CLÁUSULA TERCERA – CRITERIOS DE ELEGIBILIDAD Todos los proyectos deberán satisfacer los criterios siguientes:

The DOP study should consist of monitoring for Cryptosporidium or a suite of

Cryptosporidium surrogate organisms at each collection device (or device type cluster) and the

source river water. Pathogen monitoring could also include Giardia and perhaps members of the Microsporidia family (Brusseau et al. 2005). In the absence of Cryptosporidum oocyst removal data (calculated using measurable oocyst concentrations in the river and in the collection device), the DOP should use Cryptosporidium surrogate microorganisms and should demonstrate that removal of the recovered surrogate organism(s) would be similar to the removal of

Cryptosporidium oocysts (to the extent possible using the scientific literature, laboratory and/or

field studies).

Bank filtration efficiency can be meaningfully demonstrated and is permitted in a DOP only in porous media (similar in concept to slow sand filtration but without use of engineered materials, flow and flux control and active schmutzdecke management). Log removal calculations require counts per volume of the same organism in both surface water and nearby collection devices (wells). Comparison of the two values provides information on attenuation during subsurface passage. However it is important to calculate log removal only for microorganisms that are similar to Cryptosporidium oocysts. Log removal calculations for particles or organisms that significantly differ from oocysts in size, shape and porous media transport capability or have unknown original or final size and shape (and charge), such as turbidity, standard particle counts, and total algae, or larger organisms such as rotifers, crustaceans or fish are less meaningful and should not be used. Pumping wells generate turbidity in the aquifer as a result of pumping (van Beek et al 2010). Therefore, for groundwater, turbidity data are useful primarily for determining disinfectant treatment efficiency. It is not meaningful to count particles not known to originate in the surface water, as is the case for turbidity or standard particle counters.

Cryptosporidium and Cryptosporidium surrogate organism transport in porous media has

been well studied in both laboratory and field experiments (e.g., Schijven et al. 2003) and will not be detailed here. In general, microorganism and porous media grain size and shape are important parameters that govern removal efficiency together with grain coatings and water chemistry. Predictions without field measurements are highly uncertain. Thus, paired samples from surface water and ground water are necessary.

When subsurface materials are coarse grained (e.g., gravel), ground water flow is

relatively fast and bank filtration efficiency is significantly reduced. For example, in one study in a gravel aquifer (not a bank filtration site), aerobic spores traveled 90 m in about one day (Pang et al. 1998, Pang et al. 2005). For coarse grained aquifers, EPA recommends significant

additional study (increased monitoring frequency of multiple microorganisms from multiple monitoring wells) to improve removal efficiency measurement at high ground water velocity.

Surrogate microorganisms are more likely to be recovered from collection devices when the concentration in the surface water is high, such as during a diatomaceous algal bloom (e.g., Kearney, Nebraska) (Berger et al. 2002) or during high water stage. Eckert and Irmschser (2006) report E. coli recovery in Dusseldorf bank filtration wells only following a flood event.

The choice of the appropriate suite of Cryptosporidium surrogate organisms is the most important element of a DOP study. Favorable surrogate organisms should be 1) equivalent in size and shape to Cryptosporidium oocysts (i.e., 4-6 µm and slightly oblate), 2) sufficiently numerous in both ground water and surface water so as to be suitable for log removal calculations (log removal calculations require counts per volume of the same organism in both surface water and nearby collection devices/wells); and 3) sufficiently long-lived in the subsurface (at least as long- lived as oocysts) so that inactivation during subsurface passage does not significantly affect the calculation.

The identification of Cryptosporidum oocyst surrogate organisms is based primarily on similarity in size and shape. Other factors such as total net charge or charge distribution on the outer surface of the microorganism are important elements governing Cryptosporidium transport in the subsurface. However, choice of surrogate organisms based on charge or factors other than microorganism size and shape is an important research topic (Tufenkji 2007, Tufenkji et al. 2006) but the available information is insufficient for inclusion in this guidance. Exhibit 4.7 lists the size ranges of common pathogenic protozoa and surrogate bacteria.

