean; 41.5 and 148.2 mg year -1 km -2 of corn and corn silage respectively; and 0.3 and 1.3 mg year -1 km -2 of tobacco and yerba mate, respectively. In the other two-thirds (middle and lower parts) of the catchment, land use is more diverse and conventional soil management predominates in shallow soils with a rocky surface (Luvisols, Acrisols, and Regosols). The cultivated surface is imbricated in landscape and the family farmers have less than 10 ha. The annual production density of this region is 38.1 mg year -1 km -2 of soybean; 53.4 and 233.1 mg year -1 km -2 of corn and corn silage, respectively; 3.5 and 34.1 mg year -1 km -2 of tobacco and yerba mate, respectively (IBGE, 2019).

Sampling sites

A total of 18 sampling sites were selected, which were located in Guaporé River (G1, G2, G3, G4, G5, G6, G7, G8, G9, and G10), in the tributary of the Marau Stream (M1, M2, M3, and M4), Lajeado Carazinho Stream Tributary (LC1, LC2, and LC3) and the tributary of the Carazinho Stream (C). Descriptions of each site and locations are shown in Table 1 andFigure

Biofilm sampling

The first biofilm sampling campaign was conducted between 11 and 13 December 2014 spring/summer season), two days after a high precipitation event 89 mm [START_REF] Inmet | Multi-residue method for trace level determination of pharmaceuticals in solid samples using pressurized liquid extraction followed by liquid chromatography/quadrupole-linear ion trap mass spectrometry[END_REF]. By this time, the soybean crop was in V3 stage, the corn was in R1 stage, and the first leaves of flue-cured tobacco were harvested. The second biofilm sampling campaign was performed between 3 and 5 June 2015 (autumn/winter season); it registered 4 mm of precipitation during the preceding week. During this period, less than one-fifth of the total agricultural surface was cultivated with wheat, barley, and winter grassland forage. The rate of pesticide application was low compared to that of the spring/summer season (Fig. 2, data obtained from farmers). At each collection site, epilithic biofilms were obtained from submerged rocks with the aid of toothbrushes. The rock adhered material was washed with deionized water and recuperated in glass jars. The detailed sampling procedure has been published by [START_REF] Bastos | Presence of Anthropogenic Markers in Water: A Case Study of the Guaporé River Watershed, Brazil[END_REF].

The pesticides were selected according to the Brazilian registered agricultural compounds and with field investigations. Subsequently, a second selection was made according to the operational and methodological capacity of the laboratories involved.

The selected molecules and their physicochemical properties are presented in the appendix (Table A1).

Sampling points were selected in order to represent two macro-regions in the river basin, north and south regions, which are contrasting in terms of relief, land use, and agricultural management aspects.

Analytical procedure

In the laboratory, glass jars with biofilms were transferred to individual high-density polyethylene jars and frozen at -80 °C for subsequent lyophilization. After being freeze-dried, samples were homogenized in an agate mortar to obtain a representative sample for analysis. The biofilm extraction procedure was performed using an adapted method from Jelić et al. ( 2009) that is well described by [START_REF] Aubertheau | Impact of wastewater treatment plant discharge on the contamination of river biofilms by pharmaceuticals and antibiotic resistance[END_REF] The identification and quantification of the molecules were performed by ultra-high performance liquid chromatography (Thermo Fisher Scientific, Waltham, USA), coupled to a time-of-flight mass spectrometry (Impact II TM model, Bruker Daltonics, Billerica, USA) using the method Pesticide Screener (developed by Thermo Fisher Scientific, Waltham, USA). For each extract, there were three injections of 20 μL in positive and negative ionization mode. Data processing was performed using Compass Data Analysis 4.2 and Target Analysis software (Bruker, Billerica, USA).

The concentration of pesticides in biofilms was determined by adjusting the linear model used between the concentration of the standards and the peak area of each injection for each compound detected. Validation of the method was followed by the French norm NF XP T90-210 carried out through linearity, repeatability, and Cochran's test (more details on the methodology available in [START_REF] Lima | Modern agriculture" transfers many pesticides to watercourses: a case study of a representative rural catchment of southern Brazil[END_REF]. The limits of detection (LD) ranged from 0.5 to 1 ng/g. The LD and limit of quantification LQ were estimated from the background noise. In this case, the LD and LQ were calculated by determining the maximum amplitude of the signal over a distance equal to 20 times the width at mid-height of the peak corresponding to the compound searched, and these values were used in the statistical analysis. A total of 25 compounds were analyzed, namely: 2,4-D, atrazine, boscalid, carbendazim, chlorotoluron, chlorpyrifos-ethyl, cypermethrin, deltamethrin, desethyl-atrazine, dicamba, epoxiconazole, imidacloprid, iodosulfuron-methyl-sodium, isoproturon, MCPA, mesotrione, mesosulfuronmethyl, metconazole, nicosulfuron, prochloraz, prosulfuron, prothioconazole, simazine, tebuconazole, and thifensulfuron-methyl.

The methodology description for method validation and the physico-chemical properties of the pesticides studied are available in the appendixes of this work (A1, A2 and A3).

Statistical treatment

The Kolmogorov-Smirnov and Lilliefors tests were used to assess whether the concentrations of pesticides exhibited a normal distribution. Because the results did not show normal distribution, the median was used as a measure of position, and the upper and lower quartiles, interquartile range, and maximum and minimum values were used as dispersion measures. The hypothesis that the epilithic biofilms could discriminate spatial and temporal flux of pesticides from soil to aquatic ecosystems was statistically evaluated by the Mann-Whitney U nonparametric test. The STATISTICA 7.0 program was used to carry out statistical procedures.

Results and Discussion

Occurrence and concentration

Only five pesticides (cypermethrin, deltamethrin, dicamba, iodosulfuron-methyl, and MCPA) were not detected in the epilithic biofilms sampled in the Guaporé River and its tributaries. The absence of these compounds in biofilms is not proof that these compounds are not being used in agricultural fields and does not refute the possibility that the functional groups of biofilms may not have affinity with pesticides. However, the fieldwork carried out by our team demonstrates that, in this region, the use of pyrethroid insecticides is being replaced by systemic compounds, in particular imidacloprid and other neonicotinoid pesticides. In southern Brazil, pyrethroid is still used in urban sprayings and in fruit production. In addition, the herbicide dicamba is used in combination with genetically modified soybean varieties (Xtend®) that have not yet been cultivated in Brazil. Some farmers in the Guaporé catchment use these pesticides for controlling plants resistant to glyphosate.

The last two non-detected herbicides were recommended for dicotyledonous control in corn and cereal production, but they are rarely used this catchment (data obtained from farmers).

