application of toxicity tests into discharges of the pulp-paper

8/4/2019 Application of Toxicity Tests Into Discharges of the Pulp-paper

1/13

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 (floc and coliform bacteria), algae (Chlorella sp.), protozoa (Vorticella sp.), and fish (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 effluent 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 effluents 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

effluents.

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 waste-

water can influence the operational efficiency of existing

wastewater treatment facilities and cause them not to

meet the more stringent effluent standards. The conven-

tional approach to controlling harmful chemicals in the

aquatic environment is to use a set of global physical

chemical and biochemical parameters. Chemical proce-

dure alone cannot provide sufficient information on the

potential harmful effects of chemicals on the aquatic

environment (Vyryan et al., 1999). The complex nature

of effluents cannot be overcome by specific chemical

approaches (USEPA, 1981, 1996). The toxic effects of

unknown and often undetermined substances in com-

plex mixtures or with possible synergistic effects among

compounds to wastewaters can be detected only by

toxicity testing. In addition, although in some cases the

effluent 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 effluents 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 effluent 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

field involving numerous bioanalytical techniques devel-

oped and applied to organisms at different trophic levels*Corresponding author. Fax: +90-232-453-1153.

E-mail address: [email protected] (D.T. Sponza).

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


2/13

(Slabbert, 1996). Since living material responds to the

total effect of actual and potential disruptions, biologi-

cal assays have become important tools in assessing

harmful chemical activity (Blaise et al., 1988). Envir-

onmentally relevant biotests provide information on

the initial levels of damage and assist in developing

precautionary measures and strategies for environmen-tal 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 contain-

ing bacteria could be valuable screening tools for

identifying and categorizing toxic effluents 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 effluents is focused on the precautionary

principle, i.e., reduction of specific 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 effluents 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

effluent 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 waste-

waters to inland water underlie the Water Pollution

Control Regulation passed in 1988 and published on 4

September 1988 in the Official 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 effluents. Only the

fish 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 standar-

dized 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

identified in the effluent of kraft mills ranging from

simple organochlorine compounds to a high-molecular-

weight class of, chlorolignins (Dyer and Mignone, 1983;Ferguson, 1994).The chlorinated organics present in

effluent 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 approxi-

mately 65% reduction in chlorinated phenolic com-

pounds was observed in anaerobic download filters

under steady-state conditions (Ferguson, 1994; Hagg-

blom and Salkinoja-Solonen, 1991). Chlorine bleachery

effluents 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 identified as by-products of toxic phenolic com-

pounds and are said to be responsible for the mutagenicactivity found in effluents from pulp bleaching plants

(Solomon et al., 1996). In a study performed by Zaror

et al. (2001), mutagenic effects of effluent samples

containing 1,2-dihydrobenzene on Salmonella typhimur-

ium TA 100 strain were detected. Mutagenity ratios

ranging between 1.9 and 2.8 were calculated for in these

effluents (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 fish quality and quality of fish flesh

are impaired in water bodies into which chlorinated

pulping effluents are discharged (Redenbach, 1997).

Effluents containing chlorophenols and related com-

pounds are particularly problematic due to their

persistence in the environment and their high solubility

in fat. Once introduced into water ecosystems, accumu-

lation 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 effluents can cause various

disorders in fish, including sublethal chronic responses

D.T. Sponza / Ecotoxicology and Environmental Safety 54 (2003) 7486 75


3/13

and reduced tolerance to environmental factors (Dube

and Culp, 1997; Demirba-s 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 (Demirba-s et al., 1999). Eighty

percent of the organically bound chlorine reported is

present in the high-molecular-weight fraction of totalorganic 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 mem-

branes (Kingstad and Lindstrom, 1984). Past research

on pulp-paper effluents indicated that high-molecular-

weight 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 long-

term environmental problems. These substances cannot

be removed by conventional primary and secondary

treatments (Sun et al., 1989, Demirba-s et al., 1999). In

their study Slabbert and Venter (1999) found that

stream water containing the discharges of a paper mill

industry that flowed 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 bacter-

ium 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 emphasizethe incorporation of acute toxicity parameters into

Turkish Environmental Regulations to protect receiving

ecosystems. As part of the monitoring program enrich-

ment 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 effluent 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.

