Chemical resistance of gorgonian corals against fungal infections

The frequency and impact of diseases affecting corals throughout the Caribbean have been increasing but little is known about the factors promoting the emergence and outbreak of disease. A disease caused by a fungal pathogen [Aspergillus sydowii (Thom et Church)] which affects Caribbean sea fan corals provided an opportunity to examine the efficacy of coral crude extracts in disease resistance. Minimum inhibitory concentration (MIC) assays showed that of the 20 common gorgonian species in the Florida Keys, extracts from 15 species had MICs < 15 mg ml⁻¹ against A. sydowii pathogenic to sea fans. Extracts from several species in two gorgonian genera (Pseudoplexaura and Pseudopterogorgia) were among the most active, with MICs < 10 mg ml⁻¹. Gorgonia ventalina L., one of two sea fan species known to be hosts to A. sydowii in the field, had an MIC < 10 mg ml⁻¹, suggesting that complete disease resistance requires more active extracts. For the antifungal compounds to be effective in situ, they must also occur in sufficiently high concentrations in living coral tissue. For example, Pseudopterogorgia americana (Gmelin) had comparatively potent extracts but did not have sufficient concentrations in the tissue to be effective. Conversely, Plexaura homomalla Esper extracts were less potent but occurred in high enough concentrations in the tissue to be effective against A. sydowii. When potency and extract concentration are considered together (i.e. potency × concentration), several other gorgonian corals emerge as likely hosts to A. sydowii. Crude extracts from the most active gorgonian species were also effective against two geographic variants of A. sydowii pathogenic to sea fans, a non-pathogenic terrestrial strain of A. sydowii, and three strains of A. flavus Link known to be human, plant, and insect pathogens (MIC range, 7.5 to > 15 mg ml⁻¹). Although the potency in these assays did not attain a clinically significant level, the potency is comparable to a known antifungal agent, hygromycin B, which had an MIC ≤7.5 mg ml⁻¹ in our assays, highlighting the potential of these gorgonian corals for bioprospecting. Received: 29 May 1999 / Accepted: 22 May 2000     

Spatial variability in reproductive cycle of the gorgonians <Emphasis Type="Italic">Paramuricea clavata</Emphasis> and <Emphasis Type="Italic">Eunicella singularis</Emphasis> (Anthozoa,Octocorallia) in the Western Mediterranean Sea

Paramuricea clavata (Risso, 1826) and Eunicella singularis (Esper, 1794) are the most representative gorgonian species in hard bottoms sublittoral communities in the Western Mediterranean Sea. Reproductive cycles of two populations of both species were studied in two distinct locations approximately 600 km apart (Medes Islands and Cape of Palos), in order to assess interpopulation variability on a relevant geographic scale. Seasonal variation of lipid concentration levels in the gorgonian tissue was used as a tool to quantify energy storage by each studied population in order to explain possible interpopulation differences in gonadal output. Sex ratio in Medes Islands populations of both species was 1:1, while in Cape of Palos sex ratio was significantly male biased (1:7) in P. clavata, and female biased (1.7:1) in E. singularis populations. Spawning timing occurred in all cases coinciding with a marked increase in sea-water temperature in spring, and after the most successful feeding season, but comparing localities there was a clear temporal shift in the time of gametes release, appearing well linked to the shift in sea-water temperature rising in spring in both sites at the depth where populations are placed. Therefore, in this study the temperature appears as the main synchronizing factor of gonadal development within these populations. Significant differences in gonadal volume per polyp were found in both species owing mainly to differences in the number of gonads per polyp between populations, with Cape of Palos populations displaying higher values in both studied species, suggesting that the exposition to different local conditions may be reverted in a different gonadal output. But the observed patterns in lipid concentrations levels in gorgonians disable us to conclude that lipid concentration levels explain the observed differences in gonadal output found in this study.     

