Ecotoxicology and Environmental Safety 54 (2003) 7486

Application of toxicity tests into discharges of the pulp-paper industry in Turkey

Delia Teresa Sponza*
Environmental Engineering Department, Engineering Faculty, Dokuz Eylul University, Buca-Kaynaklar Campus, Izmir, Turkey Received 18 June 2001; received in revised form 1 May 2002; accepted 30 May 2002

Abstract The aim of this study was to investigate the acute toxicity of pulp-paper industry wastewater using traditional and enrichment toxicity tests and to emphasize the importance of toxicity tests in wastewater discharge regulations. Enrichment toxicity tests are novel applications and give an idea of whether there is potential toxicity or growth-limiting and -stimulating conditions. Different organisms were used such as bacteria (oc and coliform bacteria), algae (Chlorella sp.), protozoa (Vorticella sp.), and sh (Lepistes sp.) to represent four trophic levels. Furthermore, chemical oxygen demand (COD) fractionation results were compared with these tests to assess the effect of COD subcategories on the determination of possible toxicity. The pulp-paper industry results revealed acute toxicity to at least two organisms in 6 of 20 efuent samples. The toxicity test results were assessed with chemical analyses such as COD, biochemical oxygen demand (BOD), color, absorbable organic halogen (AOXs), and phenol. It was observed that the toxicity of the efuents could not be explained by using physicochemical analyses in four cases for the pulp-paper industry. The results clearly indicate that bioassay tests provide additional information on the toxicity potential of industrial discharges and efuents. r 2002 Elsevier Science (USA). All rights reserved.
Keywords: Toxicity; Conventional; Enrichment toxicity test; Pulp-paper industry

1. Introduction The deliberate discharge and accidental release of harmful chemical compounds into the environment have the potential to disrupt the structure and functioning of natural ecosystems. The toxicity of industrial wastewater can inuence the operational efciency of existing wastewater treatment facilities and cause them not to meet the more stringent efuent standards. The conventional approach to controlling harmful chemicals in the aquatic environment is to use a set of global physical chemical and biochemical parameters. Chemical procedure alone cannot provide sufcient information on the potential harmful effects of chemicals on the aquatic environment (Vyryan et al., 1999). The complex nature of efuents cannot be overcome by specic chemical approaches (USEPA, 1981, 1996). The toxic effects of unknown and often undetermined substances in complex mixtures or with possible synergistic effects among
*Corresponding author. Fax: +90-232-453-1153. E-mail address: delya.sponza@deu.edu.tr (D.T. Sponza).

compounds to wastewaters can be detected only by toxicity testing. In addition, although in some cases the efuent quality of wastewater does not violate the discharge limits, the wastewater may be toxic. Large number of chemicals are discharged into the aquatic environment for which there is no direct means of control (USEPA, 1991; Kinnersley, 1990). The conventional approach to controlling harmful chemicals in the aquatic environment is to use a set of physical chemical and biochemical parameters (Cronin et al., 1991; Trevizo and Nirmalakhandan, 1999). Since the complex nature of many efuents limits a complete assessment with chemical analysis, the toxic effects of complex mixtures on wastewater can be detected only by biological tests (Chen et al., 1999). Throughout the world, where industrial efuent and hazardous waste are growing problems, a number of biological assays have been developed and evaluated for aquatic toxicity testing (USEPA, 1989; Sponza, 1999, 2002a, b). Biological toxicity testing is now a rapidly expanding eld involving numerous bioanalytical techniques developed and applied to organisms at different trophic levels

0147-6513/03/$ - see front matter r 2002 Elsevier Science (USA). All rights reserved. PII: S 0 1 4 7 - 6 5 1 3 ( 0 2 ) 0 0 0 2 4 - 6

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(Slabbert, 1996). Since living material responds to the total effect of actual and potential disruptions, biological assays have become important tools in assessing harmful chemical activity (Blaise et al., 1988). Environmentally relevant biotests provide information on the initial levels of damage and assist in developing precautionary measures and strategies for environmental management (Blaise et al., 1988; Slabbert, 1996). In the regulations concerning the evaluation of wastewater policy the incorporation of acute and complementary chronic toxicity tests with various organisms is necessary to protect aquatic ecosystems. Conventional toxicity tests using Daphnia magna and the Microtox, Biosensor, Eclox, and Toxalert tests are routinely used to assess the toxicity of wastewater samples. In particular, enrichment toxicity tests containing bacteria could be valuable screening tools for identifying and categorizing toxic efuents together with acute toxicity tests (Wharfe and Tinsley, 1995; Slabbert and Venter, 1999). The assessment of whether a complex substance poses a risk to organisms in the environment is possible only by acute and enrichment toxicity tests (Schowanek et al., 2001). Throughout the world, the assessment of wastewater discharges or efuents is focused on the precautionary principle, i.e., reduction of specic pollutants or substances in the framework of their emission policies (Kinnersley, 1990). For instance, in The Netherlands the governmental institutes have been using acute toxicity for the assessment of complex industrial efuents since 1995 (Beckers-Maessen, 1994). This is also in use in a more or less similar way in the United States (USEPA, 1991). Direct toxicity assessment has been in use in efuent discharge regulations for surface water in the United Kingdom since 1996 (NRA, 1993, 1994, 1995; Whitehouse and Dijk, 1996; Johnson et al., 1996). In Turkey the basic principles for water quality classes and standards governing the discharges in industrial wastewaters to inland water underlie the Water Pollution Control Regulation passed in 1988 and published on 4 September 1988 in the Ofcial Gazette. The receiving water discharge standards given in this regulation consist of chemical and biochemical parameters such as biochemical oxygen demand BOD5 ; chemical oxygen demand (COD), NH4 -N, phenol, sulfur, Fe, Cr, oil, and grease for all industrial efuents. Only the sh toxicity test carried out with Lepistes sp. , based on the toxicity dilution factor (TDF), which indicates toxicity, was included in the Turkish Water Pollution and Control Regulation (Turkish Water Pollution Control Regulation, 1992). Other conventional standardized toxicity tests (for instance, bacteria, algae, Daphnia, protozoa) were not taken into consideration for industrial discharges in these practices. Although these Regulations were revised on 1 July 1999, none of the toxicity tests were incorporated into these

