Vita Nuning

Pascale Hidayati

Laurence Prudent

Laurent Asia

Franck Vassalo

Ita Torre

Agus Widowati

Agung Dhamar Sabdono

Pierre Syakti

Nuning Vita Hidayati

Pascale Prudent

Laurence Asia

Laurent Vassalo

Franck Torre

Ita Widowati

Agus Sabdono

Agung Dhamar Syakti

Pierre Doumenq

Assessment of the ecological and human health risks from metals in shrimp aquaculture environments in Central Java, Indonesia

Keywords: Sustainable aquaculture, Litopenaeus vannamei, Food safety, Trace metals, Pollution index

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

Introduction

World aquaculture production continues to increase, with shrimp farming representing one of the major aquaculture industries. The shrimp market has become the most valuable globally traded seafood commodity and had the highest production value in 2016. In the same year, FAO data classified Indonesia as the third largest aquaculture producer, with production reaching 4,950,000 tons, which accounted for 6.18% of the world's aquaculture production (FAO 2018a). In Indonesia, white-legged shrimp, also known as the king prawn (Litopenaeus vannamei), is the dominant farmed spe-cies, and it generates the highest revenue on a per kg basis compared to the other fishery commodities (KKP 2017).

The expansion and intensification of this sector have em-phasized the need for continuous monitoring and control of environmental quality, which is essential for ensuring the sus-tainability of aquaculture and the safety of the product [START_REF] Paul | Organic shrimp aquaculture for sustainable household livelihoods in Bangladesh[END_REF] based on the 14th indicator of the Sustainable Development Goals Agenda 2030, which aimed to ensure the contribution and conduct of fisheries and aquaculture towards food security and nutrition (FAO 2018b). Good water quality is also essential in aquaculture to maintain the health, optimal growth, and survival of the cultured species [START_REF] Mohanty | Water quality suitability and water use indices: useful management tools in coastal aquaculture of Litopenaeus vannamei[END_REF][START_REF] Suantika | Development of a zero water discharge (ZWD)-recirculating aquaculture system (RAS) hybrid system for super intensive white shrimp (Litopenaeus vannamei) culture under low salinity conditions and its industrial trial in commercial shrimp ur-ban farming in Gresik, East Java, Indonesia[END_REF].

Shrimp aquaculture environments face natural and human impacts from urban and suburban areas. In this study, we focused on seven important toxic metals (Cd, Cu, Co, Cr, mercury (Hg), Pb, and Zn) and three additional metals (Al, Fe, and Mn) related to their transportation. These metals can enter aquatic environments through surface runoff and from natural sources, such as geologic weathering and atmospheric inputs, or they can be derived via effluent discharge from multiple anthropogenic sources, including industrial, agricul-tural [START_REF] Gu | Identification of heavy metal sources in the reclaimed farmland soils of the pearl river estuary in China using a multivariate geostatistical approach[END_REF], mining, shrimp farming [START_REF] Lacerda | Copper emission factors from intensive shrimp aquaculture[END_REF], and domestic effluents [START_REF] Ju | Assessment of heavy metals in aquaculture fishes collected from southwest coast of Taiwan and human consumption risk[END_REF][START_REF] Sarkar | Assessment of heavy metals contam-ination and human health risk in shrimp collected from different farms and rivers at Khulna-Satkhira region, Bangladesh[END_REF].

Heavy metal contamination is becoming a major con-cern in coastal systems, which are often associated with shrimp farming activity [START_REF] Syakti | Heavy metal concentrations in natural and human-impacted sediments of Segara Anakan Lagoon, Indonesia[END_REF], due to the ability of heavy metals to persist in the environment [START_REF] Ju | Assessment of heavy metals in aquaculture fishes collected from southwest coast of Taiwan and human consumption risk[END_REF]. Heavy metals are also toxic at high concentra-tions [START_REF] Sfakianakis | Effect of heavy metals on fish larvae deformities: a review[END_REF] and could pose risks to hu-man health [START_REF] Fakhri | Systematic review and health risk assessment of arsenic and lead in the fished shrimps from the Persian gulf[END_REF][START_REF] Garcia-Hernandez | Mercury con-centrations in seafood and the associated risk in women with high fish consumption from coastal villages of Sonora, Mexico[END_REF][START_REF] Zhang | Risk assessment of total mercury and methylmercury in aquatic products from offshore farms in China[END_REF], which is why exposure must be evaluated.

In the present study, we monitored the occurrence and evaluated the state of contamination of ten metals (Al, Cd, Co, Cr, Cu, Fe, Mn, Pb, Zn, Hg) in three environmental ma-trices, i.e., water, sediment, and shrimp, from the aquaculture area in Indonesia, which is located on the northern coast of Central Java in the Brebes, Tegal, Pekalongan, and Pemalang regions. Some studies have reported that aquatic organisms tend to take metals up from their direct environment [START_REF] Núñez-Nogueira | Accumulation and regulation effects from the metal mixture of Zn, Pb, and Cd in the tropical shrimp Penaeus vannamei[END_REF][START_REF] Wang | Heavy metal pollution in coastal areas of South China: a review[END_REF]. For water samples, we investigated the occurrence of metals in the environment and evaluated their contamination and/or status in order to determine whether the quality of the aquatic environment is suitable for intended uses, i.e., aquaculture. Our evaluation based on the extent of dissolved metals in water compared acceptable or safe metal levels via direct comparisons and appropriate indexes. Sediment is a primary location for metal accumulation in the environment and is the main source of contamination in the water column due to resuspension and trapping processes associated with salinity or metal dissolu-tion through biotic/abiotic processes. As a valuable commod-ity, shrimp muscle tissue must be monitored for contaminants, e.g., heavy metals, because these animals live in water and are in contact with sediments. Several studies have documented the ability of shrimp to accumulate these metals in their tissues since many metals have the ability to form complexes with organic substances [START_REF] Jonathan | Bioaccumulation of trace metals in farmed pacific oysters Crassostrea gigas from SW Gulf of California coast, Mexico[END_REF][START_REF] Nascimento | Trace metal distribution in white shrimp (Litopenaeus schmitti) tissues from a Brazilian coastal area[END_REF][START_REF] Pourang | Tissue distribution and redistribution of trace elements in shrimp species with the emphasis on the roles of metallothionein[END_REF][START_REF] Ruelas-Inzunza | Distribution and concentration of trace metals in tissues of three penaeid shrimp species from Altata-Ensenada del Pabellón Lagoon (S.E. Gulf of California)[END_REF][START_REF] Qiu | Bioaccumulation of heavy metals both in wild and mariculture food chains in Daya Bay, South China[END_REF][START_REF] Zhang | Heavy metals in aquatic organisms of different trophic levels and their potential human health risk in Bohai Bay, China[END_REF]). These metals do not tend to be excreted and thus may be passed up the food chain to higher organisms, including humans [START_REF] Soto-Jiménez | Trophic transfer of lead through a model marine four-level food chain: Tetraselmis suecica, Artemia franciscana, Litopenaeus vannamei, and Haemulon scudderi[END_REF]. Due to its high toxicity, we also evaluated the levels of Hg in the edible muscles of the shrimp. In this study, we investigated the occurrence of metals in the environment and the contamination status and assessed the potential ecological and human health risks.

Materials and methods

Study area

Sampling campaigns were carried out in shrimp farms in four major areas located on the north coast of Central Java, i.e., the Brebes, Tegal, Pekalongan, and Pemalang regions (Fig. 1). These different regions were selected as the target study areas since they are the highest producers of shrimp in Central Java, which is characterized by water sources from different riverine systems that are among the most important in Indonesia. In each selected area, twenty ponds were selected: 4 ponds in Brebes, 4 in Tegal, 5 in Pekalongan, and 7 in Pemalang. The sample number from each site was determined based on the number of shrimp ponds in each area. Brebes Regency is a shrimp farming center in Central Java that covers 14 ha of intensive shrimp farming and 403 ha of extensive shrimp farming. However, shrimp production in Brebes has de-creased since 2016 due to environmental pressures, such as industrial waste, agricultural fertilizers, onion/shallot farming activities, industrial activities, and settlements [START_REF] Bayuaji | Environmental quality of shrimp ponds in the coastal region of Brebes Regency[END_REF]. It is well documented that there are many problems associated with heavy metal contamination in this area [START_REF] Ismanto | The association of lead levels and malondialdehyde levels of pregnant woman in coastal area of Brebes District[END_REF][START_REF] Hartini | Kadar plumbum (pb) dalam umbi bawang merah di kecamatan kersana kabupaten brebes[END_REF].

The shrimp farms in Tegal have a lowland topography with upstream rivers flowing into the Java Sea. Tegal has four rivers that pass through 16 villages (59.26%). Similar to Brebes, L. vannamei shrimp farms have shown declining shrimp production, and the two largest threats to production are the spread of shrimp white feces disease (0.76) and envi-ronmental pollution (0.67). Pemalang consists of a 363.82 km 2 wetland area and a 751.48-km 2 nonwetland area (BPS 2018c). The population of Pemalang Regency generally lives in coastal areas in city centers, and the number of people living in coastal areas accounts for 57.77% of the total population [START_REF] Wahyuningrat | Practices of illegal fishing in Pemalang region: a policy analysis[END_REF]. The area of farms in this region is 1728 ha and includes milkfish and shrimp farms. Moreover, the fisheries sector is the largest contributor to the gross re-gional domestic product in this regency [START_REF] Wahyuningrat | Practices of illegal fishing in Pemalang region: a policy analysis[END_REF]). In parallel with the contribution of the shrimp sector to the local economy of the region, there are many problems in local ecosystems caused by anthropogenic activity, including Pb and Cd contamination, which might originate from domes-tic waste in the upstream areas carried by the Comal River [START_REF] Puspita | Water quality appropriate-ness for some allotment in coastal area (case study: Pesantren and Mojo Village, Ulujami, Pemalang)[END_REF][START_REF] Yulianto | Heavy metals (Cd, Pb, Cu, Zn) concentrations in edible bivalves harvested from northern coast of Central Java, Indonesia[END_REF]) and deterioration of soil and water quality related to mangrove exploitation in this coastal region [START_REF] Sawitri | Riverine quality of wetlands in Comal River, Pemalang and Kedung Coet river, Indramayu[END_REF]. Pekalongan has a coastline along the Java Sea of approximately 6 km stretching from west to east. The coastal area of Pekalongan City has two large river estuaries, namely the Pekalongan River estuary and the Sodetan River estuary [START_REF] Rachmansyah | Characteristics, suitability, and management of land for brackishwater ponds in Pekalongan City, Central Java Province[END_REF]. In 2017, Pekalongan had a brackish farm area of 379 ha, with 752 aquaculture households in this region (BPS 2018b). More than 1500 small-scale textile industries, namely batik indus-tries, have developed rapidly in this region and may represent an important contributor of Cr enrichment in this area [START_REF] Apriliani | Influence of characteristics of entrepre-neurs, capital and labor on the success of batik industry (ind.)[END_REF].