All monitored sites in the spring/summer season present biofilms impregnated by three herbicides (2,4-D, atrazine, and simazine), and one metabolite (desethyl-atrazine), three fungicides (carbendazim, tebuconazole, and epoxiconazole), and two insecticides (imidacloprid and chlorpyrifos-ethyl). In addition, another seven herbicides (25 to 81% frequency) and four fungicides (6 to 69% frequency) were detected in biofilms occurring in both the Guaporé River and in three of its main tributaries. In autumn/winter season, only two pesticides were found at 100% of the sites monitored (2,4-D and carbendazim), but all 20 compounds were quantified in biofilms with frequency ranging from 22% (thifensulfuron-methyl) to 83% (atrazine and tebuconazole, Table 2). Some

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The maximum concentrations of herbicides, in ng g -1 of biofilms, were 17.6, 445.9, 22.3, and 59.6 for 2,4-D, atrazine, desethyl-atrazine, and simazine, respectively. The highest concentrations of insecticides were 20.3 and 14.3 in ng g -1 of biofilms for imidacloprid and chlorpyrifos-ethyl, respectively; and finally, the highest concentrations of fungicides in biofilms were 73.0, 44.6, and 35.9 ng g -1 of biofilms for carbendazim, epoxiconazole, and tebuconazole, respectively (Fig. 3). Fifty per cent of the epilithic biofilms sampled had median concentrations varying, in ng g -1 of biofilms, between 0.5 and 6.0 (2,4-D), 0.5 and 34.0 (atrazine), 0.5 and 12.0 (desethyl-atrazine), 0.5 and 10.0 (simazine), 0.5 and 5.0 (imidacloprid), 3.4 to 24.0 (carbendazim), and 0.5 and 4.0 (epoxiconazole and tebuconazole). Usually, the highest concentration of pesticides in biofilms is in accordance with the highest frequency of detection. High concentrations and loads can also be linked to catchment and climate conditions, where peaks in concentrations are observed in the summer, which is usually a rainy period with conditions known to be linked to high pollutant mobilization [START_REF] Zhang | A field study of colloid transport in surface and subsurface flows[END_REF]. In particular, sorption on dissolved organic carbon can effectively increase the mobility of some pesticides (e.g., atrazine and 2,4-D, [START_REF] Li | Effect of organic fertilizers derived dissolved organic matter on pesticide sorption and leaching[END_REF]Tang et al. 2012). The physicochemical properties of pesticides such as water solubility, octanol-water partition coefficient, dissociation (pKa), and Henry's law constant are also determining factors of leaching potential. However, in the case of preferential flow, pesticides can be rapidly transferred to groundwater irrespective of their physicochemical properties.

Vryzas (2018) reported higher pesticide concentrations in shallow groundwater from experimental boreholes compared to the concentrations in the adjacent deep groundwater (drinking or irrigation water wells). Mixed leaching mechanisms ("chromatographic" and preferential flow) have been reported by the authors to be involved in the contamination of adjacent aquifers with different water residence times.

Epilithic biofilms can adsorb pesticides by specific adsorption (inner-sphere surface complexation), by nonspecific mechanisms (physical adsorption), and by hydrophobic interaction [START_REF] Barrett | Phosphate and glyphosate mobility in soil columns amended with roundup[END_REF][START_REF] Kleber | A conceptual model of organo-mineral interactions in soils: selfassembly of organic molecular fragments into zonal structures on mineral surfaces[END_REF][START_REF] Lawrence | Sorption and metabolism of selected herbicides in river biofilm communities[END_REF][START_REF] Späth | Sorption properties of biofilms[END_REF]. Additionally, microorganisms and macroinvertebrates can uptake and accumulate pesticides, since they are in constant interaction with biofilms and are sometimes used as a source of nourishment [START_REF] Rhea | Biomonitoring in the Boulder River Watershed, Montana, USA: Metal Concentrations in Biofilm and Macroinvertebrates, and Relations with Macroinvertebrate Assemblage[END_REF]. The observed high diversity and frequency of detection of pesticides in the Guaporé catchment showed that biofilms can be used as a powerful ecological indicator of pesticide exposure, as has been proposed by [START_REF] Edwards | Applications of biofilms in bioremediation and biotransformation of persistent organic pollutants, pharmaceuticals/personal care products, and heavy metals[END_REF].

Spatial sources of pesticide contamination (soil use and management)

The frequency and the median concentration of some pesticides in epilithic biofilms allow discrimination of the upper and the middle/lower regions of the Guaporé catchment (Table 3). In the upper region, by comparison with other catchment places, biofilms were more impregnated by three herbicides (2,4-D, thifensulfuron-methyl, isoproturon), four fungicides (carbendazim, epoxiconazole, metconazole, tebuconazole), and one insecticide (imidacloprid). In addition, the interquartile range for the atrazine and simazine in the upper region was lower than that found in the middle/lower region. The upper region has a plane relief terrain with low slopes and almost 100% of the agricultural surface is cultivated on a no-tillage system with soybean and corn genetically modified. Moreover, high hydraulic connectivity between cultivated fields and watercourses was observed. In contrast, although agriculture is performed in a more declivous area and mainly under conventional tillage, the cultivated areas are imbricated in the landscape and the riparian forest is very large in the middle/lower region of the Guaporé watershed in comparison to the upper region (Table 1). 3 -Medians concentration of pesticides in epilithic biofilms sampled in Guaporé River and its tributaries, according to their sampling period (spring/summer and autumn/hiver) seasons and location (upper and middle/lower).

Pesticides

The southern region of Brazil presents a subtropical humid climate with an average annual rainfall between 1,550 and 1,700 mm, but it may even reach 2,500 mm (INMET 2019). The rain has very high erosion potential [START_REF] Didoné | Impact of no-tillage agricultural systems on sediment yield in two large catchments in Southern Brazil[END_REF]. Even several decades after the adoption of a no-tillage system, agricultural areas are still the main source of sediments to rivers in the Guaporé catchment (Tiecher et al. 2017a), as well as in other watersheds in the south of Brazil (Tiecher et al. 2018;Tiecher et al. 2017b). In Brazil, the no-tillage system is usually a non-optimal soil management system because farmers removed the terraces, and there is an insufficient amount of straw in the surface of the soil. High rates of water loss, sediment [START_REF] Deuschle | Erosion and hydrological response in no-tillage subjected to crop rotation intensification in southern Brazil[END_REF], and nutrients [START_REF] Zafar | Phosphorus seasonal sorption-desorption kinetics in suspended sediment in response to land use and management in the Guaporé catchment, Southern Brazil[END_REF] have been observed in the Guaporé catchment. In the no-tillage system, the pesticides added do not reach the soil surface because of the interception by the mulch of crop residues [START_REF] Aslam | Mulch of plant residues at the soil surface impact the leaching and persistence of pesticides: A modelling study from soil columns[END_REF], decreasing the reactivity with functional groups of inorganic and organic colloids. Moreover, due to regularity and frequent rainfall occurring in the catchment, there is a constant pesticide wash-off the superficial residues. In fact, under these conditions, it is not possible to produce crops without transferring sediment and agrochemicals to the superficial water resources. There is no doubt that certain amounts of the pesticides used in crops are transferred to water courses. The amount and frequency of pesticides reaching surface water reserves depends on the quantity added, the distribution of crops in the landscape, and the presence of physicalmechanical and/or natural barriers to the transfer of sediment and water by erosion and runoff.