regulations. In other words, toxicity limitations are not yet part of Turkish Water Pollution Control Regulations. A wide variety of chlorinated compounds have been identied in the efuent of kraft mills ranging from simple organochlorine compounds to a high-molecularweight class of, chlorolignins (Dyer and Mignone, 1983; Ferguson, 1994).The chlorinated organics present in efuent from kraft mills employing either chlorine or chlorine dioxide in the bleaching process have become a matter of concern due to their recalcitrance to biological degradation, toxicity to aquatic species, genotoxicity, and potential to accumulate in a variety of organics (Fracasso et al., 1992; Tirsch, 1989; Volskay and Grady, 1988). The environmental effect of halogenated organic pollutants has long been the subject of vigorous legislative control and research. They are not only poorly destroyed by conventional biological treatment but also reduce the effectiveness of the process. Removal of absorbable organic halogens (AOXs) has been reported as 3035% in aerobic treatment systems and 4045% in anaerobic treatment systems. An approximately 65% reduction in chlorinated phenolic compounds was observed in anaerobic download lters under steady-state conditions (Ferguson, 1994; Haggblom and Salkinoja-Solonen, 1991). Chlorine bleachery efuents are considered toxic to the environment owing their highly structured formation under chlorinated phenolic contents. By partial oxidation of lignin some soluble aromatic derivatives such as 1,2-dihydrobenzene were identied as by-products of toxic phenolic compounds and are said to be responsible for the mutagenic activity found in efuents from pulp bleaching plants (Solomon et al., 1996). In a study performed by Zaror et al. (2001), mutagenic effects of efuent samples containing 1,2-dihydrobenzene on Salmonella typhimurium TA 100 strain were detected. Mutagenity ratios ranging between 1.9 and 2.8 were calculated for in these efuents (Boncz et al., 1993). A large amount of refractory chemical oxygen demand (COD) is caused by high-molecular-weight synthetic bleaching agents and dyes. In some cases, dyes contain high levels of AOX and heavy metal concentrations due to chlorinated bleaching agents and halogen compounds (Minke and Rott, 1998). Both sh quality and quality of sh esh are impaired in water bodies into which chlorinated pulping efuents are discharged (Redenbach, 1997). Efuents containing chlorophenols and related compounds are particularly problematic due to their persistence in the environment and their high solubility in fat. Once introduced into water ecosystems, accumulation within river sediments and bioaccumulation within the tissues of organisms have been observed (Dyer and Mignone, 1983; Ferguson, 1994). Exposure to bleached kraft pulp mill efuents can cause various disorders in sh, including sublethal chronic responses

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and reduced tolerance to environmental factors (Dube and Culp, 1997; Demirbas et al., 1999). Regulatory action is now being taken worldwide to limit chlorine discharges, expressed as AOX, to less than 1:5 kg ton1 of bleached kraft pulp (Demirbas et al., 1999). Eighty percent of the organically bound chlorine reported is present in the high-molecular-weight fraction of total organic chlorine (Naru et al., 1993; Nirmalakhandan et al., 1993). Low-molecular-weight chlorolignins, in particular, are known to be toxic and mutagenic to bioaccumulate, and to penetrate through cell membranes (Kingstad and Lindstrom, 1984). Past research on pulp-paper efuents indicated that high-molecularweight compounds are not acutely toxic to biota. However, polar and high-molecular-weight constituents can be toxic to the marine animals (Robinson and Novak, 1994). Slow degradation and accumulation of high-molecular-weight chlorolignins may cause longterm environmental problems. These substances cannot be removed by conventional primary and secondary treatments (Sun et al., 1989, Demirbas et al., 1999). In their study Slabbert and Venter (1999) found that stream water containing the discharges of a paper mill industry that owed into a dam was very toxic. This toxicity seriously affected the protozoan Tetrahymena pyriformis (10% inhibition in enzyme activity), the alga Selenastrum capricomutum (53% mortality), the bacterium Esherichia coli (74% mortality), and embryos of the toad Bufo calamita (lethality 36%, deformation 54%) in this river. The aim of this study was to evaluate the toxicity of wastewater from the pulp-paper industry and emphasize the incorporation of acute toxicity parameters into Turkish Environmental Regulations to protect receiving ecosystems. As part of the monitoring program enrichment toxicity tests were performed by using bacteria to categorize toxic discharges. In addition, conventional toxicity tests were carried out to assess the toxicity of paper-mill industry efuent wastewater to four test organisms. Besides these tests, chemical analysis results and COD fractionation were compared with traditional and enrichment test results to try to uncover the reasons for wastewater toxicity.