Sample collection

Water and sediment samples were collected during the dry season (August 2017), while shrimp samples were collected in both the dry season (simultaneously with water and sedi-ment samples) and wet season (March 2018). In each pond, surface water samples were collected at three different points in the pond, i.e., the inlet (before water enter to the pond), pond (inside the pond), and outlet (after the discharge gates), using polypropylene samplers that were prewashed with 10% nitric acid and then rinsed with distilled water. The water samples from each pond were prepared as composite samples by mixing water from three subpoints at each collection site, and then, they were transferred in acid-washed plastic bottles, placed in an ice box at 4 °C, and transported to the laboratory. In the laboratory, the samples were filtered using 0.45-μm Whatman mixed cellulose ester (MCE) membrane filters, pre-served at 4 °C in 20-mL polyethylene bottles, and acidified with 1% nitric acid (HNO 3 ) to pH 1.5-2.0 until analysis.

Regarding the sediments from each pond, bulk samples representing the recently deposited material in the shrimp ponds were collected using a 10-L perforated plastic bucket at the pond siphoning system outlet, and they were collected five times with a 15-min interval to generate subsamples. Dewatering was conducted by decantation, and a composite sample for each pond was created by mixing the 5 subsamples in the same quantities. The solid mixed subsamples of sediment/sludge were immediately placed in labeled polyeth-ylene zip bags and stored in a cool box to minimize microbial degradation during transport to the laboratory. The sediment samples were subsequently freeze-dried to a constant weight (approximately 72 h), disaggregated, sieved through 2-mm stainless steel sieves, and then ground using a Retsch RM 200 agate mortar grinder. The sediments were then stored in hermetic plastic vials until analysis. Shrimp (size 15.02-32.54 g in the dry season and 15.61-31.07 g in the wet season; Electronic Supplementary Material 1, ESM 1) in the intermediate-final stage of the culture cycle (84-110 days of culture (DOC)) were collected using a non-metallic (nylon) net at different points in each pond and then immediately transferred into labeled polyethylene zip bags and stored in a cool box for transport to the laboratory. A composite sample for each pond was created by mixing to-gether the same quantity of each shrimp subsample (n = 10). The muscle tissue samples were freeze-dried to a constant weight (approximately 72 h), homogenized with a Retsch RM 1000 ultracentrifugal mill, and stored at -20 °C until processing for the metal analyses.

Sample digestion and element analysis

The sediment samples were digested using microwave-assisted acid digestion methods: ISO 11466 (1995). Briefly, approximately 0.5 g of the ground freeze-dried sediment sample was digested in a microwave digestion system (Milestone Start D) using a mixture of 7 mL of 37% hydrochloric acid (HCl) (trace metal grade) and 3.5 mL of 65% HNO 3 (trace metal grade) at 200 °C for 40 min. The obtained solutions were transferred into a 50-mL graduated flask, filtered through a 0.45-μm mixed cellulose ester membrane filter, and then stored in high-density polyethylene (HDPE) flasks at 4 °C [START_REF] Ahmed | Levels and sources of heavy metals and PAHs in sediment of Djibouti-city (Republic of Djibouti)[END_REF]. The blank and reference material digestion procedures were also conducted as described above.

The shrimp muscles were ground (Retsch ZM 1000 with tungsten blades) to pass through a 0.2-mm titanium sieve. Then, 0.5 g was weighed and placed in a Teflon digestion reactor, to which 3 mL of concentrated HCl and 6 mL of concentrated HNO 3 were added (trace metal grade acids). The samples were then digested in a microwave digestion system (Milestone Start D) and mineralized consecutively at 110 °C for 10 min, 150 °C for 10 min, and 180 °C for 5 min. The supernatant was then transferred into a 25-mL volumetric flask and filtered using a 0.45-μm MCE membrane filter.

Quality assurance and quality control

The method of analysis was validated using Canadian Certified Reference Materials (STSD-3) for the sediments and DORM-4 certified reference material for the shrimp sam-ples (National Research Council of Canada). The accuracy was checked by analyzing the certified reference material un-der the same conditions. During the analysis, a verification of the stability of the measurement was carried out every 12 to 15 samples through the measurement of a standard or these qual-ity controls (QCs), and the acceptance was carried out for a maximum drift of 5%. The blanks and standard solutions were prepared and included in the measurements to define the back-ground correction and any form of unintended contamination during the sample preparation.

The detection limits (μg L -1 ) of Al, Cd, Co, Cr, Cu, Fe, Mn, Pb, Zn, and Hg were 3.76, 0.48, 0.99, 6.21, 1.39, 115.36, 0.27, 14.94, 13.38, and 0.45 in the water samples, respectively; 1.43, 0.06, 0.06, 0.21, 0.11, 3.18, 0.12, 9.64, 0.25, and 0.02 ) of Al, Cd, Co, Cr, Cu, Fe, Mn, Pb, Zn, and Hg were 12.53, 1.61, 3.31, 20.70, 4,62, 384.53, 0.91, 49.80, 44.60, and 1.43 in the water samples, respectively; 4.77, 0.19, 0.21, 0.71, 0.35, 7.45, 0.39, 11.74, 0.82, and 0.07 in the sediment samples, respectively; and 94.06, 0.36, 0.32, 1.03, 10.50, 10.95, 0.88, 3.68, 3.14, and 0.07 in the shrimp muscles, respectively. The blank samples containing the acids used for digestion were also processed in the same way. All mineralized samples were then stored at 4 °C in HDPE flasks until analysis. The metal and trace metal concentrations in the water samples and digestion samples (except mercury) were measured using an inductively coupled plasma atomic emission spectrometry (ICP-AES) system (Horiba Jobin Yvon JY2000-2), while mercury was analyzed after reduction and cold vapor genera-tion using flame atomic absorption spectrometry (FAAS) (Thermo Scientific ICE 3000). Quality assurance, quality con-trols, accuracy, and precision were checked using the standard sediment reference materials (STSD-3, CCRMP-PCMRC, Canada) and fish protein certified reference material (DORM-4, NRC-CNRC, Canada), with accuracies within 100 ± 10%. The low RSD% of the concentration values of each metal did not exceed 10% using the proposed method, thus indicating the method precision.

Occurrence of metals in water bodies and assessment of metal water quality

The most widely used method for calculating contamination and/or pollution indexes is the Nemerow pollution index, which includes a single pollution index for a particular heavy metal (P i ) and a comprehensive pollution index (P n ) [START_REF] Dadzie | Survey and assess-ment of metals distribution in the overlying water of the representa-tive lake and rivers in Zhenjiang, China[END_REF][START_REF] Yan | Distribution and assessment of heavy metals in the surface sediment of Yellow River, China[END_REF]). The single-factor index reflects the contamination degree of every metal, while the P n reflects the overall effect of all metals on water quality.

The single-factor index method of the Nemerow pollution index (P i ) was calculated using the following equation (Gąsiorek et al. 2017):

where P i represents the single-factor index, C i is the measured concentration of metals in water, and S i is the standard value for the aquatic body as the organisms' habitat as proposed by different guidelines (ESM 2). The single-factor pollution in-dex is categorized into five grades, where P i ≤ 1 represents nonpolluted water, 1 < P i ≤ 2 signifies slight pollution, 2 < P i ≤ 3 indicates mild pollution, 3 < P i ≤ 5 represents moderate pollution, and P i ≥ 5 indicates heavy pollution [START_REF] Dadzie | Survey and assess-ment of metals distribution in the overlying water of the representa-tive lake and rivers in Zhenjiang, China[END_REF].

The comprehensive Nemerow pollution index reflects the degree of contamination from all the measured metals and is calculated as follows [START_REF] Brady | Development of a hybrid pollution index for heavy metals in marine and estuarine sediments[END_REF][START_REF] Liu | Heavy metals distribution and environmental quality assessment for sediments off the southern coast of the Shandong Peninsula, China[END_REF][START_REF] Zhang | A water quality management strategy for regionally protected water through health risk assessment and spatial distribution of heavy metal pollution in 3 marine reserves[END_REF]):

where P n represents the composite index, which indicates the degree of heavy metal pollution in the water; avg iϵI P 2 represents the average value of Pi2; and max Pi2 is the maximum value of Pi2. [START_REF] Liu | Heavy metals distribution and environmental quality assessment for sediments off the southern coast of the Shandong Peninsula, China[END_REF] categorized the Pn values as follows: Pn < 0.7, uncontaminated water; 0.7 ≤ Pn ≤ 0.7, under the threat of contamination; and Pn > 1, heavily contaminated water.

Assessment of the sediment

Sediment quality guidelines

A comparison of the measured concentrations of various con-taminants within the sediments with these guideline values will provide a basic indication of the degree of contamination and likely impact on the ecosystem. In the present study, two sets of sediment quality guidelines (SQGs), i.e., the threshold effects level (TEL), probable effects level (PEL), effects range low (ERL), and effects range median (ERM), from the Canadian sediment quality guidelines were applied to assess the possible risk from metal contamination in the sediment [START_REF] Ccme ; Cheng | Canadian environmental quality guidelines Chapter 6: Canadian sediment quality guidelines for the protection of aquatic life Canadian Council of Ministers of the Environment[END_REF].

Enrichment factor

The enrichment factor (EF) is a geochemical index used to evaluate a possible source of anthropogenic activities for trace metals using the normalization of metal concentrations by uncontaminated background values [START_REF] Dickinson | Heavy metal history from cores in Wellington arbour, New Zealand[END_REF]. The EF was calculated based on the following equation [START_REF] Gargouri | Use of sediment quality indicators for heavy metals contamination and ecological risk assessment in urbanized coastal zones[END_REF] ; Nowrouzi andPourkhabbaz 2014): where EF is the enrichment factor of an element, (C i /C Fe ) s represents the metal/Fe ratio of the sample concentrations at each sampling point, and (C i /C Fe ) r is the corresponding element/Fe ratio of the concentrations in the reference sedi-ments. Ideally, the reference sediment values for metals would be estimated from in situ sediments, although in the absence of these data, earth's crust values can sometimes be substituted as shown in [START_REF] Abrahim | Assessment of heavy metal enrichment factors and the degree of contamination in marine sediments from Tamaki Estuary, Auckland, New Zealand[END_REF]. For this study, the average shale concentration of the sedimentary rock in earth's crust [START_REF] Turekian | Distribution the elements in some major units of the earth's crust[END_REF]) was adopted as the base-line value. In this work, we employed Fe as a conservative element for geochemical normalization of the heavy metal data in the EF calculation because Fe is one of the three con-servative elements generally used [START_REF] Ganugapenta | Assessment of heavy metal pollution from the sediment of Tupilipalem coast, southeast coast of India[END_REF][START_REF] Pandey | Ecological risk assessment of soil contamination by trace elements around coal mining area[END_REF]. The six contaminants were categorized based on their enrichment factors.

Geoaccumulation index

The geoaccumulation index (Igeo) has been widely used to evaluate the degree of metal contamination or pollution in terrestrial, aquatic, and marine environments [START_REF] Kabir | Correlation assessment and monitoring of the potential pollutants in the surface sediments of Pyeongchang River, Korea[END_REF][START_REF] Muller | Index of geoaccumulation in sediments of the Rhine River[END_REF]) and is calculated using the following equation:

where Ci is the measured concentration of the examined metal (i) in the sample and Bi is the background concentration of the corresponding metal (i). In this study, we used the following as the background concentrations: 17 mg kg-1 for Co and Pb (McLennan 2001), 35 mg kg-1 for Cr, 25 mg kg-1 for Cu [START_REF] Ranjan | Trace metal distribution, assessment and enrichment in the surface sediments of Sundarban mangrove ecosystem in India and Bangladesh[END_REF], 56,000 mg kg-1 for Fe [START_REF] Taylor | Abundance of chemical elements in the continental crust: a new table[END_REF], 292.2 mg kg-1 for Mn [START_REF] Xavier | Determination of geochemical background values on a tropical es-tuarine system in a densely urban area. Case study: Capibaribe es-tuary, northeastern Brazil[END_REF]), and 59.11 mg kg-1 for Zn [START_REF] Yang | Distribution and enrichment of trace metals in surface marine sediments collected by the manned submersible Jiaolong in the Yap Trench, northwest Pacific Ocean[END_REF]. A factor of 1.5 was applied to the geochemical background concentration to minimize the pos-sible variations in the background value of a given metal due to terrigenous effects [START_REF] Lizárraga-Mendiola | Environmental assessment of an active tailings pile in the state of Mexico (Central Mexico)[END_REF][START_REF] Lu | Contamination assessment of copper, lead, zinc, manganese and nickel in street dust of Baoji, NW China[END_REF].