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 effluent is discharged into a bay (see Fig. 1).

Samples were taken from the effluent of the secondary

settling tank, which followed the biological activated

sludge treatment plant in the pulp-paper industry, and

were analyzed for chemical, biochemical, and toxicity

parameters during the 10 months from March 1998 to

January 1999. Protozoa (Vorticella sp.), algae (Chlorella

sp.), fish (Lepistes sp. with a size of 5 cm), and bacteria

(coliform and floc bacteria) were used to represent four

different trophic levels for conventional investigation of

the toxicity of effluent 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

efficiency of the biological treatment plant deteriorates.

Therefore, to test the effect of effluents, floc bacteria

were chosen as test organisms. Therefore, short-term

definitive traditional acute and enrichment toxicity testswere 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 filtered samples by using Spectroquant Kits 014541

and 014551. The precision (random error) at the 95%

confidence level was 0.1 and 1 for two sets of paired

samples. Color was measured in filtered samples as

spectral absorption intensity with a Unicam spectro-

photometer at 597 and 254 mm: pH was measured witha digital pH meter. AOX measurements were carried out

in an AOX analyzer MT-20. The values reported in the

figures 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 utiliza-tion rate (OUR) and COD profiles obtained on the

Fig. 1. Schematic configuration of full-scale treatment plant in pulp-

paper industry.

D.T. Sponza / Ecotoxicology and Environmental Safety 54 (2003) 748676


4/13

samples at a food/biomass F=M ratio of 4:9 g COD/gvolatile 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

unfiltered wastewater and diluted to have an initialCOD concentration in the range of 15002000 mg=L;and the other fed with the filtered 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. Experi-

ments 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

effluents of treatment plants. In the determination of

viable numbers of bacteria, protozoa, algae, and fish,

the effluent 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 thenumber 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 effluent

(EC50 values as w/v) were calculated. All tests were

performed in triplicate.

2.3. Toxicity to bacteria, algae, protozoa, and fish

As mentioned previously, since the numbers of floc

and coliform bacteria are significantly high and pre-

dominantly 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

effluent samples diluted with sterilized distilled water

(between 1% and 100%) by membrane filtration on

mEndo broth and incubated at 37:51C for 48 h asdescribed in 922 B (APHA/AWWA/WPCF, 1992). Floc-

forming bacteria were also isolated and counted by

membrane filtration. 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 identification and enumeration

of Chlorella sp. were done under a microscope on

filtered 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 Depart-

ment of Science Faculty for the determination of

protozoan toxicity. This microorganism was inoculated

into the effluent wastewater diluted with sterilizeddistilled water varying between 1% and 100% and

incubated for 24 h at 211C: Motility or viable cells ofVorticella sp. were assessed for lack of toxicity (Pelezar

and Chan, 1972).

To determine fish toxicity, effluent 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 tempera-

ture (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 effluent 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 effluent.

To indicate that the raw effluent was not diluted due to

the absence of toxicity the percentage concentration of

effluent wastewater was set at 100%. In an effluent

diluted 10 times, the value is 10%; that is, the effluent is

acutely toxic to 50% of the test organisms at a dilutionof 10. The assessment ranges of these tests are as

follows:

EC50 (w/v) of effluent Acute toxicity evaluation

wastewater (%)

o1 Acute toxicity

110 Moderate acute toxicity

1099 Minor acute toxicity

100 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 fishes remain alive is considered the

TDF. In other words, TDF indicates the degree of

wastewater dilutions at which the fish remain alive. In

this experiment a 2-L aquarium with sufficient aeration

and wastewater of different dilutions (with sterilized

distilled water) and fish (Lepistes sp.) was used. The



5/13

control aquarium was filled only with sterilized distilled

water. At the end of 24 and 48 h; the dilution at whichall fishes remained alive (no mortality was observed) was

accepted as the appropriate dilution (Turkish Environ-

ment Regulation, 1992).

