Targeted sampling protocol as prelude to bacterial source tracking with Enterococcus faecalis

Recent studies suggest that host origin databases for bacterial source tracking (BST) must contain a large number of isolates because bacterial subspecies change with geography and time. A new targeted sampling protocol was developed as a prelude to BST to minimize these changes. The research was conducted on the Sapelo River, a tidal river on the Georgia coast. A general sampling of the river showed fecal enterococcal numbers ranging from <10 (below the limit of detection) to 990 colony-forming units (CFU) per 100 mL. Locations with high enterococcal numbers were combined with local knowledge to determine targeted sampling sites. Fecal enterococcal numbers around one site ranged from <10 to 24,000 CFU per 100 mL. Bacterial source tracking was conducted to determine if a wastewater treatment facility at the site was responsible for this contamination. The fecal indicator bacterium was Enterococcus faecalis. Ribotyping, automated with a RiboPrinter (DuPont Qualicon, Wilmington, DE), was the BST method. Thirty-seven ribotypes were observed among 83 Ent. faecalis isolates obtained from the Sapelo River and the wastewater lagoon. Sixteen ribotypes were associated with either the river or the lagoon, and only five ribotypes (14%) were shared. Nevertheless, these five ribotypes represented 39 of the 83 Ent. faecalis isolates, almost a majority (47%). These results suggest that the fecal contamination in the river came from the wastewater treatment facility. As a prelude to BST, targeted sampling minimized subspecies changes with geography and time, and eliminated the need for a permanent host origin database by restricting BST to a small geographic area and requiring sampling to be completed in one day.     

Assessment of the 16S-23S rDNA intergenic spacer region in Enterococcus spp. for microbial source tracking

A new library-based microbial source tracking (MST) approach intended for initial application in the coastal waters of Virginia was evaluated. Host-origin isolates of Enterococcus spp. were collected from beaches and the surrounding tidewater region of Virginia and used to construct a library based on the pattern of DNA band lengths produced by the amplification of the 16S-23S rDNA intergenic spacer (IGS) region, and subsequent digestion with MboI. Initial results from small host-origin libraries (64 and 200 total isolates) with discriminant analysis (DA) and logistic regression (LR) yielded high average rates of correct classification (ARCC) for a four-source classification split (birds, dogs, sewage, and wildlife), with ARCCs ranging from 83 to 100%. However, the poor results obtained when classification was attempted on a non-library validation set (VS, ARCCs of 47 and 48%, respectively, using DA and LR) demonstrated that a library of 200 isolates was insufficient to adequately represent the diversity of the enterococci in the sampled region. An increase in the library size to 1029 total isolates was accompanied by a reduction in the ARCC of the library to 42.7% with DA and 45.7% with LR, plus similarly poor results obtained from the VS. The low correct classification rates generated by the larger known-source library were unsuitable for field application. Many reported MST methods have been based on results obtained using small host-origin libraries without external validation. Our results indicate that such an approach can be very misleading, and that larger libraries and external validation is essential for the confirmation of preliminary results.     

Identifying sources of fecal contamination inexpensively with targeted sampling and bacterial source tracking

Most bacterial source tracking (BST) methods are too expensive for most communities to afford. We developed targeted sampling as a prelude to BST to reduce these costs. We combined targeted sampling with three inexpensive BST methods, Enterococcus speciation, detection of the esp gene, and fluorometry, to confirm the sources of fecal contamination to beaches on Georgia's Jekyll and Sea Islands during calm and stormy weather conditions. For Jekyll Island, the most likely source of contamination was bird feces because the percentage of Ent. faecalis was high (30%) and the esp gene was not detected. For the Sea Island beach during calm conditions, the most likely sources of fecal contamination were leaking sewer lines and wildlife feces. The leaking sewer lines were confirmed with fluorometry and detection of the esp gene. For the Sea Island beach during stormflow conditions, the most likely sources of fecal contamination were wildlife feces and runoff discharging from two county-maintained pipes. For the pipes, the most likely source of contamination was bird feces because the percentage of Ent. faecalis was high (30%) and the esp gene was not detected. Sediments were also a reservoir of fecal enterococci for both Jekyll and Sea Islands. Combining targeted sampling with two or more BST methods identified sources of fecal contamination quickly, easily, and inexpensively. This combination was the first time targeted sampling was conducted during stormy conditions, and the first time targeted sampling was combined with enterococcal speciation, detection of the esp gene, and fluorometry.     