Direct runoff and erosion of soil particles are the major pathways responsible for the transportation of pesticides to surface water. Rates of soil erosion, irrigation, precipitation, and half-life are the major factors that govern the transportation of pesticides to the surface water [START_REF] Aravinna | Use pattern of pesticides and their predicted mobility into shallow groundwater and surface water bodies of paddy lands in Mahaweli river basin in Sri Lanka[END_REF]). The compounds with low K ow and K oc , and high mobility were in more variable concentrations in biofilms sampled in spring/summer season (simazine, carbendazim, and imidacloprid). However, pesticides with moderate adsorption capacity in colloids (K ow 3.0 to 5.0, [START_REF] Lewis | An international database for pesticide risk assessments and management[END_REF] were also found in high concentrations in biofilms (tebuconazole, epoxiconazole, and chlorpyrifos-ethyl). Pesticides are transferred from soil to watercourse, mostly in soluble form by runoff from cultivated fields, but also adsorbed by inorganic and organic colloids in eroded sediment.

The transport of pesticides through preferential water flow to macropores to tile drainage plays an important role in the rapid transport of pesticides to surface waters (Tang et al. 2012;Vymazal and Březinová 2015). When these pesticides enter the water bodies, desorption from colloids occurs, releasing them to the aqueous phase [START_REF] Aravinna | Use pattern of pesticides and their predicted mobility into shallow groundwater and surface water bodies of paddy lands in Mahaweli river basin in Sri Lanka[END_REF]). The water flow velocity also plays an important role in the accumulation of these compounds in biofilms. Chaumet et al. (2019a) showed higher accumulation of diuron at lower flow velocity compared to the higher flow for raw biofilms.

Temporal use of pesticides (applicationtransfer to watercoursedecompositionresilience) All 20 pesticides detected were detected in epilithic biofilms in both seasons. However, the concentrations of five herbicides (2,4-D, atrazine, desethyl-atrazine, simazine, and nicosulfuron), three fungicides (carbendazim, epoxiconazole, and tebuconazole), and one insecticide (imidacloprid) were higher in the spring/summer than in the autumn/winter season (Table 3, statistical analysis is available in Table A4. These results were coherent with the phase of development of soybean, corn, and tobacco plants and the period of pesticide application. The maximum herbicide consumption occurs from October to December, the period in which they are used in pre-sowing and post-emergence of genetically modified soybean and corn (> 90% of herbicides consumption in the Guaporé catchment). Also, although in smaller quantities, herbicides are used in pre-transplantation and during the development of tobacco during the spring/summer season. The fungicides and insecticides were used more intensely between December and February, mainly in soybean and tobacco production (Fig. 2).

After February, pesticide application is reduced drastically in the Guaporé catchment. The tobacco harvest ends in mid-February and many farmers sow corn or silage production, but without the use of fungicide and insecticides. The soybean harvest takes place from mid-March until the end of April. More than 90% of the agricultural area remains fallow after the harvest of soybean and maize. The sowing of winter crops, wheat, colza, and barley starts in June, after the second sampling of epilithic biofilms. The median concentrations of atrazine, carbendazim, and imidacloprid, for example, were drastically reduced between the first (spring/summer) and the second (autumn/winter) sampling: 38.0 to 0.5, 25.0 to 4.7, and 4.1 to 0.5 ng g -1 of biofilms, respectively (Table 3, statistical analysis is available in appendix Table A5). The concentration of many pesticides in biofilms sampled in autumn/winter was lower than the LD (between 0.5 and 1 ng g -1 ). The capacity of the epilithic biofilms to biodegrade pesticides is evident and pointed out in various studies [START_REF] Feckler | Impacts of Contaminants on the Ecological Role of Lotic Biofilms[END_REF][START_REF] Sabater | Monitoring the effect of chemicals on biological communities. The biofilm as an interface[END_REF]Staley et al. 2015) that attribute pesticide removal by biofilms to biodegradation, not to sorption. [START_REF] Carles | Meta-analysis of glyphosate contamination in surface waters and dissipation by biofilms[END_REF], through a study of exposure of biofilms to glyphosate in mesocosm, were able to define that the biological decomposition of the glyphosate pesticide was performed in approximately 13 days, in the case of low concentration of this pesticide in water (< 10 µg L -1 ).

The Guaporé River and its tributaries can be classified as turbulent systems. The area presents a wellpreserved riparian forest, especially in the middle/lower region of the catchment (Table 1). Villeneuve et al.

(2010) showed that in turbulent mesocosms, the biofilms were more diversified in comparison to static environments. Additionally, the relation between the autochthonous primary production and the heterotrophic organisms in biofilms is modified with direct exposure of rivers to the sun which may alter the autotrophic/heterotrophic proportion in biofilms [START_REF] Feckler | Impacts of Contaminants on the Ecological Role of Lotic Biofilms[END_REF]. Furthermore, the average temperature of the water is also adequate to optimal microbiota development in the winter season (17.9 and 22.5 ºC in autumn/winter and spring/summer samplings, respectively).

However, the soil is a reservoir of pesticides, and it can transfer the compounds to the surface and groundwater ecosystems [START_REF] Sassine | Spatial distribution of triazine residues in a shallow alluvial aquifer linked to groundwater residence time[END_REF]) slowly and for a long time. For example, in Brazil, the use of the herbicide triazines is authorized (2.5 x 10 4 mg of atrazine consumed in 2017, the fifth most used pesticide), and the persistence in biofilms in the autumn/winter season is reasonable. The storage and remobilization from soil and the continuing legal applications guarantee the flux of pesticides from higher to lower landscape position, and the water can be a constant pesticide source to the biofilms. The carbendazim and imidacloprid persistence in biofilms sampled in July, especially in the upper region of the Guaporé catchment, are probably being used for the control of end-cycle maladies (Cercospora kikuchii and Septoria glycines) and sucking insect pests in soybeans. These pesticides are widely used until a few days before the soybean harvest. Tien et al. ( 2013) evaluated the maximum pesticide removal capacity in river biofilms and showed that natural river biofilms from different seasons were able to remove different pesticides with rates ranging from 99.6 to 41.2%. The authors observed different dissipation rates and degradation capacities in biofilms from different seasons which might be due to the colonization of microbial species with different pesticide degradation capacity within river biofilms and might differ with the variation of pesticide concentration in river water associated with the lower use of pesticides in different seasons.

