**5. Water quality testing**

The water purification system is a multiple layer system designed to progressively purify water at each step. The resulting dialysis water needs to meet minimum criteria for chemical and microbiological characteristics. These criteria form the basis of the definition for standard quality and ultrapure quality dialysis water. There are several available guidelines specifying the minimum allowable standards for water quality. (eg U.S. Association for the Advancement of Medical Instrumentation (AAMI) and the European best practice guidelines (EBPG). The ISO (international organisation for standardisation) is an international collaboration of national standard bodies (ISO member bodies). Minimum water testing parameters from the ISO are summarised below. The ISO recommends at least annual testing for chemical con‐ taminants and quarterly testing for microbiological contaminants (Table 2) [9]. Ultimately a schedule of water testing must achieve two goals. Firstly, testing must ensure that a high quality of dialysis water is being delivered at the end of the filtration process. Perturbations in feed water quality and breakthrough in upstream filters do not usually result in fouling of the dialysis water, as there is a fair degree of redundancy built into each filter. Hence, even if, for example, a post-WRO sample reveals an elevated bacterial load, a well maintained further downstream ultrafilter should still prevent this exposure to the patient. Secondly, testing at different points in the circuit enables troubleshooting and localization of the problem area. As in the example above, the problem would be proximal to the WRO testing port and hence the carbon filters would be backwashed and the RO disinfected. A suggested schedule for water testing frequency in the home environment in outlined below. *(see Table 3)*.

There are 4 main points of water testing. (see table 3 and Figure 2). Water samples are best sent to an experienced testing facility for all water analyses.



#### **Table 3.** Schedule for water testing

in the example above, the problem would be proximal to the WRO testing port and hence the carbon filters would be backwashed and the RO disinfected. A suggested schedule for water

There are 4 main points of water testing. (see table 3 and Figure 2). Water samples are best sent

testing frequency in the home environment in outlined below. *(see Table 3)*.

to an experienced testing facility for all water analyses.

232 Updates in Hemodialysis

**Table 2.** ISO recommendations for water quality in dialysis water [20]


**Figure 10.** Example of a standard water analysis report

#### **5.1. Chemical testing**

*Chlorine* is a strong oxidising agent used in the disinfection of municipality drinking water. Inadequately treated dialysis water can result in significant patient exposure to chlorine. Here ferrous iron (Fe2+) in haemoglobin is converted ferric iron (Fe3+) resulting in the formation of methaemoglobin, haemolysis and anaemia [21, 22]. Due to its toxic nature and abundance, testing for chlorine occurs twice daily in dialysis units. This is performed using indicator test strips or testing tablets, which cause a colour change in the presence of chlorine. Electronic chlorimeters are also used and these are serviced regularly. In the home setting, patients are trained to perform a chlorine check before any dialysis session is initiated. In addition, trained water technicians visiting the home for maintenance reasons, also check chlorine levels. Chlorine testing is not routinely performed in the lab, as the samples need to be tested within 4 hours of collection due to the instability of the chlorinated compounds.

*Aluminium* has been associated with renal bone disease, anaemia and dementia [1). Although measurable, serum levels are not a good marker of organ concentration and proper water treatment is the only preventative measure possible. Other disease associations with contam‐ inants are summarised in the table 3. Gas or liquid chromatography with mass spectrometry are powerful tools used in analytical chemistry to separate, identify and quantify compounds in a liquid and can also be used as a screening test to detect contaminants in water samples. It is important to realise that chemicals detected in ultrapure samples tested at the end of the circuit may have originated not only from the feed water but also the plumbing, membranes or heated plastics, within the circuit.

*Trihalomethanes* levels are not specified in the ISO guidelines but are part of routine testing at certain facilities. (also see carbon filters above). These are a group of organic chemicals that are formed by the water disinfection process, by the reaction of chlorine with organic matter eg decaying plant and vegetable matter. Exposure to trihalomethanes in drinking water has been associated with liver damage, carcinogenicity and adverse reproductive effects [10, 23, 24]. Our institution currently tests for THM's in our water circuit between carbon tanks. The Australian drinking water guideline specifies a maximum concentration of trihalomethanes not greater than 0.25g/l [250ppb) [25]. The EPA (Environmental protection agency) in the USA specifies a lower limit of 0.08g/l ie 80 parts per billion (ppb). There is no specification for THM levels in dialysis water. In Australia, dialysis facilities use a level of 1/10th of the EPA drinking water guideline. It is believed that the presence of THM's greater than 8ppb usually heralds the imminent breakthrough of chloramines and/or chlorine post carbon. Levels greater than 8ppb trigger an action protocol resulting in swapping out and rotation of the carbon tanks, which can be performed on a semi elective basis.