Individualized water source as an indicator of attitudes about water management and conservation in humid regions

Public perceptions about water quantity and water as a common pool resource are understudied in humid regions. As water demand increases, the need to more closely manage water, even in humid areas, will increase, requiring better understanding how people perceive their water supply, how they view paying for water conservation and how water user characteristics influence attitudes. A survey finds correlations between utilizing an individual water source (e.g. well or spring) and attitudes toward water management and conservation. Compared to respondents with a shared water source, those with an individual source believe they are segregated from regional water concerns. They are less willing to pay for water management or conservation measures and less supportive of any government intervention in water management. These results suggest that planners and water managers may face resistance to conservation policies or any policy based on the idea of water as a common pool resource.     

Wastewood as a source of woodfuel

Land clearing and forest sector residues, notably the wastewood generated on large timber plantations, can provide a sizeable and hitherto neglected source of woodfuel. This article highlights experience in Malawi where wastewood from pine plantations is converted into charcoal that is sold to residential, industrial and agro-industrial users. Similar initiatives proposed in other countries of sub-Saharan Africa indicate that comprehensive utilization of wastewood resources could help to reduce regional and local imbalances.     

Cupric ion activity in peat soil as a toxicity indicator for maize

Copper phytotoxicity in soils is difficult to assess because Cu accumulates at and damages roots, and is not readily transferred to shoots. Soil chemical properties strongly influence Cu speciation, so that total soil Cu alone is not a broadly useful indicator of potential toxicity to plants. The present study measured free Cu2+ activity in Cu-enriched peat soils using the ion selective electrode. The soil Cu2+ activity was related to the severity of phytotoxicity as measured by several indicators in a maize (Zea mays L.) bioassay, including leaf chlorosis, root stunting, and reduced shoot growth and Fe concentration. A soil Cu2+ activity of 10(-7.0) to 10(-7.5), corresponding to total Cu of about 275 mg/kg in the peat soil, caused phytotoxicity in maize seedlings. It is proposed that Cu2+ activity is more directly related to phytotoxic effects than other soil tests, such as extractions with strong acids or chelating agents, because it is the free Cu2+ in soil solution that has the most direct toxic effects on roots. There was very limited uptake of Cu into maize shoots, and even when Cu2+ activity and total soil Cu were raised into the extreme toxicity range of 10(-5) and 4,000 mg/ kg, respectively, shoot Cu remained less than 35 mg/kg. These results indicate the inadequacy of the USEPA risk assessment of potential for Cu toxicity to crops amended with sewage sludge, which assumed a no-effect level of maize shoot Cu of 40 mg/kg.     

Barium as a potential indicator of phosphorus in agricultural runoff

In many catchments, anthropogenic input of contaminants, and in particular phosphorus (P), into surface water is a mixture of agricultural and sewage runoff. Knowledge about the relative contribution from each of these sources is vital for mitigation of major environmental problems such as eutrophication. In this study, we investigated whether the distribution of trace elements in surface waters can be used to trace the contamination source. Water from three groups of streams was investigated: streams influenced only by agricultural runoff, streams influenced mainly by sewage runoff, and reference streams. Samples were collected at different flow regimes and times of year and analyzed for 62 elements using ICP-MS. Our results show that there are significant differences between the anthropogenic sources affecting the streams in terms of total element composition and individual elements, indicating that the method has the potential to trace anthropogenic impact on surface waters. The elements that show significant differences between sources are strontium (p < 0.001), calcium (p < 0.004), potassium (p < 0.001), magnesium (p < 0.001), boron (p < 0.001), rhodium (p = 0.001), and barium (p < 0.001). According to this study, barium shows the greatest potential as a tracer for an individual source of anthropogenic input to surface waters. We observed a strong relationship between barium and total P in the investigated samples (R(2) = 0.78), which could potentially be used to apportion anthropogenic sources of P and thereby facilitate targeting of mitigation practices.     

