Identifying human and livestock sources of fecal contamination in Kenya with host-specific Bacteroidales assays

Microbial source tracking to distinguish between human, livestock and wildlife fecal pollution using molecular techniques is a rapidly evolving approach in many developed countries, but has not previously been applied on the African continent. DNA extracts from cow, donkey, and human fecal specimens and raw domestic sewage samples collected in Kenya were tested against five existing quantitative PCR assays designed to detect universal (2), human-specific (2), and cow-specific (1) fecal Bacteroidales genetic markers. Water samples from the River Njoro in Kenya were evaluated using the five tested Bacteroidales markers and a multi-species assay for Cryptosporidium in a preliminary exploration of fecal pollution sources and health risks in this watershed. Diagnostic sensitivity on the validation set varied from 18 to 100% for the five assays while diagnostic specificity was 100%. Of the 2 universal assays, Total Bacteroidales [Dick, L.K, Field, K.G., 2004. Rapid estimation of numbers of fecal Bacteroidetes by use of a quantitative PCR assay for 16S rRNA genes. Appl. Environ. Microbiol. 70, 5695-5697] showed lower generic fecal diagnostic sensitivity, at 55%, than BacUni-UCD, at 100%, in detecting fecal markers on the 42-sample validation set. Human-specific assay HF183 demonstrated 65% sensitivity overall, and 80% on the human sewage samples, compared to 18% overall and 0% sewage for human-specific assay BacHum-UCD. Cow-specific assay BacCow-UCD had 94% sensitivity. Testing of 18 water samples indicates cows are a likely predominant source of fecal contamination in the Njoro Watershed (78% prevailing rate). Probabilistic assessment of human assay results indicates at most three of the river water samples contained human Bacteroidales. Cryptosporidium spp. markers were detected in samples from nine of the 12 sampling locations. Evidence suggesting widespread contamination by cow feces and Cryptosporidium in the Njoro watershed raises serious concerns for human and animal health.     

Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources

The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation.     

Rapid decay of host-specific fecal Bacteroidales cells in seawater as measured by quantitative PCR with propidium monoazide

We investigated the persistence of feces-derived Bacteroidales cells and their DNA in seawater under natural conditions using an optimized chemical method based on co-extraction of nucleic acids with propidium monoazide (PMA), which interferes with PCR amplification of molecular markers from extracellular DNA and dead bacterial cells. The previously validated Bacteroidales assays BacUni-UCD, BacHum-UCD, BacCow-UCD, and BacCan-UCD were utilized to determine concentrations of Bacteroidales genetic markers targeting all warm-blooded animals, humans, cows and dogs, specifically, over a period of 24 d. Microcosms containing mixed feces in dialysis tubing were exposed to seawater under flow-through conditions at ambient temperature in the presence and absence of sunlight. Using a two-stage plus linear decay model, the average T₉₉ (two-log reduction) of host-specific Bacteroidales cells was 28 h, whereas that of host-specific Bacteroidales DNA was 177 h. Natural sunlight did not affect the survival of uncultivable Bacteroidales cells and their DNA with the exception of the BacCow-UCD marker. Bacteroidales DNA, as measured by quantitative PCR (qPCR) without PMA, persisted for as long as 24 d at concentrations close to the limit of detection. Culturable Enterococcus cells were detected for only 70 h, whereas Enterococcus cells measured by qPCR with and without PMA persisted for 450 h. In conclusion, measuring Bacteroidales DNA without differentiating between intact and dead cells or extracellular DNA may misinform about the extent of recent fecal pollution events, particularly in the case of multiple sources of contamination with variable temporal and spatial scales due to the relatively long persistence of DNA in the environment. In contrast, applying qPCR with and without PMA can provide data on the fate and transport of fecal Bacteroidales in water, and help implement management practices to protect recreational water quality.     