Fig. 1. Schematic conguration of full-scale treatment plant in pulppaper industry.

were analyzed for chemical, biochemical, and toxicity parameters during the 10 months from March 1998 to January 1999. Protozoa (Vorticella sp.), algae (Chlorella sp.), sh (Lepistes sp. with a size of 5 cm), and bacteria (coliform and oc bacteria) were used to represent four different trophic levels for conventional investigation of the toxicity of efuent wastewater. Floc bacteria are responsible for the degradation of organic compounds in wastewater and for settling the sludge consisting of these bacteria in activated sludge and settling tank, respectively. In the absence of these bacteria the efciency of the biological treatment plant deteriorates. Therefore, to test the effect of efuents, oc bacteria were chosen as test organisms. Therefore, short-term denitive traditional acute and enrichment toxicity tests were performed, compared, and assessed. Furthermore, COD fractionation tests were performed to determine the effect of COD subcategories on toxicity of industrial wastewater. 2.1. Chemical and biochemical analysis BOD5 was measured as described in 5210 B by following Standard Methods (APHA/AWWA/WPCF, 1992). COD and phenol concentrations were determined on ltered samples by using Spectroquant Kits 014541 and 014551. The precision (random error) at the 95% condence level was 0.1 and 1 for two sets of paired samples. Color was measured in ltered samples as spectral absorption intensity with a Unicam spectrophotometer at 597 and 254 mm: pH was measured with a digital pH meter. AOX measurements were carried out in an AOX analyzer MT-20. The values reported in the gures are the averages of measurements for three samples. Readily biodegradable COD was determined with the method suggested by Ekama et al. (1986). Heterotrophic yield Y was evaluated by comparing oxygen utilization rate (OUR) and COD proles obtained on the

2. Materials and methods The pulp-paper industry treatment plant consists of a bar screening unit, an equalization tank, a primary settling tank, a biological treatment plant (conventional aerobic activated sludge system) following the chemical treatment, and a secondary settling tank. The treated chlorinated efuent is discharged into a bay (see Fig. 1). Samples were taken from the efuent of the secondary settling tank, which followed the biological activated sludge treatment plant in the pulp-paper industry, and

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samples at a food/biomass F =M ratio of 4:9 g COD/g volatile suspended solid (VSS). Inert soluble COD were determined according to the experimental procedure proposed by Germirli et al. (1991). The inert COD test involved two aerated batch reactors, of 3-L volumetric capacity: one fed with the unltered wastewater and diluted to have an initial COD concentration in the range of 15002000 mg=L; and the other fed with the ltered wastewater, having the same dilution. The microbial seed was obtained from the Pakmaya aerobic activated sludge treatment plant, operated under steady-state conditions with the same wastewater for 1 month in a laboratory-scale activated sludge reactor. The microbial seed was adjusted for an initial biomass concentration of 40 mg mixed liquor suspended solid (MLSS) in both reactors. Samples were drawn periodically from the mixed liquor and were analyzed for total and soluble COD. Experiments were continued until the observation of a stable COD level with no appreciable biomass activity. 2.2. Traditional acute toxicity tests These bioassays were performed to detect the relative toxicity of wastewater to selected organisms taken from efuents of treatment plants. In the determination of viable numbers of bacteria, protozoa, algae, and sh, the efuent wastewater samples were diluted and inoculated with the aforementioned microorganisms in a special medium containing all necessary substances for growth (APHA/AWWA/WPCF, 1992). The initial concentration of organisms was measured, then the number of organisms was monitored for 24 and 48 h of exposure, and the concentrations that affected 50% of the organisms tested in different volumes of efuent (EC50 values as w/v) were calculated. All tests were performed in triplicate. 2.3. Toxicity to bacteria, algae, protozoa, and sh As mentioned previously, since the numbers of oc and coliform bacteria are signicantly high and predominantly responsible for the organic degradation, two groups of bacteria were chosen to be used in the toxicity tests. The coliform bacteria were isolated from the efuent samples diluted with sterilized distilled water (between 1% and 100%) by membrane ltration on mEndo broth and incubated at 37:51C for 48 h as described in 922 B (APHA/AWWA/WPCF, 1992). Flocforming bacteria were also isolated and counted by membrane ltration. FF proteasepeptone yeast broth absorbent pads were used as medium and incubated at 271C for 3 days as described by Dugan and Lundgren (1968). For algal toxicity, the identication and enumeration of Chlorella sp. were done under a microscope on

ltered and immersion coated membranes following 3 days of incubation at 211C in algal medium-absorbed membranes (Pelezar and Chan, 1972). Vorticella sp. was taken from the Zoology Department of Science Faculty for the determination of protozoan toxicity. This microorganism was inoculated into the efuent wastewater diluted with sterilized distilled water varying between 1% and 100% and incubated for 24 h at 211C: Motility or viable cells of Vorticella sp. were assessed for lack of toxicity (Pelezar and Chan, 1972). To determine sh toxicity, efuent wastewater taken from the treatment plants was diluted with sterilized distilled water in certain volume percentages (between 1% and 100%) and mortality of Lepistes sp. was monitored after 48 h of incubation at ambient temperature (APHA/AWWA/WPCF, 1992). 2.4. Assessment of acute toxicity test results Acute toxicity was assessed by recording the number of viable organisms in different dilution ratios carried out with sterilized distilled water depending on the volume percentage of efuent wastewater (Canton, 1991; Tonkes et al., 1999). All results are expressed as EC50 values, that is, the concentration that affected 50% of the organisms tested in different volumes of efuent. To indicate that the raw efuent was not diluted due to the absence of toxicity the percentage concentration of efuent wastewater was set at 100%. In an efuent diluted 10 times, the value is 10%; that is, the efuent is acutely toxic to 50% of the test organisms at a dilution of 10. The assessment ranges of these tests are as follows: EC50 (w/v) of efuent Acute toxicity evaluation wastewater (%) o1 110 1099 100 Acute toxicity Moderate acute toxicity Minor acute toxicity No acute toxicity

