Iodineminho Study: Iodine Supplementation and Prevalence of Iodine Deficiency in Pregnant Women.

CONTEXT: Iodine is necessary for the proper brain development. The prevalence of iodine deficiency in Portuguese pregnant women led the health authorities, in 2013, to recommend iodine supplementation for women in preconception, throughout pregnancy and during lactation. OBJECTIVE: To assess the impact of iodine supplementation initiated in the preconception or the first trimester of pregnancy on the prevalence of iodine deficiency and maternal thyroid status. METHODS: An observational prospective cohort study that follows thyroid function and iodine status of women recruited in preconception or in the first trimester of pregnancy. RESULTS: The median urinary iodine concentration (UIC) was significantly higher among women taking iodine supplements (no-supplement group UIC=63µg/L; supplement group UIC =100µg/L, p = 0.002) but still below the levels recommended by the World Health Organization. Only 15% of pregnant women had adequate iodine status and 17% showed UIC < 50 µg/l. There was no influence of whether iodine supplementation started in preconception or in the 1st trimester of gestation (UIC preconception group: 112µg/L vs UIC pregnancy group: 91µg/L, p = 0.569). In the 1st trimester of pregnancy, total thyroxine levels were lower and free triiodothyronine levels were higher in non-supplemented women. Thyroglobulin levels were lower in women who started iodine supplementation in preconception compared to non-supplemented women and women who started iodine supplementation during gestation. CONCLUSION: In the Minho region of Portugal, fertile women have insufficient iodine intake. Additional public health measures are needed since the current recommendations for iodine supplementation for pregnancy are unsatisfactory to achieve an adequate iodine status.

Iodine intake assessment in the staff of a Porto region university (Portugal): the iMC Salt trial.

PURPOSE: Iodine deficiency disorder (IDD) is an ongoing worldwide recognized problem with over two billion individuals having insufficient iodine intake. School-aged children and pregnant women are often target groups for epidemiological studies, but there is a lack of knowledge on the general adult population. The aim of this study was to assess the iodine status among a Portuguese public university staff as a proxy for the adult working population. METHODS: The population study covered 103 adults within the iMC Salt randomized clinical trial, aged 24-69 years. Urinary iodine concentration was measured spectrophotometrically using the Sandell-Kolthoff reaction. Iodine food intake was assessed using a 24-h dietary recall. The contribution of discretionary salt to the iodine daily intake was assessed through 24-h urinary sodium excretion (UIE) and potentiometric iodine determination of household salt. RESULTS: The mean urine volume in 24 h was 1.5 L. The median daily iodine intake estimated from 24-h UIE was 113 µg/day, being lower among women (p < 0.05). Only 22% of participants showed iodine intake above the WHO-recommended cutoff (150 µg/day). The median daily iodine intake estimated from the 24-h dietary recall was 58 µg/day (51 and 68 µg/day in women and men, respectively). Dairy, including yoghurt and milk products, were the primary dietary iodine source (55%). Iodine intake estimated from 24-h UIE and 24-h dietary recall was moderately correlated (Spearman rank correlation coefficient r = 0.34, p < 0.05). The average iodine concentration in household salt was 14 mg I/kg, with 45% of the samples below the minimum threshold preconized by WHO (15 mg I/kg). The contribution of discretionary salt to the daily iodine intake was around 38%. CONCLUSION: This study contributes new knowledge about iodine status in Portuguese working adults. The results revealed moderate iodine deficiency, particularly in women. Public health strategies and monitoring programs are needed to ensure iodine adequacy in all population groups.

Iodine Availability through Iodized Salt in Portugal: 2010-2021 Sales Evolution and Distribution.

Salt iodization programs are considered the most cost-effective measures to ensure adequate iodine intake in iodine-deficient populations. Portuguese women of childbearing age and pregnant women were reported to be iodine-deficient, which led the health authorities, in 2013, to issue a recommendation for iodine supplementation during preconception, pregnancy and lactation. In the same year, iodized salt became mandatory in school canteens. Of note, no regulation or specific programs targeting the general population, or the impact of iodized salt availability in retailers, are known. The present study analyzed iodized salt supermarket sales from 2010 to 2021 from a major retailer, identifying the proportion of iodized salt in total salt sales and its distribution in mainland Portugal. Data on iodine content were collected through the nutritional label information. Of a total of 33 salt products identified, 3 were iodized (9%). From 2010 to 2021, the weighted sales of iodized salt presented a growing tendency, reaching the maximum of 10.9% of total sales (coarse plus fine salt) in 2021. Iodized salt reached a maximum of 11.6% of total coarse salt in 2021, a maximum of 2.4% of the total fine salt in 2018. The overall sales of iodized salt and their contribution to iodine intake are extremely low, prompting additional studies to understand the consumer's choice and awareness of the benefits of iodized salt.