Photosynthesis, respiration and calcification of a zooxanthellate scleractinian coral under submerged and exposed conditions

An experimental set-up was designed to investigate photosynthesis, respiration and calcification of zooxanthellate scleractinian corals under submerged and exposed conditions. The results of experiments to determine the effect of exposure to air on the metabolism of Stylophora pistillata (Esper, 1797) revealed that: (1) maximum gross photosynthesis ( p _{g max}) is 3.6 times higher in water than in air; (2) an indicator of photoadaptation (I _k ) does not reveal any difference between water and air; (3) the difference between submerged and aerial respiration is not statistically significant; (4) aerial photosynthesis–irradiance (P–I) curves display lower initial slopes (α) than aquatic P–I curves; (5) there is no calcification in air. Received: 7 February 1997 / Accepted: 4 March 1997     

Calcium uptake and net calcification rates in the octocoral Eunicella papillosa

This paper presents the results of a preliminary study on the uptake of ⁴⁵Ca in the mesoskeleton of the gorgonian Eunicella papillosa (Octocorallia). Hourly seawater aliquots over 12 h periods displayed considerable fluctuations of activity with time, reflecting a disappearance and reappearance of tracer during the experiment. Analysis of total coenenchyme mass showed an initial rapid but fluctuating uptake of tracer and a progressive decrease with time. Distinct differences in the calcification rate of specific growth regions of the coral were detected, the rate being higher in branch tips than in lower branch regions. Chase experiments on colonies previously incubated for 24 h revealed that up to 45% of the tracer taken up during labelling were returned to the seawater. A comparison of the tracer content of the coenenchyme and the calcite spicules revealed that up to 70% of the calcium taken up remained in the coral tissue. Quantification of isotopic exchange phenomena with dead corals and isolated spicules emphasized the importance of the live coral tissue as a barrier and regulator of Ca uptake. The results are discussed in the light of the limitations of radioisotope techniques for the determination of calcification rates, and an attempt to compare rates with data in the literature is made.     

Degradation and proliferation of zooxanthellae in planulae of the hermatypic coral Stylophora pistillata

Zooxanthellae in different stages of two opposite processes, degradation and proliferation, were found in the planulae of hermatypic corals. The formation of new zooxanthellae is balanced by degraded zooxanthellae in newly released planulae. The number of dividing zooxanthellae and degraded zooxanthellae during the day amounted to approximately 2 to 3% of the standing stock. In settled planulae and particularly in motionless planulae of Stylophora pistillata (Esper, 1797), the degraded zooxanthellae outnumbered proliferous zooxanthellae. The proliferation and degradation of zooxanthellae and the extrusion of degraded remnants of zooxanthellae are significantly phased. Swimming planulae are more autotrophic than motionless planulae. The physiological parameters of settled planulae with exoskeleton are similar to those of adult polyps. The significance of zooxanthella degradation in the vital functions of planulae is discussed. We suggest that the degradation of zooxanthellae in planulae occurs by the digestion of symbionts by host cells. Received: 5 March 1997 / Accepted: 6 August 1997     

Antimicrobial activity of extracts of Caribbean gorgonian corals

Extracts of 39 species of Caribbean gorgonians were tested for antimicrobial activity against 15 strains of marine bacteria. The bacteria consisted of three opportunistic pathogens, Vibrio parahaemolyticus, Leucothrix mucor, and Aerococcus viridans, and 12 strains isolated from either healthy or decayed gorgonians. Overall, only 15% (79 out of 544) of the tests resulted in antibacterial activity with 33% (13 out of 39) of the gorgonians inhibiting only one bacterial strain and 23% (9 out of 39) showing no activity. The extracts of four Pseudopterogorgia species showed relatively high levels of activity, inhibiting 43 to 86% of the bacterial strains. The potency of the active Pseudopterogorgia species was variable, however, and three additional Pseudopterogorgia species were inactive against all bacterial strains. With the exception of one sensitive strain, Vibrio species were resistant to gorgonian metabolites. Our results indicate that organic extracts of most Caribbean gorgonians do not possess potent, broad-spectrum antibacterial activity inhibitory to the growth of opportunistic marine pathogens and bacteria associated with healthy and decayed gorgonian surfaces. These findings suggest that the inhibition of bacterial growth is not the primary ecological function of gorgonian secondary metabolites and that bacteria may not be important selective agents in the evolution of gorgonian secondary chemistry.     