Bacterial source tracking guides management of boat head waste in a coastal resort area

Fecal contamination of water bodies causes a public health problem and economic loss. To control such contamination management actions need to be guided by sound science. From 2007–2009 a study was undertaken to determine the sources of fecal bacteria contamination to the marine waters adjoining the Town of Wrightsville Beach, North Carolina, USA. The research effort included sampling for fecal coliform and Enterococcus bacteria, sampling for optical brighteners, dye studies, and use of molecular bacterial source tracking techniques including polymerase chain reaction (PCR) and terminal restriction fragment polymorphism (T-RFLP) fingerprinting of the Bacteroides–Prevotella group. Of the 96 samples collected from nine locations during the study, the water contact standard for Enterococcus was exceeded on 13 occasions. The T-RFLP fingerprint analyses demonstrated that the most widespread source of fecal contamination was human, occurring in 38% of the samples, with secondary ruminant and avian sources also detected. Optical brightener concentrations were low, reflecting a lack of sewage line leakage or spills. A lack of sewer leaks and lack of septic systems in the town pointed toward discharge from boat heads into the marine waters as the major cause of fecal contamination; this was supported by dye studies. Based on these data, the Town initiated action to have the U.S. Environmental Protection Agency declare the coastal waters (out to 3 nautical miles), the nearby Atlantic Intracoastal Waterway and its tributaries a no-discharge zone (NDZ) to alleviate the human fecal pollution. The Town garnered supporting resolutions from other local communities who jointly petitioned the North Carolina Department of Environmental and Natural Resources. This State regulatory agency supported the local government resolutions and sent an application for an NDZ to the EPA in April 2009. The EPA concurred, and in February 2010 the coastal waters of New Hanover County, NC, became the first marine area on the U.S. eastern seaboard between Delaware and the Florida Keys to be declared a no-discharge zone.     

Poultry litter ash as a potential phosphorus source for agricultural crops

Maryland will impose restrictions on poultry litter application to soils with excessive P by the year 2005. Alternative uses for poultry litter are being considered, including burning as a fuel to generate electricity. The resulting ash contains high levels of total P, but the availability for crop uptake has not been reported. Our objective was to compare the effectiveness of poultry litter ash (PLA) and potassium phosphate (KP) as a P source for wheat (Triticum aestivum L.) in acidic soils, without and with limestone application. Two acidic soils (pH 4.25 and 4.48) were studied, unlimed or limed to pH 6.5 before cropping. The PLA and KP were applied at 0, 39, and 78 kg P ha(-1), after which wheat was grown. Limestone significantly increased wheat yield, but the P sources without limestone did not. The two P sources were not significantly different as P fertilizer. At the 78 kg P ha(-1) rate, wheat shoot-P concentrations were 1.10 and 1.12 g kg(-1) for the PLA treatment compared with 0.90 and 0.89 g kg(-1) for KP in the nonlimed and limed soils, respectively. Trace element concentrations in wheat shoots from the PLA treatment were less than or equal to KP and the control. The low levels of water-soluble P and metals in the soils and the low metal concentrations in wheat suggest that PLA is an effective P fertilizer. Further studies are needed to determine the optimum application rate of PLA as a P fertilizer.     

Roofing as a source of nonpoint water pollution

Sixteen wooden structures with two roofs each were installed to study runoff quality for four commonly used roofing materials (wood shingle, composition shingle, painted aluminum, and galvanized iron) at Nacogdoches, Texas. Each roof, either facing NW or SE, was 1.22 m wide x 3.66 m long with a 25.8% roof slope. Thus, there were 32 alternatively arranged roofs, consisting of four roof types x two aspects x four replicates, in the study. Runoff from the roofs was collected through galvanized gutters, downspouts, and splitters. The roof runoff was compared to rainwater collected by a wet/dry acid rain collector for the concentrations of eight water quality variables, i.e. Cu(2+), Mn(2+), Pb(2+), Zn(2+), Mg(2+), Al(3+), EC and pH. Based on 31 storms collected between October 1997 and December 1998, the results showed: (1) concentrations of pH, Cu, and Zn in rainwater already exceed the EPA freshwater quality standards even without pollutant inputs from roofs, (2) Zn and Cu, the two most serious pollutants in roof runoff, exceeded the EPA national freshwater water quality standards in virtually 100% and more than 60% of the samples, respectively, (3) pH, EC, and Zn were the only three variables significantly affected by roofing materials, (4) differences in Zn concentrations were significant among all roof types and between all roof runoff and rainwater samples, (5) although there were no differences in Cu concentrations among all roof types and between roof runoff and rainwater, all means and medians of runoff and rainwater exceeded the national water quality standards, (6) water quality from wood shingles was the worst among the roof types studied, and (7) although SE is the most frequent and NW the least frequent direction for incoming storms, only EC, Mg, Mn, and Zn in wood shingle runoff from the SE were significantly higher than those from the NW; the two aspects affected no other elements in runoff from the other three roof types. Also, Zn concentrations from new wood-shingle roofs were significantly higher than those from aged roofs of a previous study. The study demonstrated that roofs could be a serious source of nonpoint water pollution. Since Zn is the most serious water pollutant and wood shingle is the worst of the four roof types, using less compounds and materials associated with Zn along with good care and maintenance of roofs are critical in reducing Zn pollution in roof runoff.     