Major Stressors Favoring Cholera Trigger and Dissemination in Guinea-Bissau (West Africa).

Cholera remains a heavy burden worldwide, especially in Sub-Saharan African countries, which account for the majority of the reported cases on the continent. In this study, a 27-year retrospective analysis of cholera epidemics in Guinea-Bissau was performed in order to highlight major stressors fueling the trigger and dissemination of the disease. Although the role of environmental factors did not always have the same degree of importance for the onset of epidemics, a cholera seasonal pattern was clearly perceived, with most of the reported cases occurring during the wet season. The generated theoretical hypothesis indicated rainfall above climatological average, associated with a lack of WASH (water, sanitation and hygiene) infrastructure, and the occurrence of concomitant epidemics in neighboring countries as the key indicators for optimal conditions for cholera to thrive in Guinea-Bissau. Warmer air temperature, the increase in sea surface temperature, and the decrease in salinity in the coastal areas may also contribute to the emergence and/or aggravation of cholera events. Prediction of the conditions favorable for cholera growth and identification of risk pathways will allow the timely allocation of resources, and support the development of alert tools and mitigation strategies.

Can non-fortified marine salt cover human needs for iodine?

Iodine deficiency remains a worldwide problem with two billion individuals having insufficient iodine intake. Universal salt iodisation was declared by UNICEF and WHO as a safe, cost-effective, and sustainable way to tackle iodine deficiency. In Portugal, the few studies available unravel an iodine status below the WHO guidelines for pregnant women and school-aged children. In the present study, the iodine levels of household salt consumed in Portugal was assessed, for the first time. Non-iodised (median 14 ppm) and fortified (median 48 ppm) marine salt samples showed iodine levels lower than the minimum and above the maximum threshold recommended by non-mandatory Portuguese law and WHO recommendations, respectively. This study calls attention to the fact that marine salt per se, in spite of containing a natural high amount of iodine, requires further fortification in order to be used as an effective tool to deal with iodine insufficiency.

River water analysis using a multiparametric approach: Portuguese river as a case study.

The Ave River in northern Portugal has a history of riverbanks and water quality degradation. The river water quality was assessed by physicochemical, biological (macroinvertebrates) and microbiological (Enterococcus spp. and Escherichia coli) parameters in six locations (A-F, point A being the nearest to the source) throughout its course during a year. Epilithic biofilms were studied through polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Antimicrobial susceptibility testing helped with selecting isolates (n = 149 E. coli and n = 86 enterococci) for further genetic characterization. Pursuant to physicochemical and macroinvertebrates-based parameters, the river water was of reasonable quality according to European legislation (Directive 2000/60/EC). However, the microbiological analysis showed increased fecal contamination downstream from point C. At point D, four carbapenem-resistant E. coli isolates were recovered. Paradoxically, point D was classified as a point of 'Good Water Quality' according to macroinvertebrates results. Point F presented the highest contamination level and incidence of multidrug-resistant (MDR) isolates in the water column (13 MDR enterococci out of 39 and 33 MDR E. coli out of 97). Epilithic biofilms showed higher diversity in pristine points (A and B). Thus, biological and microbiological parameters used to assess the water quality led to divergent results; an outcome that reinforces the need for a holistic evaluation.

Monitoring glucose, calcium, and magnesium levels in saliva as a non-invasive analysis by sequential injection multi-parametric determination.

