LIU Huili LIU Hongling Yu Hongxia[*] Jin Hongjun

State Key Laboratory of Pollution Control and Resource Reuse

School of the Environment, Nanjing University, Nanjing, P.R. China, 210093

Abstract: The toxicity identification evaluation (TIE) procedures were the effective tools for characterizing and identifying toxicants in complex mixtures. The toxicities to Daphnia magna of industrial effluents, leaching liquor from a land-fill site of solid wastes and complex mixture of industrial effluents and municipal sewage were conducted with TIE methods. The TIE results revealed the key toxicants and their contribution rate to toxicity in deferent effluents and biosolid, which were suggested to be paid more attention for their discharge. The TIE researches should be developed in many ways in the future.

Key words: Toxicity identification evaluation; Industrial effluent; Leaching liquor; Municipal sewage

Introduction

Today, water pollution caused by toxic complex effluents is more and more serious; the simple, quick and accurate methods to identify the toxicants are in urgent demand. The conventional identification method is sample’s chemical analysis, which often emphasize on 129 “priority pollutants” ^[1-6]. But the problems of this method are that the controlling toxicants often are at relatively low concentrations in effluents, and now no aquatic toxicity data available for most of the detected chemicals in effluents.

The U.S. Environmental Protection Agency (EPA) has developed a set of procedures designed to characterize, identify, and confirm the causes of toxicity in acutely toxic complex effluents ^[7-11]. Effluent can contain thousands of chemicals, but usually only a few chemicals are responsible for any observed toxicity. The goal of EPA’s toxicity-based method is to separate the toxicant from the nontoxic components by using the response of an aquatic organism along with fractionation techniques. These toxicity identification evaluation (TIE) procedures have been successfully applied in numerous studies concerning the separation and identification of toxic compounds from complex mixtures of chemicals. This method enables direct relationships to be more easily establish between toxicants and measured analytical data, thereby avoiding the problems inherent with chemical-specific approaches to liming toxicity. It is quite useful for identifying causes of a water pollution accident ^[12-16]. But the application of the toxicity identification evaluation (TIE) procedures in China is not at large ^[17-21].

This paper is to introduce the toxicity identification evaluation (TIE) procedures and some applications in industrial effluents, leaching liquor from a land-fill site of solid wastes and complex mixture of industrial effluents and municipal sewage. At last, the future of TIE is foreshowed.

1. Materials and methods

1.1 Test organism

In TIE procedures, the test organism is Daphnia magna which is £ 24-h-old.Upon arrival of the sample 24-h and 48-h toxicity tests were conducted on the sample with Daphnia magna. Test chambers were 30-ml glass beakers. Daphnia test volumes were 10 ml. Five animals were exposed in each chamber. All toxicity tests were conducted at 20±2°C with one or two replicates, depending on the TIE phases, and a photoperiod of 16:8-h light: dark. The test end point was death. To establish LC50 values, all tests were set up using 50% serial dilutions.

1.2 TIE Procedures

The toxicity identification evaluation (TIE) procedures include three phases ^[7-10]. Phase I consists of methods to identify the physical and chemical nature of the constituents that cause acute toxicity. Phase I results are intended as a first step in identifying toxicants; however, the data generated can be used to develop specific treatment methods to remove toxicity without identifying specific toxicants. Phase II describes procedures such as fractionation schemes and associated analytical methods to identify the toxicants. Phase III describes procedures to confirm the presence of the suspected toxicants.