2.5. Toxicity dilution factor Toxicity dilution volume indicates the volume of wastewater that is diluted with dilution water. Toxic effects can be determined proportionally with the dilution volume in which the wastewater is diluted with a dilution liquid. Accordingly, the minimum dilution volume in which all shes remain alive is considered the TDF. In other words, TDF indicates the degree of wastewater dilutions at which the sh remain alive. In this experiment a 2-L aquarium with sufcient aeration and wastewater of different dilutions (with sterilized distilled water) and sh (Lepistes sp.) was used. The

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control aquarium was lled only with sterilized distilled water. At the end of 24 and 48 h; the dilution at which all shes remained alive (no mortality was observed) was accepted as the appropriate dilution (Turkish Environment Regulation, 1992). 2.6. Enrichment toxicity tests The enrichment test is based on the growth of Enterobacter aerogenes in a chemically dened minimal growth medium. The presence of a toxic agent or a growth-promoting substance will alter the 48-h population by decreasing or increasing it 20% or more when compared with the control. Depending on their concentration some unknown toxic chemicals cause mortality in microbial populations (APHA/AWWA/WPCF, 1992). Twenty-one-milliliter wastewater samples taken from efuents of the pulp-paper industry were added to asks B, C, D, and E containing different substrate media. Table 1 summarizes the volumes of carbon, nitrogen, and efuent samples in asks A, B, C, D, and E. Flask A was the control and contained sufcient amounts of nitrogen, carbon, and phosphorus sources and distilled water and no wastewater. All asks were also inoculated with 1 mL of pure E. aerogenes bacteria isolated from biological treatment plants of the industries. The total liquid volume of the asks were 30 mL: Appropriate dilutions were carried out in the E. aerogenes suspension to arrive at a specic density range between 30 and 100 viable cells per milliliter in every ask. Cell densities below this range result in ratios that are not consistent, while densities above 100 cells=mL result in decreased sensitivity to nutrients. All asks were incubated at room temperature for 1 week. At the end of the incubation period the number of E. aerogenes was counted by ltering from membrane lters. All tests were done in triplicate. The compositions of the sodium citrate, salt mixture, and phosphate buffer solutions that were used as growth media in enrichment tests were as follows (concentrations of constituents are given in parentheses as g/L for 1000 mL distilled water): Na3 C6 H5 O7 2H2 O (0.58) for

the sodium citrate solution; NH4 2 SO4 (1.2) for the ammonium sulfate solution; MgSO4 7H2 O (0.52), CaCl2 2H2 O; FeSO4 7H2 O (0.46), NaCl (5.0) for the salt mixture; and KH2 PO4 (2.70), KH2 PO4 2:70; MgSO4 2:8 for the phosphate buffer solution. pH was adjusted to 7:373 with 1 N NaOH in all solutions. 2.7. Assessment of enrichment toxicity test results The enrichment tests were assessed based on the ratio of E. aerogenes number in the asks (B) to that in the control ask (A) as summarized below (APHA/ AWWA/WPCF, 1992): 1. If the B/A ratio is 0.8 to 1.2, no toxic substances are present, but the conditions are growth limiting. 2. If the B/A ratio is less than 0.8, the water contains toxic substances. 3. The B/A ratio could go as high as 3.0 from 1 .2. For ratios C/A, D/A, and E/A a value in excess of 1.2 indicates the presence of available nitrogen or carbon sources, or both, for bacterial growth. In other words, growth-stimulating substances or conditions are present. 2.8. Statistical analysis Differences in sensitivity scores between microorganisms determined in conventional acute toxicity tests were examined by performing a nonparametric Kruskal Wallis test followed by a MannWhitney U test (Conover, 1971; Siegel, 1956). The KruskalWallis test was used to compare the sensitivities of toxicity responses between bacteria, algae, sh, and protozoa. The Mann Whitney U test was used to evaluate the relationship between paired microorganism groups. All results are reported at a signicance level of Pp0:10 (Zar, 1984). The statistical package used for analysis was SPSSWIN in Windows (Norussis, 1986). Multiple regression analysis between y and x variables was performed using the SPSSWIN in Windows. The multiple regression analysis was used to determine the correlations between x and y variables. The linear correlation was assessed

Table 1 Characterization of enrichment toxicity tests for control (A), nutrient- and carbon-sufcient (B), nitrogen- and carbon-decient (C), nitrogendecient (D), and carbon-decient (E) asks Medium Sodium citrate Ammonium sulfate Salt solution Phosphate buffer Pulp-paper industry efuent Distilled water Total volume Flask A (control) (mL) 2.5 2.5 2.5 1.5 21 30 Flask B (mL) 2.5 2.5 2.5 1.5 21 30 Flask C (mL) 2.5 1.5 21 5 30 Flask D (mL) 2.5 2.5 1.5 21 2.5 30 Flask E (mL) 2.5 2.5 1.5 21 2.5 30

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with r2 : r2 is the correlation coefcient and reects statistical signicance between dependent and independent variables.