2.6. Enrichment toxicity tests

The enrichment test is based on the growth of

Enterobacter aerogenes in a chemically defined minimal

growth medium. The presence of a toxic agent or a

growth-promoting substance will alter the 48-h popula-

tion by decreasing or increasing it 20% or more when

compared with the control. Depending on their con-

centration some unknown toxic chemicals cause mor-

tality in microbial populations (APHA/AWWA/WPCF,

1992). Twenty-one-milliliter wastewater samples taken

from effluents of the pulp-paper industry were added to

flasks B, C, D, and E containing different substratemedia. Table 1 summarizes the volumes of carbon,

nitrogen, and effluent samples in flasks A, B, C, D, and

E. Flask A was the control and contained sufficient

amounts of nitrogen, carbon, and phosphorus sources

and distilled water and no wastewater. All flasks were

also inoculated with 1 mL of pure E. aerogenes bacteria

isolated from biological treatment plants of the indus-

tries. The total liquid volume of the flasks were 30 mL :Appropriate dilutions were carried out in the E.

aerogenes suspension to arrive at a specific density

range between 30 and 100 viable cells per milliliter in

every flask. 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 flasks were incubated at room temperature for 1

week. At the end of the incubation period the number of

E. aerogenes was counted by filtering from membrane

filters. 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 (concentra-

tions of constituents are given in parentheses as g/L for

1000 mL distilled water): Na3C6H5O7 2H2O (0.58) for

the sodium citrate solution; NH42SO4 (1.2) for theammonium sulfate solution; MgSO4 7H2O (0.52),CaCl2 2H2O; FeSO4 7H2O (0.46), NaCl (5.0) for thesalt mixture; and KH2PO4 (2.70), KH2PO42:70;MgSO42:8 for the phosphate buffer solution. pH wasadjusted 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 flasks (B) to that in the

control flask (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 1.2indicates 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 microorgan-

isms determined in conventional acute toxicity tests were

examined by performing a nonparametric Kruskal

Wallis test followed by a MannWhitney U test (Con-

over, 1971; Siegel, 1956). The KruskalWallis test was

used to compare the sensitivities of toxicity responses

between bacteria, algae, fish, and protozoa. The Mann

Whitney U test was used to evaluate the relationship

between paired microorganism groups. All results are

reported at a significance 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-sufficient (B), nitrogen- and carbon-deficient (C), nitrogen-

deficient (D), and carbon-deficient (E) flasks

Medium Flask A Flask B Flask C Flask D Flask E

(control) (mL) (mL) (mL) (mL) (mL)

Sodium citrate 2.5 2.5 2.5

Ammonium sulfate 2.5 2.5 2.5

Salt solution 2.5 2.5 2.5 2.5 2.5

Phosphate buffer 1.5 1.5 1.5 1.5 1.5

Pulp-paper industry effluent 21 21 21 21

Distilled water 21 5 2.5 2.5

Total volume 30 30 30 30 30



6/13

with r2: r2 is the correlation coefficient and reflectsstatistical significance between dependent and indepen-

dent 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, andAOX coupled with the results of biological toxicity

assays. It is important to determine and characterize the

wastewater discharge entering these ecosystem from

industrial effluents. For this purpose receiving medium

discharge standards contain some parameters such as

COD, BOD5; pH, and total suspended solids (TSSs) forpulp-paper effluents according to Turkish wastewater

regulations (Turkish Water Pollution Control Regula-

tion, 1998). Table 2 summarizes the Turkish receiving

water discharge standards for pulp-paper industry

effluents. 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 effluent in

this regulation. The regulations should cover theseparameters due to the carcinogenic and toxic effects of

AOX, phenol, and dye.

3.1. Biochemical characterization of pulp-paper industry

effluents

The variations in BOD5; COD, and COD=BOD5 insamples of pulp-paper industry effluents 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

effluents 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 effluent samples, COD concentrations ranged

from 400 to 1400 mg=L and exceeded the dischargestandards. This could be attributed to toxic substances

in the biological treatment plant that caused bacteria to

die, resulting in low COD removal efficiency. From the

COD and BOD5; the COD=BOD5 ratio for each of theeffluent samples was calculated for the sampling period.