No single Cryptosporidium surrogate organism is best. Each organism has strengths and weaknesses. Multiple surrogates should be analyzed initially to ascertain which surrogate suite is best suited to the DOP at that site. Examples of surrogates include total aerobic bacterial spores (e.g., Bacillus subtilus), anaerobic bacterial spores (e.g., Clostridium perfringens and/or

Clostridium bifermentans), total coliform, E. coli, enterococci bacteria, bacteriophage (e.g., Bacteroides phage), coliphage (male-specific and somatic), diatoms (Reilly et al. 2005) at the

genus or species level, turbidity, particle counting and microscopic particulate analysis (MPA) (U.S. EPA 1992, AWWA 1990). EPA recommends monitoring for at least three or four surrogate

LT2ESWTR Toolbox Guidance Manual 4-40 April 2010 organisms using paired surface water and ground water samples to calculate log removal

efficiency.

As with any monitoring program, there is a trade-off between monitoring frequency and information cost. The cost for each Cryptosporidium surrogate assay varies between $50 and $250. EPA recommends that the less expensive assays, such as total aerobic spores, total

coliform, and enterococci be performed more frequently and the more expensive assays, such as MPA, be performed at a lesser frequency.

Exhibit 4.7 Size of Pathogenic Protozoa and Surrogate Bacteria

Protozoa Size (µm) Surrogate Bacteria Size (µm)

Cryptosporidium parvum oocyst (Xiao et al., 2000) 4.2-5.6 Total Coliform (Holt, 1986) ~0.5-6.0

Giardia lamblia cyst (WHO, 2004)

8-12 Escherichia coli (vegetative cell form)

(Foppen and Schijven, 2006)

1.1-6.0

Cyclospora sp. (Mota et al., 2000)

8-10 Clostridium perfringens (vegetative cell form) (Holt, 1986)

2-19

Microsporidia

(Brusseau et al., 2005)

1-5 Clostridium perfingens spore (Lund and Peck, 1994)

0.3-0.4 Clostridium bifermentans

(vegetative cell form) (Holt, 1986)

1-11

Clostridium bifermentans spore (Brock and Madigan, 1991)

1.2 Bacillus subtilus

(vegetative cell form) (Holt, 1986)

2-5

Bacillus subtilus spore

(Rice et al., 1996 and P. Payment, personal communication

0.5-0.8

Cryptosporidium oocysts are slightly oblate with a length-to-width ratio that ranged, in

one study, from 1.04 to 1.33 (Xiao, 2000). Aerobic spores are typically slightly oblate as well but smaller than oocysts, ranging from 0.5-0.8 microns in diameter as compared with 4-6 microns for oocysts. Bacterial vegetative cells of E. coli are slightly larger than aerobic spores but

significantly differ in length to width ratios (2.0-6.0 µm × 1.1-1.5 µm, Foppen and Schijven 2006). Futhermore, vegetative cells produce extracellular polymers, particularly if these cells form biofilms, and these polymers may significantly alter passage characteristics in the subsurface. The bacterial spore form is significantly longer lived in the environment (and especially the subsurface) than the vegetative cell form.

EPA recently completed a laboratory study (12 laboratories) of the total aerobic spore method. The study used natural ground water from a deep confined aquifer in Montana and Ohio River surface water. The ground water was analyzed to insure that it was devoid of (but not

sterile) aerobic spore forming bacteria. Aerobic spores (from BioBall) with well-defined counts but subject to variability were spiked in the ground water samples. Split surface water samples of unknown variability were also prepared. One ground water and one surface water sample was sent to each laboratory for multiple assay.

Laboratory performance was evaluated by comparing mean assay values separately for ground water and surface water. Spiked ground water sample variability among the twelve laboratories using Youden’s Laboratory Ranking Test (Youden 1969) did not identify any outlying laboratories. Surface water mean values showed two outlying laboratories (one high outlying value) but the range of mean values was about a factor of five different between the high (23,962 CFU/100 ml) and (low 4,370 CFU/100 ml) values. Spore removal in this guidance is typically an assessment of whether surface water spore counts are diminished by a factor of one hundred when measured at a well. A factor of five difference is acceptable variability, assuming other information does not contradict that assessment.

The laboratory study shows that the aerobic spore laboratory method can be reproducibly performed by different laboratories and also provides acceptable recoveries of spores from spiked water samples. Because aerobic spores are: 1) relatively cheap and easy to measure, 2)

identifiable without unacceptable laboratory error, 3) long-lived in the environment, 4) similar in shape to oocysts (albeit slightly smaller), and 5) do not produce extracellular polymers, EPA recommends aerobic spores, if present in large numbers in the surface water at the DOP site, as the most useful Cryptosporidium surrogate organism. Aerobic spores have long been recognized as a useful measure of surface water influence on and hygienic quality of ground water (e.g., Schubert 1975). Aerobic spores are also commonly used to assess the performance of engineered filtration systems (e.g., Mazoua and Chauveheid 2005).