Railway-Associated Attractants as Potential Contaminants for Wildlife

Environmental Management - Grizzly bears (Ursus arctos) appear to be attracted to natural and anthropogenic forage along railways, which may increase collision vulnerability, but also potentially...     

Selection of a water-extractable phosphorus test for manures and biosolids as an indicator of runoff loss potential

The correlation of runoff phosphorus (P) with water-extractable phosphorus (WEP) in land-applied manures and biosolids has spurred wide use of WEP as a water quality indicator. Land managers, planners, and researchers need a common WEP protocol to consistently use WEP in nutrient management. Our objectives were to (i) identify a common WEP protocol with sufficient accuracy and precision to be adopted by commercial testing laboratories and (ii) confirm that the common protocol is a reliable index of runoff P. Ten laboratories across North America evaluated alternative protocols with an array of manure and biosolids samples. A single laboratory analyzed all samples and conducted a separate runoff study with the manures and biosolids. Extraction ratio (solution:solids) was the most important factor affecting WEP, with WEP increasing from 10:1 to 100:1 and increasing from 100:1 to 200:1. When WEP was measured by a single laboratory, correlations with runoff P from packed soil boxes amended with manure and biosolids ranged from 0.79 to 0.92 across all protocol combinations (extraction ratio, filtration method, and P determination method). Correlations with P in runoff were slightly lower but significant when WEP was measured by the 10 labs (r=0.56-0.86). Based on laboratory repeatability and water quality evaluation criteria, we recommend the following common protocol: 100:1 extraction ratio; 1-h shaking and centrifuge 10 min at 1500xg (filter with Whatman #1 paper if necessary); and determining P by inductively coupled plasma-atomic emission spectrometry or colorimetric methods.     

Broiler litter as a micronutrient source for cotton: concentrations in plant parts

Analytically, poultry litter contains nearly all essential micronutrients but the extent of phytoavailability of these nutrients and whether cotton (Gossypium hirsutum L.) and other crop plants can receive adequate amounts of these nutrients from litter is not fully known. The objective of this research was to determine whether cotton receives sufficient amounts of Fe, Cu, Mn, and Zn from litter and estimate the efficiency of cotton in extracting these metal nutrients from litter in the absence of any other source of the micronutrients. The greenhouse research used plastic pots filled with approximately 11 kg of a 2:1 (v/v) sand to vermiculite growing mix. Cotton (cv. Stoneville 474) was grown in the pots fertilized with broiler litter at rates of 30, 60, 90, or 120 g pot(-1) in a factorial combination with four supplemental nutrient solution (NS) treatments. The nutrient solutions consisted of full Hoagland's nutrient solution (NS-full); a solution of the macronutrients N, P, K, Ca, and Mg (NS-macro); a solution of the micronutrients Fe, Zn, Mn, Cu, B, and Mo (NS-micro); and water (NS-none). Based on tissue nutrient analysis, a one-time broiler litter application supplied adequate amounts of Fe, Cu, and Mn to bring the concentration of these nutrients in upper leaves within published sufficiency ranges. Zinc, with <17 mg kg(-1) concentration in the upper leaves, was the only micronutrient below the established sufficiency range regardless of the rate of applied litter. Cotton extracted Fe and Mn more efficiently than Cu or Zn, removing as much as 8.8% of Fe and 7.2% of Mn supplied by 30 g litter pot(-1). In contrast, the extraction efficiency was 1.7% for Cu and 1.9% for Zn. Roots accumulated 58% of the total absorbed Fe and 64% of Cu, and leaves accumulated 32% of the Fe and only 13% of the Cu supplied by litter. In contrast, only 16% of the total absorbed Mn and 23% of Zn accumulated in roots while leaves accumulated 64% of the total Mn and 37% of Zn. These results demonstrate that broiler litter is a valuable source of the metal nutrients supplying Fe, Cu, and Mn in full and Zn in part, but a very large fraction of the litter-supplied metal nutrients remained in the growing mix.     