Temperate reefs in a changing ocean: skeletal carbonate mineralogy of serpulids

We present a review of the published data about serpulid skeletal carbonate geochemistry, augmented with new data from the Southern Hemisphere. We know something about skeletal carbonate mineralogy of 15 % of extant species (n = 52); and about half of extant genera (n = 25). Serpulid worm tubes vary in their skeletal mineralogy from entirely aragonitic (about 24 % of species) to entirely high-Mg calcite (40 %) to mixtures of the two. Mg in calcite ranges from 7 to 15 wt% MgCO₃, with a mean of 11 wt% MgCO₃. Little mineralogical variation within individuals or species can be found in aragonitic specimens, whereas high-Mg calcitic species show somewhat more variability in both calcite and Mg content, and those with mixed mineralogies are highly variable. These three groups correspond broadly with currently accepted clades. Given this strong phylogenetic signal, we analysed the data using phylogenetically independent contrasts, a statistical approach that separates genotypic from phenotypic variability; we found that variations which might be ascribed to environment were generally weak. The mineralogy of serpulid tubes makes them particularly vulnerable to ocean chemistry changes. While some serpulids appear to be able to adjust their tube mineralogy in order to adapt to sea-water chemistry, overall strength and elasticity may be sacrificed when they do. The biodiverse reef habitat provided by serpulids in some temperate regions may be the only complex solid habitat available, and loss or compromise of these temperate reefs will most likely have deleterious flow-on effects on temperate benthic communities.     

Molecular genetic variation in tarpon (<Emphasis Type="Italic">Megalops atlanticus</Emphasis> Valenciennes) in the northern Atlantic Ocean

The tarpon (Megalops atlanticus) is a highly valued game fish and occasional food fish in the eastern and western Atlantic Ocean. Tarpon have a high capacity for dispersal, but some regional biological differences have been reported. In this study we used two molecular genetic techniques—protein electrophoresis of nuclear DNA loci, and restriction fragment length polymorphism analysis of the mitochondrial DNA (mtDNA)—to assess this species population genetic structure in the eastern (coastal waters off Gabon and Sierra Leone, Africa) and western (coastal waters off Florida, Caribbean Sea) Atlantic Ocean north of the equator. Genetic differentiation was observed between tarpon from Africa and tarpon from the western Atlantic Ocean. A unique allele and haplotype, significant differences in allozyme allele and mtDNA haplotype frequencies between the African and western Atlantic samples, and significant F_ST analyses suggest that levels of gene flow between tarpon from these two regions is low. Among the western Atlantic Ocean collections, genetic diversity values and allele and haplotype frequencies were similar. AMOVA analyses also showed a degree of genetic relatedness among most of the western Atlantic Ocean collections: however, some significant population structuring was detected in the allozyme data. A regional jackknifed F_ST analysis indicated the distinction of the Costa Rica population from the other western Atlantic populations and, in pairwise analyses, F_ST values tended to be higher (i.e., genetic relatedness was lower) when the Costa Rican sample was paired with any of the other western Atlantic samples. These data suggest that Costa Rican tarpon could be partially isolated from other western Atlantic tarpon populations. Ultimately, international cooperation will be essential in the management of this species in both the eastern and western Atlantic Ocean.Communicated by P.W. Sammarco, Chauvin     

Application of eggshell waste for the immobilization of cadmium and lead in a contaminated soil

Liming materials have been used to immobilize heavy metals in contaminated soils. However, no studies have evaluated the use of eggshell waste as a source of calcium carbonate (CaCO₃) to immobilize both cadmium (Cd) and lead (Pb) in soils. This study was conducted to evaluate the effectiveness of eggshell waste on the immobilization of Cd and Pb and to determine the metal availability following various single extraction techniques. Incubation experiments were conducted by mixing 0–5% powdered eggshell waste and curing the soil (1,246 mg Pb kg^?1 soil and 17 mg Cd kg^?1 soil) for 30 days. Five extractants, 0.01 M calcium chloride (CaCl₂), 1 M CaCl₂, 0.1 M hydrochloric acid (HCl), 0.43 M acetic acid (CH₃COOH), and 0.05 M ethylendiaminetetraacetic acid (EDTA), were used to determine the extractability of Cd and Pb following treatments with CaCO₃ and eggshell waste. Generally, the extractability of Cd and Pb in the soils decreased in response to treatments with CaCO₃ and eggshell waste, regardless of extractant. Using CaCl₂ extraction, the lowest Cd concentration was achieved upon both CaCO₃ and eggshell waste treatments, while the lowest Pb concentration was observed using HCl extraction. The highest amount of immobilized Cd and Pb was extracted by CH₃COOH or EDTA in soils treated with CaCO₃ and eggshell waste, indicating that remobilization of Cd and Pb may occur under acidic conditions. Based on the findings obtained, eggshell waste can be used as an alternative to CaCO₃ for the immobilization of heavy metals in soils.     