The MPA method counts spores but these are fungal spores and not bacterial spores. Bacterial spore assay requires, at present, a culture step that is not currently part of the MPA method. MPA simply concentrates particulates and counts them. An aerobic spore assay standard method is available (APHA 2004). EPA recommends that unused aerobic spore sample be refrigerated (4 degrees C.). These refrigerated samples may then be re-assayed up to 24 hours post-receipt of the sample at the laboratory so that additional and differing dilutions can be conducted to reanalyze samples that are reported as “Too Numerous to Count” (TNTC).

Aerobic spore data have been collected from several recent studies at potential bank filtration sites (e.g., Weiss et al. 2005, Vogel et al. 2005, Gollnitz et al. 2004, Gollnitz et al. 2005, Partinoudi and Collins 2007, Gollnitz et al. 2007). It is important to differentiate sites that may be described as riverbank filtration sites but are not recognized as GWUDI by the state. For example, both Lincoln, NE (Vogel et al. 2005) and Cincinnati, OH (Gollnitz et al. 2004) field sites were studied in great detail using very sophisticated methods despite not being regulated as GWUDI. Thus, high aerobic spore log removal at these sites is expected because they are regulated as ground water rather than as surface water. Finally, at least one laboratory counted aerobic spore colonies in ground water without use of a dissecting microscope (in contrast to APHA 2004) (Partinoudi and Collins 2007). Partinoudi and Collins (2007) did not use a microscope so they report a high aerobic spore detection limit (<30 CFU/100 ml). Based on

LT2ESWTR Toolbox Guidance Manual 4-42 April 2010 unpublished data from Casper, WY and results reported in Locas et al. (2008), Schubert (1975) and Rice et al (1999), the aerobic spore natural background concentration is about 10 CFU/100 ml or less. Based on these studies, values higher than 10 CFU/100 ml may be considered to have some surface water influence. Thus, a high detection limit makes it difficult to differentiate native and surface water-influenced ground water.

The ability to produce environmentally-resistant aerobic spores is fairly limited in the bacterial world. Current methodology recovers almost exclusively those aerobic spores from the genus Bacillus. There are many species of Bacillus and all produce aerobic spores. Members of this genus are found naturally in all environments, including surface waters and especially in soil. It is assumed that, when found in surface water, aerobic spores found naturally in soil are washed into surface water by natural processes. As soil bacteria, aerobic spore populations in surface water are expected to comprise a diverse bacterial population, representing all aerobic spore taxa within the surface water watershed. The aerobic spore population in a ground water sample may be 1) similarly as taxonomically diverse as the surface water population, 2) taxonomically less diverse than the surface water population because some spore taxa have favorable properties (e.g., charge) for subsurface passage while other taxa are more likely to attach to aquifer solids, or 3) taxonomically diverse or not but representative only of the spore population in the soil in the immediate vicinity of the wellhead.

Gollnitz et al. (2005) suggest that ground water aerobic spore samples exhibit “endospore monocultures” and also suggest that these “monocultures” explain the instances when collection devices exhibit negative (low) removal efficiency. (Given the high uncertainty in log removal calculations, the difference between negative and low removal efficiency is not significant.) “Monocultures” implies that all of the cells recovered on the growth medium are of the same strain and possible clonal, being genetically identical. This would ordinarily imply that the organisms were growing either in the groundwater itself or in the sample once it was collected. Some type of genetic profiling analysis would need to be run in order to document all of the cells recovered as clonal. To date no such data has been reported. A more likely explanation for a sample yielding Bacillus colonies that are morphologically similar is laboratory contamination. Thus, any suggestion that, in the absence of genetic profile or speciation data, colonies are “monocultures,” should be recognized as premature and possibly incorrect. Finally, as discussed above, low taxa diversity in a ground water sample (when recognized by speciation data)

provides no information on log removal by subsurface passage.