Sequence-based source tracking of Escherichia coli based on genetic diversity of beta-glucuronidase

High levels of fecal bacteria are a concern for recreational waters; however, the source of contamination is often unknown. This study investigated whether direct sequencing of a bacterial gene could be utilized for detecting genetic differences between bacterial strains for microbial source tracking. A 525-nucleotide segment of the gene for beta-glucuronidase (uidA) was sequenced in 941 Escherichia coli isolates from the Clinton River-Lake St. Clair watershed, 182 E. coli isolates from human and animal feces, and 34 E. coli isolates from a combined sewer. Environmental isolates exhibited 114 alleles in 11 groups on a genetic tree. Frequency of strains from different genetic groups differed significantly (p < 0.03) between upstream reaches (Bear Creek-Red Run), downstream reaches, and Lake St. Clair beaches. Fecal E. coli uidA sequences exhibited 81 alleles that overlapped with the environmental set. An algorithm to assign alleles to different host sources averaged approximately 75% correct classification with the fecal data set. Using the same algorithm, the percent of environmental isolates assignable to humans decreased significantly between Bear Creek-Red Run (30 +/- 3%) and the beaches (17 +/- 2%) (p < 0.05). Birds accounted for approximately 50% of assignable environmental isolates. For combined sewer isolates, the same algorithm assigned 51% to humans. These experiments demonstrate differences in the frequency of different E. coli strains at different locations in a watershed, and provide a "proof in principle" that sequence-based data can be used for microbial source tracking.     

Environmental and production consequences of using alum-amended poultry litter as a nutrient source for corn

Field trials were established to compare alum-treated poultry litter (ATPL), normal poultry litter (NPL), and triple superphosphate (TSP) as fertilizer sources for corn (Zea mays L.) when applied at rates based on current litter management strategies in Virginia. Trials were established in the Costal Plain and Piedmont physiographic regions near Painter and Orange, VA, respectively. Nitrogen-based applications of ATPL or NPL applied at rates estimated to supply 173 kg of plant-available nitrogen (PAN) ha(-1) resulted in significantly lower grain yields than treatments receiving commercial fertilizer at the same rate in 2000 and 2001 at Painter. These decreases in grain yield at the N-based application rates were attributed to inadequate N availability, resulting from overestimates of PAN as demonstrated by tissue N concentrations. However, at Orange no treatment effects on grain yield were observed. Applications of ATPL did not affect Al concentrations in corn ear-leaves at either location. Exchangeable soil Al concentrations were most elevated in treatments receiving only NH4NO3 as an N source. At N-based application rates, the ATPL resulted in lower Mehlich 1-extractable P (M1-P) and water-extractable soil phosphorus (H2O-P) concentrations compared to the application of NPL. A portion of this reduction could be attributed to lower rates of P applied in the N-based ATPL treatments. Runoff collected from treatments which received ATPL 2 d before conducting rainfall simulations contained 61 to 71% less dissolved reactive phosphorus (DRP) than treatments receiving NPL. These results show that ATPL may be used as a nutrient source for corn production without significant management alterations. Alum-treated poultry litter can also reduce the environmental impact of litter applications, primarily through minimizing the P status of soils receiving long-term applications of litter and reductions in runoff DRP losses shortly after application.     

Iron Ore Industry Emissions as a Potential Ecological Risk Factor for Tropical Coastal Vegetation