Analytical comparison of nine PCR primer sets designed to detect the presence of Escherichia coli/Shigella in water samples

The analytical performance of 9 different PCR primer sets designed to detect Escherichia coli and Shigella in water has been evaluated in terms of ubiquity, specificity, and analytical detection limit. Of the 9 PCR primer sets tested, only 3 of the 5 primer sets targeting uidA gene and the primer set targeting tuf gene amplified DNA from all E. coli strains tested. However, of those 4 primer sets, only the primer set targeting the tuf gene also amplified DNA from all Shigella strains tested. For the specificity, only the primer sets targeting the uidA gene were 100% specific although the primer sets targeting 16S rRNA, phoE, and tuf genes only amplified Escherichia fergusonii as non-specific target. Finally, the primer set targeting the 16S-ITS-23S gene region, was not specific as it amplified DNA from many other Enterobacteriaceae species. In summary, only the assay targeting the tuf gene detected all E. coli/Shigella strains tested in this study. However, if it becomes important to discriminate between E. coli and E. fergusonii, assays targeting the uidA gene would represent a good choice although none of them were totally ubiquitous to detect of the presence of Shigella strains.     

沥青混合料低温性能评价指标研究

通过对现有评价沥青混合料低温性能的试验方法和评价指标的对比，论述了低温弯曲和弯曲蠕变试验是较为经济可行的试验方法，破坏能是较好地评价沥青混合料低温性能的指标，并应用低温弯曲和弯曲蠕变试验所得数据，提出了破坏能的简单计算方法。     

Characterization of fecal concentrations in human and other animal sources by physical,culture-based,and quantitative real-time PCR methods

The characteristics of fecal sources, and the ways in which they are measured, can profoundly influence the interpretation of which sources are contaminating a body of water. Although feces from various hosts are known to differ in mass and composition, it is not well understood how those differences compare across fecal sources and how differences depend on characterization methods. This study investigated how nine different fecal characterization methods provide different measures of fecal concentration in water, and how results varied across twelve different fecal pollution sources. Sources investigated included chicken, cow, deer, dog, goose, gull, horse, human, pig, pigeon, septage and sewage. A composite fecal slurry was prepared for each source by mixing feces from 6 to 22 individual samples with artificial freshwater. Fecal concentrations were estimated by physical (wet fecal mass added and total DNA mass extracted), culture-based (Escherichia coli and enterococci by membrane filtration and defined substrate), and quantitative real-time PCR (Bacteroidales, E. coli, and enterococci) characterization methods. The characteristics of each composite fecal slurry and the relationships between physical, culture-based and qPCR-based characteristics varied within and among different fecal sources. An in silico exercise was performed to assess how different characterization methods can impact identification of the dominant fecal pollution source in a mixed source sample. A comparison of simulated 10:90 mixtures based on enterococci by defined substrate predicted a source reversal in 27% of all possible combinations, while mixtures based on E. coli membrane filtration resulted in a reversal 29% of the time. This potential for disagreement in minor or dominant source identification based on different methods of measurement represents an important challenge for water quality managers and researchers.     

Specificity and sensitivity evaluation of novel and existing Bacteroidales and Bifidobacteria-specific PCR assays on feces and sewage samples and their application for microbial source tracking in Ireland

Three novel ruminant-specific PCR assays, an existing ruminant-specific PCR assay and five existing human-specific PCR assays, which target 16S rDNA from Bacteroidales or Bifidobacteria, were evaluated. The assays were tested on DNA extracted from ruminant (n = 74), human (n = 59) and non-ruminant animal (n = 44) sewage/fecal samples collected in Ireland. The three novel PCR assays compared favourably to the existing ruminant-specific assay, exhibiting sensitivities of 91-100% and specificities of 95-100% as compared to a sensitivity of 95% and specificity of 94%, for the existing ruminant-specific assay. Of the five human-specific PCR assays, the assay targeting the Bifidobacterium catenulatum group was the most promising, exhibiting a sensitivity of 100% (with human sewage samples) and a specificity of 87%. When tested on rural water samples that were naturally contaminated by ruminant feces, the three novel PCR assays tested positive with a much greater percentage (52-87%) of samples than the existing ruminant-specific assay (17%). These novel ruminant-specific assays show promise for microbial source tracking and merit further field testing and specificity evaluation.     