3. Results The quality of aquatic media such as rivers, bays, and inner seas have been determined largely by using a few chemical determinants namely BOD5 ; COD, color, and AOX coupled with the results of biological toxicity assays. It is important to determine and characterize the wastewater discharge entering these ecosystem from industrial efuents. For this purpose receiving medium discharge standards contain some parameters such as COD, BOD5 ; pH, and total suspended solids (TSSs) for pulp-paper efuents according to Turkish wastewater regulations (Turkish Water Pollution Control Regulation, 1998). Table 2 summarizes the Turkish receiving

water discharge standards for pulp-paper industry efuents. As can be seen from this table the COD standards for paper-mill industries vary between 300 and 800 mg=L: Some parameters such as color, AOX, and phenol have not been taken into consideration as discharge standards for paper-mill industry efuent in this regulation. The regulations should cover these parameters due to the carcinogenic and toxic effects of AOX, phenol, and dye. 3.1. Biochemical characterization of pulp-paper industry efuents The variations in BOD5 ; COD, and COD=BOD5 in samples of pulp-paper industry efuents are depicted in Fig. 2. The measured phenol levels, color, AOX concentrations, and TDF levels are illustrated in Fig. 3. Water quality analysis results revealed that the efuents sampled were violating the discharge limits on Days 1, 10, 80, 90, 100, 110, 170, 180, and 190 for COD, BOD5 ; and TDF parameters in the pulp-paper industry. In the efuent samples, COD concentrations ranged from 400 to 1400 mg=L and exceeded the discharge standards. This could be attributed to toxic substances in the biological treatment plant that caused bacteria to die, resulting in low COD removal efciency. From the COD and BOD5 ; the COD=BOD5 ratio for each of the efuent samples was calculated for the sampling period. Higher COD=BOD5 ratios reect less biodegradability of organic substances. Signicantly, many of the efuent samples also exhibited low BOD values giving rise to COD=BOD5 ratios of 3 and 5 on Days 1,10, 80, 90, 100, 170, 180, and 190. The efuents contained considerable amounts of low or nonbiodegradable COD and compounds on the days mentioned above. Low biodegradability of some organics, high phenol content, and high color levels and other unknown pollutants in wastewater increase COD
7 6 COD/BOD5 ratio 5 4 3 2 1 0

Table 2 Receiving water discharge standards in pulp-paper mill industrya Parameter COD (mg/L) BOD5 (mg/L) TSS (mg/L) pH Cr6 (mg/L) Total Cr (mg/L) TDF NH4 -N (mg/L) S2 (mg/L) Oil and grease (mg/L) Phenol (mg/L) Free chlorine (mg/L) Total hydrocarbon (mg/L)
a b

Paper millb 800 300 50 69 Not 2 8 Not Not Not Not Not Not

taken into consideration

taken taken taken taken taken taken

into into into into into into

consideration consideration consideration consideration consideration consideration

Composite sample taken in 24 h: Cellulose and paper production.

1600 COD, BOD5 concentrations (mg/liter) 1400 1200 1000 800 600 400 200 0 1 30 60 90 Days 120 150 180

COD (mg/liter)

BOD5 (mg/liter)

COD/BOD5 ratio

Fig. 2. Variation of COD and BOD concentrations in pulp-paper industry efuents.

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Fig. 3. Variation of chemical parameters and TDF in pulp-paper industry efuents.

concentration in efuents. Throughout these experiments, high COD concentrations in the efuent were accompanied by high TDFs r2 0:94; P 0:005: Potential toxicity is a suitable description of these efuents. The same results were observed for phenol and the permissible levels were also exceeded. Color and AOX levels in the efuent samples were higher on Days 1, 10, 80, 90, 180, and 190 on which acute toxicity was observed (see Figs. 2 and 3). The multiple regression analysis between TDF (y dependent variable) and COD, phenol, AOX, and color (x independent variable) in the efuent samples revealed a very strong linear correlation (r2 0:94; DurbinWatson statistic 1:99; P 0:005). 3.2. Toxicity tests Toxicity analyses were conducted in parallel including conventional and enrichment tests from March 1998 until January 1999 in the pupl-paper industry. During this period algae, bacteria, protozoa, and sh were used as test organisms representing four different trophic levels. Table 3 summarizes all acute toxicity and enrichment test results obtained during the 10-month sampling period for pulp-paper industry efuents. Throughout these analyses no signicant difference in activity was observed among the three organisms, except for protozoa. All acute toxicity tests except those with protozoa yielded positive results. The highest sensitivities were exhibited by bacteria, algae, and sh. It was found that protozoa were not sensitive. Denitive acute tests in the pulp-paper industry indicated that the EC50 (%) values of the efuents were mostly 100 for protozoa. Similar results were obtained by Slabbert and Venter (1999).

The EC50 values obtained from traditional toxicity tests were compared with B/A values obtained from enrichment toxicity tests. It was observed that the EC50 values obtained from conventional toxicity tests were in accordance with B/A ratios (the B/A ratio denes the number of bacteria grown on wastewater to that grown in control water) calculated in enrichment toxicity tests. In other words, all the results obtained from the acute denitive toxicity tests were conrmed by the results obtained from the enrichment tests in which E. aerogenes was used instead of coliform or oc bacteria, algae, sh, and protozoa. Toxicity tests performed on the efuents of this industry revealed potential toxicity depending on the chemical composition of the wastewater. This result could be attributed to phenol, color, COD, AOX, and other unknown pollutants in efuents that exceeded the discharge limits. The general classication of all efuents based on both traditional and enrichment tests can be summarized as follows: six efuents are acutely toxic, three efuents are moderately toxic, four efuents are slightly acutely toxic, and seven efuents are not acutely toxic in the pulp-paper industry. Moderate acute toxicity and minor acute toxicity were observed on certain days depending on AOX, color, and phenol concentrations and COD=BOD5 ratios (see Figs. 2 and 3 and Table 3).