The use of saliva for diagnose and surveillance of systemic illnesses, and general health has been arousing great interest worldwide, emerging as a highly desirable goal in healthcare. The collection is non-invasive, stress-free, inexpensive, and simple representing a major asset. Glucose, calcium, and magnesium concentration are three major parameters evaluated in clinical context due to their essential role in a wide range of biochemical reactions, and consequently many health disorders. In this work, a spectrophotometric sequential injection method is described for the fast screening of glucose, calcium, and magnesium in saliva samples. The glucose determination reaction involves the oxidation of the aldehyde functional group present in glucose with simultaneous reduction of 3,5-dinitrosalicylic acid (DNS) to 3-amino, 5-nitrosalicylic acid under alkaline conditions, followed by the development of colour. The determination of both metals is based on their reaction with cresolphtalein complexone (CPC), and the interference of calcium in the magnesium determination minimized by ethylene glycol-bis[ß-aminoethyl ether]-N,N,N',N'-tetraacetic acid (EGTA). The developed multi-parametric method enabled dynamic ranges of 50 - 300 mg/dL for glucose, 0.1 - 2 mg/dL for calcium, and 0.1 - 0.5 mg/dL for magnesium. Determination rates of 28, 60, 52 h-1 were achieved for glucose, calcium, and magnesium, respectively. Less than 300 µL of saliva is required for the multi-parametric determination due to saliva viscosity and inherent necessity of dilution prior to analysis. RSDs lower than 5% were obtained, and the results agreed with those obtained by reference methods, while recovery tests confirmed its accuracy.

Endemic goiter and iodine deficiency status among Guinea-Bissau school-age children.

BACKGROUND/OBJECTIVES: Iodine deficiency disorders are estimated to affect 25% of all school-age children around the world. Current policies concerning iodine deficiency disorders in many countries are not based on robust or recent national data. Our objective was to evaluate the current status of iodine nutrition in Guinea-Bissau (West Africa). SUBJECT/METHODS: A cross-sectional survey including 299 school-age children, aged 6-14 years old, from four distinct regions of Guinea-Bissau across a coast-hinterland gradient (Bolama, Bissau, Cambaju-Bafatá, and Gabú) was performed. Several iodine deficiency disorders indicators were used: total goiter rate, median urinary iodine concentration (UIC) and regional iodine content in household salt and drinking water samples. Data on the total goiter rate were obtained by thyroid ultrasound. RESULTS: Goiter was found in 73.5% (CI 95%, 68.5-78.2%) of the school-age children. We observed a median urinary iodine concentration of 110 mcg/l, with 7.3% of the studied population presenting urinary iodine concentration < 50 mcg/l. Only 12.5% of the household salt samples revealed iodine content ≥ 15 mg/kg. Median well water iodine concentration was 11.7 mcg/l. CONCLUSIONS: Guinea-Bissau presents high rates of goiter in school-age children despite satisfactory median urinary iodine concentration population levels. Salt and drinking water iodine median content cannot explain these findings. Further studies following the progress in iodine deficiency status and the presence of potential goitrogens in diet of this vulnerable population are in needed.

Development of a robust, fast screening method for the potentiometric determination of iodide in urine and salt samples.

In this work, a potentiometric flow injection method is described for the fast bi-parametric determination of iodide and iodate in urine and salt samples. The developed methodology aimed for iodine speciation with a potentially portable system (running on batteries). The iodate reduction to iodide was effectively attained in line within the same manifold. The iodide determination was accomplished in the dynamic range of 2.50×10-6-1.00×10-3M and the total iodine dynamic range, resulted from iodide plus iodate, was 3.50×10-6-2.00×10-3M. The calculated limits of detection were 1.39×10-6M and 1.77×10-6M for iodide and iodate, respectively. A determination rate of 21h-1 for the bi-parametric iodide and iodate determination was obtained for sample injection. The urine samples (RSD <5.8% for iodide and RSD <7.0% for iodate) results were in agreement with those obtained by the classic Sandell-Kolthoff reaction colorimetric reference procedure (RD <7.0%) and standard samples from Centers for Disease Control and Prevention, Atlanta, USA (CDC) international inter-laboratory EQUIP program. The developed flow method was also successfully applied to the iodide and iodate determination in salt samples (RSD <3.1% for iodate and iodide), with comparable results to conventional procedures. No significant interferences were observed interference percentage <9% for both determinations.

Nutrient variability and its influence on nitrogen processes in a highly turbid tropical estuary (Bangpakong, Gulf of Thailand).