Contamination factor and pollution load index

The contamination factor (CF) is used to determine the con-tamination status of a sediment and is the ratio of the concentration of a metal in the sediment sample to that in the back-ground. The CF was calculated using the following equation [START_REF] Hakanson | An ecological risk index for aquatic pollution control.a sedimentological approach[END_REF]:

The CF values were categorized as follows: CF < 1, low degree of contamination; 1 ≤ CF < 3, moderate degree of contamination; 3 ≤ CF ≤ 6, considerable degree of contami-nation; and CF > 6, very high degree of contamination [START_REF] Satapathy | Spatio-temporal distribution of major and trace metals in estuarine sediments of Dhamra, Bay of Bengal, India-its environmental significance[END_REF]. To estimate the overall pollution status of the samples, the pollution load index (PLI) has also been used. The PLI of a set of n polluting elements is defined as a value calculated from the geometric mean of the respective contamination factors of those elements using the following equation [START_REF] Qing | Assessment of heavy metal pol-lution and human health risk in urban soils of steel industrial city (Anshan), Liaoning, Northeast China[END_REF]:

where CF represents the contamination factor of a metal and n represents a specific metal's contamination factor. The PLI values were used to classify samples as unpolluted (PLI ≤ 1), moderately polluted (PLI = 1-3), highly polluted (PLI = 3-5), or very highly polluted (PLI > 5). To determine the contamination status in the overall zone studied, we used the following relationship:

Potential ecological risk index and integrated ecological risk index

The potential ecological risk of each metal was calculated by the Hakanson equation as previously described by Wu et al. where Eir is the coefficient of potential ecological risk from metal i, Tir is the toxicity coefficient of metal i,and Cif is the accumulation coefficient (contamination factor) of metal i. The toxic response factors used were as follows: Cu = Pb = 5, Cr = 2, Zn = 1, Co = 5, and Mn = 1 [START_REF] Hakanson | An ecological risk index for aquatic pollution control.a sedimentological approach[END_REF]). To evaluate the overall ecological risk status (integrated potential ecological risk index (RI)) of the sediment, the fol-lowing equation is widely used:

where RI is the integrated potential ecological risk factor for the metal in sediments. The terminology used to describe the ecological risk index is as previously defined [START_REF] Hakanson | An ecological risk index for aquatic pollution control.a sedimentological approach[END_REF].

Occurrence and human health risk assessment of the shrimp

Estimated daily intake

The estimated daily intake (EDI) of the different metals was evaluated based on the tissue concentrations of each metal in the shrimp and the average daily shrimp ingestion rate [START_REF] Santos | Assessment of daily intake of trace elements due to consumption of foodstuffs by adult inhabitants of Rio de Janeiro City[END_REF]). The EDI represents the estimated daily intake of metal through the consumption of shrimp by an adult human (in mg kg-1 day-1). The following relationship was used to calculatethe EDI (Liu etal. 2018):

where EF is the exposure frequency (365 days year-1), ED is the exposure duration (70 years, equivalent to the average life span as used by [START_REF] Anandkumar | Human health risk assessment and bioaccumulation of trace metals in fish species collected from the Miri coast, Sarawak, Borneo[END_REF], [START_REF] Liu | Concentration and potential health risk of heavy metals in seafoods collected from Sanmen Bay and its adja-cent areas, China[END_REF], and Keshavarzi et al. ( 2018)), IR is the ingestion rate (g day-1), Cm is the concentration of metal in the shrimp (mg kg-1 dw), WAB is the average adult body weight (kg), and TA is the average lifetime (70 years × 365 days year-1, i.e., 25,550). The daily consumption rate was set at 20.67 g person-1 day-1 as per the report of the Statistics Indonesia database for the fish and shrimp rates of consump-tion (BPS 2016), while the average adult body weight in Asia (including Indonesia) was assumed to be 57.7 kg [START_REF] Walpole | The weight of nations: an estimation of adult human bio-mass[END_REF]. A value of 4.54 was used as the conversion factor to convert the dry weight to wet weight based on a shrimp moisture content of 78% [START_REF] Qiu | Evaluation of a high protein distiller's dried grains product as a protein source in practical diets for Pacific white shrimp Litopenaeus vannamei[END_REF].

Target hazard quotient

The target hazard quotient (THQ) is defined as the maximum tolerable daily intake of a specific metal that does not result in any deleterious health effects. In the present study, we used the THQ to assess the human health risk level due to pollutant exposure from consuming metal-contaminated shrimp. The THQ was calculated as the ratio of the average daily intake of a specific chemical over a lifetime to the oral reference dose (RfD) of the trace metal. This metric is an estimate of the daily oral exposure by a human that does not have an appreciable risk of deleterious effects during a lifetime (USEPA 2002).

The following equation was used to determine the THQ [START_REF] Anandkumar | Human health risk assessment and bioaccumulation of trace metals in fish species collected from the Miri coast, Sarawak, Borneo[END_REF][START_REF] Liu | Concentration and potential health risk of heavy metals in seafoods collected from Sanmen Bay and its adja-cent areas, China[END_REF]

Hazard index

Since exposure is usually not associated with a single toxicant, the hazard index (HI) was developed from the THQs and expressed as the sum of the hazard quotients (USEPA 2018). The hazard index derived for the shrimp was deter-mined according to the following equation:

where THQi is the target hazard quotient of an individual metal and HI is the total hazard index for all metals.

In the present study, HI is the total hazard index for eight metals (Cd, Cr, Co, Cu, Mn, Pb, Zn, Hg). HI > 1 indicates a potential for an adverse effect on human health.

Carcinogenic risk

The carcinogenic risk was estimated as the incremental prob-ability of an individual developing cancer over a lifetime of exposure to a potential carcinogen by employing a target can-cer risk value (USEPA 1989). The carcinogenic health risks related to the consumption of seafood were measured based on the target cancer risk (TR), which was calculated as follows:

where CSFo is the oral carcinogenic slope factor from the Integrated Risk Information System database. For Cr and Pb, the slope values were 0.5 mg kg-1 day-1 and 0.0085 mg kg-1 day-1, respectively. According to the New York State Department of Health (NYSDOH 2007), the threshold TR values for categorizing risk are as follows: TR ≤ 10-6, lowrisk;10-4 to 10-3, moderate risk; 10-3 to 10-1, high risk; and ≥ 10-1, veryhighrisk.

Statistical analysis

A principal component analysis (PCA) was applied to the results to establish the significance at a probability of 95% for the spatial and temporal variations in the metal concentrations in the water, sediments, and shrimp. The possible relationship between the different variables was estimated by determining the correlation coefficients. A high correlation coefficient implies a strong relationship between variables, and although this value is not a measure of quantitative changes of one variable with respect to the other, it is a measure of the intensity of the association between the two variables. All statistical analyses were performed using R statistical software.

Results and discussion

The selected shrimp farms were originally constructed based on single pond operators in which the operations were modified to include reservoirs to treat water from supply canals (river, coastal, or piped from a well) before use. However, the system had no wastewater treatment system for effluents discharged from the culture ponds during operation and har-vest. Generally, the ponds were arranged in rows, with dis-charge canals on opposite sides. The farms were operated with a pond size of 1000-3000 m 2 , a shrimp density of 80-150 m -2 , a culture period of 84-110 days, a feeding period for 4-5 times, a feed conversion ratio (FCR) of 1.2-1.8, and a shrimp size of 15.02-32.54 g piece -1 . The operation applied a 3-10% water exchange periodically. The technology used for white shrimp culture consists of middle (semi-intensive) and high (intensive) levels. The shrimp farm characteristics and management activities during this study are summarized in ESM 3.

Occurrence of metals in the water and assessment of metal water quality

The concentrations of metals observed in the water samples are displayed in Fig. 2. The metal concentrations revealed that Zn and Fe were the dominant metals in shrimp ponds in Brebes and Tegal, with the concentrations ranging from 49 to 681 μg L -1 and from 38 to 169 μg L -1 for Zn, respectively, and from 68 to 279 μg L -1 and from 35 to 404 μg L -1 for Fe, respectively.

Mn and Fe were also the dominant metals in Pekalongan and Pemalang, and the concentrations ranged from 82 to 537 μg L -1 and from 123 to 2939 μg L -1 for Fe, respectively, and from 48.8 to 1428.7μg L -1 and from 39.8 to 1336.8 μg L -1 for Mn, respectively. In the present study, Pb was not detected in the water samples from any of the studied areas (LOD of 14.94 μg L -1

). The Zn concentrations showed similar trends in all regions and increased from the inlets to the ponds and eventually decreased from the ponds to the outlets. These trends indicated the application of metals in aquaculture systems.

One possible source for various metals is from foods enriched by some metals. [START_REF] Katya | Efficacy of inorganic and chelated trace minerals (Cu, Zn and Mn) premix sources in Pacific white shrimp, Litopenaeus vannamei (Boone) fed plant protein based diets[END_REF] investigated the content of trace minerals of the commercial sources of diets applied to L. vannamei and found levels of Zn in the concentration of 29.3-182 ppm. Another study conducted by [START_REF] León-Cañedo | Assessment of environmental loads of cu and Zn from intensive inland shrimp aquaculture[END_REF] found that the main source of Cu and Zn metals in a simulated culture system with L. vannamei was the diet, which accounted for 91.8% of Cu and 97.0% of Zn. Zn is known as an essential nutrient for many physiological functions, such as growth, develop-ment [START_REF] Watanabe | Trace minerals in fish nutrition[END_REF], and immune function [START_REF] Rink | Zinc-altered immune function and cytokine production[END_REF]. Similarly, [START_REF] Lin | Comparison of chelated zinc and zinc sulfate as zinc sources for growth and immune response of shrimp (Litopenaeus vannamei)[END_REF] found that zinc significantly reduced the cumulative mortality of L. vannamei against Vibrio harveyi, indicating an enhance-ment of the immune system and hence an improved resistance of the shrimp. Zn intake could reduce Cd toxicity; thus, the presence of zinc in water plays an important role for shrimp [START_REF] Brzóska | Interactions between cad-mium and zinc in the organism[END_REF].