Size and spatial structure in deep versus shallow populations of the Mediterranean gorgonian <Emphasis Type="Italic">Eunicella singularis</Emphasis> (Cap de Creus,northwestern Mediterranean Sea)

In the Western Mediterranean Sea, the gorgonian Eunicella singularis (Esper, 1794) is found at high densities on sublittoral bottoms at depths from 10 to 70 m. Shallow colonies have symbiotic zooxanthellae that deeper colonies lack. While knowledge of the ecology of the shallow populations has increased during the last decades, there is almost no information on the ecology of the deep sublittoral populations. In October and November 2004 at Cap de Creus (42°19′12″ N; 03°19′34″ E), an analysis of video transects made by a remotely operated vehicle showed that shallow populations (10–25 m depth) were dominated by small, non-reproductive colonies, while deep sublittoral populations (50–67 m depth) were dominated by medium-sized colonies. Average and maximum colony heights were greater in the deeper populations, with these deeper populations also forming larger patch sizes and more extensive regions of continuous substrate coverage. These results suggest that shallow habitats are suitable for E. singularis, as shown by the high recruitment rate, but perturbations may limit or delay the development of these populations into a mature stage. This contrasts with the deep sublittoral habitats where higher environmental stability may allow the development of mature populations dominated by larger, sexually mature colonies.     

Precipitation of aragonite by calcitic bivalves in Mg-enriched marine waters

To understand the relative importance of biological versus physicochemical control over biomineralization, we have tested if the chemical composition of the medium (i.e., the Mg/Ca ratio) can change the mineralogy of mollusk shells. The shells of mollusks are made of calcite and/or aragonite, which are by far the most common CaCO₃ polymorphs. Several species of bivalves with predominantly calcitic shells have been cultivated in artificial seawater with a Mg/Ca molar ratio within the range of 8.3–9.2, well above the present value for seawater (5.2). Four out of six species used (the scallop Chlamys varia, the oyster Ostrea edulis, the saddle oyster Anomia ephippium and the mussel Mytilus edulis) survived long enough to secrete significant amounts of calcium carbonate. The deposits (sometimes extensive) formed on the interior shell surfaces were predominantly aragonitic. Three individuals of C. varia also increased their length by adding new shell at the margin. Contrary to the internal shell deposits, these margins were high-Mg calcite. This implies that the marginal mantle is able to exert a more strict control on the secreted mineral phase than the mantle facing the internal shell surface. This is the first report on an in vivo experimentally forced switch in bivalve shell mineralogy, from calcite to aragonite due to a change in water chemistry.     

Oxygen and carbon isotope profiles from Nassa mutabilis shells (Gastropoda): accretion rates and biological behaviour

Six shells of the Gastropoda species Nassa mutabilis L. were collected in the Adriatic Sea at a depth of a few metres off the central Italian coast during 1996 and 1997. The oxygen and carbon isotope composition of spot samples collected from the shell apex to the top was measured according to well-established techniques. The results obtained allow the following conclusions: (1) the CaCO₃ of the N. mutabilis shells is essentially formed by aragonite as proved by X-ray diffraction measurements; (2) the CaCO₃ is precipitated under isotope equilibrium conditions or very close to equilibrium with the environmental water; (3) shell accretion takes place all year round, although the warm period is preferred since the accretion rates are higher during the warm half of the year and considerably reduced in winter; (4) the carbon isotope composition changes according to the age of the specimen, probably in relation to changes in its nutritional regimen; and (5) the alternation of warm and cold periods recorded by the shells allows the evaluation of their age and of the season of their birth and death. Received: 3 December 1998 / Accepted: 7 September 1999     