Biofilms as a monitoring tool at a global scale

The use of biofilms as an environmental indicator can be carried out all over the world due to their development in all aquatic ecosystems as part of the trophic network [START_REF] Aubertheau | Impact of wastewater treatment plant discharge on the contamination of river biofilms by pharmaceuticals and antibiotic resistance[END_REF][START_REF] Bastos | Presence of Anthropogenic Markers in Water: A Case Study of the Guaporé River Watershed, Brazil[END_REF][START_REF] Fernandes | Indiscriminate use of glyphosate impregnates river epilithic biofilms in southern Brazil[END_REF][START_REF] Huerta | Determination of a broad spectrum of pharmaceuticals and endocrine disruptors in biofilm from a waste water treatment plant-impacted river[END_REF]. In this study, it was possible to verify that biofilms were able to represent the anthropic pressure near the collection points, even with the use of different pesticides and times of application of agricultural crops, biofilms were able to capture pesticide compounds even in rivers located in areas with low anthropic activity and/or with low concentration of these compounds. In addition, pollution from different soil management systems can be identified through sampling and analysis of biofilms; thus, biofilms can be used as tools to evaluate the health and water quality of water bodies. Furthermore, pesticides are ubiquitous in aquatic environments and constantly interact with aquatic organisms, including those that makeup biofilms at fluctuating concentrations (Chaumet et al. 2019a). The main advantage over point sampling (water sampling) is its capacity to accumulate compounds in an integrative way because of the heterogeneity of biofilm composition, the availability of diverse sorption sites, and their probable saturation may contribute to multiple kinetics of bioaccumulation, being consecutive or staking [START_REF] Zhang | Inhibitory effects of extracellular polymeric substances on ofloxacin sorption by natural biofilms[END_REF]. [START_REF] Chaumet | Flow conditions influence diuron toxicokinetics and toxicodynamics in freshwater biofilms[END_REF] studying diuron bioaccumulation in biofilms have demonstrated a phenomenon of continuous diffusion, but it was not linearly correlated with bioaccumulation, highlighting the complex capture mechanisms operating within the biofilm matrix. The study of biofilm contamination becomes important as a basis for better exposure assessment of biofilm feeding organisms and as useful for determining the risk of trophic transfer of pesticides along the aquatic food chain. Besides, biofilms develop naturally on several surfaces, such as rock surfaces in riverbeds, not having costs for implementation, like other passive samplers that are expensive and must be installed at sampling points.

Conclusions

The epilithic biofilms sampled in the Guaporé River watershed bioaccumulated the active principles of 10 herbicides (2,4-D, atrazine, chlorotoluron, simazine, nicosulfuron, mesosulfuron-methyl, mesotrione, prosulfuron, isoproturon, thifensulfuron-methyl), 7 fungicides (boscalid, carbendazim, epoxiconazole, tebuconazole, metconazole, prochloraz, prothioconazole), 2 insecticides (chlorpyrifos and imidacloprid), and 1 metabolite (desethyl-atrazine).

The use of epilithic biofilms as integrative matrix is effective in discriminating the spatial and temporal sources of contamination independent of the physicochemical characteristics of the compounds. Additionally, by employing the detection frequency and median concentrations of pesticides in biofilms, it was possible to estimate in which of the two main cultures (soybean or maize) the pesticides were employed.

The concentrations of the main pesticides monitored were sharply reduced in the epilithic biofilms sampled some months after the application in the summer cultures, showing the high capacity of resilience and detoxification.

Fig 1 -

 1 Fig 1 -Characteristics of area and location of the sampling sites in the Guaporé River and its tributaries:(a) location of the sampling sites in the Guaporé watershed, (b) Guaporé watershed land use and (c) Guaporé watershed slopes. (RS -Brazil).

Fig 2 -

 2 Fig 2 -Relationship between the period of the year and the intensity of pesticide applied in wheat, corn and soy crops in the Guaporé watershed.

  spring and early summer rainfall events[START_REF] Pascual Aguilar | Pesticide occurrence in the waters of Júcar River, Spain from different farming landscapes[END_REF][START_REF] Pérez | Spatial and temporal trends and flow dynamics of glyphosate and other pesticides within an agricultural watershed in Argentina: Spatial-temporal and flow dynamics of pesticides in a basin[END_REF] Rabiet et al. 2010); the 230 same was observed in biofilms in the present study.231The frequency of pesticide detection in biofilms is consistent with their consumption in Brazil and 232 consequently, with use in the Guaporé catchment. Over the last few years, Brazilian farmers used 5.8 x 10 7 Mg 233 of 2,4-D, 2.5 x 10 7 Mg of atrazine, 9.4 x 10 6 Mg of imidacloprid, and 6.5 x 10 6 Mg of chlorpyrifos-ethyl234(IBAMA 2018). The three most frequently detected fungicides in biofilms are also the most widely used in 235 Brazil (tebuconazole, carbendazim, and epoxiconazole, with 4.5, 3.7, and 0.8 x 10 6 mg, respectively).

Fig 3 -

 3 Fig 3 -Spatial and temporal pesticides concentration in biofilms sampled in the Guaporé catchment

Table 1 -

 1 1, respectively.

	Sample sites				Upper Region
		G1	G2	G3	G4	G5	M1	M2	M3	M4
	Catchment area (km 2 )	1.3	123	201	267	542	13.4	165	227	256
	Distance (km)	0	6	23	32	35	-	0	10	23
					Land use (%)
	Forest	100	28	24	23	22	22	20	21	22
	Agriculture	0	64	69	71	70	72	74	72	72
	Width riparian forest	>800	15-60	15	15	15	15	15	15	15
				Slope class (%)
	(m) 0-3%	5	10	9	8	7	6	7	6	6
	3.1-12%	49	60	59	58	57	56	60	57	56
	12.1-20%	35	22	23	23	24	25	23	24	24
	20.1-45%	12	8	9	10	11	13	9	12	13
	> 45%	0	0	0	0	1	0	0	1	1
				Middle/Lower Region
	Sample sites Catchment area (km 2 )	G6 1442	G7 1505	G8 1697	G9 1850	G10 1853	LC1 LC2 LC3 30 3.2 39	C 144
	Distance (km)	85	90	106	108	110	0	-	3.2	0
					Land use (%)
	Forest	27	28	31	32	32	50	61	52	46
	Agriculture	67	66	62	61	61	42	32	40	50
	Width riparian forest	15	>60	>100	>100	>100 >100	15	>100 >100
				Slope class (%)
	(m) 0-3%	6	5	5	5	5	4	1	3	2
	3.1-12%	47	45	42	41	41	34	10	28	23
	12.1-20%	24	24	23	23	23	24	20	22	27
	20.1-45%	21	22	24	26	26	29	53	34	43
	> 45%	3	4	5	5	5	10	17	14	5

Catchment area, land use and slope class at the different sampling sites in the Guaporé River and its tributaries.

  . Five hundred milligrams of biofilm were extracted by accelerated solvent extraction equipment (ASE TM 350, Thermo Fisher Scientific Inc., Waltham, USA) at 80 °C using methanol/water (1/2, v/v) as the extraction solvent. The extracts were purified by solid-phase extraction (Autotrace TM 150, Thermo Scientific, 125 Waltham, USA) using Oasis® HLB cartridges (6 cc, 200 mg of sorbent, Waters, Milford, USA) with methanol as eluent. The extracts obtained by the Solid Phase Extraction (SPE) system were evaporated under N 2 flow for restitution to 500 µl with a methanol/water mixture (10/90, v/v). The recovered liquid was then filtered one last time using Mini-Uniprep TM filters (PVDF Filter Media with polypropylene housing, pore size 0.45 μm, Durapore®, Millipore, Billerica, USA). Each biofilm sample was analyzed twice with triplicate measurements.