Mn is ubiquitous in the environment and accounts for ap-proximately 0.1% of earth's crust, and it can also be released into water by discharge from aquaculture activities and industrial activities and the combustion of fossil fuels. [START_REF] Katya | Efficacy of inorganic and chelated trace minerals (Cu, Zn and Mn) premix sources in Pacific white shrimp, Litopenaeus vannamei (Boone) fed plant protein based diets[END_REF] found Mn as one of trace minerals in the com-mercial diet sources for L. vannamei, and the concentration ranged from 25.9 to 132.2 ppm. Mn is essential at low con-centrations but becomes lethal at higher concentrations [START_REF] Srichandan | Distribution of trace metals in surface seawater and zooplankton of the Bay of Bengal, off Rushikulya estuary, East Coast of India[END_REF]. Municipal wastewater discharges, sewage sludge, and emissions from alloys are additional sources of Mn in the environment (WHO 2004). The opera-tion of some metallurgical industries may contribute to Mn contamination in the water in this study area, since this indus-try potentially releases Mn into the environment [START_REF] Rahman | Potential ecological risk assess-ment of heavy metal contamination in sediment and water body around Dhaka export processing zone, Bangladesh[END_REF]. Some metallurgical industries are established in Brebes (BPS 2018a), Tegal (BPS 2018d), and Pekalongan (BPS 2018b).

In the case of Cu, our results showed that Cu concentra-tions (μg L -1

) generally increased from 1.20, 2.84, and 2.63 in the inlets to 1.22, 3.15, and 3.08 in the ponds in Brebes, Pekalongan, and Pemalang, respectively (ESM 4). These re-sults were slightly higher than those of [START_REF] Wang | Contamination assessments of surface water in coastal lagoon (Maluan Bay, China) incorporating biomarker responses and bioaccumulation in hepato-pancreas of exposed shrimp (Litopenaeus vannamei)-an integrative approach[END_REF], who found that the Cu concentration ranged from 0.52 ± 0.33 to 2.49 ± 0.73 μg L -1 in Maluan Bay, China, which is the habitat of L. vannamei [START_REF] Wang | Contamination assessments of surface water in coastal lagoon (Maluan Bay, China) incorporating biomarker responses and bioaccumulation in hepato-pancreas of exposed shrimp (Litopenaeus vannamei)-an integrative approach[END_REF]). The concentrations were also lower than the concentrations that may cause mor-tality and induce adverse shrimp physiological responses. Frías-Espericueta et al. (2008b) determined the survival and possible histological alterations in the gills and hepatopancre-as of L. vannamei juveniles exposed to copper sulfate and found that the mortality of shrimp juveniles during 96 h of exposure was 50% (96-h LC 50 ) at a concentration of 35.12 mg L -1 , whereas the absence of pillar cells, loss of regular structure of the cuticular epithelium, and multifocal necrosis were observed after 3 weeks of exposure to a con-centration of 1.756 mg L -1 . Cu-related histological alterations were also reported by [START_REF] Soegianto | Effects of sublethal copper concentrations on gills of white shrimp (Litopenaeus vannamei, Boone 1931)[END_REF] and [START_REF] Qian | Toxic effect of chronic waterborne copper exposure on growth, immunity, anti-ox-idative capacity and gut microbiota of Pacific white shrimp Litopenaeus vannamei[END_REF] ) [START_REF] Boyd | Risks associated with the use of chemicals in pond aquaculture[END_REF] reported that some trace metals are present as natural components in feeds, impurities in fertilizers, or active substances in pesticides. [START_REF] Katya | Efficacy of inorganic and chelated trace minerals (Cu, Zn and Mn) premix sources in Pacific white shrimp, Litopenaeus vannamei (Boone) fed plant protein based diets[END_REF] analyzed the content of trace minerals of the tradi-tional inorganic and commercial sources of diets applied to L. vannamei and showed that the Cu concentration ranged from 12.8 to 185 ppm. [START_REF] Yuan | Effects of dietary dos-age forms of copper supplementation on growth, antioxidant capacity, innate immunity enzyme activities and gene expres-sions for juvenile Litopenaeus vannamei[END_REF] reported that Cu in copper sulfate and chelated copper compounds are very effective algicides, which are applied to ponds to reduce the abundance of nuisance aquatic plants. [START_REF] Boyd | Risks associated with the use of chemicals in pond aquaculture[END_REF] reported that some trace metals are present as natural components in feeds, impurities in fertilizers, or active sub-stances in pesticides. [START_REF] Katya | Efficacy of inorganic and chelated trace minerals (Cu, Zn and Mn) premix sources in Pacific white shrimp, Litopenaeus vannamei (Boone) fed plant protein based diets[END_REF] analyzed the con-tent of trace minerals of the traditional inorganic and com-mercial sources of diets applied to L. vannamei and showed that the Cu concentration ranged from 12.8 to 185 ppm. Cu was also presented in the chemical additives (lime and chloride) applied to shrimp farms at concentrations ranging from 1.9 to 3.3 μg g -1 dw [START_REF] Lacerda | Copper emission factors from intensive shrimp aquaculture[END_REF]). However, they concluded that aquafeeds are, by far, the largest con-tributor of Cu to shrimp ponds due to the higher Cu con-centrations and the larger amount used. The total Cu load from aquafeeds and other products applied to ponds contrib-uted 194.5 g ha

-1 cycle -1 .
It is well documented that in aquaculture systems of L. vannamei, dietary Cu (along with zinc) plays an important role as a promoter of growth and immune function [START_REF] Ccme ; Cheng | Canadian environmental quality guidelines Chapter 6: Canadian sediment quality guidelines for the protection of aquatic life Canadian Council of Ministers of the Environment[END_REF]. [START_REF] Davis | Dietary copper require-ment of Penaeus vannamei[END_REF] showed that the weight of shrimp increased in response to copper supplementation, indicating a dietary copper requirement for shrimp growth and survival. Furthermore, [START_REF] Yuan | Effects of dietary dos-age forms of copper supplementation on growth, antioxidant capacity, innate immunity enzyme activities and gene expres-sions for juvenile Litopenaeus vannamei[END_REF] demonstrated that dietary chelated copper is effective at improving oxidation resistance and nonspecific immunity of L. vannamei.

To evaluate the metal contamination, the single-factor pol-lution index (P i ) and the Nemerow pollution index (P n ) were calculated, and the results are shown in Table 1, ESM 5, and ESM 6. A comparison with the grades of the P i showed that the surface water of shrimp farms was generally contaminated by Mn and Cd. The P i and P n have been broadly used to evaluate the heavy metal contamination in water and sediment. For example, [START_REF] Zhong | Risk assess-ment and prediction of heavy metal pollution in groundwater and river sediment: a case study of a typical agricultural irrigation area in Northeast China[END_REF] employed these two risk assessment models to determine the heavy metal contamination in groundwater for drinking purposes, [START_REF] Dadzie | Survey and assess-ment of metals distribution in the overlying water of the representa-tive lake and rivers in Zhenjiang, China[END_REF] applied these indexes to determine the contami-nation status of water from lake and rivers, and [START_REF] Zhang | A water quality management strategy for regionally protected water through health risk assessment and spatial distribution of heavy metal pollution in 3 marine reserves[END_REF] used these indexes to evaluate the heavy metal con-tamination of marine reserve waters. Furthermore, many au-thors have used the P i and P n indexes to evaluate heavy metal contamination in sediment [START_REF] Asare-Donkor | Hydrochemical char-acteristics of surface water and ecological risk assessment of sedi-ments from settlements within the Birim River basin in Ghana[END_REF][START_REF] Ashayeri | Geochemical characteristics, partitioning, quantitative source apportionment, and ecological and health risk of heavy metals in sediments and water: a case study in Shadegan wetland, Iran[END_REF][START_REF] Mirza | Contamination, ecological risk and source identification of metals by multivariate analysis in surface sediments of the Khouran Straits, the Persian Gulf[END_REF][START_REF] Yan | Distribution and assessment of heavy metals in the surface sediment of Yellow River, China[END_REF]). In the present study, we applied P i and P n to assess the contamination level of metals in water for shrimp as a brackish aquatic organism, and S i is the standard value for aquatic organisms. [START_REF] Kowalska | Pollution indi-ces as useful tools for the comprehensive evaluation of the degree of soil contamination-a review[END_REF] stated that P i and P n can be used to highlight which heavy metal represents the highest threat at the site. In addition, since different heavy metals may have impacts on one site, [START_REF] Yan | Distribution and assessment of heavy metals in the surface sediment of Yellow River, China[END_REF] stated that the P n index could provide a reasonable interpretation of the heavy metal contamination at each site as a whole.

With regard to the health of the shrimp ponds, for the shrimp L. vannamei, [START_REF] Boyd | Trace metals toxic at high concentrations[END_REF] and [START_REF] Prapaiwong | Trace elements in waters of inland, lowsalinity shrimp ponds in Alabama[END_REF] have proposed safe and acceptable metal levels. Our study showed that metal contamination was within the safe or acceptable levels for the shrimp ponds' carrying ca-pacities. However, Cu and combinations of various metals may represent a hazard to the juvenile shrimp (5-6.5 cm), and a previous study showed that histological effects may be observed at 2.5 μg L -1 of Cu (Fig. 2) (Frías-Espericueta et al. 2008a, b); unfortunately, such analyses are beyond the scope of our study.

Occurrence of metals in the sediment

The maximum mean ± SD, and minimum concentrations (mg kg -1 dw) of the metals in the sediments as well as a comparison with the SQGs are provided in Table 2. The metal concentrations in the shrimp pond sediment showed that Cr, Cu, Pb, and Zn were below the ERL based on the Canadian sediment quality guidelines, which were 81, 34, 46.7, and 150 mg kg -1 dw for Cr, Cu, Pb, and Zn, respectively. According to the US EPA standard, the sediment was moder-ately polluted (25-75 mg kg -1 dw) by Cr (all regions), Cu (except in the Pekalongan region), and Zn (Brebes and Tegal regions).

Enrichment factor

The EF is generally employed to explain the contribution of metals other than those of lithogenic origin and to assess the degree of anthropogenic influence. The EFs of the metals in the sediments of the study area are displayed in ESM 7. The highest EF value was recorded for manganese in all regions (indicating considerable enrichment for Pemalang), while no enrichment of Pb was observed in any region. The EF value of Zn was from 1 to 2, indicating a moderate enrichment by this element. The EF value can also be used to differentiate be-tween crustal and noncrustal sources of trace metals. Metals are considered to originate from the crust when the EF is between 0.5 and 1.5 and from an anthropogenic source when the EF is more than 1.5 [START_REF] Zhang | Riverine composition and estuarine geo-chemistry of particulate metals in China-weathering fea-tures, anthropogenic impact and chemical fluxes[END_REF]. The EF value for the Cr in Brebes was above 1.5, suggesting an anthropogenic source of Cr in this region, while in the other three regions, Cr came from natural sources. The EF values of Pb and Cu were lower than 1.5 in all studied areas, indicating that these metals originated from the crust. In contrast, Mn was considered to have originated from anthropogenic sources in all regions except Pekalongan.

According to [START_REF] Muller | Index of geoaccumulation in sediments of the Rhine River[END_REF], the metal contamination levels were classified into seven levels: uncontaminated (I geo ≤ 0), uncontaminated to moderately contaminated (0 < I geo ≤ 1), moderately contaminated (1 < I geo ≤ 2), moderately to heavily contaminated (2 < I geo ≤ 3), heavily contaminated (3 < I geo ≤ 4), heavily to extremely contaminated (4 < I geo ≤ 5), and extremely contaminated (I geo = 5) in terms of I geo .

As shown in Table 3, almost all metals had I geo index values of less than 0, which suggested that the sediments in the study area were not polluted by these metals. There was no contamination by Mn in Pekalongan, the sediments in Brebes and Tegal were uncontaminated to moderately contaminated, and those in Pemalang were moderately contaminated.

