We are trying to develop a biosensor which can detect chloroform and other trihalomethanes in water. The idea is based in the ability of a recombinant Nitrosomonas europaea strain to detect those contaminants by expressing the exogenous gen of green fluorescent protein (gfp). It is thought that the capacity to detect trihalomethanes is due to ammonia monooxigenase (AMO), the enzyme responsible for oxidizes ammonia to nitrite. It seems that AMO recognizes chloroform nonspecifically and oxidizes it to phosgene that somehow is able to activate mbla and clpb promoters and start the expression of GFP. Our aim is to transform an Escherichia coli K-12 strain with a plasmid containing the sequence that codifies for AMO and other plasmid containing mbla or clpb promoters and gfp, in order to achieve an E. coli strain which could detect chloroform and express GFP.

Introduction

Trihalomethanes (THMs) are a group of volatile substances which are produced mainly in the drinking water treatment. Adding chlorine to water to disinfect it in water treatment plants generates some disinfection byproducts (DBPs) such as trihalomethanes, halogenated acetic acids (HAA) and others. It seems that the organic matter in water from rivers and reservoirs, especially substances such as tannins (fulvic and humic acids), through the process of oxidative chlorine, are precursors of disinfection byproducts. These products, especially trihalomethanes, are associated with risks to health and to environment, and internationally linked to possible cancers such as colon and bladder, and adverse effects during pregnancy, including abortion and fetal growth retardation. One or more of methane’s hydrogen is chemically substituted by a halogen such as fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

We can get the following compounds:

CHF3 or trifluoromethane, fluoroform, Freon 23, R-23, HFC-23 CHClF2 or chlorodifluoromethane, R-22, HCFC-2 CHCl3 or trichloromethane, chloroform, methyl trichloride CHBrCl2 or bromodichloromethane CHBr2Cl or dibromochloromethane CHBr3 or tribromomethane, bromoform, methyl tribromide CHI3 or triiodomethane, iodoform, methyl triiodide

Health risks being in water

Spanish law admits that (since 1st January 2009) the presence of these compounds in tap water should not exceed 100 μg/L to be considered safe for human consumption. These values in many parts of Spain had been passing frequently in the past. In Barcelona and the metropolitan area for example, whose water supply comes mainly from two rivers, Llobregat and Ter, historically there were different qualities of water and presence of these compounds. In water coming from Llobregat river, the presence of trihalomethanes had been greater than 150 μg/L in several times, while in water of the Ter river, without observing values so high, the concentration of 100 μg/L had been overcame easily.

Water reaching a water treatment plant contains reducing agents (organic and inorganic compounds such as nitrites, ions of iron, lead and sulfur) as well as microorganisms and bacteria. Chlorine is applied in excess (about 2 mg /L) to oxidize these compounds and kill bacteria and also to remain a residual amount of chlorine in the water pipes. Its utility is to continue disinfecting the water after the treatment plant and until it reaches the consumer. This excess of chlorine reacts with organic compounds to form trihalomethanes.

Several studies concluded that all water supply systems using free chlorine in its treatment contain at least 4 THMs in treated water: chloroform, bromodichloromethane, bromoform and dibromochloromethane.

Chlorine + Organic Matter ==> Trihalomethanes

Health risk

More recently it has been suggested that the accumulation of trihalomethanes in the water is considered a risk to health and the environment, and even carcinogenic.

Trihalomethanes have been linked in epidemiological studies with an increased risk of urinary bladder cancer, damage to liver and kidney, lung and breast cancer. The effect on human health can occur after long-term of continuous intake.

A report by the Public Health Agency of Barcelona, entitled "Health in Barcelona 2006," shows that it has been detected a maximum of 156.6 micrograms per liter of this carcinogenic substance produced by the drinking water treatment when it is captured in rivers. It should be remembered that the consumption of this substance for over 20 years has inevitably health problems and cancer is one of them.

Water is our source of life, is necessary and irreplaceable, we must maintain proper control of the quality of our waters, working in the health of citizens.

Legislation

According to the rules of the European Union States the concentrantion of trihalomethanes should not exceed one hundred micrograms per liter of water for consumption. In Spain, Real Decreto 140/2003 of 7th February 2003 puts the limits of contamination legally permitted. Thus, the content of THMs that can have tap water from 1st January 2009 is reduced to 100 μg/L while in the United States the Environmental Protection Agency (USEPA) has established a legal maximum of 80 μg/L.

Prevention

Treatments used for the sanitation of water are ozonation and chlorination. Chlorination is the one used in most countries due to the low cost of hypochlorite and its highly efficiency, and ensures an adequate disinfection of drinking water. In Europe, Mediterranean countries and the United Kingdom use chlorine gas in general, while the Nordic countries and Germany rejected the aroma and flavor using other products. Trihalomethanes are volatile compounds that can enter the human body by ingestion through the consumption of tap water, inhalation of vapors released into the showers or dermal adsorption during bathing or showering. So the best way to prevent their possible effects on our body is their direct elimination in the water used. At this point we must distinguish between potable water and water in the shower or bath.

Swimming pools

Swimming pool water needs to be disinfected to keep swimmers out from infections caused by microbial pathogens. Traditionally, sodium hypochlorite (NaClO) has been widely used for this purpose due to its moderate stability. However, the use of this compound has been reported to produce various halogenated organic compounds such as disinfection by-products (DBPs), since organic materials from various sources (perspiration, urine, mucus, skin particles, hair, lotion, etc.) are released into swimming pool water by swimmers. In addition, most pool waters are supplied with chlorinated surface water already contaminated with DBPs. The variations in the concentrations of DBPs in pool water have been suggested to depend on several factors: (a) the number of swimmers in pools; (b) the chlorine dose; (c) the bromide content; (d) the extent of outgasing of volatile DBPs; and (e) the use of DBPs-containing water (mostly chlorine-treated surface water) for pool water supply. The major products of disinfection using HBrO and HClO were bromoform (CHBr3) and chloroform (CHCl3), respectively. The addition of urine into the mixture containing humic material significantly reduce the overall formation of THMs, and this is attributed to the depletion of active free residual chlorine from the formation of less reactive chloramines. Air quality in indoor swimming pool facilities has emerged as an area concern with respect to human health. If there is not good ventilation in the buildings or swimming pool, it will cause more carcinogenesis from inhalation exposure to THMs. It is well known that THMs are volatile substances that can vaporize from water to environmental air depending on many variables, such as vapor pressure, water solubility, the water air contact area, etc.

Chloroform is a highly volatile compound that can be inhaled in swimming pool environments and also readily absorbed through the skin.