3.3. Sensitivity of toxicity tests To compare the toxicity tests, a sensitivity index was constructed by taking into consideration only the results of traditional acute toxicity tests for each organism used. A score of 1 was assigned to the most sensitive test for each sample and 5 to the least

D.T. Sponza / Ecotoxicology and Environmental Safety 54 (2003) 7486 Table 3 Toxicity test results for pulp-paper industry efuents: EC50 valuesa Days EC50 value (% w/v) Coliforms 1 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 0.6 0.5 40 40 39 12 99 100 0.5 0.3 0.3 6 89 98 69 98 100 0.40 0.14 67 Floc bacteria 0.17 0.19 88 63 58 9 88 100 0.2 0.05 0.3 4 70 97 89 99 100 0.12 0.6 27 Fish 1.0 1.0 90 79 40 22 77 100 0.7 0.1 0.05 3 100 86 100 90 99 0.8 0.7 18 Algae 0.19 0.22 98 68 56 34 100 100 0.5 0.42 23 8 100 95 34 99 70 0.5 0.12 19 Protozoa 12 100 100 100 87 87 89 100 87 0.56 85 100 89 100 45 100 75 90 100 99 Enrichment test results B/A 0.26 0.19 2.34 1.8 1.24 1.04 1.98 2.01 0.3 0.4 0.45 0.9 2.19 2.65 0.88 2.58 1.97 0.40 0.3 0.88 Potential toxicity Potential toxicity Growth stimulation Growth-limiting nutrients Growth-limiting nutrients Growth-limiting nutrients Growth stimulation Growth stimulation Potential toxicity Potential toxicity Potential toxicity Growth-limiting nutrients Growth stimulation Growth stimulation Growth-limiting nutrients Growth stimulation Growth stimulation Potential toxicity Growth-limiting nutrients Growth-limiting nutrients Conventional test results General results

81

Acute toxicity Acute toxicity No acute toxicity Moderate toxicity Minor acute toxicity Moderate toxicity No acute toxicity No acute toxicity Acute toxicity Acute toxicity Acute toxicity Moderate toxicity No acute toxicity No acute toxicity Minor acute toxicity No acute toxicity No acute toxicity Acute toxicity Minor acute toxicity Minor acute toxicity

Note. Comparison of enrichment and conventional toxicity test results give an idea of the toxicity level on sampling days in pulp-paper industry efuents and are in accordance with the determination of toxicity subcategories. a Values are means. n 3:

sensitive. Table 4 summarizes the sensitivity assessment for every organism used in acute toxicity tests. KruskalWallis test statistics revealed that protozoa had higher EC50 values and sensitivity scores than the other groups of microbes. Coliform and oc bacteria in pulp-paper industry efuents had similar toxicity responses and mortality. The EC50 values measured for oc bacteria was higher than those for protozoa and this difference was signicant. Results of statistical analysis and the signicance of the relationships between sensitivity scores of all organisms used in the toxicity tests are summarized in Table 5. According to the statistical analysis results, in a sample comparison of sensitivity among all trophic levels, the protozoan test seemed to be less sensitive than bacterial, algal, and sh tests in both industries. In other words, protozoan (Vorticella sp.) were found to be very resistant. The toxicity data indicate that the coliform and oc bacteria and sh tests were the most sensitive overall, although they were not so sensitive on some days. For instance, it was observed that the coliform test was not so sensitive on Days 90 and 180 in the leather industry and on Days 160 and 190 in the textile industry. Figure 4 illustrates the variation in sensitivity score with the type of toxicity through 190 days of sampling. The data in Table 3 indicate that with the exception of Days 1, 50, 80, and 90, all test organisms did not exhibited toxicity simultaneously to the efuent samples.

Table 4 Toxicity test results for pulp-paper industry efuents: sensitivitya Days Assessment of sensitivity Coliforms 1 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 Sum 4 3 1 1 1 2 4 1 2 3 2 3 2 4 2 2 4 2 2 4 49 Floc bacteria 1 1 3 2 4 1 2 1 1 1 2 2 1 3 3 3 4 1 3 3 42 Fish 3 4 2 4 2 3 1 1 3 2 1 1 3 1 5 1 3 4 4 1 49 Algae 2 2 4 3 3 4 5 1 2 4 3 4 3 2 1 3 1 3 1 2 53 Protozoa 5 5 5 5 5 5 3 1 4 5 4 5 2 5 4 4 2 5 5 5 83

Note. The toxicity test results indicated that the bacteria are the most sensitive and the bacteria are the most resistant microorganisms for pulp-paper industry efuents. a Values are means. n 3:

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Table 5 Comparison of organisms used in conventional toxicity tests by MannWhitney U test Coliforms Coliforms Floc bacteria N.S MW-UT 0:345 P 0:10 Algae N.S MW-UT 0:194 P 0:10 N.S MW-UT 0:572 Pp0:10 Protozoa S MW-UT 8:540 Po0:10 S MW-UT 10:654 Pp0:05 S MW-UT 9:863 Pp0:05 Fish N.S MW-UT 0:975 P 0:10 N.S MW-UT 0:662 Pp0:10 N.S MW-UT 0:401 Pp0:10 N.S MW-UT 0:508 Pp0:05

Floc bacteria

Algae

Protozoa

Note. Bacteria, algae, and protozoa have similar toxicity response to pulp-paper industry efuents, while protozoa have higher sensitivity scores. MW-UT, MannWhitney U-test statistic; S, sensitive; NS, not sensitive.

6 5 Sensitivity scores of organisms 4 3 2 1 0

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190

3 2.5 2 1.5 1 0.5 0 Days Coliform bacteria Protozoan Not acutely toxic Floc bacteria Fish Minor acutely toxic Algae Acutely toxic Moderately toxic Variation of toxicity

Fig. 4. Relationships between sensitivity scores and toxicity level in all organisms used for toxicity tests through 190 days of sampling.