An I geo value of less than 0 indicates a lithogenic origin, while a positive I geo index indicates the enrichment of metals of nonlithogenic origin. The results suggest that Mn was gen-erally enriched by anthropogenic sources while the other metals were of lithogenic origin.

Contamination factor and pollution load index

Based on the CF value (ESM 8), Fe and Pb showed a low degree of contamination in all regions and Mn contaminated the sediment in all regions (moderate degree of contamination in Brebes, Tegal, and Pekalongan and considerable degree of contamination in Pemalang). The PLI value of Pekalongan was less than 0, indicating a low degree of contamination, while that of Brebes, Tegal, and Pekalongan indicated a mod-erate degree of contamination, with values of 1.11 ± 0.08. The PLI indicated a moderate degree of contamination of the study zone (Fig. 3).

Potential ecological risk index

The potential ecological risk index for metal was proposed by [START_REF] Hakanson | An ecological risk index for aquatic pollution control.a sedimentological approach[END_REF] to evaluate the potential ecological risk posed by the metals in sediments based on the toxicity of heavy metals and the response of the environment. ESM 9 shows the value of the ecological risk factor (E i ) and the RI of the sediment. Generally, the highest RI value among the trace metals in the sediments was that of Cu followed by that of Pb, although both still indicated low potential ecological risk.

Occurrence of metals in the shrimp

Generally, there were significant differences (p > 0.05) in the heavy metal concentrations (dry weight) in the whole body tissue of shrimp of different sizes. The pattern of metal Pemalang -0.29 ±0.17 -0.97 ±0.17 -0.48 ±0.17 Among the ten metals, Al presented the highest level in all regions, followed by Fe and Zn. A similar tendency was observed in the wet and dry seasons. [START_REF] Wu | Heavy metal (Pb, Co, Cd, Cr, Cu, Fe, Mn and Zn) concentrations in harvest-size white shrimp Litopenaeus vannamei tissues from aquaculture and wild source[END_REF] found that Fe and Zn were the two most concentrated metals in the muscle tissue of wild L. vannamei, and the order of concentration was Fe > Zn > Cu > Cr > Mn.

In the present study, the Cu concentration (4.08 ± 2.98 mg kg -1 dw) was relatively similar to that reported for the muscle tissues of L. vannamei by [START_REF] Lacerda | Fate of copper in intensive shrimp farms: bioaccumulation and deposition in pond sediments[END_REF], which ranged from 23.2 to 63.4 μg g -1 dw. Previously, [START_REF] Páez-Osuna | Trace metals in the Mexican shrimp Penaeus vannamei from estuarine and marine environments[END_REF] reported Cu concentrations in the muscle tissue of this species in natural populations that varied from 22.7 to 27.5 μg g -1 dw. The metal concentration in L.

vannamei was compared with that of samples from dif-ferent countries as shown in Table 4. Cu, Fe, Mn, and Zn are essential oligo-elements for L. vannamei [START_REF] Frías-Espericueta | Toxicity of metal mixtures to the Pacific white shrimp Litopenaeus vannamei postlarvae[END_REF]), although high concentrations may affect metabolism. Dietary Cu impacts the composition of gut microbiota and subsequently affects the immune response and growth. For instance, the dietary Cu required to meet the growth and immune functions of L. vannamei has been estimated at approximately 30 mg kg -1 [START_REF] Ayisi | Recent studies toward the development of practical diets for shrimp and their nu-tritional requirements[END_REF]. At the higher level of accumulation in the shrimp muscle tended to be in the same decreasing order in Tegal and Pekalongan, in which Al and Fe were the most accumulated and Co was the least accumulated. [START_REF] Wu | Metallothionein induction and heavy metal accumulation in white shrimp Litopenaeus vannamei exposed to cadmium and zinc[END_REF] showed that Cd mostly accumulated in the followed by the gills and muscle. Overall, the descending order of the concentrations of the metals analyzed in this study during the wet season was as follows:

Brebes: Al > Zn > Fe > Cu > Mn > Cr > Hg Tegal: Al > Fe > Zn > Cu > Mn > Cr > Pb > Hg > Cd > Co Pekalongan: Al > Fe > Zn > Cu > Mn > Cr > Pb > Hg > Co > Cd Pemalang: Al > Fe > Zn > Cu > Mn > Cr > Hg > Pb > Cd > Co A similar tendency was observed for metal accumulation during the dry season as follows: Brebes: Al > Zn > Fe > Cu > Mn > Cr > Hg > Cd > Co > Pb Tegal: Al > Fe > Zn > Cu > Mn > Pb > Cr > Hg > Co > Cd Pekalongan: Al > Fe > Zn > Cu > Mn > Pb > Cr > Hg > Co > Cd Pemalang: Al > Fe > Mn > Zn > Cu > Cr > Hg > Pb > Cd > Co Generally, the metal concentrations in the shrimp in the dry season were slightly higher than those in the wet season as shown in Pekalongan (ESM 10).

mg kg

-1

, the survival of L. vannamei has been shown to be slightly depressed as reported by [START_REF] Davis | Dietary copper require-ment of Penaeus vannamei[END_REF]. The average concentrations (μg g -1 dw) of Cu in the stud-ied shrimp muscle in the Brebes, Tegal, Pekalongan, and Pemalang regions were 26.9 ± 8.72, 27.3 ± 2.6, 25.6 ± 4.8, and 36.5 ± 5.3 during the wet season, respectively, and 42.6 ± 2.6, 37.3 ± 6.9, 31.1 ± 5.05, and 31.6 ± 6.5 during the dry season, respectively. [START_REF] Wu | Effects of cadmium and zinc on oxygen con-sumption, ammonium excretion, and osmoregulation of white shrimp (Litopenaeus vannamei)[END_REF] found that Zn affects the gill function of this shrimp species. [START_REF] Frías-Espericueta | Toxicity of metal mixtures to the Pacific white shrimp Litopenaeus vannamei postlarvae[END_REF] studied the effect of exposure to a mixture of Cd-Zn on L. vannamei and showed an antagonistic effect on this species, which aligned with the results of [START_REF] Barata | Toxicity of binary mixtures of metals and pyrethroid insecticides to Daphnia magna Straus. Implications for multi-substance risks assessment[END_REF], who empha-sized that there is a high probability of competition for protein binding sites because Zn, Cu, and Cd are inducers of metallo-thionein synthesis. Thus, the combined effects of Cd and Zn may be expected to be antagonistic.

Concerning Hg, the average concentrations (μg g -1 dw) of Hg in shrimp ranged from 0.16 ± 0.04 (Pemalang) to 1.35 ± 0.89 (Brebes). The Hg source may have originated from the drainage basin (industrial, agricultural, and domestic effluents), where Hg is trapped and accumulates in a mangrove tidal creek, which may be a potential source of contamination in adjacent aquaculture sites [START_REF] Costa | Mercury distri-bution in a mangrove tidal creek affected by intensive shrimp farm-ing[END_REF], or the source may have originated from the shrimp farms because of the large amounts of feed applied during breeding. [START_REF] Lacerda | Mercury emission factors from intensive shrimp aquaculture and their relative importance to the Jaguaribe River estuary, NE Brazil[END_REF] estimated an EF from intensive shrimp farming of 83.5 mg Hg ha

-1 cycle -1
, which is comparable to the Hg emissions from urban wastewaters (200 mg ha -1

) and solid waste disposal (400 mg ha -1 year -1

) from cities located in the NE Brazil.

Estimated daily intake

To evaluate whether the metal levels found in the shrimp samples were safe for consumers, a comparison was per-formed with their tolerable intake. The calculated daily intake of the metals through the consumption of shrimp was below the acceptable daily intake suggested by the joint FAO/WHO Expert Committee on Food Additives and ATSDR for almost all samples with the exception of Cr during the dry season in Tegal and Pemalang (ESM 11). Based on this result, it can be recommended that the consumption of average amounts of the analyzed shrimps does not pose any health risk for shrimp consumers.

Based on the Indonesia Statistics Center database, the con-sumption rate of fresh fish and fish products in Central Java was 20.67 g person -1 day -1 (BPS 2016). By considering the mean body weight of Asian adults as 57.7 kg [START_REF] Walpole | The weight of nations: an estimation of adult human bio-mass[END_REF], the daily intake of metals was calculated. A linear relationship is observed based on the weight applied to esti-mate the EDI and THQ values in Taiwan by [START_REF] Ju | Assessment of heavy metals in aquaculture fishes collected from southwest coast of Taiwan and human consumption risk[END_REF], which was 57.3 kg for female adults aged 19-65 years old.

Target hazard quotient and hazard index

The THQ and HI were calculated to assess the potential risks due to metals from the consumption of shrimp raised in aquaculture ponds. In both the wet season and dry season, the THQ was less than 1, indicating that there was no obvious health risk from the intake of the individual metals. The hazard index value of below 1 also suggested no potential adverse effect from the combined metals from shrimp consumption through-out the year. However, the HI value tended to be higher in the dry season than the wet season except for Brebes (ESM 12). Mercury had a significant influence on the HI value in this region.

Pb had the highest THQ value during the dry season in Pekalongan, followed by Cr. It is well known that Pekalongan is one of the batik industrial cities in Indonesia [START_REF] Rukayah | Public participa-tion in branding road corridor as shopping window or batik industry at Pekalongan[END_REF] and that batik production has become the dominant activity among the local inhabitants [START_REF] Hadiyanto | Environmental analysis of the impacts of batik waste water polution on the quality of dug well water in the batik industrial center of Jenggot Pekalongan City[END_REF]. This industry produces wastewater that contains heavy metals, such as lead, chromium, and copper as a result of the production process [START_REF] Puspita | Kemampuan tumbuhan air sebagai agen fitoremediator logam berat kromium (Cr) yang terdapat pada limbah cair industri batik[END_REF][START_REF] Rashidi | Batik industry synthet-ic wastewater treatment using nanofiltration membrane[END_REF][START_REF] Setiyono | Pengendalian kromium (Cr) yang terdapat di limbah batik dengan metode fitoremediasi[END_REF]. Synthetic dyes contain-ing Pb and Cr are widely used during the coloring process of batiks to increase the bonding strength between the dyes and fabrics [START_REF] Hadiyanto | Environmental analysis of the impacts of batik waste water polution on the quality of dug well water in the batik industrial center of Jenggot Pekalongan City[END_REF]. [START_REF] Madusari | Analisis Kandungan Timbal (Pb) ,Cadmium (Cd) pada Air dan Ikan Bandeng (Chanos chanos) di Tambak Kota dan Kabupaten Pekalongan[END_REF] detected the toxic metals Pb and Cd in the water column and Chanos chanos from a pond in this region. It was assumed that those metals came from the riverine system as the water source for the aquaculture system. Anandriyo Suryo and Indah (2013) reported that the rivers in Pekalongan have been polluted. Because of the limitation of actual wastewater treatment plant facilities, they estimated that the wastewater in Pekalongan that cannot be treated amounted to 3600 m 3 per day from 632 home batik industries in 2011. As the result, wastewater entered into the riverine system. Furthermore, the local societies and batik entrepreneurs lack an awareness of the riverine environments, which leads to worse river pollution.