Staley, Z. R., Harwood, V. J., & Rohr, J. R. (2015). A synthesis of the effects of pesticides on microbial persistence in aquatic ecosystems. Critical Reviews in Toxicology,45(10), https://doi.org/10.3109/10408444.2015.1065471 Tang, G., Yao, J., Zhang, X., Lu, N., & Zhu, K. Y. (2018). Comparison of gene expression profiles in the aquatic midge (Chironomus tentans) larvae exposed to two major agricultural pesticides. Chemosphere, 194, 745-754. https://doi.org/10.1016/j.chemosphere.2017.12.040 Tiecher, T., Caner, L., Minella, J. P. G., Pellegrini, A., Capoane, V., Rasche, J. W. A., et al. (2017a)


Jean-Pierre Gattuso
email: gattuso@obs-vlfr.fr

Phillip Williamson

Carlos M Duarte

Alexandre K Magnan

The potential for ocean-based climate action: negative emissions technologies and beyond

Keywords: ocean-based solutions, blue carbon, bioenergy with carbon capture and storage, ocean fertilization, ocean alkalinization, climate action

The effectiveness, feasibility, duration of effects, co-benefits, disbenefits, cost effectiveness and governability of four ocean-based negative emissions technologies (NETs) are assessed in comparison to eight other ocean-based measures. Their role in revising UNFCCC Parties' future Nationally Determined Contributions is discussed in the broad context of ocean-based actions for both mitigation and ecological adaptation. All measures are clustered in three policyrelevant categories (Decisive, Low Regret, Concept Stage). None of the ocean-based NETs assessed are identified as Decisive at this stage. One is Low Regret (Restoring and increasing coastal vegetation), and three are at Concept Stage, one with low to moderate potential disbenefits (Marine bioenergy with carbon capture and storage) and two with potentially high disbenefits (Enhancing open-ocean productivity and Enhancing weathering and alkalinization).

Ocean-based NETs are uncertain but potentially highly effective. They have high priority for research and development.

INTRODUCTION

Implementing the Paris Agreement is a formidable challenge. In pathways limiting global warming to 1.5 • C with no or limited overshoot as well as in pathways with a higher overshoot, CO 2 emissions are reduced to net zero globally around 2050 (IPCC, 2018). This would require far-reaching and unprecedented transitions in all sectors, and also large-scale use of negative emissions technologies (NETs), that is removal of greenhouse gases from the atmosphere by deliberate human activities.Yet the trade-offs for food production and nature conservation limit carbon dioxide removal by most land-based approaches [START_REF] Boysen | Trade-offs for food production, nature conservation and climate limit the terrestrial carbon dioxide removal potential[END_REF].

In that context, it is timely to assess the opportunities offered by the ocean to reduce the causes and also the consequences of climate change, globally and locally. Recent papers and reports have reviewed several potential measures [START_REF] Gattuso | Ocean solutions to address climate change and its effects on marine ecosystems[END_REF] Because the Ocean, 2019; National Academies of Sciences Engineering and Medicine, 2018;[START_REF] Gesamp | High level review of a wide range of proposed marine geoengineering techniques[END_REF][START_REF] Hoegh-Guldberg | The ocean as a solution to climate change: five opportunities for action[END_REF]. Countries have so far only made limited use of ocean-related measures for tackling climate change and its impacts through their Nationally Determined Contributions (NDC) under the Paris Agreement [START_REF] Gallo | Ocean commitments under the paris agreement[END_REF]. Among the 161 initial NDCs submitted in 2014-2015, 70 mentioned marine issues, most frequently as components of adaptation action or in regard to climate impacts. Just over a third (59) also included ocean-related mitigation measures. The only ocean-based negative emission approach specifically mentioned is the conservation and restoration of coastal blue carbon ecosystems (i.e. mangroves, saltmarshes and seagrasses). The 5-year revisions of NDCs that need to be made before the re-scheduled COP26 in 2021 (26th Conference of the Parties of the United Nations Framework Convention on Climate Change, UNFCCC), offer an opportunity to adopt more ocean-inclusive mitigation and adaptation strategies.

Here we assess four ocean-based NETs involving carbon dioxide removal (Marine bioenergy with carbon capture and storage, Restoring and increasing coastal vegetation, Enhancing open-ocean productivity, Enhancing weathering and alkalinization) and put them in the broader context of ocean-based measures to support climate policies, especially greenhouse gas mitigation and ecological adaptation. These carbon dioxide removal pathways are the most documented in the literature. Other pathways are not evaluated here and it is likely that new methods or hybrids between the methods presented here will emerge. Building on recently published material [START_REF] Gattuso | Ocean solutions to address climate change and its effects on marine ecosystems[END_REF], this Policy Brief brings two innovations. It assesses marine-biomass-fueled bioenergy with carbon capture and storage (Marine BECCS) and goes a step further by grouping these NETs and a wide range of other options into policy clusters with the aim of guiding decisions on priority areas of action. [START_REF] Gattuso | Ocean solutions to address climate change and its effects on marine ecosystems[END_REF] published a comprehensive expert assessment of ocean-based measures to reduce climate change and its impacts based on a review of 862 publications and expert judgment. Here we focus on NETs and consider eight criteria: (1) effectiveness to increase net carbon uptake, (2) effectiveness to reduce ocean warming, ocean acidification, and sea level rise; (3) feasibility, covering both technological readiness and lead time until full potential effectiveness; (4) duration of effects; (5) cost effectiveness; (6) co-benefits; (7) disbenefits and (8) governability from an international perspective. Full information is available in the supplementary material of [START_REF] Gattuso | Ocean solutions to address climate change and its effects on marine ecosystems[END_REF] and in Supplementary Material SM3.

OCEAN NEGATIVE EMISSIONS TECHNOLOGIES

Marine bioenergy with carbon capture and storage

Considering the area, water and nutrient requirements leads to the tentative conclusion that macroalgae (seaweed) based BECCS could be a better long-term option than woody biomass energy crops, because achievable productivity is higher, freshwater demands are eliminated, and nutrient demands may be lower if efficient recycling systems can be developed [START_REF] Lenton | The global potential for carbon dioxide removal[END_REF]. Here we do not consider hybrid bioenergy with carbon capture and storage approaches to focus on marine-biomass-fueled BECCS. The latter is expected to involve three overarching steps: increasing carbon uptake by macroalgal aquaculture; converting the biomass to biofuel; and deploying carbon capture (at power stations) and long-term geological storage. [START_REF] Capron | Restoring pre-industrial CO 2 levels while achieving sustainable development goals[END_REF] review recent progress by US researchers in using macroalgae for biofuel.

The effectiveness of Marine BECCS to increase carbon uptake is scored high. Its effectiveness to moderate ocean climate drivers is also scored high given the close relationship between CO 2 and ocean warming, acidification and sea level rise. However, feasibility is considered low because components of this approach have not all been demonstrated at scale and may require a decade or more of research and development to prove capacity, cost effectiveness and safety. Cost effectiveness (based on a single estimate) is high, at 26 US$ per t CO 2 removed for marine biomass fueled BECCS.

Climate-relevant alternatives to using macroalgae for BECCS would be to develop methods for deep water disposal of the additional biomass generated through macroalgal aquaculture (Williamson et al., 2021) and the manufacturing of long-lived products, such as plastic, such as the manufacturing of long-lived products, such as plastic, that displace products generating emissions, thereby avoiding emissions, or enhancing soil fertility while contributing to carbon sequestration through use as biochar.