Overall toxicity for the aforementioned days was to be expected for the efuent samples associated with high COD, AOX, phenol, and color (see Figs. 2 and 3). Fig. 4 illustrates the relationship between the sensitivity score of all the organisms and the variation of toxicity based on conventional test results. The toxicity scores of microorganisms increased when toxicity was not observed. Contrarily, decreases in sensitivity scores were determined while toxicity was not shown. 3.4. Sensitivity ranking in the pulp-paper industry To explain the sensitivity of biotest results based on toxicity, a table was constructed ranking the order of toxicity. Organisms representing four different trophic levels were classied according to traditional test results. Toxicity response and sensitivity ranking are assessed in

Table 6. The acute toxicity classication of an efuent should always be based on the results of testing all trophic levels at least once. For this reason, two groups of bacteria (decomposers), algae (primary producers), protozoa (primary consumers), and sh (secondary consumers), which represent four different trophic levels, were classied in terms of sensitivity. The overall results are summarized in Table 6 for coliform and oc bacteria, algae, protozoa and sh. To only ve efuents no toxicity response was elicited in coliform bacteria. Of the remaining 15 efuents, the potential acute toxicity to at least one or two of the ve trophic levels could be totally explained by using physical and chemical information. In most cases, this was attributed to COD, phenol, color, and AOX concentrations as mentioned earlier for the pulp-paper industry (see Figs. 2 and 3). Only in the cases of efuents

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on Days 30 and 40 could this not be explained solely on the basis of the data on discharges made available in this industry. Of all 14 efuents eliciting acute toxicity, the toxicity to oc bacteria could be explained on the basis of the chemical and toxicity data. Of all 20 efuents, 11 efuents elicited acute toxicity in algae, 10 efuents were toxic to sh, and 17 efuents were toxic to protozoa (see Fig. 2). As only one trophic level (bacteria) was used in the enrichment tests, sensitivity was not assessed. The results of this research indicate that discharges from the treatment plants display different toxicity response from day to day. It can be concluded that the acute toxicity and enrichment tests produced valuable and additional information on the toxic characteristics of discharges when compared with chemical analysis alone. In particular, the data obtained from the enrichment toxicity tests provided information on nutrient deciencies and growth-stimulating conditions besides potential toxicity. It has become clear that the chemical-specic approach produces enough information for only a limited number of complex efuent samples. It is important to note that the precise chemistry of efuents is not known very well except for COD, AOX, phenol, and color analysis. Therefore, it is not possible to speculate on the mode of the toxic action due to the difculties involved in the detailed characterization of

the chemical content of wastewater. However, all efuents that are studied for the presence of acute toxicity should be characterized as thoroughly as possible by physical and chemical analysis. This is needed to gain more insight into the possible causes of toxicity, and should always be performed using the same efuent sample as that used for toxicity testing. 3.5. Relationships between COD subcategories and toxicity For industrial wastewaters with low COD=BOD5 ratios, the following comments are relevant: The wastewater contains very slowly biodegradable organics, the majority of organic matter is nonbiodegradable (refractory), and the wastewater contains some inhibitory compounds such as heavy metals and toxic organics that decrease the efciency of biological treatment in the treatment plant and in the receiving aquatic ecosystems when discharged. Biological treatment in the plant may have great impact in the case when inhibitors are identied. To determine the assumptions given above the COD subcategories should be researched. As mentioned above, COD fractionation analysis was done to determine the effect of COD subcategories such as soluble inert COD, soluble slowly biodegradable COD, and soluble readily biodegradable COD on toxicity test results for efuents. Table 7 summarizes the COD fractionation data on different days. The toxicity can be explained by the low level of soluble inert COD and the large amount of soluble slowly biodegradable COD on Days 20 and 150. As can be seen in Table 7, the slowly hydrolyzable COD caused the discharge limits to be violated, but did not cause toxicity, indicating the organics that should be hydrolyzed in pulp-paper efuents.

Table 6 Organism Sensitivity rank Very toxic Coliforms Floc bacteria Algae Fish Protozoa 6 6 5 4 2 Moderately toxic 3 1 3 3 1 Slightly toxic 4 4 3 3 1 Not toxic 5 9 9 10 16

Note. Sensitivity ranking indicated that the pulp-paper industry discharges exhibited toxicity on some days. Fifteen efuents were toxic to coliforms, 11 efuents were toxic to oc bacteria, 11 efuents were toxic to algae, 10 efuents were toxic to sh, and 4 efuents were toxic to protozoa. Table 7 COD fractionation results for pulp-paper industry efuents Total COD (mg/L) Soluble COD (mg/L) Soluble inert microbial product COD (mg/L) 20 33

4. Discussion Some of the substances used for bleaching have high COD concentrations and can affect toxicity even though they may be degraded via an efuent treatment process.

Soluble inert COD (mg/L)

Readily hydrolyzable COD (mg/L) 60 232

Slowly hydrolyzable COD (mg/L) 380 360

Sampling days/toxicity

Pulp

880 890

550 650

90 25

10 Yes 150 No

Note. Although discharge limits exceeded the standards, toxicity was not determined on Days 20 and 150. This result may be explained by the low level of inert soluble COD and large amount of soluble slowly biodegradable COD, which violated the discharge limits did not cause toxicity, indicating the organics that should be hydrolyzed in the pulp-paper efuents. On the contrary, while the COD concentrations did not exceed discharge limits, toxicity was determined due to the high level of inert COD in wastewater.