Target cancer risk

Chromium in the white-legged shrimp tissue is the primary risk for human health [START_REF] Wu | Heavy metal (Pb, Co, Cd, Cr, Cu, Fe, Mn and Zn) concentrations in harvest-size white shrimp Litopenaeus vannamei tissues from aquaculture and wild source[END_REF]. Cr showed a moderate cancer risk (TR = 10

-4 ) for the exposed population in all regions, in both the wet season and dry season (ESM 13). While Pb indicated a low cancer risk during the wet season in all regions, the risk increased from low to mod-erate during the dry season in Tegal and Pekalongan as shown by the value of 10 -5 (between 10 -6 and 10 -4 ) stipulated by the New York State Department of Health (NYSDOH 2007).

Predominant metals in different environmental matrices

The PCA results correlating the extent of the metals in water are presented in Fig. 4. The cumulative variance contribution of PC1 was 44%. PC1 had high loadings and strong correla-tions with Al and Fe (R = 0.79) (Fig. 4a). The Brebes, Pekalongan, and Pemalang districts were correlated with Al and Fe concentrations (ESM 14a). This result confirmed the important contribution of the riverine system to the major metal contaminants rather than anthropogenic inputs, such as the precipitation of aerosol particles released by industrial ac-tivities [START_REF] Ye | Investigation of submicron aerosol characteristics in Changzhou, China: composition, source, and comparison with co-collected PM2.5[END_REF]) and the use of fertilizer in agriculture in the upstream areas [START_REF] Comero | Characterization of the Danube River sediments using the PMF multivariate approach[END_REF]). PC2 accounted for 22.1% of the variations and had high loadings of Zn and Mn, although a low correlation was observed between them (R = 0.12) (Fig. 4a). The most prominent metals at the Tegal district stations were Cu and Cd, which were moderately correlated (R = 0.46). In this district, which has the densest suburban population, the presence of Cu and Cd can be associated with industrial activities, i.e., fishing, farming, and shipyard activ-ity (De [START_REF] Witte | Chemical analyses of dredged spoil disposal sites at the Belgian part of the North Sea[END_REF]).

In the sediment, we observed that PC1 accounted for 44.1% of the variance and had high loadings on Cu and Cr as well as Pb and Zn, while PC2 elucidated 18% of the total variance and had a high loading of Mn (Fig. 4). The occur-rence of Zn was correlated with Cu and Pb (both R = 0.73), and Cr was correlated with Al (R = 0.73) and Pb (R = 0.65) (ESM 14b). Zn and Cr were correlated, with R = 0.60. Al showed a strong correlation with Pb (R = 0.55) and Zn (R = 0.50), while Fe and Mn were also strongly correlated with Co (R = 0.56) (ESM 14b). Figure 4 b shows that the sediment from Pekalongan and Tegal was characterized by high concentrations of Cu, Cr, Pb, and Zn. In fact, Pekalongan is the area known for batik textiles [START_REF] Rukayah | Public participa-tion in branding road corridor as shopping window or batik industry at Pekalongan[END_REF], which are processed with Cr dye; thus, Cr ends up as a byproduct and an environmental contaminant in the riverine system. As shown in Fig. 4 a, b, and c, Al, Cd, Co, and Fe were associated with the second component, which may indicate that the sources of those metals in the sediment were similar and likely from natural sources. This finding was confirmed by the use of several indexes, with the EF, I geo , and CF showing that the Al, Cd, Co, and Fe concentrations in the sediment at some sampling sites were lower than the background concentrations.

Concerning the shrimp, PC1 accounted for 27.86% of the total variance and PC2 accounted for 24.7%. Figure 4 c shows that the Al concentration was highly correlated with the Fe (R = 0.94), Mn (R = 0.64), and Zn (R = 0.54) concentrations. The points in the figure represent the soil samples (n = 40). Many points were close, which indicates that these samples had less variability but were adequately discriminated. Figure 4 c also shows that the Cd concentrations had the same higher magnitude as the eigenvalues of Cu, Co, and Zn, which formed a group that was not attributed to a specific site. Figure 4 c and ESM 14c show that the Fe concentrations were highly correlated with the Mn (R = 0.66), Pb (R = 0.54), and Zn (R = 0.53) concentrations, whereas the Mn and Hg concen-trations were not correlated despite sharing the same eigen-values. ESM 12 shows that Pb and Cr were the most influen-tial to the THQ, especially in Tegal.

In the present study, the metal environmental status of L. vannamei in aquaculture systems in Indonesia has been described for the first time. This study used a comprehensive pollution index and demonstrated that aquaculture pond water is probably contaminated by Mn and Zn from natural sources. Regarding the sediment, our findings from various indexes (I geo , CF, and PLI) showed that the sediments were not polluted by the studied metals and were considered to present a low ecological risk. Despite the occurrence of many heavy metals in shrimp, Zn remained a source of concern because it was found in excess in the L. vannamei muscle.

Conclusions

Here, we investigated the ecological and human health risks of heavy metals in the water and sediment in aquaculture environments. The results showed that the occurrence of heavy metals in water was dependent on the site. The results suggested that riverine systems contributed to higher concentra-tions of Fe and Mn. The nonessential element Cd was observed in the shrimp ponds as a trace element in all regions, with the highest level found in Pekalongan (3.15 ± 0.33 μg L -1 ). From the water assessment, the greatest poten-tial ecological hazard was Mn based on the P n value.

Concerning the sediment, Cr, Cu, Zn, Co, and Pb showed similar spatial variation trends with decreasing concentrations. Their high-value area was located in Brebes, and the lowest value occurred in Pekalongan. The risk assessment for sedi-ment using the SQG results suggested that the ecological risk of heavy metals at the studied sites was low. However, the EF, I geo , CF, and PLI values for Mn showed a medium-high envi-ronmental risk. In shrimp, the accumulation of Al, Fe, Zn, Cu, and Mn showed similar trends at all stations and low values of Hg, Pb, Cd, and Co were observed. The findings of this re-search also revealed that the lack of risk associated with con-sumption if we assumed that protein daily intake per capita was approximately 20 g person -1 day -1 for all studied heavy metals. Furthermore, the THQ and HI were higher in the dry season, and our findings demonstrated that Pb and Cr influ-enced the THQ while Hg significantly influenced the HI val-ue. This research provided an adequate overview of the heavy metal contamination in different environmental matrices, al-though further study is necessary to design the conceptual framework model for heavy metal contamination in an aqua-culture field to better mitigate the hazards and risks in sustain-able aquaculture.

Fig. 1

 1 Fig. 1 Study sites. The Brebes (1-4), Tegal (5-8), Pemalang (9-15), and Pekalongan regencies (16-20)

  ): Oral RfDs (mg kg-1 day-1) of 0.001, 0.14, and 0.3 were used for Cd, Mn, and Zn, respectively, and these values were based on US EPA guidelines (USEPA 2018): 0.03 was used for Co (Finley et al. 2012), 0.003 was used for Cr (Liu et al. 2018; Zhaoetal. 2016; Yabanli and Alparslan2015), 0.02 was used for Cu (Ahmed et al. 2015), 0.004 was used for Pb, and 0.0003 was used for Hg (Yabanli and Alparslan 2015); these values were used to assess the exposure hazards from shrimp consumption. THQ values of < 1 indicate that adverse health effects are unlikely to occur, and THQ values of > 1 indicate that the consumer population has potential health risks.

Fig. 2

 2 Fig. 2 Metal concentrations (μg L -1 ) of a Fe and Mn, b Cd and Cu, and c Al and Zn in the surface water of shrimp ponds on the northern coast of Central Java

(

  .016 -1,33±0.019 -0.84±0.019 -0.25±0.57 -1.51±0.29 -0.51±0.15

  Igeo ≤ 1), class 1 (uncontaminated to moderately contaminated)

Fig. 3

 3 Fig. 3 Pollution load index (PLI) of the metals in the sediments of the studied area

Fig. 4

 4 Fig. 4 Principal component analysis of the metals in a water, b sediment, and c shrimp

Table 1

 1 Single-factor pollution index for six metals in the surface water of shrimp farm

	Region	sub-site	min & max	Cd	status	Cr	status	Range of Pi value Cu status Mn	status	Pb	status	Zn	status
		Inlet	min max	0.00 4.80	np mop	0.00 0.00	np np	0.32 0.89	np np	5.60 10.07	hp hp	0.00 0.00	np np	0.50 4.04	np mop
	Brebes	pond	min max	0.00 4.88	np mop	0.00 0.00	np np	0.00 3.75	np mop	0.89 1.85	np sp	0.00 0.00	np np	0.98 13.62	np hp
		outlet	min max	0.00 4.27	np mop	0.00 2.02	np mp	0.60 2.47	np mp	7.99 34.33	hp hp	0.00 0.00	np np	0.66 2.84	np mp
		Inlet	min max	0.00 4.48	np mop	0.00 0.00	np np	0.00 0.30	np np	0.16 5.31	np hp	0.00 0.00	np np	0.88 1.30	np sp
	Tegal	pond	min max	0.00 3.25	np mop	0.00 0.00	np np	0.00 0.43	np np	0.48 1.45	np sp	0.00 0.00	np np	0.76 3.38	np mop
		outlet	min max	0.00 3.44	np mop	0.00 0.00	np np	0.00 0.20	np np	0.90 27.30	np hp	0.00 0.00	np np	0.84 3.70	np mop
		Inlet	min max	1.56 3.93	sp mop	0.00 0.00	np np	0.00 2.08	np mp	0.09 42.19	np hp	0.00 0.00	np np	0.00 5.40	np hp
	Pemalang	pond	min max	1.96 4.76	sp mop	0.00 0.00	np np	0.24 5.68	np hp	0.40 13.37	np hp	0.00 0.00	np np	1.50 7.64	sp hp
		outlet	min max	0.00 5.09	np hp	0.00 0.00	np np	0.00 4.19	np mop 23.03 0.57	np hp	0.00 0.00	np np	0.44 5.74	np hp
		Inlet	min max	2.20 3.30	mp mop	0.00 0.00	np np	0.29 0.51	np np	0.35 2.42	np mp	0.00 0.00	np np	0.60 1.48	np sp
	Pekalongan	pond	min max	2.65 3.52	mp mop	0.00 0.00	np np	0.25 2.44	np mp	0.49 14.29	np hp	0.00 0.00	np np	0.46 3.28	np mop
		outlet	min max	0.63 3.34	np mop	0.00 0.00	np np	0.00 0.57	np np	0.52 5.44	np hp	0.00 0.00	np np	0.28 2.10	np mp
	np nonpollution (green), sp slight pollution (pink), mp mild pollution (yellow), mop moderate pollution (blue), hp heavy pollution (red)

Table 2

 2 Comparison between the sediment quality guidelines (SQGs) and metal concentrations (mg kg -1 dry weight) in the shrimp pond sediment

	Region	Cr	Cu	Pb	Zn
	Brebes
	Mean	55.5 ± 0.8	31.8 ± 4.2	13.9 ± 1.2	93.7 ± 1.6
	Min	54.7	27	12.2	91.6
	Max	56.4	37.2	15	95.3
	Tegal
	Mean	46.6 ± 4.53	51.825 ± 9.11	14.1 ± 1.11	99 ± 4.86
	Min	39.9	40	12.7	92.3
	Max	49.8	61.3	15.4	103.8
	Pekalongan
	Mean	32.48 ± 0.9	21.08 ± 3.1	9.11 ± 2.04	75 ± 8.24
	Min	31.2	19.4	7.6	70.8
	Max	33.6	26.6	12.7	89.7
	Pemalang
	Mean	38.16 ± 6.51	27 ± 3.32	9.84 ± 1.65	76 ± 8.02
	Min	28.5	22	8.3	67
	Max	48	33.3	12.9	90
	Canadian sediment quality guideline
	ERL	81	34	46.7	150
	ERM	370	270	218	410
	TEL	52.3	18.7	30.2	124
	PEL	160	108	112	271
	US EPA sediment quality guideline
	Not polluted	< 25	< 25	< 40	< 90
	Moderately polluted	25-75	25-50	40-60	90-200
	Heavily polluted	> 75	> 50	> 60	> 200

Table 3

 3 Geoaccumulation index (Igeo) for the metal concentrations at the 20 stations

Table 4

 4 Comparison of the mean concentrations (mg kg -1 dw) of trace metals reported for shrimp worldwide (muscle)

	Location Species	Al	Cd	Co	Cu	Cr	Fe	Mn	Pb	Zn	Hg	Ni	Rb	Ref

Acknowledgments The authors thank Elodie Vermeille, Wulandari Sarasati, Jamaludin, Rizqy Firmansyah, Helmi Mubarak, and Jessica for their support and help during the sampling campaign and during sample processing in the laboratory. Finally, we also thank the reviewers for their helpful comments and suggestions.