Restoring and increasing coastal vegetation

Restoring and increasing coastal vegetation ("blue carbon" ecosystems) aims at enhancing CO 2 uptake and avoiding further emissions. Here we look at the implementation of this measure at the global scale, by restoring a high proportion of the human-induced degraded salt marshes, mangroves and seagrasses habitats of the planet.

Conservation and restoration of blue carbon ecosystems support and enhance CO 2 sequestration, whilst also reducing emissions associated with habitat degradation and loss [START_REF] Mcleod | A blueprint for blue carbon: toward an improved understanding of the role of vegetated coastal habitats in sequestering co-2[END_REF][START_REF] Pendleton | Estimating global "blue carbon" emissions from conversion and degradation of vegetated coastal ecosystems[END_REF][START_REF] Duarte | Assessing the co-2-capture potential of seagrass restoration projects[END_REF][START_REF] Marbà | Impact of seagrass loss and subsequent revegetation on carbon sequestration and stocks[END_REF][START_REF] Howard | Clarifying the role of coastal and marine systems in climate mitigation[END_REF][START_REF] Hamilton | Global carbon stocks and potential emissions due to mangrove deforestation from 2000 to 2012[END_REF]. The theoretical effectiveness of such measures at the global scale is limited by the maximum area that can be occupied by these habitats, with their maximum global carbon burial estimated to be 234 Tg C per year for salt marshes, mangroves and seagrasses combined [START_REF] Mcleod | A blueprint for blue carbon: toward an improved understanding of the role of vegetated coastal habitats in sequestering co-2[END_REF]. Assuming that historic losses of blue carbon ecosystems (50% for mangroves since the 1940s, 29% for seagrass since 1879, 25% for salt marshes since the 1800s; [START_REF] Mcleod | A blueprint for blue carbon: toward an improved understanding of the role of vegetated coastal habitats in sequestering co-2[END_REF]Waycott et al., 2009) are rapidly reversed through restoration, the maximum cumulative carbon burial would be 26 Pg C until 2100, equivalent to about 2.5 years of current anthropogenic emissions. This is a theoretical upper estimate because of likely constraints on implementation. The effectiveness to increase carbon uptake is therefore scored very low, noting also that increased methane (CH 4 ) emissions may fully or partly negate the climatic benefits of CO 2 removal [START_REF] Al-Haj | A synthesis of methane emissions from shallow vegetated coastal ecosystems[END_REF][START_REF] Rosentreter | Concerns and uncertainties relating to methane emissions synthesis for vegetated coastal ecosystems[END_REF]. Carbonate formation or dissolution in blue carbon habitats will also affect the capacity of these habitats to remove atmospheric carbon; although dissolution may dominate, thereby increasing removal [START_REF] Saderne | Role of carbonate burial in blue carbon budgets[END_REF], these processes have not yet been explored sufficiently to provide global estimates.

Like carbon uptake, the effectiveness to moderate warming, acidification and sea level rise is limited by the maximum area possibly occupied by these habitats, hence it is scored very low. Feasibility is high as blue carbon approaches are well tested and deployed around the world (Howard et al., 2014) but the full delivery of the benefits at their maximum global capacity will require years to decades to be achieved.

Protection of blue carbon habitats must be multi-decadal in order to be effective, and successful restoration also requires a long term commitment (e.g., [START_REF] Duarte | Assessing the co-2-capture potential of seagrass restoration projects[END_REF][START_REF] Marbà | Impact of seagrass loss and subsequent revegetation on carbon sequestration and stocks[END_REF][START_REF] Reynolds | Ecosystem services returned through seagrass restoration[END_REF] -with the assumption that future temperature increases can be kept below 2 • C (under higher warming, blue carbon ecosystems are at risk, and their stored carbon is likely to be lost; [START_REF] Bindoff | Changing ocean, marine ecosystems, and dependent communities[END_REF]. The duration of the effect is scored permanent, with those provisos. At median costs of 240, 30,000 and 7800 US$ per t CO 2 , respectively for mangroves, saltmarshes and seagrass habitats, the cost effectiveness (for climate mitigation) is collectively scored very low [START_REF] Siikamaki | Global economic potential for reducing carbon dioxide emissions from mangrove loss[END_REF][START_REF] Bayraktarov | The cost and feasibility of marine coastal restoration[END_REF][START_REF] Narayan | Coastal wetlands and flood damage reduction: using risk industry-based models to assess natural defenses in the Northeastern USA (London: Lloyd's Tercentenary Research Foundation[END_REF], recognising that there are large differences in restoration costs between the three ecosystems (and at the local level), and that the cost effectiveness of restoration for the delivery of other ecosystem services could nevertheless be very high.

Enhancing open-ocean productivity

Enhancing open-ocean productivity (hereafter Fertilization) is based on nutrients addition directly or indirectly to increase CO 2 drawdown by phytoplankton. Such methods may appear technologically feasible, as it has been shown for iron/macronutrient addition, as well as for artificial upwellings [START_REF] Pan | Research progress in artificial upwelling and its potential environmental effects[END_REF]. For iron addition, modelling studies show a maximum effect on atmospheric CO 2 of 15 to 45 ppmv in 2100 (Zeebe and Archer, 2005;[START_REF] Aumont | Globalizing results from ocean in situ iron fertilization studies[END_REF][START_REF] Keller | Potential climate engineering effectiveness and side effects during a high carbon dioxide-emission scenario[END_REF]. Ocean fertilization with macro-nutrients (N, P) has a higher theoretical potential of 1.5 PgC yr-1 [START_REF] Harrison | Global negative emissions capacity of ocean macronutrient fertilization[END_REF], about 50 times more than the maximum simulated effects of Southern Ocean iron fertilization [START_REF] Oschlies | Side effects and accounting aspects of hypothetical large-scale southern ocean iron fertilization[END_REF]; however, very large quantities of macronutrients are needed and the proposed scaling of this technique seems unrealistic (Williamson and Bodle, 2016). For artificial upwelling (upward pumping of nutrient-rich deep waters), the intended carbon removal by increased productivity [START_REF] Lovelock | Ocean pipes could help the earth to cure itself[END_REF]) may be matched by the undesirable release of CO 2 from the deeper water [START_REF] Shepherd | Geo-engineering might cause, not cure, problems[END_REF][START_REF] Dutreuil | Impact of enhanced vertical mixing on marine biogeochemistry: lessons for geo-engineering and natural variability[END_REF]Yool et al., 2009). The potential impact of artificial upwellings on nitrogen fixation, and hence on natural carbon sequestration is controversial [START_REF] Fennel | Widespread implementation of controlled upwelling in the north pacific subtropical gyre would counteract diazotrophic n 2 fixation[END_REF][START_REF] Karl | Nitrogen fixation-enhanced carbon sequestration in low nitrate, low chlorophyll seascapes[END_REF]. Some modelling studies indicate that net CO 2 drawdown is theoretically possible if upwelling rates are increased in appropriate locations in 2100 for RCP8.5. Because of the many associated uncertainties, the overall effectiveness of Fertilization to increase carbon uptake is scored low.