1.2.1 TIE Phase I –Toxicity Characterization

This phase is accomplished by chemically or physically manipulating the sample to neutralize, alter, or remove specific groups of chemicals. Bioassay tests are then conducted on the manipulated sub-samples to assess which treatments have removed or mitigated toxicity. Tests undertaken included initial toxicity test, baseline toxicity test, pH adjustment test, pH adjustment/aeration test, pH adjustment/filtration test, pH adjustment/C₁₈ solid-phase extraction (SPE) test, EDTA chelation test, oxidant reduction test and graduated pH test. The purpose of initial toxicity test was to determine the initial toxicity of effluent and to set desired test concentrations for the subsequent tests. Baseline toxicity tests were performed on successive days with each set of manipulation tests, and used to detect any change in whole effluent toxicity from initial toxicity and provide baseline toxicity value for comparison to the manipulated effluent. EDTA addition could indicate the presence of acutely toxic cationic metals. pH adjustment/filtration consisted of sample aliquots at pHi (initial pH), pH3 and pH11, which were allowed to stand for periods of time required to complete aeration and filtration. Sample pHs were adjusted to pH 3 and 11 by the addition of reagent-grade HCl and NaOH, respectively. After sample manipulations (aeration, filtration) at altered pH, the samples were readjusted to pHi by the addition of NaOH and HCl, respectively, period to testing. Solid-phase extraction (SPE), with a C₁₈ column of the effluent at pHi, pH 3 and pH 11 also was performed to detect the presence of nonpolar organic compounds causing toxicity. Two aliquots of sample (one after passage of 25 ml and one corresponding to the final 150 ml of sample ) were tested for toxicity to determine whether the column’s removal capacity had been exceeded. The graduated pH test at pHs of 6, 7 and 8 indicates the presence of pH-dependent toxicants such as ammonia or metals. The test pHs were altered through the addition of HCl and NaOH. By observing the alteration of effluent toxicity, the classes of chemicals causing the whole effluent toxicity could be identified.

1.2.2 TIE Phase II –Toxicity Identification

The objective of phase II TIE procedures was to identify specific toxicants within the classes of compounds characterized by phase I TIE using chemical analysis methods such as GC/MS, HPLC-MS, AAS and ICP-AES, etc., all of which relying on tracking the toxicity of the effluent throughout the analytical procedures. In some cases, a tentatively identified causative toxicant can provides sufficient information to eliminate or control a sample‘s toxicity to an acceptable level.

1.2.3 TIE Phase III –Toxicity Confirmation

In Phase III, the goal is to confirm the suspected key toxicants causing the effluent toxicity identified in phase II with specific approaches, such as correlation approach, spiking approach, species sensitivity approach, mass balance approach, and deletion approach. Phase III evaluations provide the "weight of evidence" that causative toxicants have been accurately identified. Upon the successful completion of Phase III, sufficient data will be available to assure that causative toxicants have been accurately identified.

1.3 Statistical analysis

Median immobilization concentrations were calculated for all aqueous toxicity tests, including those in phase I, II and III of TIE. LC50 values for toxicity tests were calculated with the Trimmed Spearman-Karber (TSK) Program Version 1.5^[22]. For certain comparisons, effluent toxicity was converted to toxic units (TU) by diving 100% by the LC50 of effluent. For specific chemicals, TU was calculated by dividing the concentration of chemical in the sample by its LC50 to Daphnia magna which was cited from relevant literature or gained from the laboratory toxicity test.

2 Application of TIE

2.1 TIE for complex mixture of industrial effluents and municipal sewage

There is growing community concern over the soil quality following the application of the reclaimed biosolid. How to dispose of large amounts of surplus sludge is a serious problem worldwide in sewage treatment. This TIE case study is about a municipal sewage treatment plant in ChangZhou, Jiangsu Province, which receives mainly domestic wastewater with industrial inputs, such as chemical, pharmaceutical, pesticide, and leather plants. The wastewater treatment process at the wastewater treatment plant involves aerobic and anaerobic oxidation of raw sewage (Table 1). The surplus sludge was disposed by a dehydration process before applying the reclaimed solid directly as a fertilizer for trees and cotton production. The wastewater treatment plant services many industrial factories such persistent toxicants remaining in the reclaimed solids may pollute the soil and receiving waters upon application to the farming land. To address this serious issue, TIE procedures were also performed on the sludge from the treatment plant. Through this case study, we describe the sequence of TIE testing used to characterize, identify, and confirm organic compounds in the influent and effluent wastewaters that cause acute toxicity to Daphnia magna. Furthermore the applicability of the sludge in agriculture as a fertilizer is evaluated.

Table 1 Information of the municipal sewage treatment plant

Items Data Treatment Capacity of the Plant 40,000 t/d Treatment Process A-A-O and Traditional Activated Sludge Method

Mixed Ratio of the complex mixture

35% Industrial Effluents

65% Municipal Sewages
2.1.1 Phase I Toxicity characterization

The median immobilization concentrations and TU mean values for all unmanipulated and manipulated effluents were listed in Table 2. In the phase I TIE test, lower toxicity values were obtained at pH3 and pH6.98 than at pH11, and suggest that suspected toxicants may be acid based chemicals. The addition of EDTA and Na₂S₂O₃ showed no reduction in toxicity of the influent, suggesting few metals and oxidants present. However, the C₁₈ SPE treatment appeared to retain most toxic compounds on the column and reduced toxicity in all manipulations. This result indicated that chemical(s) causing acute toxicity were most likely organic compounds.