Anaerobic spores are also recommended as surrogate microorganisms because these microorganisms, like aerobic spores, are small (0.3-1.2 µm), spherical, and long-lived. Riverbank filtration studies in the Netherlands (e.g., Shijven et al. 2003, Medema and Stuyfzand 2002) used spores of sulfide-reducing Clostridia (SSRC) and Clostridium bifermentans spores as

Cryptosporidium surrogates in studies of the Rhine and Meuse Rivers. However, anaerobic

spores probably do not have a significant presence in surface water unless there are significant upstream sewage discharges to surface water, as there are in the Rhine and Meuse. Thus, the utility of anaerobic spores at a DOP site where surface water quality is typically very good is limited.

Total coliform bacteria are vegetative cells that, like Bacillus sp., originate largely as soil bacteria, and are found at high density in surface water. Total coliform density in wells has been used for GWUDI determination in bank filtration settings. Price et al., 1999 show that higher total coliform density occurs in horizontal collector well #5 from January to April, during highest flow conditions in the river. Thus, well #5 is used primarily during summer months when water use demand is high and river flow is low. However, using measurement of aerobic spores (or carboxylated microspheres, Metge et al. 2007) in addition to measurement of total coliform vegetative cells might provide a differing assessment because aerobic spores should more efficiently passage through the subsurface and may be present in significant density before January or after April.

Diatoms are a specialized group of marine and freshwater algae that all produce a rigid cell wall (frustule) composed of silica. There are 58 freshwater diatom genera (AWWA 1995). Diatoms are counted separately from other algae in a MPA (U.S. EPA 1992). The MPA method only counts whole diatoms; diatom fragments are not considered. Exhibit 4.8 shows the size and shape of some common freshwater diatoms. Diatoms vary in size (e.g., from 4-10 microns to 60 or 70 microns). Smaller diatoms may be transported through sand and other porous media at rates similar to oocysts. Larger diatoms may, if they have large length to width ratios, orient

themselves in the ground water flow field so that the long axis is parallel to the flow direction, which also may allow them to pass through sand and other porous media. Diatoms are

photosynthesizing algae that require light to maintain their green chlorophyll. After about 6 months residence time in the subsurface, the green color will fade. (Susan Boutros, EPA GWUDI Determination Presentation, Denver CO, verbal communication on unpublished laboratory experiments with diatoms placed in a refrigerator).

Exhibit 4.8 Size of Some Common Fresh Water Diatoms

Diatom Size (length x width) (µm) Shape

Stephanodiscus hantzchii 10 × 5-8 Cylindrical (Hendricks et al., 2000)

Synedra acus 60-70 × 3-4 Needle (Hendricks et al., 2000)

Cyclotella meneghiniana 5 × 3 Cylindrical (Hendricks et al., 2000)

Cyclotella pseudostelligera 4-10 Centric (Reilly et al., 2005) Fragilaria crotonensis 40-170 × 2-4 Pennate (Reilly et al., 2005)

Aulacoseira granulata 4-30 Centric (Reilly et al., 2005)

Asterionella formosa 40-80 × 1.3-6 Pennate (Reilly et al., 2005)

Nitzschiia palea 15-70 × 2.5-5 Pennate (Reilly et al., 2005)

Because most diatoms are larger than Cryptosporidium oocysts (see section 4.7.2), diatom occurrence in a well signals that oocysts, like diatoms, could also be present in inadequately filtered drinking water from a well. Diatoms occurrence is subject to less

uncertainty because the rigid frustule is not likely to be sufficiently deformable to pass through smaller pores, unlike most biological particles. Thus, one or more whole diatom tests, identified in well water, and counted using the MPA or another method, are particularly meaningful data. Some diatom species are also identifiable using immunoassay methods (Walker et al. 2005),

LT2ESWTR Toolbox Guidance Manual 4-44 April 2010 although the detection limit is high (500 cells per liter) and thus not well suited for porous media groundwater sites where the diatom count is expected to be significantly lower.

EPA recommends weekly or biweekly aerobic spore samples plotted on a graph together with monthly diatom data from MPA and river stage values to evaluate bank filtration efficiency. The spore data are a measure of bank filtration efficiency which should decrease with increasing river stage (i.e., high spore occurrence in a well at high river stage). For a regulated river (e.g., upstream dams and reservoirs) the correlation between aerobic spore recovery in wells and river stage might be muted or non-existent. Diatom data are used to validate the aerobic spore data; weeks or months with high spore recovery in wells should also have accompanying diatom occurrence in well water.