Identification of the sources of fecal coliforms in an urban watershed using antibiotic resistance analysis

Bacteria such as fecal coliforms are used as indicators of fecal pollution in natural waters. These bacteria are found in the feces of most wild and domestic animals and thus provide no information as to the source of fecal contamination, yet identification of indicator bacteria sources allows improved risk assessment, remediation, and total daily maximum load (TDML) assessment of environmental waters. This bacterial source tracking study was initiated in order to identify the dominant source(s) of fecal contamination in the urban watershed of Stevenson Creek in Clearwater, Florida. Five sites that represent areas where routine monitoring has previously shown high levels of fecal coliforms were sampled over 7 months. Fecal coliforms were enumerated by membrane filtration, and antibiotic resistance analysis was used to "fingerprint" a subset of randomly selected isolates and statistically match them to fingerprints of fecal coliforms from known sources (the library). A field test of the classification accuracy of the library was carried out by isolating fecal coliforms from the soil and waters surrounding a failing onsite wastewater treatment and disposal system (OSTDS). The vast majority of the isolates were classified into the human category. The major sources of fecal pollution in Stevenson Creek over the course of the study were wild animal, human, and, to a lesser extent, dog. Overall, wild animal feces were identified as the dominant source when fecal coliform levels were high, but when fecal coliform levels were low, the dominant source was identified as human. The results of this study demonstrate that the sources of fecal indicator bacteria within one urban watershed can vary substantially over temporal and spatial distances.     

Multiple lines of evidence to identify the sources of fecal pollution at a freshwater beach in Hamilton Harbour, Lake Ontario

Multiple microbial source-tracking methods were investigated to determine the source of elevated Escherichia coli levels at Bayfront Park Beach in Hamilton Harbour, Lake Ontario. E. coli concentrations were highest in wet foreshore sand (114,000 CFU/g dry sand) and ankle-depth water (177,000 CFU/100mL), declining rapidly in deeper waters. Many gull and geese droppings were enumerated each week on the foreshore sand within 2m of the waterline. Both antimicrobial resistance analysis and rep-PCR DNA fingerprinting of E. coli collected at the beach and nearby fecal pollution sources indicated that E. coli in sand and water samples were predominantly from bird droppings rather than from pet droppings or municipal wastewater. Both methods indicated a trend of decreasing bird contamination, and increasing wastewater contamination, moving offshore from the beach. When foreshore sand was treated as a reservoir and secondary source of E. coli, waterborne E. coli were found to be more similar to sand isolates than bird or wastewater isolates out to 150 m offshore. Multiple lines of evidence indicated the importance of bird droppings and foreshore sand as primary and secondary sources of E. coli contamination in beach water at Bayfront Park.     

Cross-phylum extrapolation of the Daphnia magna chronic biotic ligand model for zinc to the snail Lymnaea stagnalis and the rotifer Brachionus calyciflorus

We investigated if the chronic zinc biotic ligand model (BLM) developed earlier for the arthropod Daphnia magna could be extrapolated to predict chronic ecotoxicity of zinc as a function of water chemistry to two species from other phyla, i.e. the mollusc Lymnaea stagnalis and the rotifer Brachionus calyciflorus. We chronically exposed these two species to zinc in six natural surface waters. These water covered a wide range of pH (6.8-8.3), dissolved organic carbon (1.2-12.7 mg/L) and Ca (8.8-118 mg/L). Across all waters tested, the 28d-EC10s (200-1629 μg Zn/L) and EC50s (382-2026 μg Zn/L) for L. stagnalis spanned a 8.1-fold and 5.3-fold range, respectively. The 2d-EC10s (142-550 μg Zn/L) and 2d-EC50s (195-1104 μg Zn/L) for B. calyciflorus spanned a 3.9-fold and 5.7-fold range, respectively. The data indicated that higher pH and higher concentrations of Ca and DOC were generally associated with lower toxicity (higher ECx values). Furthermore, the chronic Zn BLM for D. magna, when calibrated only to reflect the intrinsic sensitivity of L. stagnalis and B. calyciflorus, was able to predict all ECx values with a less than 1.6-fold error, which demonstrates that the chronic D. magna Zn BLM can be extrapolated to other invertebrate phyla. This lends further support to the use of the chronic Zn BLM to account for bioavailability of zinc in aquatic risk assessment and the derivation of environmental quality standards.     