Estuarine ecosystems in SE Asia have been poorly studied when compared to other tropical environments. Important gaps exist particularly in the understanding of their biogeochemical function and contribution to global change. In this work we looked into N-turnover in the water column and sediments of the Bangpakong estuary (13°N). A seasonal sampling program was performed along the salinity gradient covering different stretches of the estuary (68km). Key physical and chemical characteristics were also monitored in order to unravel possible environmental controls. Results showed the occurrence of active denitrification in sediments (5.7-50.9nmol N-N2/(cm(3)·hr)), and water column (3.5-1044pmol N-N2/(cm(3)·hr)). No seasonal or spatial variability was detected for denitrification potential in sediment samples. However, in the water column, the denitrification activity peaked during the transition season in the downstream sites coinciding with high turbidity levels. Therefore, in that period of the year, the water column compartment may be an important contributor to nitrate reduction within the estuary. The rather low nitrification rates detected were not always measurable, probably due to the reduced oxygen content and high siltation. This study is one of the few dealing simultaneously with sediments and water column processes in a highly turbid tropical estuary. Therefore, it emerges as a valuable contribution for the understanding of the dynamics of the nitrogen cycle in tropical environments by exploring the role of estuarine N microbial activity in reducing the effects of increased nitrogen loads.

Detection and Quantification of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in Coastal Waters of Guinea-Bissau (West Africa).

V. cholerae, V. parahaemolyticus, and V. vulnificus are recognized human pathogens. Although several studies are available worldwide, both on environmental and clinical contexts, little is known about the ecology of these vibrios in African coastal waters. In this study, their co-occurrence and relationships to key environmental constraints in the coastal waters of Guinea-Bissau were examined using the most probable number-polymerase chain reaction (MPN-PCR) approach. All Vibrio species were universally detected showing higher concentrations by the end of the wet season. The abundance of V. cholerae (ISR 16S-23S rRNA) ranged 0-1.2 × 10(4) MPN/L, whereas V. parahaemolyticus (toxR) varied from 47.9 to 1.2 × 10(5) MPN/L. Although the presence of genotypes associated with virulence was found in environmental V. cholerae isolates, ctxA+ V. cholerae was detected, by MPN-PCR, only on two occasions. Enteropathogenic (tdh+ and trh+) V. parahaemolyticus were detected at concentrations up to 1.2 × 10(3) MPN/L. V. vulnificus (vvhA) was detected simultaneously in all surveyed sites only at the end of the wet season, with maximum concentrations of 1.2 × 10(5) MPN/L. Our results suggest that sea surface water temperature and salinity were the major environmental controls to all Vibrio species. This study represents the first detection and quantification of co-occurring Vibrio species in West African coastal waters, highlighting the potential health risk associated with the persistence of human pathogenic Vibrio species.

Salt marsh sediment characteristics as key regulators on the efficiency of hydrocarbons bioremediation by Juncus maritimus rhizospheric bacterial community.

Mitigation of petroleum hydrocarbons was investigated during a 5-month greenhouse experiment, to assess the rhizoremediation (RR) potential in sediments with different characteristics colonized by Juncus maritimus, a salt marsh plant commonly found in temperate estuaries. Furthermore, the efficiency of two bioremediation treatments namely biostimulation (BS) by the addition of nutrients, and bioaugmentation (BA) by addition of indigenous microorganisms, was tested in combination with RR. The effect of the distinct treatments on hydrocarbon degradation, root biomass weight, and bacterial community structure was assessed. Our result showed higher potential for hydrocarbon degradation (evaluated by total petroleum hydrocarbon analysis) in coarse rhizosediments with low organic matter (OM), than rhizosediments with high OM, and small size particles. Moreover, the bacterial community structure was shaped according to the rhizosediment characteristics, highlighting the importance of specific microbe-particle associations to define the structure of rhizospheric bacterial communities, rather than external factors, such as hydrocarbon contamination or the applied treatments. The potential for hydrocarbon RR seems to depend on root system development and bacterial diversity, since biodegradation efficiencies were positively related with these two parameters. Treatments with higher root biomass, and concomitantly with higher bacterial diversity yielded higher hydrocarbon degradation. Moreover, BS and BA did not enhance hydrocarbons RR. In fact, it was observed that higher nutrient availability might interfere with root growth and negatively influence hydrocarbon degradation performance. Therefore, our results suggested that to conduct appropriate hydrocarbon bioremediation strategies, the effect of sediment characteristics on root growth/exploration should be taken into consideration, a feature not explored in previous studies. Furthermore, strategies aiming for the recovery of bacterial diversity after oil spills may improve the efficiency of hydrocarbon biodegradation in contaminated salt marsh sediments.