Higher COD=BOD5 ratios reflect less biodegradabilityof organic substances. Significantly, many of the effluent

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 effluents contained considerable amounts of low

or nonbiodegradable COD and compounds on the days

mentioned above. Low biodegradability of some or-

ganics, high phenol content, and high color levels and

other unknown pollutants in wastewater increase COD

0

200

400

600

800

1000

1200

1400

1600

1 30 60 90 120 150 180

Days

COD,

BOD5concen

trations

(mg/liter)

0

1

2

3

4

5

6

7

COD/BOD5ra

tio

COD (mg/l iter) BOD5 (mg/li ter) COD/BOD5 ratio

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

Table 2

Receiving water discharge standards in pulp-paper mill industrya

Parameter Paper millb

COD (mg/L) 800

BOD5 (mg/L) 300

TSS (mg/L) 50

pH 69

Cr6 (mg/L) Not taken into consideration

Total Cr (mg/L) 2

TDF 8

NH4-N (mg/L) Not taken into consideration

S2 (mg/L) Not taken into consideration

Oil and grease (mg/L) Not taken into consideration

Phenol (mg/L) Not taken into consideration

Free chlorine (mg/L) Not taken into consideration

Total hydrocarbon (mg/L) Not taken into consideration

aComposite sample taken in 24 h:bCellulose and paper production.



7/13

concentration in effluents. Throughout these experi-

ments, high COD concentrations in the effluent were

accompanied by high TDFs r2 0:94; P 0:005:Potential toxicity is a suitable description of these

effluents. The same results were observed for phenol

and the permissible levels were also exceeded. Color and

AOX levels in the effluent 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

effluent 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 fish 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 effluents.

Throughout these analyses no significant 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 fish. It was found

that protozoa were not sensitive. Definitive acute tests in

the pulp-paper industry indicated that the EC50 (%)

values of the effluents 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 EC 50values obtained from conventional toxicity tests were in

accordance with B/A ratios (the B/A ratio defines 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

definitive toxicity tests were confirmed by the results

obtained from the enrichment tests in which E.

aerogenes was used instead of coliform or floc bacteria,

algae, fish, and protozoa.Toxicity tests performed on the effluents 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 effluents that exceeded the

discharge limits.

The general classification of all effluents based on

both traditional and enrichment tests can be summar-

ized as follows: six effluents are acutely toxic, three

effluents are moderately toxic, four effluents are slightly

acutely toxic, and seven effluents 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

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



8/13

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 theother groups of microbes. Coliform and floc bacteria in

pulp-paper industry effluents had similar toxicity

responses and mortality. The EC50 values measured

for floc bacteria was higher than those for protozoa and

this difference was significant. Results of statistical

analysis and the significance 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 fish tests in both industries. In other

words, protozoan (Vorticella sp.) were found to be very

resistant. The toxicity data indicate that the coliform and

floc bacteria and fish 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 effluent samples.

Table 3

Toxicity test results for pulp-paper industry effluents: EC50 valuesa

Days EC50 value (% w/v) Enrichment Conventional General

test results test results results

Coliforms Floc bacteria Fish Algae Protozoa B/A

1 0.6 0.17 1.0 0.19 12 0.26 Potential toxicity Acute toxicity10 0.5 0.19 1.0 0.22 100 0.19 Potential toxicity Acute toxicity

20 40 88 90 98 100 2.34 Growth stimulation No acute toxicity

30 40 63 79 68 100 1.8 Growth-limiting nutrients Moderate toxicity

40 39 58 40 56 87 1.24 Growth-limiting nutrients Minor acute toxicity




80 0.5 0.2 0.7 0.5 87 0.3 Potential toxicity Acute toxicity

90 0.3 0.05 0.1 0.42 0.56 0.4 Potential toxicity Acute toxicity

100 0.3 0.3 0.05 23 85 0.45 Potential toxicity Acute toxicity





150 98 99 90 99 100 2.58 Growth stimulation No acute toxicity160 100 100 99 70 75 1.97 Growth stimulation No acute toxicity

170 0.40 0.12 0.8 0.5 90 0.40 Potential toxicity Acute toxicity

180 0.14 0.6 0.7 0.12 100 0.3 Growth-limiting nutrients 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

effluents and are in accordance with the determination of toxicity subcategories.aValues are means. n 3:

Table 4

Toxicity test results for pulp-paper industry effluents: sensitivitya

Days Assessment of sensitivity

Coliforms Floc bacteria Fish Algae Protozoa

1 4 1 3 2 5

10 3 1 4 2 5

20 1 3 2 4 5

30 1 2 4 3 5

40 1 4 2 3 5

50 2 1 3 4 5

60 4 2 1 5 3

70 1 1 1 1 1

80 2 1 3 2 4

90 3 1 2 4 5

100 2 2 1 3 4

110 3 2 1 4 5

120 2 1 3 3 2130 4 3 1 2 5

140 2 3 5 1 4

150 2 3 1 3 4

160 4 4 3 1 2

170 2 1 4 3 5

180 2 3 4 1 5

190 4 3 1 2 5

Sum 49 42 49 53 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 effluents.aValues are means. n 3:



9/13

Overall toxicity for the aforementioned days was to be

expected for the effluent 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 ob-

served. 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 classified according to traditional test results.

Toxicity response and sensitivity ranking are assessed in

Table 6. The acute toxicity classification of an effluent

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 fish (secondary

consumers), which represent four different trophic

levels, were classified in terms of sensitivity. The overall

results are summarized in Table 6 for coliform and floc

bacteria, algae, protozoa and fish.

To only five effluents no toxicity response was elicited

in coliform bacteria. Of the remaining 15 effluents, the

potential acute toxicity to at least one or two of the five

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 effluents

Table 5

Comparison of organisms used in conventional toxicity tests by MannWhitney U test

Coliforms Floc bacteria Algae Protozoa Fish

Coliforms N.S N.S S N.S

MW-UT 0:345 MW-UT 0:194 MW-UT 8:540 MW-UT 0:975P 0:10 P 0:10 Po0:10 P 0:10

Floc bacteria N.S S N.SMW-UT 0:572 MW-UT 10:654 MW-UT 0:662Pp0:10 Pp0:05 Pp0:10

Algae S N.S

MW-UT 9:863 MW-UT 0:401Pp0:05 Pp0:10

Protozoa N.S

MW-UT 0:508Pp0:05

Note. Bacteria, algae, and protozoa have similar toxicity response to pulp-paper industry effluents, while protozoa have higher sensitivity scores.

MW-UT, MannWhitney U-test statistic; S, sensitive; NS, not sensitive.

0

1

2

3

4

5

6

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

Days

Sensitivityscoresof

organisms

0

0.5

1

1.5

2

2.5

3

Variationoftoxicity

Coliform bacteria Floc bacteria Algae

Protozoan Fish Acutely toxic

Not acutely toxic Minor acutely toxic Moderately toxic

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



10/13

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 effluents eliciting acute toxicity, the

toxicity to floc bacteria could be explained on the basis

of the chemical and toxicity data. Of all 20 effluents, 11

effluents elicited acute toxicity in algae, 10 effluents were

toxic to fish, and 17 effluents were toxic to protozoa (seeFig. 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 deficiencies and growth-stimulating conditions

besides potential toxicity. It has become clear that the

chemical-specific approach produces enough informa-

tion for only a limited number of complex effluent

samples.

It is important to note that the precise chemistry of

effluents 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

difficulties involved in the detailed characterization of

the chemical content of wastewater. However, all

effluents 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

effluent sample as that used for toxicity testing.

3.5. Relationships between COD subcategories and

toxicity

For industrial wastewaters with low COD=BOD5ratios, the following comments are relevant: The waste-

water contains very slowly biodegradable organics, the

majority of organic matter is nonbiodegradable (refrac-

tory), and the wastewater contains some inhibitory

compounds such as heavy metals and toxic organics that

decrease the efficiency 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

identified. 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 effluents. 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 biode-

gradable COD on Days 20 and 150. As can be seen inTable 7, the slowly hydrolyzable COD caused the

discharge limits to be violated, but did not cause

toxicity, indicating the organics that should be hydro-

lyzed in pulp-paper effluents.

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 effluent treatment process.