Using SWAT, Bacteroidales microbial source tracking markers,and fecal indicator bacteria to predict waterborne pathogen occurrence in an agricultural watershed

Developing the capability to predict pathogens in surface water is important for reducing the risk that such organisms pose to human health. In this study, three primary data source scenarios (measured stream flow and water quality, modelled stream flow and water quality, and host-associated Bacteroidales) are investigated within a Classification and Regression Tree Analysis (CART) framework for classifying pathogen (Escherichia coli 0157:H7, Salmonella, Campylobacter, Cryptosporidium, and Giardia) presence and absence (P/A) for a 178 km² agricultural watershed. To provide modelled data, a Soil Water Assessment Tool (SWAT) model was developed to predict stream flow, total suspended solids (TSS), total N and total P, and fecal indicator bacteria loads; however, the model was only successful for flow and total N and total P simulations, and did not accurately simulate TSS and indicator bacteria transport. Also, the SWAT model was not sensitive to an observed reduction in the cattle population within the watershed that may have resulted in significant reduction in E. coli concentrations and Salmonella detections. Results show that when combined with air temperature and precipitation, SWAT modelled stream flow and total P concentrations were useful for classifying pathogen P/A using CART methodology. From a suite of host-associated Bacteroidales markers used as independent variables in CART analysis, the ruminant marker was found to be the best initial classifier of pathogen P/A. Of the measured sources of independent variables, air temperature, precipitation, stream flow, and total P were found to be the most important variables for classifying pathogen P/A. Results indicate a close relationship between cattle pollution and pathogen occurrence in this watershed, and an especially strong link between the cattle population and Salmonella detections.     

Error analysis of in situ field tests of fenestration net energy performance

The need for information about fenestration net energy performance under realistic conditions is discussed and the method of measuring it in a “field test” on a complete building is reviewed. A detailed consideration of the energy flows entering the energy balance on a building space adjacent to a fenestration system and the effect of random measurement errors on the determination of fenestration performance is presented. Estimates of the error magnitudes are made for prototypical field tests utilizing a residential and a moderate-sized commercial building. In both cases, it is shown that these errors make it difficult to isolate the fenestration performance even for relatively low-performance windows (1 – 2 times the resistance of single glazing). It is concluded that whole-building measurements are not well adapted to measuring the performance of advanced fesestration system (thermal resistance 2 – 10 times that of single glazing or shading coefficient less than 0.7).     

Comparison of the efficacy of an existing versus a locally developed metabolic fingerprint database to identify non-point sources of faecal contamination in a coastal lake

A comparison of the efficacy of an existing large metabolic fingerprint database of enterococci and Escherichia coli with a locally developed database was undertaken to identify the sources of faecal contamination in a coastal lake, in southeast Qld., Australia. The local database comprised of 776 enterococci and 780 E. coli isolates from six host groups. In all, 189 enterococci and 245 E. coli biochemical phenotypes (BPTs) were found, of which 118 and 137 BPTs were unique (UQ) to host groups. The existing database comprised of 295 enterococci UQ-BPTs and 273 E. coli UQ-BPTs from 10 host groups. The representativeness and the stability of the existing database were assessed by comparing with isolates that were external to the database. In all, 197 enterococci BPTs and 179 E. coli BPTs were found in water samples. The existing database was able to identify 62.4% of enterococci BPTs and 64.8% of E. coli BPTs as human and animal sources. The results indicated that a representative database developed from a catchment can be used to predict the sources of faecal contamination in another catchment with similar landuse features within the same geographical area. However, the representativeness and the stability of the database should be evaluated prior to its application in such investigation.     

