Method repeatability for measuring Enterococcus in southern California beach sands.

AIMS: A recent study that evaluated 22 methods for enumerating faecal indicator bacteria in sand recommended standardization to a preferred method, but all researchers involved in that study had extensive experience in processing sand samples. The purpose of this study was to evaluate how well the recommended method can be transferred to laboratories without such experience. METHODS AND RESULTS: Eight southern California laboratories that rarely measure bacteria in sand processed six sand and three water samples in replicates to assess repeatability. Among-laboratory variability was found to be less than within-laboratory variability, with no significant differences in results among any of the laboratories. Moreover, within-laboratory variability was comparable between the sand and water samples, indicating that the elution procedure added little additional method error even when performed by laboratories without prior experience. CONCLUSIONS: The simple extraction method for enumerating Enterococcus in beach sands was easily transferable to and repeatable among laboratories with little or no prior experience. SIGNIFICANCE AND IMPACT OF THE STUDY: The demonstrated success of technology transfer will further demonstrate the success of method standardization and adoption, aiding in understanding of how sands affect surface water quality.

Temporal and spatial variability of fecal indicator bacteria in the surf zone off Huntington Beach, CA.

Fecal indicator bacteria concentrations measured in the surf zone off Huntington Beach, CA from July 1998-December 2001 were analyzed with respect to their spatial patterns along 23 km of beach, and temporal variability on time scales from hourly to fortnightly. The majority of samples had bacterial concentrations less than, or equal to, the minimum detection limit, but a small percentage exceeded the California recreational water standards. Areas where coliform bacteria exceeded standards were more prevalent north of the Santa Ana River, whereas enterococci exceedances covered a broad area both north and south of the river. Higher concentrations of bacteria were associated with spring tides. No temporal correspondence was found between these bacterial events and either the timing of cold water pulses near shore due to internal tides, or the presence of southerly swell in the surface wave field. All three fecal indicator bacteria exhibited a diel cycle, but enterococci rebounded to high nighttime values almost as soon as the sun went down, whereas coliform levels were highest near the nighttime low tide, which was also the lower low tide.

Comparison of bacterial indicator analysis methods in stormwater-affected coastal waters.

Membrane filtration (MF) and multiple tube fermentation (MTF) have been used for decades to measure indicator bacteria levels in beach water samples, but new methods based on chromogenic substrate (CS) technology are becoming increasingly popular. Only a few studies have compared results among these methods and they have generally been based on samples collected from a limited number of sites during dry weather. In this study, samples were collected from 108 sites the day after a major rainstorm, and three indicator bacteria (total coliforms (TCs), fecal coliforms (FCs) or E. coli, and enterococci (EC)) were each measured using MF, MTF, and CS. Sampling sites were selected using a stratified random design, stratified by open sandy beach, rocky shoreline, and beach areas near urban runoff outlets. The CS results were found to be highly correlated with both MF and MTF for all three indicators regardless of whether the samples were taken along open shoreline or near a runoff outlet. While correlated, TC values were higher using the CS method, consistent with other studies that have demonstrated false positives with this method. FC values were 12% lower with CS, reflecting the specificity of the CS method for E. coli rather than for the entire FC group. No significant differences were observed for EC, although some differences were observed within specific laboratories. Differences for all of these indicators were small enough that, when assessed categorically, there was more than 90% agreement between CS methods and either MF or MTF methods as to whether State of California Beach Water Quality Standards were met or exceeded.

Comparison of total coliform, fecal coliform, and enterococcus bacterial indicator response for ocean recreational water quality testing.

In July 1999, California's ocean recreational bacterial water quality standards were changed from a total coliform (TC) test to a standard requiring testing for all three bacterial indicators: TC, fecal coliforms (FC), and enterococci (EC). To compare the relationship between the bacterial indicators, and the effect that changing the standards would have on recreational water regulatory actions, three regional studies were conducted along the southern California shoreline from Santa Barbara to San Diego, California. Two studies were conducted during dry weather and one following a large storm event. In each study, samples were collected at over 200 sites which were selected using a stratified random design, with strata consisting of open beach areas and rocky shoreline, and areas near freshwater outlets that drain land-based runoff. During the dry weather studies, samples were collected once per week for 5 weeks. For the storm event study, sampling occurred on a single day about 24 h following the storm. The three indicator bacteria were measured at each site and the results were compared to the single sample standards (TC > 10,000; FC > 400 and EC > 104 MPN or cfu/100 ml). EC was the indicator that failed the single sample standards most often. During the wet weather study, 99% of all standard failures were detected using EC, compared with only 56% for FC, and 40% for TC. During the Summer Study, EC was again the indicator that failed the single sample standards most often, with 60% of the failures for EC alone. The increased failure of the EC standard occurred consistently regardless of whether the sample was collected at a beach or rocky shoreline site, or at a site near a freshwater outlet. Agreement among indicators was better during wet weather than during dry weather. During dry weather, agreement among indicators was better near freshwater outlets than along open shoreline. Cumulatively, our results suggest that replacement of a TC standard with an EC standard will lead to a five-fold increase in failures during dry weather and a doubling of failures during wet weather. Replacing a TC standard with one based on all three indicators will lead to an eight-fold increase in failures. Changes in the requirements for water quality testing have strong implications for increases in beach closures and restrictions.

