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= Toxicity Identification Evaluation (TIE) =

Introduction
The National Pollutant Discharge Elimination System (NPDES) was enacted in 1972 under what is now known as the Clean Water Act (CWA). NPDES requires that parties discharging into water sources of the United States have a permit which describes protective actions, monitoring procedures and limits of discharge. If monitoring indicates an undesirable level of pollutant, the party can initiate a Toxicity Reduction Evaluation (TRE), which aims to identify the causes of effluent toxicity, isolate the source and evaluate control options and confirm that the reduction in toxicity complies with the NPDES requirements.

Because effluent toxicity is usually composed of multiple chemicals, it can be extremely difficult to determine which chemicals are causing the observed toxicity. It is also difficult to choose which chemicals to analyze in a sample of effluent, since testing for all chemicals would be extremely expensive and equipment is limited in its abilities to test for a multitude of chemicals. A Toxicity Identification Evaluation (TIE) can be conducted as part of the TRE; a TIE consists of three phases: characterization, identification and confirmation. Each phase uses organismal response to detect the presence of toxicants in the effluent, in other words, acute toxicity tests are used to analyze toxic components of the effluent. Tests conducted in the first phase are based on a series of manipulations which characterize physiochemical attributes of the effluent. These tests include modifications of pH, filtration, aeration, oxidation, reduction, and chelation. Once effluent toxicity is found to be caused by a group of chemicals, Phase 2 of TIE is used to identify the specific chemicals causing toxicity. Phase 3 of TIE uses procedures such as correlation of toxicity with chemical concentration, toxicity mass balance, spiking experiments and additional test species to confirm identified toxicants. TIE has been used to examine the toxicity of pesticides in aquatic systems. It has been used to confirm methyl parathion and carbofuran in the Sacramento River, pyrethroid in agricultural run-off , and to examine sediment toxicity in the lower Santa Maria River.

TIE Preliminaries
There are a few things to note prior to the initiation of any TIE for accurate toxicity test results. Test organisms are chosen carefully because there is a variability of sensitivities between test species to different groups of toxicants. Age, availability, size, lifespan, habitat, and amount of toxicological data of potential test species, are all crucial in the decision to which test organisms are used for which tests.

Whole effluent toxicity methods (WET) are standard USEPA toxicity test methods put into place for the testing of effluent discharged into United States waters and must be followed for regulatory purposes under the CWA. The USEPA recommends that the lab considers historic records of the site-specific effluent to aid in gauging the dilution series. Dilution series in this context is a sequence of related, increasing concentrations of an effluent sample diluted with water of specific characteristics resembling the receiving waters of the site the sample came from. It can either be synthetic or from the site out of the mixing zone where the effluent mixes with the receiving waters. Typically, dilution water will have similar pH, salinity, and hardness to ensure healthy growth and reproduction of the test species.

Additionally, care needs to be taken when assessing effluent received by brackish or marine waters. Altering the salinity of the effluent sample could unintentionally cause harm to saltwater test organisms if the source of salinity is not sodium chloride. Sodium Chloride is needed for osmoregulation of sodium and chloride in marine organisms. It is important to note this because not all measuring devices can specify which dissolved solids are in an effluent sample, so freshwater species are often used in phase I and II of TIE to reduce potentially added stress.

Quick transport and storage under 4 degrees Celsius of effluent samples are important to retain the integrity of the original effluent collection site. Effluent samples can oftentimes either change in toxicity or change toxicity source over time, so immediate attention is required upon reception of samples.

Initial Toxicity Test
The initial toxicity test is performed the first day the whole effluent sample arrives in the lab, where a known fraction of this original sample is exposed to live test organisms. Two replicates are done with a negative control and whole effluent concentrations of 100%, 50%, 25%, 12.5%, and 6.25%. Standard parameters are measured and recorded to compare with adjusted tests later in the process. Test organisms exposed to this unaltered whole effluent sample are then counted after 24 hours and used to create a general Lethal Concentration 50. Identifying the lethal concentration 50 (LC50) is a critical step because it will determine the concentrations for the dilutions series in the baseline toxicity test

Baseline Toxicity Test
The baseline toxicity test is used as a comparison from the original effluent sample to the altered test result portions of the sample. Concentrations for the dilution series in this test are based on the LC50 results from the initial toxicity test. Two series of concentrations will be set up, with the first being 0.5, 1, 2, and 4 times the 24- hour LC50 of the effluent sample generated from the initial toxicity test. This will also be the dilution series for most of the following characterization tests. The purpose for this first baseline test is as the control, to be repeated with each characterization test and others throughout phases II and III of the TIE process. The second test will be the same dilution series of the initial toxicity test to make comparisons between the original effluent and the test results from each of the characterization tests. This test is to monitor any temporal change in toxicity of the original effluent. The test organisms need to be uniform in age, species, and exposure time.