Fertilization has no direct effect on sea level rise nor on ocean warming hence its effectiveness to reduce those drivers is low. Acidification could be ameliorated in the short term in the upper ocean (by CO 2 removal), but enhanced in the long term in the ocean interior (by CO 2 release); the overall effectiveness is considered to be low. Effects would happen through increased carbon uptake but there is limited scope for enhanced ocean productivity and increased carbon uptake due to biological and physicalchemical constraints (Williamson and Bodle, 2016). Mesoscale variability in the strength of different processes responsible for long-term carbon removal [START_REF] Boyd | Should we fertilize oceans or seed clouds? no one knows[END_REF]) make carbon accounting highly uncertain. Furthermore, once Fertilization is started, it would need to be done continuously; if not, a large part of the sequestered carbon will be returned to the atmosphere on decadal timescales (e.g., [START_REF] Aumont | Globalizing results from ocean in situ iron fertilization studies[END_REF]. Ocean Fertilization is considered to have negative consequences for 8 Sustainable Development Goals (SDGs), and a combination of both positive and negative consequences for 7 SDGs (Honegger et al., in press). The cost of iron fertilization has been estimated at 22-119 and 457 US$ per t CO 2 sequestered [START_REF] Harrison | A method for estimating the cost to sequester carbon dioxide by delivering iron to the ocean[END_REF]. There are lower cost estimates for Fertilization using macronutrients, at around US$ 20 per t CO 2 [START_REF] Jones | The cost of carbon management using ocean nourishment[END_REF], but scalability is questionable and monitoring costs are excluded. The median of all estimates is 230 US$ per t CO 2 , indicating very low cost effectiveness.

Sample et al.

Ocean-based Negative Emissions Technologies

Enhancing weathering and alkalinization

Enhancing weathering and alkalinization (hereafter Alkalinization) is the addition of natural or manmade alkalinity (ground carbonate or silicate rocks, such as olivine or basalt), to enhance CO 2 removal and/or carbon storage. Adding huge amounts of alkalinity globally could, in theory, substantially mitigate atmospheric CO 2 without elevating biogeochemical properties significantly beyond naturally occurring levels [START_REF] Ilyina | Assessing the potential of calciumbased artificial ocean alkalinization to mitigate rising atmospheric co-2-and ocean acidification[END_REF][START_REF] Keller | Potential climate engineering effectiveness and side effects during a high carbon dioxide-emission scenario[END_REF].

Since the carbon uptake capacity of this approach is very high, without a determined limit [START_REF] Ciais | Carbon and other biogeochemical cycles[END_REF], its effectiveness to reduce ocean warming and acidification is also very high, with impacts on sea level via surface air temperature [START_REF] González | Impacts of artificial ocean alkalinization on the carbon cycle and climate in earth system simulations[END_REF]. Regional Alkalinization could be effective in protecting coral reefs against acidification (but not warming; [START_REF] Feng | Could artificial ocean alkalinization protect tropical coral ecosystems from ocean acidification?[END_REF]. There is technological readiness to add alkalinity to the ocean, at least at small scales. What is needed is research and testing to acquire adequate knowledge about the environmental co-benefits and disbenefits. Feasibility is also impeded by lack of infrastructure to mine or produce, process and distribute alkalinity at large scales.

Feasibility is therefore scored very low.

A unit of alkalinity added would sequester CO 2 essentially permanently, hence the duration of the effect is scored very high. Some of the inorganic carbon could precipitate as CaCO 3 , releasing CO 2 and reducing the carbon storage and alkalinity co-benefits afforded. The addition of alkalinity would probably have to be continued for decades to centuries (or longer) to have a substantial impact on atmospheric CO 2 [START_REF] Keller | Potential climate engineering effectiveness and side effects during a high carbon dioxide-emission scenario[END_REF].

The cost of various ocean alkalinity carbon storage technologies is largely speculative at this stage. [START_REF] Renforth | Engineering challenges of ocean liming[END_REF] indicated a range of 72-159 US$ per t CO 2 taken up. This range reflects the extraction, calcination, hydration, and surface ocean dispersion costs at a global scale (including transportation). In the case of direct addition of alkaline minerals to the ocean (i.e., without calcination), the cost is 20-50 US$ per t CO 2 [START_REF] Harvey | Mitigating the atmospheric co-2-increase and ocean acidification by adding limestone powder to upwelling regions[END_REF][START_REF] Köhler | Geoengineering impact of open ocean dissolution of olivine on atmospheric co-2-, surface ocean ph and marine biology[END_REF][START_REF] Renforth | Assessing ocean alkalinity for carbon sequestration[END_REF]. Overall, at 10-190 US$ per t CO 2 , the cost effectiveness is moderate.

POLICY-RELEVANCE OF NEGATIVE EMISSION TECHNOLOGIES

Understanding the policy relevance of NETs requires putting them into a broader context of global and/or local climate action. Here we consider 12 ocean-related measures to enhance both global mitigation and local coastal ecological adaptation1 (Fig. 2). These options can be clustered into three overarching policyrelevant clusters (Decisive, Low Regret, Concept Stage) that are defined in Panel A of Fig. 2 according to their state of implementation, effectiveness to reduce climate-related ocean drivers globally, effectiveness to reduce impacts/risks locally, and potential co-benefits and disbenefits (i.e. associated adverse impacts and other undesirable consequences, including opportunity costs).

Cluster 1 -The only ocean-based Decisive measure that we have identified addresses the causes of climate change: Marine renewable energy. It includes energy from offshore winds, tides, waves, ocean currents as well as thermal gradients. This action has the theoretical potential to meet all global electricity requirements, although requiring massive infrastructure development. Whilst some local adverse impacts are inevitable, these can be minimised.

Ocean-based Negative Emissions Technologies

Cluster 2 -Low Regret measures provide both climatic and non-climatic benefits, with few disbenefits.

For example, Conservation measures can protect carbon-rich coastal ecosystems from direct human disturbance and loss, and play an important role in limiting local climate impacts. Similarly, Restoring and enhancing coastal vegetation supports ecological adaptation whilst providing storm protection, contributing to food security, and enhancing biodiversity. It can also increase carbon uptake (i.e. addressing the causes of climate change), at levels that may be locally and/or nationally significant. Nevertheless, because of the limited total area for restoring such blue carbon ecosystems and also associated methane emissions, this action can only make a very small contribution to climate mitigation at the global scale [START_REF] Ipcc ; H.-O. Pörtner | Summary for policymakers[END_REF][START_REF] Al-Haj | A synthesis of methane emissions from shallow vegetated coastal ecosystems[END_REF]. Pollution reduction in coastal waters removes contaminants and excess nutrients that impair ecosystem function, thereby supporting ecosystem-based adaptation. Reducing atmospheric pollution from shipping can also, to a limited degree, address the causes of climate change. However, Low Regret measures are not cost-free, requiring well-informed planning and effective coordination over a wide range of spatial and temporal scales.