Table 2 Result of Phase I Toxicity Characterization

Manipulation to effluent pH 24h LC₅₀ (%) TU Original pHi^a 20.31 4.92

pH adjustment pH3 48.11 2.08 pHi 30.78 3.25 pH11 23.33 4.29

pH adjustment/filtration pH3 46.11 2.17 pHi >100 <1.0 pH11 20.31 4.92

pH adjustment/aeration pH3 >100 <1.0 pHi 44.54 2.25 pH11 23.33 4.29 EDTA addition pHi 30.78 3.25 Na₂S₂O₃ reduction pHi 30.78 3.25

Graduated pH pH6 26.79 3.73 pH7 30.78 3.25 pH8 23.33 4.29

pH adjustment/C₁₈SPE pH3 >100 <1.0 pHi >100 <1.0 pH11 77.11 1.30

pHi^a, original effluent pH, 6.98

2.1.2 Phase II Toxicity Identification

In phase II TIE, where the effluent was concentrated on C₁₈ SPE columns and the fractions tested, toxicity occurred in three fractions such as 25%, 50% and 75% methanol/water fractions. Toxic fractions were combined and concentrated using 200-mg C₁₈ SPE columns. Toxicity testing revealed that toxicity was retained in the concentrate. The concentrate was injected into a GC/MS system and identified predominately as 2-propyl-bezaldehyde oxmie at a concentration of 1.50 mg/L.

2.1.3 TIE Phase III –Toxicity Confirmation

In order to confirm 2-propyl-bezaldehyde oxmie as a key toxicant contained in the effluent, a mass balance approach in Phase III TIE was performed in the early stages of Phase II as well before toxicants identified at all. The mass balance tests were conducted to ascertain whether toxicity in the toxic fractions was equal to that of the total fractions that was eluted by the C₁₈ SPE columns. It was possible that some chemicals contributing to toxicity in the original sample had not eluted from the C₁₈ column or may have been partially eluted into these fractions. Their toxicity might thus be difficult to observe. In this study, TUs removed by C₁₈ SPE columns were essentially the same as total fractions and toxic-fractions add-back tests, and no toxicity was detected with the nontoxic-fractions add-back test (Table 3). High correlation with TUs in the individual fractions and TUs in the add-back test supported evidence of high separation and recovery of toxicants using the C₁₈ SPE column. The mass balance tests showed high agreement with the original samples.

Table 3 Results^a of the mass balance confirming suspended toxicants

Test Type 24-h TUs Test Type 24-h TUs Original sample 3.25 toxic fractions 2.09 Post-column 1.41 Nontoxic-fractions NT^b C₁₈column removal 1.84 all-fractions 1.62

^aResults from literature (Cheng et al., 2001).

^bNT, no toxicity.

The calculated 48h LC50 of 2-propyl-bezaldehyde oxmie to Daphnia magna is 0.51 mg/L, using a commercial software package ^[24]. This software is a specific one for predicting toxicity and physicochemical parameters of organic pollutants. Analysis by GC/MS of the effluent determined the concentration of 2-propyl-bezaldehyde oxmie to be 1.50 mg/L. From these results the toxicity unit contributing to the effluent was calculated as 2.94. The toxicity contribution of 2-propyl-bezaldehyde oxmie can also be expressed as a percentage and was calculated as 62%. According to the requirements of TIE procedures, if the toxicity contribution of the suspected toxicant is over 60%, it can then be considered as a key toxicant. These results indicate greater toxicity of 2-propyl-bezaldehyde oxmie in comparison to the 48h TUs, 4.76 of the effluent confirms that it is a key toxicant in the effluent. The results also suggest that further attention needs to be given to receiving effluents, which may contain such compounds in order to avoid detrimental effects to microorganisms in primary activated sludge treatment plants.