Reproductive toxicity of nitroaromatics to the cricket, Acheta domesticus

The effect of TNT (2,4,6-trinitrotoluene) and its metabolites, 2,4-dinitrotoluene (2,4-DNT), 2-amino-4,6-dinitrotoluene (2A-DNT), and 4-amino-2,6-dinitrotoluene (4A-DNT) on cricket (Acheta domesticus) reproduction was evaluated. We previously used crickets to assess the toxicity of a nitramine explosive (RDX) and its metabolites. It is common to find that while much information on the environmental impact of the parent compound is available in the literature, such is often not the case for the degradation metabolites of the parent compound. In some instances, these metabolites are as toxic (or more so) as the parent compound and we hypothesized that this might be the case for TNT. The presence of TNT and its metabolites in sand (10 µg/g) did not adversely affect cricket egg production, but adversely affected hatching of cricket eggs as compared to controls. However, there were no differences in hatching success among TNT and metabolite treatment groups. Hatching success of cricket eggs in soil or following topical exposure decreased as concentrations of TNT and its metabolites increased. The relative toxicity of TNT and its metabolites in soil generally followed the trend of TNT < 2A-DNT < 4A-DNT < 2,4-DNT. In addition, toxicity appeared to be higher in sand than in sandy loam soil or in the topical exposure test. After 45 days of exposure in sandy loam soil, the EC₂₀ (20% effect concentration), EC₅₀ (50% effect concentration), and EC₉₅ (95% effect concentration) were 14, 116, and 10,837 µg/g for TNT: 1.7, 32, and 16,711 µg/g for 2A-DNT: 1.9, 9, and 296 µg/g for 4A-DNT: and 0.4, 5.7, and 1437 µg/g for 2,4-DNT. Overall, results suggest that parent TNT and metabolites are toxic to cricket eggs at relatively high concentrations and these toxic effects are manifested as a decrease in hatching success.     

Evaluation of host-specific Bacteroidales 16S rRNA gene markers as a complementary tool for detecting fecal pollution in a prairie watershed

Our ability to identify and eliminate fecal contamination of water, now and in the future, is essential to reduce incidences of waterborne disease. Bacterial source tracking is a recently developed approach for identifying sources of fecal pollution. PCR primers designed by Bernhard and Field [Bernhard, A.E., Field, K.G., 2000a. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA. Appl. Environ. Microbiol. 66(10), 4571-4574] and Dick et al. [Dick, L.K., Bernhard, A.E., Brodeur, T.J., Santo Domingo, J.W., Simpson, J.M., Walters, S.P., Field, K.G., 2005. Host distributions of uncultivated fecal Bacteroidales bacteria reveal genetic markers for fecal source identification. Appl. Environ. Microbiol. 71(6), 3184-3191] for the detection of human (HF183), pig (PF163) and ruminant (CF128) specific Bacteroidales 16s rRNA genetic markers were tested for their suitability in detecting fecal pollution in Saskatchewan, Canada. The sensitivity and specificity of these primers were assessed by testing eight raw human sewage samples and 265 feces from 12 different species in Saskatchewan. The specificity of each primer set was ≥94%. The accuracy of HF183 and PF163 to distinguish between the different species was 100%, whereas CF128 cross-reacted with 22% of the pig feces. Occurrence of the host-specific Bacteroidales markers and the conventional indicator Escherichia coli in relation to several enteropathogens was investigated in 70 water samples collected from different sites along the Qu'Appelle River (Saskatchewan, Canada). Human and ruminant fecal markers were identified in 41 and 14% of the water samples, respectively, whereas the pig marker was never detected in the river water. The largest concentrations in E. coli counts were concomitant to the simultaneous detection of HF183 and CF128. Thermotolerant Campylobacter spp., Salmonella spp. and Shiga toxin genes (stx1 and stx2)-positive E. coli (STEC) were detected in 6, 7 and 63% of the water samples, respectively. However, none of the stx positive water samples were positive for the E. coli O157:H7 gene marker (uidA). Odds ratios analysis suggests that CF128 may be predictive for the presence of Salmonella spp. in the river investigated. None of the fecal indicators were able to confidently predict the presence of thermotolerant Campylobacter spp. and STEC.     