Decadal and shorter period variability of surf zone water quality at Huntington Beach, California.

The concentration of fecal indicator bacteria in the surf zone at Huntington Beach, CA, varies over time scales that span at least 7 orders of magnitude, from minutes to decades. Sources of this variability include historical changes in the treatment and disposal of wastewater and dry weather runoff, El Niño events, seasonal variations in rainfall, spring-neap tidal cycles, sunlight-induced mortality of bacteria, and nearshore mixing. On average, total coliform concentrations have decreased over the past 43 years, although point sources of shoreline contamination (storm drains, river outlets, and submarine outfalls) continue to cause transiently poor water quality. These transient point sources typically persist for 5-8 yr and are modulated by the phase of the moon, reflecting the influence of tides on the sourcing and transport of pollutants in the coastal ocean. Indicator bacteria are very sensitive to sunlight therefore, the time of day when samples are collected can influence the outcome of water quality testing. These results demonstrate that coastal water quality is forced by a complex combination of local and external processes and raise questions about the efficacy of existing marine bathing water monitoring and reporting programs.

Generation of enterococci bacteria in a coastal saltwater marsh and its impact on surf zone water quality.

Elevated levels of enterococci bacteria, an indicator of fecal pollution, are routinely detected in the surf zone at Huntington State and City Beaches in southern California. A multidisciplinary study was carried out to identify sources of enterococci bacteria landward of the coastline. We find that enterococci bacteria are present at high concentrations in urban runoff, bird feces, marsh sediments, and on marine vegetation. Surprisingly, urban runoff appears to have relatively little impact on surf zone water quality because of the long time required for this water to travel from its source to the ocean. On the other hand, enterococci bacteria generated in a tidal saltwater marsh located near the beach significantly impact surf zone water quality. This study identifies a potential tradeoff between restoring coastal wetlands and protecting beach water quality and calls into question the use of ocean bathing water standards based on enterococci at locations near coastal wetlands.

Dietary fatty acid composition induces comparable changes in cardiolipin fatty acid profile of heart and brain mitochondria.

The fatty acid profile of cardiolipin (CL) from brain and cardiac mitochondria was measured to determine whether CL isolated from these two tissue sources responded similarly to alterations in dietary fat composition. Male Wistar rats were fed 20% (w/w) diets containing 2 to 12% (w/w) 18:2n-6 for four weeks. Despite higher baseline levels of CL 18:2n-6 in cardiac (54 +/- 1% of total fatty acids) compared to brain (13 +/- 1%) mitochondria, CL 18:2n-6 levels increased in proportion to dietary 18:2 levels. The degree of change in 18:2n-6 was comparable with both tissues showing an approximate 1.5- to 2-fold increase. The time course of changes in CL fatty acid profile was examined in a subsequent experiment in which animals were fed 20% (w/w) fat diets containing either 3 or 15% alpha-linoleate. Changes in cardiac CL 18:1, 18:2n-6, and 22:6n-3 levels were observed within one week of feeding. While statistically significant differences were not observed in brain CL until the second week of feeding, the time course did not differ substantively from that observed in heart. The results from this study suggest that while baseline fatty acid profile of cardiac and neural CL differ, mitochondria from both tissues show comparable sensitivity to changes in dietary fat composition. Furthermore, it would appear that the turnover rate of fatty acids in CL is similar in both tissues.

Dietary fat composition and age affect synaptosomal and retinal phospholipid fatty acid composition in C57BL/6 mice.