Oxidation-Reduction
This test determines whether or not the toxicity of an effluent can be contributed to a redox reaction by adding sodium thiosulfate, a strong reducing agent, to the effluents and observing toxicity. Chlorine, ozone, chlorine dioxide, monochloramine, dichloramine, bromine, and iodine are examples of chemicals which can be reduced by sodium thiosulfate. There are two ways to carry out the oxidation-reduction test. One is to add a gradient of thiosulfate concentrations to chambers that have 100% effluent concentrations. It is important that the concentrations of thiosulfate added are less than the thiosulfate LC50 for the test organism being used to ensure the accuracy of results. The organisms that are recommended are Daphnia magna, Ceriodaphnia dubia, and Pimephales promelas. The other way to carry out the oxidation-reduction test is to set up a matrix of three thiosulfate concentrations (based on the test organism’s LC50) and three effluent concentrations. It is recommended that the thiosulfate concentrations are: .5x LC50, .25x LC50, and .125x LC50 and that the effluent concentrations are 25%, 50% and 100% effluent. Usually, the addition of thiosulfate will not eliminate all of the toxicity, but it will significantly reduce it if it is composed of effluents. If the toxicity in the chambers with thiosulfate is less than the baseline (control with no thiosulfate added) then it is evident that oxidants are contributing to effluent toxicity. Not only is thiosulfate a reducing agent for forms of chlorine compounds, but it can also be a chelating agent for metals such as cadmium, copper, silver, and mercury. Reduced toxicity observed in the oxidant reduction tests can also be contributed to the complexation of thiosulfate and these metals. To determine whether or not the toxicity is due to oxidants or cationic metals, the EDTA Chelation test can be carried out.

EDTA
EDTA is an acidic chelating agent. The most common metals that form compounds with EDTA are aluminum, barium, cadmium, cobalt, copper, iron, lead, manganese, nickel, strontium, and zinc. EDTA does not complex with anionic metals. Like sodium thiosulfate, EDTA is toxic in high enough concentrations. Hardness affects EDTA toxicity more than salinity does, so the concentration of EDTA for a certain effluent should prioritize hardness measurements. The EDTA LC50 in lab water will be lower than in effluent water, which is why it is necessary to determine the LC50 using lab water. There are two ways you can run an EDTA test. One is to add a gradient of EDTA to six chambers composed of 100% effluent or 4x the LC50. The point of this test is to determine which concentration of EDTA will reduce metal toxicity, and a high concentration of effluent is used because it ensures accuracy of results. The second test is an effluent dilution test. In this case, as with the oxidant reduction test, different concentrations of EDTA are added to a gradient of effluent dilutions. Figuring out which EDTA concentrations to use can be difficult. Not only can the EDTA LC50 be used to determine the concentration of EDTA, but hardness and salinity can be accounted for as referenced earlier. When using hardness as a measurement to determine EDTA concentrations, the most common approach is to measure the hardness of the 4x-24h LC50 concentration and use titration to find the concentration of EDTA needed to produce an endpoint. The other option is to measure calcium and magnesium in the 100% effluent sample and calculate the amount of EDTA needed to bind with those molecules. The additive number of Ca and Mg mols will equal the mols of EDTA require for chelation. The last option to calculate the concentrations of EDTA to add is to use the EDTA LC50. This is the most commonly used approach and is what allows for an EDTA gradient approach. As was previously mentioned, the LC50 should take hardness and salinity into account. Once the concentrations of EDTA are calculated using one of the above methods, the EDTA gradient test can be conducted. This test requires 7 chambers with 100% effluent which will receive a gradient of EDTA concentrations which you have previously calculated. A control is required. The effluent dilution test requires three sets of three effluent concentrations which are 100%, 50%, 25% or 4xLC50, 2xLC50, and 1x LC50. A baseline test will be used as the control and will be compared to. A gradient of EDTA .2 mL, .05 mL, and .0125 mL is added to the gradient of effluent dilutions in a matrix. Organisms should be added in a careful fashion. It is recommended that the samples are refrigerated overnight before toxicity test begin, this allows the metal to complex with the EDTA before organisms are added. Because EDTA is acidic, pH also needs to be monitored after EDTA addition and organisms can only be added once pH is stable and equal to the initial value. If the time until mortality is shorter in the treatments than in the control, a lower concentration of EDTA gradients is often used, if the time to mortality is unphased by the addition of EDTA, then the test is repeated with a higher concentration of EDTA.

pH Adjustment Test
The pH adjustment test is the first pH test done in the lab for phase 1 TIE. In this test, a large volume of effluent is collected. One volume of the sample is raised to pH 11 and another volume is lowered to pH 31. From there, the samples are all taken back to their initial pH and toxicity tests are ran. The pH adjustment test is used to determine if toxicity of the effluent changes when pH alone is manipulated, as pH can influence many aspects of chemical properties, particularly metals, including but not limited to ionization, solubility, and membrane permeability. The pH adjustment test provides a solution for several other tests done as part of TIE. pH adjustment tests have been conducted for many purposes, including to examine municipal solid waste landfills.