Kinetic model of thermophilic l-lactate fermentation by Bacillus coagulans combined with real-time PCR quantification

A simple l-lactate fermentation of organic wastes at pH 5.5 and 55 °C under nonsterile conditions using Bacillus coagulans can be suitable for l-lactate fermentation of garbage. A mathematical model that simulated the lactate fermentation characteristics of B. coagulans was developed by focusing on the inhibitory effects of substrate, lactate (product) and NaCl, and bacterial growth. Basic fermentation experiments were performed using simple substrates to derive fundamental parameters of growth rate and inhibition effects. The model was then applied to fermentations using simple substrates and artificial kitchen garbage in order to verify its applicability. Microbial concentration, a key state variable of the model was measured using both real-time polymerase chain reaction (PCR) and traditional methods. The results of these methods were compared for experimental cases in which only soluble substrates were used. B. coagulans concentrations were suitably measured using real-time PCR, even when traditional measurement methods for microbial concentrations cannot be used. The results indicate that the developed model and biomass measurement can be used to evaluate lactate fermentations using both simple and complex substrates. These proposed methods would be useful for developing a new bacterial function-based mathematical model for more complex acid fermentations.     

Modeling the contribution of point sources and non-point sources to Thachin River water pollution

Major rivers in developing and emerging countries suffer increasingly of severe degradation of water quality. The current study uses a mathematical Material Flow Analysis (MMFA) as a complementary approach to address the degradation of river water quality due to nutrient pollution in the Thachin River Basin in Central Thailand. This paper gives an overview of the origins and flow paths of the various point- and non-point pollution sources in the Thachin River Basin (in terms of nitrogen and phosphorus) and quantifies their relative importance within the system. The key parameters influencing the main nutrient flows are determined and possible mitigation measures discussed.The results show that aquaculture (as a point source) and rice farming (as a non-point source) are the key nutrient sources in the Thachin River Basin. Other point sources such as pig farms, households and industries, which were previously cited as the most relevant pollution sources in terms of organic pollution, play less significant roles in comparison. This order of importance shifts when considering the model results for the provincial level. Crosschecks with secondary data and field studies confirm the plausibility of our simulations. Specific nutrient loads for the pollution sources are derived; these can be used for a first broad quantification of nutrient pollution in comparable river basins. Based on an identification of the sensitive model parameters, possible mitigation scenarios are determined and their potential to reduce the nutrient load evaluated.A comparison of simulated nutrient loads with measured nutrient concentrations shows that nutrient retention in the river system may be significant. Sedimentation in the slow flowing surface water network as well as nitrogen emission to the air from the warm oxygen deficient waters are certainly partly responsible, but also wetlands along the river banks could play an important role as nutrient sinks.     

Oceanic sources of copper to the atlantic aerosol

Copper has been determined in a series of aerosols collected on a north-south Atlantic Ocean transect and its distribution has been related to those of Na, the marine source indicator, and Al, the crustal source indicator. Average Cu EF_sea values for a number of sample groups, calculated with respect to ambient surface sea water, range from a maximum of 60 × 10³ for aerosols collected off the U.K. coast to a minimum of 3.2 × 10³ for those from the remote South Atlantic westerlies. However, Cu can undergo fractionation in the micro-layer at the air/sea interface leading to sea-salts having enhanced Cu concentrations relative to bulk sea water. From the relationship between the concentrations of Cu and Na in the complete Atlantic aerosol data set, a minimum Cu/Na ratio was identified which was equivalent to a Cu enrichment of ∼1.9 × 10³, relative to bulk sea water. This enrichment is similar to those estimated by other workers from the BIMS experiments (∼ 0.8 × 10³), and from cascade impactor size-fractionated data (∼ 1.4 × 10³. Using the relative sea water enrichment factor of 1.9 × 10³, the oceanic contribution of Cu to the total Cu in the Atlantic aerosol was calculated and was compared with that having a crustal source and that from a third “excess” source, which was probably mainly pollutant. It was shown that in remote areas of the Atlantic, where the influence of continental sources is decreased, oceanic Cu can make up over half of the total Cu in the aerosol.