In the coastal zone of the Espírito Santo state, Brazil, fragments of restinga, which form a natural ecosystem, share their space with an increasing number of iron ore industries. The iron ore dust and SO(2) originating from the industry processing activities can interfere with the vegetation of the adjacent ecosystems at various levels. This study was undertaken in order to evaluate the effects of industry emissions on representative members of the restinga flora, by measuring physiological and phenological parameters. Foliar samples of Ipomoea pes caprae, Canavalia rosea, Sophora tomentosa, and Schinus terebinthifolius were collected at three increasing distances from an ore industry (1.0, 5.0, and 15.0 km), and were assessed for their dust deposition, chlorophyll, and Fe content. Phenological monitoring was focused on the formation of shoots, flowers, and fruits and was also performed throughout the course of a year. The results showed that the edaphic characteristics and the mineral constitutions of the plants were affected by industry emissions. In addition, the chlorophyll content of the four species increased with proximity to the industry. Phenological data revealed that the reproductive effort, as measured by fruit production, was affected by emissions and S. tomentosa was the most affected species. The use of an integrative approach that combines biochemical and ecological data indicates that the restinga flora is under stress due to industry emissions, which on a long-term basis may put the ecosystem at risk.     

Dissolution of phosphate in a phosphorus-enriched ultisol as affected by microbial reduction

Phosphorus dissolution often increases as soils become more reduced, but the mechanisms are not fully understood. The objectives of this research were to determine rates and mechanisms of P dissolution during microbial reduction of a surface soil from the North Carolina Coastal Plain. Duplicate suspensions of silt + clay fractions from a Cape Fear sandy clay loam (fine, mixed, semiactive, thermic Typic Umbraquult) were reduced in a continuously stirred redox reactor for 40 d. We studied the effects of three treatments on P dissolution: (i) 2 g dextrose kg(-1) solids added as a microbial carbon source at time 0 d; (ii) 2 g dextrose kg(-1) solids split into three additions at 0, 12, and 26 d; and (iii) no added dextrose. After 40 d of reduction, concentrations of dissolved reactive phosphorus (DRP) were similar for all treatments and increased up to sevenfold from 1.5 to 10 mg L(-1). The initial rate of reduction and dissolution of DRP was significantly greater for the 0-d treatment. A linear relationship (R(2) = 0.79) was found between DRP and dissolved organic carbon (DOC). Dissolved Fe and Al and pH increased, suggesting the formation of aqueous Fe- and Al-organic matter complexes. Separate batch experiments were performed to study the effects of increasing pH and citrate additions on PO(4) dissolution under aerobic conditions. Increasing additions of citrate increased concentrations of DRP, Fe, and Al, while increasing pH had no effect. Results indicated that increased dissolved organic matter (DOM) during soil reduction contributed to the increase in DRP, perhaps by competitive adsorption or formation of aqueous ternary DOM-Fe-PO(4) or DOM-Al-PO(4) complexes.     

Evaluating sewage-associated JCV and BKV polyomaviruses for sourcing human fecal pollution in a coastal river in Southeast Queensland, Australia

In this study, the host-sensitivity and host-specificity of JC virus (JCV) and BK virus (BKV) polyomaviruses were evaluated by testing wastewater and fecal samples from nine host groups in Southeast Queensland, Australia. The JCV and BKV polyomaviruses were detected in 63 human wastewater samples collected from primary and secondary effluent, suggesting high sensitivity of these viruses in human wastewater. In the 81 animal wastewater and fecal samples tested, 80 were polymerase chain reaction (PCR) negative for the JCV and BKV markers. Only one sample (out of 81 animal wastewater and fecal samples) from pig wastewater was positive. Nonetheless, the overall host-specificity of these viruses to differentiate between human and animal wastewater and fecal samples was 0.99. To our knowledge, this is the first study in Australia that reports on the high specificity of JCV and BKV polyomaviruses. To evaluate the field application of these viral markers for detecting human fecal pollution, 20 environmental samples were collected from a coastal river. In the 20 samples tested, 15% (3/20) and 70% (14/20) samples exceeded the regulatory guidelines for Escherichia coli and enterococci levels for marine waters. In all, five (25%) samples were PCR positive for JCV and BKV, indicating the presence of human fecal pollution in the coastal river investigated. The results suggest that JCV and BKV detection using PCR could be a useful tool for identifying human-sourced fecal pollution in coastal waters.     

‘Reserves’ as a leading indicator to future mineral production

The factors which influence the size and value of mineral reserves are discussed and an example is given of a more rigorous system for measuring supplies of non-renewable resources.The extent to which reserves and resources of minerals can be used as leading indicators of future mineral production is examined and examples of the dangers of over-reliance on this factor are described.Key aspects, such as confidence limits, differences in the economic exploitability, the dynamic nature of reserves, and their variability in grade, are examined in some detail using the South African coal industry as an example.