Funding information This study received financial support from the Indonesian Endowment Fund for Education (LPDP), the Ministry of Finance, the Directorate General of Higher Education (DIKTI), and the Ministry of Research, Technology and Higher Education of the Republic of Indonesia, provided through the framework of the BUDI-LN program and a doctoral research grant.


Jeremy Vanhelst

Laurent Beghin

Dominique Turck

Julien Labreuche

Stéphanie Coopman

Frédéric Gottrand

Delphine Ley

Jérémy Vanhelst

Laurent Béghin

Daily physical activity patterns in children and adolescents

Introduction

Inflammatory bowel diseases (IBD) encompass Crohn's disease (CD), ulcerative colitis (UC), and IBD-unclassified (IBD-U). They represent a worldwide health-care problem with continually increasing incidence, especially in industrialized countries and among youth [START_REF] Ghione | Dramatic increase in incidence of ulcerative colitis and Crohn's disease (1988-2011): A population-based study of French adolescents[END_REF][START_REF] Ng | Worldwide incidence and prevalence of inflammatory bowel disease in the 21 st century: a systematic review of population-based studies[END_REF].

Physical activity (PA) refers to any bodily movement produced by the contraction of skeletal muscles that increase energy expenditure above a basal level [START_REF] Caspersen | Physical activity, exercise, and physical fitness: definitions and distinctions for health-related research[END_REF]. PA is widely recognized as an important determinant of health in youth [START_REF] Pedersen | Exercise as medicine -evidence for prescribing exercise as therapy in 26 different chronic diseases[END_REF]. The US Department of Health and Human Services recently recommended that children and adolescents aged 6-17 years should engage in 60 minutes or more of moderate-to-vigorous physical activity (MVPA) daily [START_REF]Physical Activity Guidelines for Americans[END_REF]. It has been shown that 60 min of consistent, daily MVPA reduces the risks of many chronic diseases, such as non-communicable diseases and other adverse health outcomes including bone health [START_REF] Cale | Exercise recommendations for young people: An update[END_REF]. In contrast, a sedentary lifestyle, referring to any waking behavior characterized by a low level of energy expenditure (≤1.5 metabolic equivalents of task [METs]) while sitting, reclining, or lying, is associated with adverse health consequences and increases morbidity and mortality in adulthood [START_REF] Saunders | Screen time and health indicators among children and youth: current evidence, limitations and future directions[END_REF].

Several IBD symptoms, such as abdominal pain, diarrhea, and fatigue, as well as poor nutritional status and depression may reduce PA and increase sedentary status, with potential negative impacts on these children's health status [START_REF] Sawczenko | Presenting features of inflammatory bowel disease in Great Britain and Ireland[END_REF][START_REF] Narula | Exercise and inflammatory bowel disease[END_REF]. A recent systematic review showed that data on PA in children and adolescents with IBD are scarce [START_REF] Bourdier | Physical activity and sedentary levels in children with juvenile idiopathic arthritis and inflammatory bowel disease. A systematic review and meta-analysis[END_REF]. Only three studies in the past decade have assessed PA patterns in pediatric patients with IBD [START_REF] Mählmann | Psychological wellbeing and physical activity in children and adolescents with inflammatory bowel disease compared to healthy controls[END_REF][START_REF] Walker | Sedentary time and screen-based sedentary behaviors of children with a chronic disease[END_REF][START_REF] Werkstetter | Lean body mass, physical activity and quality of life in paediatric patients with inflammatory bowel disease and in healthy controls[END_REF]. However, methodological issues including limited sample size, type of accelerometer used, or use of an inappropriate epoch length to assess PA limit the strengths of their results.

Our study aim was to assess daily PA patterns measured objectively in a large sample of children and adolescents with IBD compared with healthy age-and sex-matched controls.

Methods

Participants

The current analyses use data from two prospective studies assessing PA, which used the same methods to assess samples of children and adolescents with IBD and healthy children and adolescents [START_REF] Vanhelst | Awareness of wearing an accelerometer does not affect physical activity in youth[END_REF][START_REF] Vanhelst | Physical activity is associated with improved bone health in children with inflammatory bowel disease[END_REF]. From a total sample of 162 children and adolescents (84 patients with IBD and 78 healthy controls), 120 children and adolescents (60 patients with IBD vs. 60 healthy controls) could be age-and sex-matched for inclusion in these analyses.

The inclusion criteria for patients with IBD were: (i) IBD diagnosis (CD, UC, IBD-U) for at least 6 months ( 16); (ii) age 10-18 years; (iii) assent form signed by the patient and informed consent signed by parents (or legal guardian); and (iv) no simultaneous inclusion in another biomedical study. Exclusion criteria were: (i) any associated acute or chronic disease known to influence PA (e.g., cardiovascular/respiratory disease, sprain, fracture, arthritis, severe skin lesions); (ii) any acute infection event over the past 14 days; (iii) no patent assent or parent/legal guardian consent; and (iv) (for female patients) a positive baseline blood-based pregnancy test.

The inclusion criteria for healthy control children and adolescents were: (i) age-(± 1 year) and sex-matched with a patient with IBD; (ii) informed assent signed by the participant and consent by their parents (or legal guardian); (iii) absence of any acute or chronic disease known to influence PA (e.g., cardiovascular/respiratory disease, musculoskeletal pain); (iv) no acute infection over the past 14 days; and (v) no simultaneous inclusion in another biomedical study. Sample characteristics of patients with IBD and healthy controls are reported in Table 1.

Before beginning the study, its objectives were carefully explained to each participant and their parents/guardian. Written informed assent was obtained from the participant and consent from the parents (or guardian) by the study physician.

Clinical assessment

Each patient and healthy control was required to pass a medical examination to exclude pathologies that would have constituted a contraindication to study participation. Body weight was measured using an electronic scale, with the participant wearing light clothing and without shoes, to the nearest 0.1 kg. Height was measured without shoes to the nearest 0.1 cm using a standard physician's scale. Body mass index (BMI) was calculated as weight divided by height squared (kg/m 2 ).

For patients with IBD, blood samples were collected by venipuncture during the physical examination by a study nurse. Hematocrit, C-reactive protein, albumin, and erythrocyte sedimentation rate were measured to assess inflammatory status and calculate disease activity.

The Pediatric CD Activity Index (PCDAI) was used for patients with CD and the Pediatric Ulcerative Colitis Activity Index (PUCAI) for patients with UC [START_REF] Hyams | Development and validation of a pediatric Crohn's disease activity index[END_REF][START_REF] Turner | Development, validation, and evaluation of a pediatric ulcerative colitis activity index: a prospective multicenter study[END_REF]. No IBD-U score was used. Remission was defined as scores <10 and mild activity as scores 10-27.5 on the PCDAI and 10-34 on the PUCAI. Values >27.5 on the PCDAI and >34 on the PUCAI indicated moderate-to-severe activity. Specific IBD drug therapy data were collected. Body composition (fat mass and fat free mass) was measured using dual-energy X-ray absorptiometry (DEXA; Hologic Corp., Discovery Type, Software 12.6, Bedford, MA).

Patients were scanned in the supine position at high resolution. Fat mass and fat free mass were determined based on whole body scan analyses.

Physical activity measurements

The methods for measuring PA have been described in detail elsewhere [START_REF] Vanhelst | Quantification de l'activité physique par l'accélérométrie [Physical activity assessment using accelerometry[END_REF]. Briefly, daily PA patterns were assessed by a triaxial accelerometer, an objective measure of PA in youth [START_REF] Vanhelst | Awareness of wearing an accelerometer does not affect physical activity in youth[END_REF]. This triaxial accelerometer measures 46×33×15 mm and weighs 19 g. The GT3X accelerometer has been validated for measuring PA against physiological markers of PA, such as oxygen consumption and heart rate. The epoch interval for the GT3X monitor was set at 1 s. A computer was used to initialize and synchronize the accelerometer, which was calibrated based on each patient's age, height, weight, and position for measurement using ActiLife software (version 6.13.4; ActiGraph, Pensacola, FL).

The accelerometer was worn beneath clothing on the lower back, fastened with an elastic belt and adjustable buckle. Participants were asked to follow their normal daily routine and to remove the accelerometer during sport contacts, swimming, showering, and bathing. The accelerometer recorded activity for 4 consecutive days (2 weekdays and 2 weekend days) and was removed at night. Accelerometer data were uploaded from the monitor to a computer after the completed registration period (4 days) using the same ActiLife software. Consistent with consensus recommendations for assessing PA in youth, patients who did not report at least 3 days with a minimum of 10 h of PA per day were excluded from analyses [START_REF] Rich | Quality control methods in accelerometer data processing: defining minimum wear time[END_REF]. Time spent in sedentary activities, light PA, moderate PA, and vigorous PA was based on cut-off points of 0-180, 181-757, 758-1112, and >1112 counts per 15 sec -1 , respectively, as previously established in laboratory conditions [START_REF] Romanzini | Calibration of ActiGraph GT3X, actical and RT3 accelerometers in adolescents[END_REF]. PA patterns were analyzed according to weekdays (Monday-Friday) and weekends. Data were averaged and expressed in counts•min -1 .

Log diary

For quality control of accelerometry in both groups, participants were asked to complete a diary during the 4-day period when they wore the accelerometer [START_REF] Ottevaere | The use of accelerometry in adolescents and its implementation with non-wear time activity diaries in free-living conditions[END_REF]. Waking, going to bed, and times when the accelerometer was put on and taken off were recorded daily on a standardized, preprinted recording sheet. Participants were instructed to report why the accelerometer was removed (e.g., sleeping, contact sport, water-based activities). The sport activities performed after removal of the device were classified based on the Compendium of Energy Expenditures for Youth and time spent in moderate or vigorous PA was added to the participant's record. Activities from 4-6 METs were defined as moderate PA and activities with ≥6 MET were defined as vigorous PA [START_REF] Ridley | Development of a compendium of energy expenditures for youth[END_REF]. Nonwear time each day was calculated by summing the durations noted on the log diary when the accelerometer was removed at night or for other activities.