Possible control mechanism of cell motility in the gorgonian Eunicella cavolinii

In a previous study it was demonstrated that a lectin controls cell-cell interaction in the gorgonian Eunicella cavolinii (Koch) as a negative modulator. Now we describe the procedure to purify this lectin to homogeneity; its molecular weight is 23 400. The homologous proteoglycans were identified as positive modulators of cell-cell (and/or cell substrate) interaction. The purified single proteoglycan aggregates were 1200±700 nm long and the distance between the attachment points of the proteoglycan subunits was about 45 nm. The glycosaminoglycan residues of the gorgonian proteoglycans were identified as hyaluronic acid (35.5%), heparan sulfate (47.9%) and dermatan sulfate (14.1%). Binding studies with immobilized homologous proteoglycan preparations revealed that gorgonian cells attach to this substratum presumably via its glycosaminoglycans. Quantitative determinations of these two modulators of cell-cell recognition in the three regions of individual colonies revealed concentration gradients these concentration differences reflect a lower cell motility in the polyp-bearing branches and a higher motility of the cells in the polyp-free stem and in the basis, is discussed.     

Elevated CO2 alters larval proteome and its phosphorylation status in the commercial oyster, Crassostrea hongkongensis

Ocean acidification (OA) is beginning to have noticeable negative impact on calcification rate, shell structure and physiological energy budgeting of several marine organisms; these alter the growth of many economically important shellfish including oysters. Early life stages of oysters may be particularly vulnerable to OA-driven low pH conditions because their shell is made up of the highly soluble form of calcium carbonate (CaCO₃) mineral, aragonite. Our long-term CO₂ perturbation experiment showed that larval shell growth rate of the oyster species Crassostrea hongkongensis was significantly reduced at pH < 7.9 compared to the control (8.2). To gain new insights into the underlying mechanisms of low-pH-induced delays in larval growth, we have examined the effect of pH on the protein expression pattern, including protein phosphorylation status at the pediveliger larval stage. Using two-dimensional electrophoresis and mass spectrometry, we demonstrated that the larval proteome was significantly altered by the two low pH treatments (7.9 and 7.6) compared to the control pH (8.2). Generally, the number of expressed proteins and their phosphorylation level decreased with low pH. Proteins involved in larval energy metabolism and calcification appeared to be down-regulated in response to low pH, whereas cell motility and production of cytoskeletal proteins were increased. This study on larval growth coupled with proteome change is the first step toward the search for novel Protein Expression Signatures indicative of low pH, which may help in understanding the mechanisms involved in low pH tolerance.     

Respiratory quotient and calcification of Nautilus macromphalus (Cephalopoda: Nautiloidea)

Respiration and calcification were investigated in the ectocochleate cephalopod Nautilus macromphalus Sowerby. Specimens were collected off New Caledonia, in October 1991, and kept at the Nouméa Aquarium until December 1991. The respiratory quotient and calcification rate of 5 individuals were measured during 14 short term incubations (63 to 363 min). Oxygen uptake was recorded with a polarographic oxygen sensor. CO₂ flux and calcification were calculated from changes in pH and alkalinity (alkalinity-anomaly technique). Several methods were used to compute the respiratory quotient (RQ); a functional regression indicated an RQ of 0.74. CaCO₃ exchange rates were linearly related to respiratory quotient, calcification occurring in individuals with a low RQ. CaCO₃ uptake from the surrounding water was noncontinuous. From the highest CaCO₃ uptake, maximum growth rate was estimated as 7.1 mg shell wt h^- (=61 g yr^-1).     

A morphological and structural study of the larval shell from the abalone <Emphasis Type="Italic">Haliotis tuberculata</Emphasis>

The larval shell of the marine gastropod Haliotis tuberculata was investigated by polarised light microscopy, scanning electron microscopy, Raman microspectroscopy and infra-red spectroscopy. Trochophore and veliger larval sections were used for histological examination of the growing shell and each larval stage was related to the shell development and the appearance of calcified formations. We determined the stage of initial calcification by specific staining combined with polarised light examination. The shell of 30-h-old pre-veliger larvae was found to be mineralized, confirming that calcification occurred before larval torsion. Using both infra-red and Raman spectroscopy, we showed that CaCO₃ deposition occurred at the pre-veliger stage and that the mineral phase initially deposited was essentially composed of aragonite.     

