enteric viruses and protozoa. Further, concerns for newly emerging pathogenic organisms which may arise from nonhuman reservoirs (e.g., pathogenic E. coli, Cryptosporidium parvum, and Giardia lamblia) have led to the questioning of the use of indicators that arise primarily from fecal inputs. In a recent study, it was concluded that coliform bacteria are adequate indicators for the potential presence of pathogenic bacteria and viruses but are inadequate as an indicator of the presence of waterborne protozoa. Waterborne disease outbreaks have also occurred in drinking water systems that have not violated their micro- bial water quality standards (Craun et al., 1997).
Given the limitations in using coliform organisms as indicators of potential contamina- tion by wastewater, attention has now focused on the use of bacteriophages as an indicator organism and more specifically as indicators of enteric viruses. Bacteriophages are viruses that can infect prokaryotic cells. There are six major families of bacteriophages, five of which are DNA-based and one of which is RNA-based. Of the five DNA-based bacteriophages, three are double stranded and two are single stranded. Bacteriophages that infect E. coli are known as coliphages. Coliphages that attach directly to the cell wall are known as somatic.
Coliphages that infect only male strains of E. coli (possess pilli) are known as male-specific (F1) coliphages. The source of male-specific phages is thought only to be feces.
Within the male specific family there are four serotypes. Groups II and III are primarily of human origin whereas groups I and IV are of animal origin, with the exception of pigs, which may harbor groups II and III. Interest in using coliphages as an indicator of enterovirses is based on the fact that the phages are approximately the same size as most enteric viruses of interest (e.g., polio), are of fecal origin, and are always present in raw municipal wastewater.
Coliphages have been used extensively in disinfection studies (see Sec. 12–9 in Chap. 12).
Evolving Pathogenic Microorganisms
In recent years there has been a disturbing increase in the number of disease outbreaks in the United States and in many other parts of the world, especially in light of the fact that it was thought that a number of endemic contagious diseases had been controlled or eliminated (only smallpox to date) (Levins et al., 1994). The bacteria Legionella pneumophila, the causative agent in Legionnaire’s disease, is ubiquitous and is found in drinking water, waste- water, and reclaimed wastewater, is an example of a disease causing organism that has been identified relatively recently (Levins et al., 1994). The significance of the identification of new disease organisms, disease outbreaks, and the reemergence of old diseases is that the concern for public health must remain the primary objective of wastewater management.
2–10 TOXICITY
Wastewater can contain a variety of constituents, many of which can cause adverse impacts if discharged to the environment. Toxicity is a measure of the degree to which single or multiple constituents that may be present in untreated and treated wastewater can cause adverse impacts (damage) to human and animal health, sensitive aquatic biota, and ecosys- tems. To provide a general introduction to the subject of toxicity and toxicity testing, it will be useful to consider the following topics: (1) sources of toxicity in untreated and treated wastewater, (2) the evolution and application of toxicity testing, (3) toxicity testing proce- dures, (4) the analysis of toxicity test results, (5) the application of toxicity test results, and (6) methods that can be used to identify specific toxicity constituents.
Sources of Toxicity
The sources of toxicity in untreated and treated wastewater are derived from the constitu- ents added during usage, treatment, and disinfection with chemical agents.
Constituents Added During Usage. Where a separate wastewater collection system is used, constituents added during usage can include (1) physical properties such as elevated temperature and TDS; (2) inorganic nonmetallic constituents such as ammonia and hydrogen sulfide; (3) metallic constituents such as chromium, mercury, and silver;
(4) aggregate organic constituents such as cleaning and personal care products; and (5) individual organic compounds such as identified in Table 2–16. Where a combined wastewater collection system is used, an additional source of toxicity is from runoff. Con- stituents in runoff that can cause toxicity include pesticides and nutrients from yards, city landscaping, and agricultural lands, and heavy metals and organic and inorganic (e.g., salt) constituents from streets and highways.
Constituents Added During Treatment. Constituents added during treatment that can result in toxicity issues can include flocculent aids that contain contaminants, chemicals to precipitate phosphorus that contain contaminants, chemicals added to control foaming and frothing, and chemicals added to control algae growths.
Constituents Added During Disinfection. One of the most important sources of toxicity is from the disinfection byproducts formed during the disinfection of treated effluent with chemicals such as chlorine, chlorine dioxide, and ozone. The formation and control of chlorine, chlorine dioxide, and ozone disinfection byproducts is considered in Secs. 12–3, 12–4, and 12–6 in Chap. 12, respectively.
Evolution and Application of Toxicity Testing
Until the latter part of the twentieth century, pollution control measures were focused primarily on conventional pollutants (such as oxygen-demanding materials, suspended solids, etc.) which were identified as causing water quality degradation. During the past 30 y, increased attention has been focused on the control of toxic substances, especially those contained in wastewater treatment plant discharges. The national policy prohibiting the discharge of toxic pollutants in toxic amounts is documented in Section 101(a) (3) of the federal Clean Water Act. Because it is not economically feasible to determine the specific toxicity of each of the thousands of potentially toxic substances in complex effluents, whole effluent toxicity testing using aquatic organisms is a direct, cost- effective means of determining effluent toxicity. Whole effluent toxicity testing involves the introduction of appropriate bioassay organisms into test aquariums (see Fig. 2–37) containing various concentrations of the effluent in question and observing their responses. The whole effluent test procedure is used to determine the aggregate toxicity of unaltered effluent discharged into receiving waters. Toxicity is the only parameter measured.
Even though the focus of this section is on effluent toxicity, it should be noted that toxicity testing has number of other applications, including
1. Assess the suitability of environmental conditions for aquatic life.
2. Establish acceptable receiving water concentrations for conventional parameters (such as dissolved oxygen, pH, temperature, salinity, or turbidity).