The purpose of this study was (i) to determine whether dietary fat-induced differences in neural and retinal membranes occur when dietary fat treatment is implemented in aged animals and (ii) to characterize the effect of long-term differences in dietary fat on neural and retinal membrane composition. For the first objective, young (six-week-old) and old (95-week-old) mice were randomly assigned to beef tallow (TAL) or soybean oil (SBO) diets for eight weeks. For the second objective, young (four-week-old) mice consumed either TAL or SBO diets for 99 weeks. Young and old mice challenged with a change in dietary fat for an eight-week period showed both diet and age effects on neural and retinal phospholipid fatty acid composition (P < 0.05). In addition, significant diet by age interactions were evident. In mice that consumed TAL and SBO diets throughout their life, only retinal phosphatidylethanolamine (PE) 18:2n-6 and neural phosphatidylserine 22:5n-6, PE 18:2n-6 and phosphatidylcholine 18:2n-6 differed between dietary treatments (P < 0.05). Neither the unsaturation index nor the n-6/n-3 ratio was affected by diet. Neural and retinal phospholipid fatty acid composition were responsive to changes in dietary fat even when the treatment was implemented beyond developmental or post-weanling stages. In contrast, when mice consumed TAL or SBO diets throughout their life, fewer differences in phospholipid fatty acid composition were detected, suggesting that the effect of the dietary treatment was mitigated by aging.

Dietary fat-induced changes in protein and carbohydrate selection are not explained by alterations in neuronal membrane fatty acid composition.

We previously showed changes in protein and carbohydrate selection in response to qualitative differences in dietary fat. Alterations in macronutrient selection were specifically related to changes in dietary saturated fat, but not to relative or absolute differences in dietary essential fatty acids. Three experiments were conducted to determine if changes in specific fatty acids in bulk phase neural membranes were associated with differences in macronutrient selection. For each experiment, specific fatty acid profiles were achieved by blending dietary fat sources. Rats consumed 20% (w/w) fat diets varying only in their fatty acid composition. After 2 weeks, rats were challenged with a selection paradigm. Each experiment showed a significant effect of dietary fat on neural membrane fatty acid composition (p less than 0.05) and alterations in individual fatty acids were correlated with changes in dietary fatty acids (p less than 0.05). However, dietary fat had no consistent effect with respect to which particular neural membrane fatty acids were modified, and there was no correlation between changes in specific membrane fatty acids and macronutrient selection. These findings suggest that alteration of specific fatty acids in bulk phase neural membranes do not mediate macronutrient selection behavior.

Protein and carbohydrate selection respond to changes in dietary saturated fatty acids but not to changes in essential fatty acids.

We previously reported differences in protein and carbohydrate selection patterns in post-weanling rats fed beef tallow or soybean oil-based diets. Two experiments were designed to determine the characteristic of the dietary fat which mediates the selection behavior. For each experiment, dietary fat was 20% (w/w) of diets and fatty acid profiles were obtained by blending fat sources. Rats were randomly assigned to diets (24% protein, 40% carbohydrate) which varied only in fatty acid composition. After 2 weeks, rats selected from 2 diets with the fat composition previously fed, but varying in their protein and carbohydrate composition (55% protein, 4% carbohydrate and 5% protein, 61% carbohydrate). Experiment 1 was designed to test the effect of relative (omega 6: omega 3 ratios of 1 and 20) and absolute (15% or 4% omega 6, 0.7% or 0.2% omega 3) differences in essential fatty acids on macronutrient selection patterns. Differences in dietary essential fatty acids had no effect on energy intake or the proportion of energy consumed as protein and carbohydrate. Experiment 2 examined the effect of differences in the level of saturated fat (3-10% diet (w/w] on protein and carbohydrate selection. Animals selecting from diets with higher levels of saturated fat consumed more energy as protein and less as carbohydrate than rats selecting from diets with lower levels of saturated fat (p less than 0.0001). Regression analysis was used to examine the relationship between percent protein or carbohydrate energy and classes of dietary fat. The strongest relationship existed between percent dietary saturated fat and percent protein or carbohydrate energy (p less than 0.0001). Polyunsaturated:saturated fat ratio was also weakly associated with percent protein and carbohydrate energy (p less than 0.05). Polyunsaturated, monounsaturated, omega 6 and omega 3 fatty acids were not significantly related to percent protein or carbohydrate energy. These results indicated that protein and carbohydrate selection patterns are altered in response to qualitatively different dietary fatty acids, and that the amount of saturated fat in the diet is the important characteristic of dietary fat mediating the behavioral alteration.

Blood and tissue tocopherol levels in rats following intraperitoneally administered alpha-tocopheryl acetate.