Standard quality dialysis water has minimum microbiological and contaminant concentra‐ tions, defined by the ISO [9). (see table 3). **Ultrapure water** has more stringent microbiological criteria specifying a lower bacterial load and endotoxin concentration. Although ultrapure water can now be easily produced with the regular use of ultrafilters, the more stringent microbiological criteria make testing a more involved and lengthy process for instance longer culture times. Microbiological testing involves testing for bacteria and endotoxin and is discussed further below.

#### **5.2. Microbiological testing**

**5.1. Chemical testing**

234 Updates in Hemodialysis

**Figure 11.** Colour based chlorine test strips

or heated plastics, within the circuit.

*Chlorine* is a strong oxidising agent used in the disinfection of municipality drinking water. Inadequately treated dialysis water can result in significant patient exposure to chlorine. Here ferrous iron (Fe2+) in haemoglobin is converted ferric iron (Fe3+) resulting in the formation of methaemoglobin, haemolysis and anaemia [21, 22]. Due to its toxic nature and abundance, testing for chlorine occurs twice daily in dialysis units. This is performed using indicator test strips or testing tablets, which cause a colour change in the presence of chlorine. Electronic chlorimeters are also used and these are serviced regularly. In the home setting, patients are trained to perform a chlorine check before any dialysis session is initiated. In addition, trained water technicians visiting the home for maintenance reasons, also check chlorine levels. Chlorine testing is not routinely performed in the lab, as the samples need to be tested within

*Aluminium* has been associated with renal bone disease, anaemia and dementia [1). Although measurable, serum levels are not a good marker of organ concentration and proper water treatment is the only preventative measure possible. Other disease associations with contam‐ inants are summarised in the table 3. Gas or liquid chromatography with mass spectrometry are powerful tools used in analytical chemistry to separate, identify and quantify compounds in a liquid and can also be used as a screening test to detect contaminants in water samples. It is important to realise that chemicals detected in ultrapure samples tested at the end of the circuit may have originated not only from the feed water but also the plumbing, membranes

*Trihalomethanes* levels are not specified in the ISO guidelines but are part of routine testing at certain facilities. (also see carbon filters above). These are a group of organic chemicals that are formed by the water disinfection process, by the reaction of chlorine with organic matter eg decaying plant and vegetable matter. Exposure to trihalomethanes in drinking water has been associated with liver damage, carcinogenicity and adverse reproductive effects [10, 23, 24]. Our institution currently tests for THM's in our water circuit between carbon tanks. The Australian drinking water guideline specifies a maximum concentration of trihalomethanes

4 hours of collection due to the instability of the chlorinated compounds.

Microbiological analysis involves testing for bacteria and endotoxins. Endotoxins are the lipopolysaccharide layer of the gram-negative bacterial cell walls. However it is worth noting that bacterial culture and endotoxin testing do not exclude all possibility of bacterial contam‐ ination. For example, they do not detect gram-positive cell wall components e.g. peptidogly‐ can, or bacterial fragments, unculturable organisms or dead bacteria [26], all of which may still elicit an inflammatory response when introduced into the blood stream. The clinical relevance of this is unknown.

Various factors can affect the ability of bacteria to be cultured. These include the type of culture medium, length of incubation and temperature, all of which are specified in the ISO guideline [9). The method of collection is also standardized by the ISO. Sampling ports are cleaned with alcohol, connectors soaked in ethanol to prevent contamination and water is allowed to run for the first 30 seconds. The [9) guidelines also recommend that samples be assayed within 4 hours of collection. The guideline recommends that this time can be extended by refrigeration, up to 24rs. However this is not easily achievable especially considering the remote location of many of the home dialysis patients.

Potential organisms that contaminate the water circuit are accustomed to a nutrient poor environment hence necessitating special consideration. These include using a nutrient poor culture medium (e.g. Reasoners agar [R2A]), the use of membrane filtration for low colony count detection, an incubation temperature of between 17 and 23 degrees Celsius and an incubation period of 7 days [27]. The concentration of contaminants at which action should be instituted is set at 50% of the maximum allowable level; that is intervention is required when 50% of the allowed maximum is reached.

Endotoxin testing is performed using the Limulus amoebocyte lysate (LAL) assay. Here serum (amebocytes) removed from the horseshoe crab (limulus) is exposed to endotoxin. This activates a proteolytic cascade in the crab serum resulting in the formation of a gel like substance. An artificial substrate added to the lysate is also proteolytically cleaved and the liberated protein can be measured by its ability to absorb light, which is the basis of the chromogenic test kits. The test can also be quantified by its ability to form a clot or produce turbidity. It is important to note that the LAL assay does not detect endotoxin < 8000 Da.