Iodine speciation in coastal and inland bathing waters and seaweeds extracts using a sequential injection standard addition flow-batch method.

The present work describes the development of a sequential injection standard addition method for iodine speciation in bathing waters and seaweeds extracts without prior sample treatment. Iodine speciation was obtained by assessing the iodide and iodate content, the two inorganic forms of iodine in waters. For the determination of iodide, an iodide ion selective electrode (ISE) was used. The indirect determination of iodate was based on the spectrophotometric determination of nitrite (Griess reaction). For the iodate measurement, a mixing chamber was employed (flow batch approach) to explore the inherent efficient mixing, essential for the indirect determination of iodate. The application of the standard addition method enabled detection limits of 0.14 µM for iodide and 0.02 µM for iodate, together with the direct introduction of the target water samples, coastal and inland bathing waters. The results obtained were in agreement with those obtained by ICP-MS and a colorimetric reference procedure. Recovery tests also confirmed the accuracy of the developed method which was effectively applied to bathing waters and seaweed extracts.

Water bags as a potential vehicle for transmitting disease in a West African capital, Bissau.

BACKGROUND: Street vendors of chilled packaged water have an increasing role in meeting the drinking water demand of people on the move in developing nations. Hygienic conditions can be questionable, and water quality screening scarce or non-existent. METHODS: In order to ascertain the quality of the packaged water sold by street vendors in Bissau, the capital of the Western African country Guinea-Bissau, water bags were screened in 2011 and during the 2012 cholera outbreak for key physical, chemical and microbiological parameters. RESULTS: Water used to fill the hand-filled hand-tied bags originated from communal tap water and melted ice. All samples (n=36) were microbiologically contaminated, although levels showed a pronounced variability (e.g. 7-493 372 cfu 250 ml(-1) for Escherichia coli). In 2012, the fecal contamination levels increased (p<0.05), and Vibrio cholerae was detected in all water bags obtained from the neighborhood where the outbreak started. CONCLUSION: Findings showed that all packaged water samples were unfit for human consumption and during the 2012 cholera outbreak represented a potential vehicle for the spread of the disease. The design of measures to decrease the risk associated to the consumption of highly contaminated chilled water is clearly required.

Analysis of the bacterial community composition in acidic well water used for drinking in Guinea-Bissau, West Africa.

Potable water is a resource out of reach for millions worldwide, and the available water may be chemically and microbiologically compromised. This is particularly acute in Africa, where water-networks may be non-existent or restricted to a small fraction of the urban population, as in the case of Guinea-Bissau, West Africa. This study was carried out seasonally in Bolama (11°N), where unprotected hand-dug wells with acidic water are the sole source of water for the population. We inspected the free-living bacterial community dynamics by automated rRNA intergenic spacer analyses, quantitative polymerase chain reaction and cloning approaches. The results revealed a clear seasonal shift in bacterial assemblage composition and microbial abundance within the same sampling site. Temperature, pH and turbidity, together with the infiltration and percolation of surface water, which takes place in the wet season, seemed to be the driving factors in the shaping and selection of the bacterial community and deterioration of water quality. Analysis of 16S rDNA sequences revealed several potential pathogenic bacteria and uncultured bacteria associated with water and sediments, corroborating the importance of a culture-independent approach in drinking water monitoring.

Development of autochthonous microbial consortia for enhanced phytoremediation of salt-marsh sediments contaminated with cadmium.