The correction or maintenance of blood and tissue alpha-tocopherol (alpha-Toc) levels by intraperitoneally administered all-rac-alpha-tocopheryl acetate (alpha-Tac) was compared with RRR- alpha-tocopherol (alpha-Toc) in vitamin E-depleted and control rats. Rats received 1.3 TE vitamin E daily for 7 days. alpha-Tac was detected in plasma of one-third of alpha-Tac-treated rats 24 hr after the first treatment, although not in subsequent samplings. Both alpha-Tac and alpha-Toc increased tocopherol levels in plasma and liver of E-deprived rats, while little or no change was observed in adipose tissue and brain. Similarly, control rats treated with alpha-Tac or alpha-Toc had significantly greater (p less than 0.05) plasma and liver alpha-Toc levels at day 3 and day 7 than did saline-treated rats. There was no significant difference in adipose alpha-Toc levels among treatment groups of control rats. The results of this study suggest that alpha-Tac is rapidly hydrolyzed to its biologically active alcohol form and results in similar effects to that of intraperitoneally administered alpha-Toc.

Dietary guidelines for prevention of cancer: are they justified?

Although dietary guidelines for reduction of cancer risk have been issued and well publicized, there is still controversy within the scientific community about whether such guidelines are appropriate. Given that the scientific evidence of a causal role for diet in carcinogenesis is suggestive but not conclusive, is it more responsible to promulgate guidelines that might be ineffective or to delay communicating findings to the public until all the evidence is in-a process that could take decades? Because the public is already familiar with guidelines intended to minimize the risk of cancer, how should they be promoted by health professionals given the strength of the evidence on which they are based?

Effects of dietary fatty acid composition on macronutrient selection and synaptosomal fatty acid composition in rats.

Our previous work showed that rats consuming 20% (wt/wt) lard diets selected more protein and less carbohydrate than did rats fed 20% (wt/wt) soybean oil diets. To determine if this effect is specific to saturated fat source and to measure changes in fatty acid composition of synaptosomal phospholipids, male Wistar rats were fed a 20% (wt/wt) soybean oil or tallow diet with 24% protein and 48% carbohydrate for 2 wk. Rats then self-selected from two diets with the same fat previously fed but different protein and carbohydrate composition (5% protein, 67% carbohydrate and 55% protein, 17% carbohydrate). Tallow-fed rats selected more protein and less carbohydrate than did soybean oil-fed rats. Dietary fat also affected fatty acid composition of synaptosomal phospholipids, phosphatidylcholine being most affected and phosphatidylinositol most resistant. Later studies determined if selection differences were due to taste. When rats self-selected without prior exposure to diets, tallow-fed rats selected more protein and less carbohydrate than did soybean oil-fed rats during the last 2 wk but not the first 2 wk. When rats selected from diets with identical protein and carbohydrate composition but different dietary fat, rats consumed slightly greater amounts of the tallow diet at each protein level. The relative intake of tallow and soybean oil diets, however, did not vary significantly with dietary protein treatment. These results confirm that rats fed saturated fat diets select more protein and less carbohydrate than do rats fed polyunsaturated fat diets and suggest this difference is not due to taste factors.

Vitamin E and selenium status of patients receiving short-term total parenteral nutrition.

In a prospective protocol, plasma tocopherols, selenium (Se), Se-dependent glutathione peroxidase, platelet aggregation and erythrocyte hemolysis were measured in 23 control subjects, and 15 patients receiving total parenteral nutrition (TPN), before and after 2 wk of TPN unsupplemented with vitamin E and Se. The results indicate that short-term TPN did not alter status of these nutrients. However, TPN patients had significantly lower plasma levels of Se (p less than 0.01) and alpha-tocopherol (p less than 0.01) relative to control subjects. Low plasma levels, with no attendant decrease in function, suggest a marginal depletion. In view of this, and considering the low amount of vitamin E and Se supplied by the TPN solutions, supplementation with these nutrients is recommended.

Selenium and vitamin E stability in parenteral solutions.

Stability of alpha-tocopherol acetate and selenium in amino acid/dextrose solutions with SoluZyme or MVI-1000 vitamin injections was evaluated following exposure to fluorescent lighting and room temperature, and after flowing through an infusion apparatus. The stability of selenium in parenteral solutions for a 10-wk period was also determined. In each condition no significant loss of alpha-tocopherol acetate or selenium was observed. It was concluded that alpha-tocopherol acetate and selenium as selenious acid are stable in parenteral solutions and no significant loss occurs during delivery to patients.