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Low biodegradability gives rise to increased COD concentration in efuents and increased pressure on pulp-paper companies to use ecochemicals in processing. As mentioned in the introduction, within the paper-mill industry a broad range of chemicals such as chlorophenols and aromatic derivatives that vary in toxicity are in use. However, limited studies have been undertaken on the toxicity of the discharges of this industry using conventional and rapid toxicity tests (Hayes et al., 1999). Similarly, little work has been carried out on bleaching and kraft-mill products (including chlorine, aromatic derivatives, phenols, and dyes) using conventional toxicity tests in Turkey. A severe problem with the decolorization of dye-containing bleaching process discharges is that this would pose a serious hazard if released to the aquatic environment untreated. On the other hand, AOX have been known to violate discharge standards because of the high concentration of the organic binder chlorine in pulp-paper efuents (Dyer and Mignone, 1983; Ferguson, 1994). The biodegradability of an organic substance is a measure of the speed and completeness of its biodegradation by microorganisms. If the BOD5 =COD ratio is between 0.1 and 0.5, the substance is slightly less difcult to degrade (Landner, 1994). This indicates the presence of potential toxicity in the case of direct discharge into the receiving medium. In other words, if COD=BOD5 ratios vary between 5 and 9, the wastewater is toxic and the biodegradability is reduced as reported by Tchobanoglous and Burton (1991). Although in some cases the efuent wastewater quality was ensured, the toxicity test results indicates potential toxicity. This indicates that toxicity tests should be incorporated into discharge standard regulations. For instance although COD levels, phenol concentrations, and TDF values were lower than permissible discharge limits in the pulp-paper industry on some days, the toxicity test results demonstrated the presence of acute and minor acute toxicity. This is already a difcult task for conventional Turkish standards in terms of acceptability and applicability. It is to be concluded that the reasons for this are a lack of knowledge of the composition of, and a lack of acute toxicity data on, the substances that are known to be present. The impact of industrial efuents on river systems has been noticeable for at least the last 20 years. Water quality problems have arisen from toxic organic and inorganic pollutants which originated from a lack of processing and the mode of operation of the treatment plant. It should, however, be pointed out that the bacterial, algal, and sh tests are the most sensitive tests for determination of toxicity in pulp-paper efuents. These tests are reference standards used worldwide for toxicity testing and represent one of the trophic level tests

required in toxicity evaluation. Fish, algal, and bacterial growth inhibition tests have been found to be the most suitable for regulatory and management purposes in paper-mill and metal plating efuents (Slabbert and Venter, 1999). From Table 4, it would appear that the sh and algal tests would be a potential surrogate since their overall sensitivity is only slightly lower than that of the bacterial test. However, both traditional and enrichment tests are affected by the presence of color, COD, AOX, and phenol, in paper-mill industry efuent samples. For the test in protozoa, which displayed resistance, greater sensitivity would be obtained by using coliforms or oc bacteria, sh, and algae, which are more appropriate sensitive cultures for pulp-paper industry efuents. COD fractionation should be regarded as the required complement to the total COD parameter within conventional characterization. Since conventional characterization of pollution (total COD concentration) alone cannot provide information on the biological biodegradability or inert percentage of wastewater, COD fractionation should be performed. It has also been reported that conventional pollution characterization (in terms of COD) alone is not adequate in providing knowledge about COD fractions. COD fractionation provides information on the biodegradability of a substrate or inert fraction of COD (Orhon et al., 1998). Since treatment plants are not controlled by the depletion of readily biodegradable organics, slowly biodegradable COD is the major role-limiting component for heterotrophic microorganisms in biological treatment plants. Furthermore, inert COD, which could contain inhibitory and toxic substances, should be taken into account (Germirli et al., 1991).

5. Conclusions Studies on wastewater efuents indicated that all toxicity tests have a viable role to play in water quality monitoring and control in rural areas. The studies demonstrated that there is no single method that can constitute a comprehensive approach to aquatic life protection. For this reason, toxicity tests containing sensitive microorganisms should be applied in battery form so those tests can complement each other and chemical analysis. Authorities from environmental agencies will propose the application of direct acute toxicity assessment for assessing the impact of discharges into receiving waters. According to the results of this study, the bacterial, sh, and algal toxicity tests could be developed as promising techniques for control of industrial wastewater discharges to receiving aquatic ecosystems. Furthermore, results of this study indicate that enrichment toxicity tests are practical, cost-effective, and accurate in the determination of potential

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effects. The acute toxicity classication of an efuent should always be based on the results of testing at least once at trophic levels. All efuents that are studied for the presence of acute toxicity should be characterized as thoroughly as possible by physical and chemical analysis. A methodology for wastewater characterization employing a single substance or a specic chemical substance cannot yield convenient results for determination of the wastewater toxicity. AOX, phenol, and inert COD originating from pulppaper processing discharges to receiving media should be decreased by the application of clever technology in the treatment plant, thus preventing toxicity in the receiving media. Despite the overall decrease in COD concentrations, AOX and inert COD originating from dyestuffs and nonbiodegradable organics are still at the highest levels. For environmentally signicant discharges of complex organics where not all important constituents can be individually identied and numerically limited, a clearly dened toxicity limit should be specied, along with the appropriate form of toxicity test to be used, and the minimum frequency with which it should be applied. Toxic efuents emitted from wastewater treatment plant installations should be strongly controlled. Although the COD concentrations exceeded discharge limits, toxicity was not determined on some days in pulp-paper industry efuents. This can be explained by the large amount of readily biodegradable COD and low level of inert COD in wastewaters. Although the discharge limits were not exceeded, toxicity was determined for the aforementioned industry efuents. This could be attributed to the large amount of slowly biodegradable or inert COD in efuent wastewater. Toxicity tests should be incorporated into receiving water discharge standards and the existing receiving water discharge standards should be reviewed to include toxicity tests. The authorities should take measures to reduce urgently and drastically the total quantity of dangerous toxic substances before they reach the aquatic environment.

Acknowledgments The authors thank the Dokuz Eyl. University Reul search Fund for Financial Support (Grant 092298.01.34).

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