2.1.4 TIE on the water extracts from the dehydrated sludge

We also conducted TIE on the water extracts from the dehydrated sludge. When the dehydrated sludge was stored outdoors for different times its toxicity increased with increasing sludge storage duration. TIE procedures, therefore, were performed to identify the toxicity causes. The TIE phase I results suggest that the suspected toxicants involved were grouped into two classes of chemicals. Class 1 included organic compounds such as phenols, anilines, and indoles and class 2 covered metals (Cd, Pb, Cr, Cu, Zn, Ni, K, Ca, Na, Mg, Al, Fe, Mn, Ba, Ti, Ce, V, Mo, and Sn). During phase II testing, sludge samples stored outdoors for 1, 8, 32, and 64 days were analyzed qualitatively with GC/MS. The results indicate that substituted aromatic compounds were gradually being degraded into aromatic acids and even alkyl acids with increasing storage duration, which means the dehydrated sludge was not stable. These organic acids are known to be detrimental to the soil. The increase in metal concentrations upon sludge storage reveals that metals can easily be released from the sludge and into the surrounding environment and suggests that the dehydrated sludge should not be directly used as a fertilizer for agriculture although its nutrition compounds are enough (N 3.524%, P 3.957%, K 0.5905%). It is recommended that the dried biosolid be composted until stability occurs and at the same time be treated to reduce the concentrations of metals. The use of TIE phase III testing is likely to be limited because of the unstable nature of the sludge and toxicity confirmation would be very difficult to ascertain. Future research will further refine phase III toxicity testing of the sludge.

2.2 TIE for industrial effluents and leaching liquor from a land-fill site of solid wastes

In recent years, the impacts of complex effluents to aquatic ecosystems have been getting much more serious with the development of industry in China. The effluent discharge limits in China now emphasize the general physico-chemical parameters as well as a few priority toxic pollutants among thousands of chemicals. Toxics release can not be effectively controlled, and some of treated effluents, which were considered to have met the requirements of the national discharge limits, were still highly toxic to aquatic organisms based on bioassay results. Therefore, the TIE approach is very useful for toxic discharge supervision to identify the real toxicants causing whole effluent toxicity in China. It is also helpful to develop cost-effective effluent treatment and resource recovery technology for the toxicity reduction.

In our research, the effluent types are various from containing metals, non polar and polar organic compounds to volatile toxic organic compounds. According to the requirements of TIE procedures, if the toxicity contribution of the suspected toxicant is over 60%, it can then be considered as a key toxicant. Therefore, the chemicals listed in Table 4 were confirmed the key toxicity ones due to their toxicity contributions from 77.5% to 109.4%. These results suggested that we should pay attention to these chemical discharges.

Table 4 TIE results of industrial effluents and leaching liquor

Sources

Identified toxic compounds Contribution rate to toxicity (%)

Land-Fill Site of Solid Wastes

Copper

Zinc

40.6%

68.8%

Metalhurgy Plant

Cr₂O₇^2- 86.7%

Electroplating Plant

Copper

Cr₂O₇^2-

2.6%

96.4%

Huafa Chemical Plant

Benzopyrone

Phenol

44.6%

32.9%

Petrochemical Company

Phenols, Anilines,

Naphthalene 87.5%

Coal Gasification Plant

Ammonia

Volatile phenols 90%

Nitrogen Fertilizer Plant

CN^- 94.8%

Jintian Chemical Plant

Anilines 100%

Nanjing Chemical Plant

Benzenes and Naphthalenes 90.0%

3. Future of TIE

TIE methods have been found to be effective tools for characterizing and identifying toxicants in samples of effluents, sediments, ambient waters, and other complex mixtures, but its’ application studies on trace pollution sources of toxic compounds in the drinking water sources and interstitial water have some shortage. On the other hand, the standard procedures are not simple and rapid enough in practice, especially in the latter two phases. We also should simplify procedures of chemical analysis and reduce the cost. The TIE progress also can encourage us to develop the cost-effective treatment process and the technology of the resource recovery for the toxicity reduction of an effluent. Except for Daphnia magna used in TIE, we also could develop some other organisms such as Brachydanio rerio, Oryzias latipes and Luminescent bacteria according to their sensitivity for different concentration of toxicants in practice.

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[*] Corresponding author

E-mail address: yuhx@nju.edu.cn