Cluster 3 -Concept Stage measures are illustrated by the NET Marine BECCS, and some forms of Assisted evolution (alterations to species and genetics). The former would use cultivated macroalgae (or possibly microalgae) as the biomass source for bioenergy. Such measures have potential but their feasibility and cost-effectiveness for climatic benefits have yet to be demonstrated. Several measures have potentially high disbenefits, as identified for Fertilization, Alkalinization and the sunlight reflection techniques of marine Cloud brightening and Surface albedo enhancement (e.g. covering the ocean surface with reflective foam). Whilst all these approaches have a very large theoretical potential to address climate change globally, only Fertilization has been investigated through field experiments (although not carried out for climate mitigation purposes), with limited success. Much more attention needs to be given to their governance and public acceptability before they can be considered for implementation as climate policy responses. This agrees with and extends the conclusion of [START_REF] Boyd | Should we fertilize oceans or seed clouds? no one knows[END_REF] for marine geoengineering.

The effectiveness of measures such as Assisted evolution to support ecological adaptation critically depends on the environmental and societal contexts of their implementation. For example, while some Assisted evolution tools could have local benefits, others such as the spread of genetically alien populations have serious risks.

The global governability of NETs refers in this study to the potential capability of the international community (both nation states and international non-state actors) to implement them, managing associated conflicts and harnessing mutual benefits, It is therefore a key dimension to be considered when discussing the potential role of NETs within a broader range of climate actions. In our assessment, global governability is considered moderate to very high for more than half of the measures considered (Fig. 1), but low to very low for Fertilization and Alkalinisation as well as for Assisted evolution and the two SRM options (Cloud brightening, Surface Albedo enhancement). Marine BECCS score is moderate: although this measure could have high co-benefits and could be implemented at modest scale without having to rely on international cooperation, wider scale implementation could raise serious national and international complications, hence potentially requiring new governing arrangements and accounting, monitoring, and verification entities. Alkalinization and Fertilization score very low, but for different reasons: concerns regarding their potential adverse environmental impacts result in low public acceptability, reflected in political rejection of such methods (and legal constraints) by the London Convention/London Protocol 2 , the UN Convention on Biological Diversity, and other bodies. These findings align with recent conclusions that "marine geoengineering" challenges the ability of the international law system, as it stands now, to govern the implementation of these ideas [START_REF] Mcgee | Geoengineering the oceans: an emerging frontier in international climate change governance[END_REF].

CONCLUSION AND ACTIONABLE RECOMMENDATIONS

Other than by eliminating nearly all anthropogenic greenhouse gas emissions, there is no single measure to dramatically accelerate progress in achieving the net zero target of the Paris Agreement. Nevertheless, opportunities exist for all coastal countries to implement a wide range of ocean-based climate actions, including negative emission approaches. Guidelines and actionable recommendations are required to address the urgency of climate action. Marine geoengineering techniques (e.g., [START_REF] Boyd | Should we fertilize oceans or seed clouds? no one knows[END_REF] and other ocean-based measures need to be assessed and ranked (e.g., [START_REF] Gattuso | Ocean solutions to address climate change and its effects on marine ecosystems[END_REF]. Here we build on previous assessments to provide a first attempt to define policy-relevant clusters for climate action, both

  global mitigation and local ecological adaptation. None of the four ocean-based NETs assessed are Decisive, one is Low Regret (Restoring and increasing coastal vegetation), and three are at Concept Stage with either low to moderate potential disbenefits (Marine bioenergy with carbon capture and storage) or with high potential disbenefits (Enhancing open-ocean productivity and Enhancing weathering and alkalinization). The next iteration of more ambitious NDCs should scale up ocean-based climate action by prioritising the implementation of Decisive and Low Regret measures, and urgently improving knowledge on Concept Stage measures. Decisive and Low Regret measures are both key priorities for implementation because, on the one hand, the full implementation of Decisive measures will not completely eliminate coastal risks while, on the other hand, the effectiveness and security of Low Regret conservation/nature-based solutions are being put at risk by increasing climate change and rising sea level. Concept Stage measures require further scientific investigations because the full implementation of proven measures runs the risk of falling short of providing enough cost effective NETs capacity.Balancing ocean-based measures also calls for securing robust national-to-local enabling conditions and enhanced international support for climate action. This is important because the majority of ocean-inclusive NDCs are conditional on external financing and support. They were discussed at COP25, referred to the "Blue COP", and could be, with nature-based solutions, one of the priority concerns for COP26 and the subsequent Global Stocktake in 2023. Enhancing the enabling conditions can be achieved by (i) strengthening the formal recognition of the ocean-climate nexus and the ocean as a provider of solutions for climate change, and (ii) facilitating the UNFCCC Party delegations' understanding of the role of ocean-related measures and how to include them in the next generation of NDCs.

Figure 1 .

 1 Figure 1. Fig. 1-Assessment of ocean negative emissions approaches (in bold) compared with other ocean-based measures. Based on Gattuso et al. (2018), except for marine BECCS (see SM3), with the following changes: Vegetation and Alkalinization are only considered at global scale except the co-benefits and disbenefits which are considered at local scale. Two criteria are newly defined by combining criteria assessed earlier. Feasibility is the mean of Technological readiness and Lead time until full potential effectiveness. Effectiveness to reduce OW/OA/SLR (ocean warming, ocean acidification and sea level rise) is the mean of the effectiveness to Moderate warming, Moderate acidification and Moderate sea level rise. Grey tiles indicate criteria not assessed for the corresponding measure. Further information on the measures and criteria are available in Supplementary Information SM1 and SM2.

This is a provisional file, not the final typeset article

Societal adaptation measures such as infrastructure-based or community-based adaptation, risk reduction policies improvement, and the relocation of people, assets and economic activities are not included.

As regulated by the London Protocol, with an amendment prohibiting such action unless constituting legitimate scientific research authorised under permit. That amendment has not yet legally entered force.This is a provisional file, not the final typeset article

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ACKNOWLEDGMENTS

Thanks are due to the coauthors of the papers by Gattuso et al. (2018) and Gattuso et al. (2019). This work is a product of The Ocean Solutions Initiative (http://bit.ly/2xJ3EV6) and the STORISK project (https://bit.ly/2YMhfZX). Comments from L. Kapsenberg and three anonymous reviewers have significantly improved an earlier version of this paper.

FUNDING

The Ocean Solutions Initiative is financially supported by the Prince Albert II of Monaco Foundation, the Veolia Foundation, the IAEA Ocean Acidification International Coordination Centre and the French Facility for Global Environment, and the STORISK project (French National Research Agency, ANR-15-CE03-0003). The ANR programme "Investissements d'avenir" (ANR-10-LABX-14-01) provides support to Iddri.

DATA AVAILABILITY STATEMENT

No new data was generated in this policy brief.

CONFLICT OF INTEREST STATEMENT

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.

AUTHOR CONTRIBUTIONS

All co-authors contributed ideas, JPG wrote the first draft which was then finalized by all co-authors.

Ocean-based Negative Emissions Technologies

Waycott, M., Duarte, C., Carruthers, T., Orth, R., Dennison, W., Olyarnik, S., et al. (2009)