Microbial assisted phytoremediation is a promising, though yet poorly explored, new remediation technique. The aim of this study was to develop autochthonous microbial consortia resistant to cadmium that could enhance phytoremediation of salt-marsh sediments contaminated with this metal. The microbial consortia were selectively enriched from rhizosediments colonized by Juncus maritimus and Phragmites australis. The obtained consortia presented similar microbial abundance but a fairly different community structure, showing that the microbial community was a function of the sediment from which the consortia were enriched. The effect of the bioaugmentation with the developed consortia on cadmium uptake, and the microbial community structure associated to the different sediments were assessed using a microcosm experiment. Our results showed that the addition of the cadmium resistant microbial consortia increased J. maritimus metal phytostabilization capacity. On the other hand, in P. australis, microbial consortia amendment promoted metal phytoextraction. The addition of the consortia did not alter the bacterial structure present in the sediments at the end of the experiments. This study provides new evidences that the development of autochthonous microbial consortia for enhanced phytoremediation of salt-marsh sediments contaminated with cadmium might be a simple, efficient, and environmental friendly remediation procedure. CAPSULE ABSTRACT: Development of autochthonous microbial consortia resistant to cadmium that enhanced phytoremediation by salt-marsh plants, without a long term effect on sediment bacterial diversity.

Potential of phytoremediation for the removal of petroleum hydrocarbons in contaminated salt marsh sediments.

Degradation of petroleum hydrocarbons in colonized and un-colonized sediments by salt marsh plants Juncus maritimus and Phragmites australis collected in a temperate estuary was investigated during a 5-month greenhouse experiment. The efficiency of two bioremediation treatments namely biostimulation (BS) by the addition of nutrients, and bioaugmentation (BA) by addition of indigenous microorganisms was tested in comparison with hydrocarbon natural attenuation in un-colonized and with rhizoremediation in colonized sediments. Hydrocarbon degrading microorganisms and root biomass were assessed as well as hydrocarbon degradation levels. During the study, hydrocarbon degradation in un-colonized sediments was negligible regardless of treatments. Rhizoremediation proved to be an effective strategy for hydrocarbon removal, yielding high rates in most experiments. However, BS treatments showed a negative effect on the J. maritimus potential for hydrocarbon degradation by decreasing the root system development that lead to lower degradation rates. Although both plants and their associated microorganisms presented a potential for rhizoremediation of petroleum hydrocarbons in contaminated salt marsh sediments, results highlighted that nutrient requirements may be distinct among plant species, which should be accounted for when designing cleanup strategies.

Bioremediation potential of microorganisms from a sandy beach affected by a major oil spill.

The aim of this work was to evaluate the bioremediation potential of microorganisms from intertidal sediments of a sandy beach affected by a major oil spill 7 years before and subject to chronic petroleum contamination since then. For that, the response of microorganisms to a new oil contamination was assessed in terms of community structure, abundance, and capacity to degrade hydrocarbons. Experiments were carried out under laboratory-controlled conditions by mixing sediment with crude oil with three different nitrogen supplementations in 50 ml serum bottles under constant shake for 15 days. Autochthonous microorganisms were able to respond to the new oil contamination by increasing their abundance (quantified by DAPI) and changing the community structure (evaluated by DGGE). This response was particularly clear for some specific bacterial groups such as Pseudomonas, Actinomycetales, and Betaproteobacteria. These communities presented an important potential for hydrocarbon degradation (up to 85 % for TPHs and 70 % for total PAHs), being the biodegradation stimulated by addition of an appropriate amount of nitrogen.

Influence of different salt marsh plants on hydrocarbon degrading microorganisms abundance throughout a phenological cycle.

The influence of Juncus maritimus, Phragmites australis, and Triglochin striata on hydrocarbon degrading microorganisms (HD) in Lima River estuary (NW Portugal) was investigated through a year-long plant life cycle. Sediments un-colonized and colonized (rhizosediments) by those salt marsh plants were sampled for HD, total cell counts (TCC), and total petroleum hydrocarbons (TPHs) assessment. Generally, TCC seemed to be markedly thriving by the presence of roots, but without significant (p > 0.05) differences among rhizosediments. Nevertheless, plants seemed to have a distinct influence on HD abundance, particularly during the flowering season, with higher HD abundance in the rhizosediments of the fibrous roots plants (J. maritimus < P. australis < T. striata). Our data suggest that different plants have distinct influence on the dynamics of HD populations within its own rhizosphere, particularly during the flowering season, suggesting a period of higher rhizoremediation activity. Additionally, during the vegetative period, plants with fibrous and dense root system tend to retain hydrocarbons around their belowground tissues more efficiently than plants with adventitious root system. Overall results indicate that fibrous root plants have a higher potential to promote hydrocarbons degradation, and that seasonality should be taken into account when designing long-term rhizoremediation strategies in estuarine areas.