pH Adjustment/Filtration Test
This is the second test performed as part of the pH adjustment tests and involves filtering the pH adjusted samples from the pH Adjustment Test. By filtering the pH 3, pH 11, and the test pH samples, and performing toxicity tests with them, a better understanding can be made about whether the toxicant sorbs to particles. This information is valuable for filter feeders, like cladocerans, and other organisms that interact with sediment. The test also examines whether changes in pH affect the likelihood of toxicants to sorb to sediment. Positive pressure filtration is recommended over vacuum filtration, and glass-fiber filters are usually used. Blanks are created using dilution water for all three samples. Prior to conducting toxicity tests on day two, the pH 3 and pH 11 blanks should be readjusted to the original pH of the effluent. If the effluent has lots of solid particles, it may be possible to centrifuge the sample and filter only the supernatant, or several rounds of filtration may be required.

pH Adjustment/Aeration Test
This test is performed to determine if volatile compounds contribute to effluent toxicity. Sparging with air is the primary method for performing this experiment because it also allows for oxidation, although nitrogen sparging can be performed in addition to air. pH 3, pH 11, and the sample pH solutions are sparged along with their blanks. After sparging, a 24-hour LC50 is determined from the effluent sample and toxicity tests are ran at 4x,2x,1x, and .5x that LC50. Prior to running the toxicity test on day two, the pH 3 and pH 11 solutions must be brought back to the pH of the original sample. Once toxicity tests are completed, a series of steps are performed to determine if toxicity resulted from sparging, oxidation, or sublation.

Extraction
Solid phase extraction (SPE) is a two day multistep procedure resulting in the extraction of C 18. C 18 columns are prepared using washes of methanol and pure water. Then, three solutions are added to the prepared columns. One solution is composed of unaltered dilution water at its original pH, one solution with added HCl at pH 3, and one solution with added NaOH at pH 9. After running the solutions, a sample is taken. Then, three more solutions are run through the same columns. These solutions are composed of unaltered dilution water at its initial pH, a solution with added HCl at pH 3 and a solution with added NaOH at pH 9. A sample is collected from each of the columns at two points after a certain volume of the solutions have been filtered through. These samples are run through toxicity tests on day two.

Graduated pH Test
The Graduated pH test is used to see if the toxicity is dependent upon pH sensitive compounds. Three gradual effluent tests are run, each using a different pH level. These gradual tests look to see if the pH is causing increases, decreases or removal of the target toxicity. Tests for toxicity are run on test organisms, five for each one of the test pH levels. Once the tests are run, survival readings are taken after 24, 48, 72 and 96 hours. Survival readings are taken at LC50 levels for the organisms. During these tests, buffers are used to keep the pH levels constant, and pH levels should be recorded once all of the organisms in the test have died.

Phase II: Identification
Phase II testing is designed to aid in the identification of causes of acute and chronic toxicity stemming from nonpolar organic chemicals, metals, ammonia, surfactants and chlorine Polar organic chemicals are not identified during the Phase II test. Once the toxicity cause is determined, Phase III confirms that identification is correct. Approaches for identification are as follows: nonpolar organics testing, ammonia testing, metal testing and chlorine testing. In nonpolar organics testing, the goal is to isolate the nonpolar organic compounds from the non toxic compounds through fractionation and then identity by comparing concentrations to toxicity values. Ammonia testing can identify ammonia as the toxic compound by taking advantage of the relationship between ammonia and pH changes. The testing involves a graduated pH test, a column test and an air-stripping test. For metal testing, the addition of EDTA should reduce the toxicity of the sample in the presence of cationic metals. Chlorine testing can identify chlorine as a toxicant by measuring TRC (total residual chlorine) levels.

Phase III: Confirmation
In some circumstances, characterization and identification from Phase I and Phase II testing may produce incorrect conclusions. Phase III tests are designed to indicate variability of toxicants and confirm toxicants of interest. This phase is crucial in remediation. Most of the tests in Phase III contribute information on chronic and acute toxicity. Approaches for confirmation are as follows: correlation approach, species symptoms, species sensitivity, spiking, mass balance and deletion. The correlation approach is used to show consistency between the concentration of the identified toxicant and the effluent toxicity. The species symptom approach compares symptoms of organisms exposed to effluent to symptoms of organisms exposed to the identified toxicant to evaluate whether or not the symptoms are the same. The species sensitivity approach analyzes toxicity of the identified toxicant to at least 2 different species and the toxicity of the effluent to at least two different species. If the toxicity ratios are the same for the identified toxicant compared to the effluent then confirmation would be supported. A spiking approach focuses on toxicity in the effluent after the identified toxicant is spiked. If the identified toxicant is causing effluent toxicity you would expect to see increased toxicity as the concentration of the identified toxicant increases. The Mass Balance approach requires three toxicity tests: all fraction, toxic fraction, and non-toxic fraction tests. If the toxicant of interest is causing toxicity you should see toxicity in your toxic fraction, intermediate levels of toxicity in the all-fraction tests and no toxicity in your non-toxic fraction tests. The dilution approach works for removable chemicals; if toxicity tests indicate a decrease in toxicity after the chemical of interest is deleted then confirmation is supported.