Respiration as a function of oxygen concentration in intertidal barnacles

Respiration as a function of oxygen concentration was studied in two species of intertidal barnacles: Balanus amphitrite amphitrite ¹ (Darwin) and B. tintinnabulum tintinnabulum (L.). A critical oxygen tension was observed in both species below which the respiratory regulation broke down. In B. amphitrite amphitrite the critical oxygen tension was 2.5 ml O₂/l, and in B. tintinnabulum tintinnabulum 3.5 ml O₂/l. Species differences and habitat relations were observed in both species in their respiratory adaptation to oxygen concentration. B. amphitrite amphitrite which inhabits oxygen-deficient areas was able to regulate to much lower concentrations than B. tintinnabulum tintinnabulum which inhabits oxygen-rich open intertidal regions.     

Vitamine B12 et phytoplancton dans l'Océan Antarctique. Distribution et approche expérimentale

In late summer (Antiprod I cruise, March 1977), antarctic waters of the southern part of the Indian Ocean contain low amounts of dissolved Vitamin B₁₂: 1 ng l^-1 on average. However, higher concentrations were recorded in two areas: in the subantarctic convergence zone and between 50° and 57°S, with values greater than 3,5 ng l^-1. Despite nutrient-rich waters, primary production was low and the phytoplankton sparse. Abundance of diatoms was related, to a certain extent, to the concentration of Vitamin B₁₂. An experimental approach (Antiprod II cruise, February and March 1980) was applied to (1) artificially enriched natural sea water, and (2) cultures of two diatom species isolated from antarctic waters, Nitzschia turgiduloides and Chaetoceros sp. The experiments demonstrated that antarctic diatoms in general do not require any of the three investigated vitamins for growth. However, Vitamin B₁₂ has a slightly stimulating effect on growth. This may explain the observed relation between phytoplankton density and vitamin concentration in the Antarctic Ocean. This experimental approach elucidates the minor role of vitamins in the primary production of antarctic waters and provides indirect evidence of the major importance of turbulence as a limiting factor.     

Mitochondrial DNA sequence variation and phylogeography of oceanic insects (Hemiptera: Gerridae: Halobates spp.)

Relatively few insects have invaded the marine environment, and only five species of sea skaters, Halobates Eschscholtz (Hemiptera: Gerridae), have successfully colonized the surface of the open ocean. All five species occur in the Pacific Ocean, H. germanus White also occurs in the Indian Ocean, whereas H. micans Esch- scholtz is the only species found in the Atlantic Ocean. We sequenced a 780 bp long region of the mitochondrial cytochrome oxidase subunit I gene (COI) for a total of 66 specimens of the five oceanic Halobates species. Our purpose was to investigate the genetic variation within species and estimate the amount of gene flow between populations. We defined 27 haplotypes for H. micans and found that haplotype lineages from each of the major oceans occupied by this species are significantly different, having sequences containing five to seven unique base substitutions. We conclude that gene flow between populations of H. micans inhabiting the Atlantic, Pacific, and Indian Ocean is limited and hypothesize that these populations have been separated for 1 to 3 million years. Similarly, there may be limited gene flow between H. germanus populations found in the Pacific and Indian Ocean and between H. sericeus populations inhabiting the northern and southern parts of the Pacific Ocean. Finally, we discuss our findings in relation to recent hypotheses about the influence of oceanic diffusion on the distribution and population structure of oceanic Halobates spp. Received: 29 July 1999 / Accepted: 23 November 1999     

Potential fluoride contamination in the drinking water of Marks Nagar,Unnao district,Uttar Pradesh,India

This study was conducted in the summer season (May, 2007). The fluoride concentration along with other physico-chemical parameters in ground water samples was determined in Marks Nagar of Unnao district, Uttar Pradesh (India), since it is the only source of drinking water for the villagers. The fluoride concentration in the water varied from 0.8 to 13.9 mgl⁻¹ with a mean of 4.02 mgl⁻¹. The correlation analysis revealed that fluoride had a positive correlation with pH, CO₃, HCO₃, and sodium adsorption ratio (SAR), whereas a negative correlation with Ca and Mg was found. A soil profile was also dug in the area to assess depth-wise fluoride content in the soil. The soil samples and underneath calcium carbonate (CaCO₃) concretion were analyzed for fluoride content. The percent of soluble fluoride to total fluoride in the soil varied from 25.15 to 4.76% down the soil profile. The soluble fluoride was found to decrease with the increase in the clay content in the soil. The total leachable fluoride in CaCO₃ concretions was found to be 6.08%. It was inferred from this study that the soil and underneath layer of CaCO₃ concretions may be the potential source of fluoride contamination in the shallow drinking water sources of the area.