Outcome measures

The primary outcome was the difference in time spent (in min.day -1 ) in each PA level (sedentary, moderate, vigorous and MVPA) during the week, assessed by accelerometer, between IBD pediatric patients and healthy controls.

Secondary outcomes were the differences between IBD pediatric patients and healthy controls in: (i) time spent (min.day -1 ) in each PA level (sedentary, moderate, vigorous and MVPA) during weekdays and weekend days; (ii) time spent in sedentary activity (min.day -1 ) by accelerometer according to sex, IDB diagnosis and disease intensity; (iii) time spent in MVPA (min.day -1 ) by accelerometer according to sex, IDB diagnosis and disease intensity.

Statistical analysis

Quantitative variables are expressed as mean (± standard deviation) in the case of normal distribution or median (range) otherwise and categorical variables are expressed as frequencies and percentages. Distribution normality was assessed using histograms and the Shapiro-Wilk test. PA parameters (i.e., sedentary, moderate, vigorous, moderate-to-vigorous) assessed during all days, weekdays, and weekend days were compared between patients with IBD (cases) and their age-and sex-matched healthy controls using paired Student's t-tests.

Normality of intrapair differences was checked and the magnitude of differences was assessed by calculating the mean differences (cases vs. controls) based on 95% confidence intervals (CIs). Initial analyses included all patients with IBD, while further analyses were based on sex, disease activity, and IBD type. Heterogeneity in case-control differences in PA parameters (measured during all days) according to the key subgroups was assessed by including the corresponding multiplicative interaction term in a linear mixed regression model with matched sets as the random effect. Two-tailed testing was conducted and an α-level of p < 0.05 was considered statistically significant. Data were analyzed using SAS software (version 9.4; SAS Institute, Cary, NC).

Results

The participant characteristics are presented in Table 1. The mean ages of the 60 patients with IBD (30 male patients) and 60 controls were 14.3 ± 2.3 years and 13.8 ± 2.1 years, respectively. No between-group differences were observed with respect to sex, age, height, weight, or BMI. Mean disease duration was 34 months (range, 4-122). Patient data on disease activity, medical treatment, and biological markers are also presented in Table 1. According to the PCDAI and PUCAI scales, three-quarters of the patients were in remission and onequarter had mild disease activity. There were no patients with moderate-to-severe activity. All participants wore the accelerometer during at least 3 days and had a minimum of 10 h of PA per day. No participant performed PA after removal of the accelerometer (e.g., swimming, contact sports) according to their diaries. Therefore, data analyses were performed on the accelerometer data alone.

Comparisons of time spent in different PA levels between patients with IBD and healthy matched controls are presented in Table 2. There was no difference according to each intensity or during weekdays vs. weekend days between patients with IBD and controls. The percentage of participants fulfilling the recommended 60 min MVPA daily was 31.7% in patients with IBD and 38.3% in healthy controls (p = 0.43).

Figures 1 and2 show comparisons of PA levels between patients with IBD and healthy controls. In addition, disease activity and diagnosis type (CD or UC) were considered in patients with IBD. For sedentary level, there was no significant heterogeneity according to sex, IBD diagnosis or disease activity (Figure 1). For time spent in MVPA (Figure 2), significant heterogeneity was found according to sex. Male patients with IBD spent significantly less time in MVPA compared with healthy controls (mean difference, -16.2 min•day-1 ; 95% CI: -28.--3.9) while no significant difference was observed between female patients with IBD and healthy controls (mean difference, 8.2 min•day -1 ; 95% CI: -4.1-20.5).

Discussion

Our study shows that children and adolescents with inactive or mildly active IBD have similar sedentary and PA patterns compared with their age-and sex-matched healthy peers. The only significant difference was observed in male patients with IBD, among whom MVPA was significantly reduced compared with their healthy counterparts.

The available PA pattern data among pediatric patients with IBD can be summarized as [START_REF] Mählmann | Psychological wellbeing and physical activity in children and adolescents with inflammatory bowel disease compared to healthy controls[END_REF][START_REF] Walker | Sedentary time and screen-based sedentary behaviors of children with a chronic disease[END_REF][START_REF] Werkstetter | Lean body mass, physical activity and quality of life in paediatric patients with inflammatory bowel disease and in healthy controls[END_REF]: (i) children and adolescents with active IBD have lower PA than those in IBD remission [START_REF] Mählmann | Psychological wellbeing and physical activity in children and adolescents with inflammatory bowel disease compared to healthy controls[END_REF]; (ii) sedentary time or prevalence of TV watching and computer or video game usage (i.e., all activities combined) is similar between children and adolescents with IBD and controls [START_REF] Walker | Sedentary time and screen-based sedentary behaviors of children with a chronic disease[END_REF]; and (iii) although the differences in PA duration and number of steps between pediatric patients with IBD and controls did not reach statistical significance, female patients with IBD had a significantly shorter duration of total and moderate-intensity PA [START_REF] Werkstetter | Lean body mass, physical activity and quality of life in paediatric patients with inflammatory bowel disease and in healthy controls[END_REF].

Methodological issues may explain some of the discrepancies between the above-mentioned studies and the present study. A primary limitation is the small sample sizes in the three abovementioned studies, N = 15, N = 22, and N = 39, respectively [START_REF] Mählmann | Psychological wellbeing and physical activity in children and adolescents with inflammatory bowel disease compared to healthy controls[END_REF][START_REF] Walker | Sedentary time and screen-based sedentary behaviors of children with a chronic disease[END_REF][START_REF] Werkstetter | Lean body mass, physical activity and quality of life in paediatric patients with inflammatory bowel disease and in healthy controls[END_REF]. A second limitation is the methodology used to assess PA patterns in patients with IBD and controls.

Walker et al. used two accelerometer models (Uniaxial GT1M ActiGraph and triaxial GT3X

accelerometer) within the same study [START_REF] Walker | Sedentary time and screen-based sedentary behaviors of children with a chronic disease[END_REF]. Mählmann et al. used the Fitbit Flex to assess PA patterns with 1 min epoch setting [START_REF] Mählmann | Psychological wellbeing and physical activity in children and adolescents with inflammatory bowel disease compared to healthy controls[END_REF]. However, a 2 or 5 s epoch is usually preferred for assessing PA in children and adolescents because most of their spontaneous activities are markedly short and do not exceed 1 min [START_REF] Baquet | Improving physical activity assessment in prepubertal children with high-frequency accelerometry monitoring: a methodological issue[END_REF]. In addition, the accuracy of measurement by Fitbit Flex for activity duration varies considerably by activity type and is poor for complex sets of activity, such as a run embedded within 2 walking segments [START_REF] Dorn | Automatic identification of physical activity type and duration by wearable activity trackers: A validation study[END_REF].

In our study, male patients with IBD were significantly less active compared with healthy controls (mean difference, -16.2 min•day -1 for MVPA level), while there was no difference in sedentary time. No significant difference was found for female patients with IBD. The difference of 16 min represents 35% of the time spent in MVPA in male controls and is likely biologically relevant. Indeed, there is a dose-response association between MVPA and mortality rate in adults and metabolic syndrome in children [START_REF] Renninger | Associations between accelerometry measured physical activity and sedentary time and the metabolic syndrome: A meta-analysis of more than 6000 children and adolescents[END_REF][START_REF] Ekelund | Dose-response associations between accelerometry measured physical activity and sedentary time and all cause mortality: systematic review and harmonised meta-analysis[END_REF]. This may be related to low PA levels in female controls (mean difference of 35.4% between male and female controls). It is well known that PA in developed countries is lower in female patients than in male patients [START_REF] Ruiz | Objectively measured physical activity and sedentary time in European adolescents: the HELENA study[END_REF][START_REF] Sallis | A review of correlates of physical activity of children and adolescents[END_REF][START_REF] Dumith | Physical activity change during adolescence: a systematic review and a pooled analysis[END_REF][START_REF] Matthews | Amount of time spent in sedentary behaviors in the United States, 2003e2004[END_REF]. In addition, the decline in PA observed with age is more important in female patients than in male patients [START_REF] Ortega | Objectively measured physical activity and sedentary time during childhood, adolescence and young adulthood: a cohort study[END_REF].

Although only one third of patients with IBD fulfilled the recommended 60 min MVPA daily, this is not significantly different from that observed in healthy controls. Several authors have highlighted the benefits of PA for health status in patients with IBD [START_REF] Pedersen | Exercise as medicine -evidence for prescribing exercise as therapy in 26 different chronic diseases[END_REF][START_REF] Sawczenko | Presenting features of inflammatory bowel disease in Great Britain and Ireland[END_REF][START_REF] Narula | Exercise and inflammatory bowel disease[END_REF].

Implementing a physical education curriculum could help promote better health in patients with IBD who are in remission or have mild disease.

The current study has both strengths and limitations. Strengths include a large sample of both pediatric patients with IBD and age-and sex-matched healthy controls. However, no sample size calculation could be performed; therefore, a lack of adequate statistical power to detect significant differences, especially heterogeneity in subgroup analyses, cannot be excluded. With our sample size, a minimum effect size (standardized mean difference) of 0.43 can be detected with a paired t-test at a 0.05 significance level. A false positive difference regarding the multiple testing issue also cannot be excluded. Another strength is the use of a strong objective method to assess PA patterns (i.e., a triaxial accelerometer with appropriate epoch length for adolescents). However, it cannot be excluded that variations in weather conditions (e.g., wind, rain, sunshine) during PA assessments (not recorded) may have affected our results [START_REF] Béghin | Influence of meteorological conditions on physical activity in adolescents[END_REF]. The study is not without limitations. Parental education levels or socioeconomic status were not collected. These parameters may play a role in adolescents' daily PA patterns and their attitudes about healthy lifestyles [START_REF] Beets | Social support and youth physical activity: the role of provider and type[END_REF]. Another limitation is that the majority of our IBD patients were either in remission or had mild disease. Therefore, they were probably more prone to have a PA higher than that of IBD patients with moderate-tosevere disease.

Overall, pediatric IBD patients in remission or with mild disease have similar PA patterns compared with their healthy counterparts. The prevalence of IBD patients fulfilling the recommended 60 min a day of MVPA is low, but not significantly different from healthy controls. It is critical that we further evaluate the health benefits of PA programs aimed at increasing PA in pediatric IBD patients in remission or with mild disease. Data are needed on PA in pediatric IBD patients with moderate-to-severe disease.

Figure 1 .

 1 Figure 1. Differences in time spent in sedentary activity (min.day -1 ) between IBD patients

Figure 2 .

 2 Figure 2. Differences in time spent in moderate-to-vigorous physical activity (min.day -1 )

  The study was approved by the Research Ethical Committee (Comité Protection des Personnes, Nord-Ouest IV, Lille, France) and French research competent authority (Agence Nationale de Sécurité du Médicament et des Produits de Santé, Paris, France). All procedures were performed according to the ethical standards of the 1975 Declaration of Helsinki, as revised in 2013, and the European Union's

	Guidelines for Good Clinical Practice. According to the General Data Protection Regulation,
	data collection was approved by the French personal informatics competent authority
	(Commission Nationale de l'Informatique et des Libertés). Studies were registered in
	clinicaltrials.gov (NCT02844101; NCT02341742).

Table 1 .

 1 Characteristics of IBD patients and healthy controls

	IBD Patients	Healthy controls
	(N=60)	(N=60)

Acknowledgments

The authors thank the participants for enrolling in the study.