3. Study the effects of water quality parameters on wastewater toxicity.
4. Assess the toxicity of wastewater to one or more freshwater, estuarine, or marine test organisms.
5. Establish relative sensitivity of a group of standard aquatic organisms to effluent as well as standard toxicants.
6. Assess the degree of wastewater treatment needed to meet water quality requirements.
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7. Determine the effectiveness of wastewater treatment methods.
8. Establish permissible effluent discharge rates.
9. Determine compliance with federal and state water quality standards and water quality criteria associated with NPDES permits (Standard Methods, 2012).
Such tests provide results that are useful in protecting human health, aquatic biota, and the environment from impacts caused by the release of constituents found in wastewater into surface waters. Toxicity identification, in which the constituents or compounds responsible for the observed toxicity are delineated, is another important aspect of toxicity assessment.
Toxicity Testing
Terms commonly encountered when considering the conduct of toxicity tests and the analy- sis, interpretation, and application of test results are summarized in Table 2–29. Because the terms reported in Table 2–29 are subject to change as new and improved methods of toxicity testing are developed, it is imperative that the latest version of Standard Methods and related U.S. EPA protocols be reviewed before undertaking any toxicity testing.
Toxicity tests are classified according to (1) duration (short-term, intermediate-term, or long-term); (2) method of adding test solutions (static, recirculation, renewal, or flow- through); (3) type of test (in vitro, tests in petri dishes or test tubes, or in vivo, toxicity tests using the whole organism); and (4) purpose (NPDES permit requirements, mixing zone determinations, etc). In vitro toxicity testing has been validated widely in recent years.
Even though organisms vary in sensitivity to effluent toxicity, the U.S. EPA has docu- mented that (1) toxicity of effluents correlate well with toxicity measurements in the receiving waters when effluent dilution was measured, and that (2) predictions of impacts from both effluent and receiving water toxicity tests compare favorably with ecological community responses in the receiving waters. The U.S. EPA has conducted nationwide tests with freshwater, estuarine and marine ecosystems. Methods include both acute as Figure 2–37
Typical setup used to conduct of whole effluent toxicity tests where mortality is the test end point.
Table 2–29
Terms used in evaluating the effects of contaminants on living organismsa, b
Term Description
Acute toxicity Exposure that will result in significant response shortly after exposure (typically a response is observed within 48 or 96 h)
Chronic toxicity Exposure that will result in sublethal response over a long-term, often 1/10 of the life span or more Chronic value (ChV) Geometric mean of the NOEC and LOEC from partial
and full cycle tests and early-life-stages tests Cumulative toxicity Effects on an organism caused by successive
exposures
Dose Amount of a constituent that enters the test organism
Effective concentration (EC) Constituent concentration estimated to cause a specified effect in a specified time period (e.g., 96-h EC50)
Exposure time Time period during which a test organism is exposed to a test constituent
Inhibiting concentration (IC) Constituent concentration estimated to cause a specified percentage inhibition or impairment in a qualitative function
In vitro Tests conducted in glass petri dishes or test tubes In vivo (in life) Toxicity tests conducted using the whole organism Lethal concentration (LC) Constituent concentration estimated to produce death
in a specified number of test organisms in a specified time period (e.g., 96-h LC50)
Lowest-observed-effect concentration (LOEC)
Lowest constituent concentration in which the measured values are statistically different than the control Maximum allowable toxicant
concentration (MATC)
Constituent concentration that may be present in receiving water without causing significant harm to productivity or other uses
Median tolerance limit (TLm) An older term used to denote the constituent concen- tration at which at least 50 percent of the test organ- isms survive for a specified period of time. Use of the term “median tolerance limit” has been superseded by the terms median lethal concentration (LC50) and median effective concentration (EC50)
No observed-effect concentration (NOEC)
Highest constituent concentration at which the measured effects are no different from the control Sublethal toxicity Exposure that will damage organism, but not cause
death
Toxicity Potential for a test constituent to cause adverse effects on living organisms
Whole effluent toxicity (WET) The total (or aggregate) toxicity effect of treated effluent measured directly in a toxicity test
a Adapted from Hughes (1996) and Standard Methods (2012).
b It should be noted that the terms given in this table apply only to aquatic organisms and are, for the most part, distinct from the terms used for animals and humans.
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well as chronic exposures. Typical short-term chronic toxicity test methods are reported in Table 2–30. Detailed contemporary testing and analysis protocols are summarized in Standard Methods (2012) and in U.S. EPA publications (U.S. EPA, 1985b, c, d, e).
Analysis of Toxicity Test Results
Methods used to analyze both short-term (acute) and long-term (chronic) toxicity data are considered in the following discussion.
Acute Toxicity Data. The median lethal concentration (LC50) when mortality is the test end point, or median effective concentration (EC50) when a sublethal effect (e.g., immobilization, fatigue in swimming, “avoidance”) is the end point, is typically used to define acute toxicity (Stephen, 1982). A typical bioassay setup using fish where mortality is the test endpoint is shown on Fig. 2–37. A fish swimming chamber is used to assess sublethal effects. A fish is placed in a chamber where the flow-through velocity can be increased until the fish is swept out of the chamber. The washout velocity for fish exposed to a specific compound can be compared to the washout velocity for the control fish.
Because the LC50 value is the median value, it is important to provide some infor- mation on the variability of the test population. The LC50values can be determined graphically or analytically using the Spearman Karber, moving average, binomial, and probit methods. The 95 percent confidence limits are usually specified. Most standard Table 2–30