Table 6

Organism Sensitivity rank

Very toxic Moderately

toxic

Slightly

toxic

Not toxic

Coliforms 6 3 4 5

Floc bacteria 6 1 4 9

Algae 5 3 3 9

Fish 4 3 3 10

Protozoa 2 1 1 16

Note. Sensitivity ranking indicated that the pulp-paper industry

discharges exhibited toxicity on some days. Fifteen effluents were

toxic to coliforms, 11 effluents were toxic to floc bacteria, 11 effluents

were toxic to algae, 10 effluents were toxic to fish, and 4 effluents were

toxic to protozoa.

Table 7

COD fractionation results for pulp-paper industry effluents

Total COD

(mg/L)

Soluble COD

(mg/L)

Soluble inert

microbial

product COD

(mg/L)

Soluble inert

COD (mg/L)

Readily

hydrolyzable

COD (mg/L)

Slowly

hydrolyzable

COD (mg/L)

Sampling

days/toxicity

Pulp 880 550 20 90 60 380 10 Yes

890 650 33 25 232 360 150 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 effluents. 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.



11/13

Low biodegradability gives rise to increased COD

concentration in effluents 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 chlorophe-

nols and aromatic derivatives that vary in toxicity are in

use. However, limited studies have been undertaken onthe 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 conven-

tional 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 effluents (Dyer

and Mignone, 1983; Ferguson, 1994).

The biodegradability of an organic substance is a

measure of the speed and completeness of its biode-

gradation by microorganisms. If the BOD5=COD ratiois between 0.1 and 0.5, the substance is slightly less

difficult 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 waste-water is toxic and the biodegradability is reduced as

reported by Tchobanoglous and Burton (1991).

Although in some cases the effluent wastewater

quality was ensured, the toxicity test results indicatespotential toxicity. This indicates that toxicity tests

should be incorporated into discharge standard regula-

tions. 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 difficult 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 effluents 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 fish tests are the most sensitive tests for

determination of toxicity in pulp-paper effluents. 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 effluents (Slabbert and

Venter, 1999). From Table 4, it would appear that the

fish and algal tests would be a potential surrogate since

their overall sensitivity is only slightly lower than that ofthe bacterial test. However, both traditional and

enrichment tests are affected by the presence of color,

COD, AOX, and phenol, in paper-mill industry effluent

samples. For the test in protozoa, which displayed

resistance, greater sensitivity would be obtained by using

coliforms or floc bacteria, fish, and algae, which are

more appropriate sensitive cultures for pulp-paper

industry effluents.

COD fractionation should be regarded as the required

complement to the total COD parameter within

conventional characterization. Since conventional char-

acterization 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 characteriza-

tion (in terms of COD) alone is not adequate in

providing knowledge about COD fractions. COD

fractionation provides information on the biodegrad-

ability 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 compo-

nent for heterotrophic microorganisms in biological

treatment plants. Furthermore, inert COD, which couldcontain inhibitory and toxic substances, should be taken

into account (Germirli et al., 1991).

5. Conclusions

Studies on wastewater effluents 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 dis-

charges into receiving waters. According to the results of

this study, the bacterial, fish, 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-effec-

tive, and accurate in the determination of potential



12/13

effects. The acute toxicity classification of an effluent

should always be based on the results of testing at least

once at trophic levels. All effluents 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 characteriza-

tion employing a single substance or a specific chemicalsubstance cannot yield convenient results for determina-

tion of the wastewater toxicity.

AOX, phenol, and inert COD originating from pulp-

paper 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 significant dis-

charges of complex organics where not all important

constituents can be individually identified and numeri-

cally limited, a clearly defined toxicity limit should be

specified, along with the appropriate form of toxicity

test to be used, and the minimum frequency with which

it should be applied. Toxic effluents emitted from

wastewater treatment plant installations should be

strongly controlled.

Although the COD concentrations exceeded dis-

charge limits, toxicity was not determined on some

days in pulp-paper industry effluents. 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 industryeffluents. This could be attributed to the large amount

of slowly biodegradable or inert COD in effluent

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.ul University Re-

search Fund for Financial Support (Grant 092298.01.34).

References

APHA/AWWA/WPCF, 1992. Standard methods for the examination

of water and wastewater, 16th Edition. Washington, DC.

Beckers-Maessen, C.M.H., 1994. Toxicity tests in WWO law

regulatory framework. RIZA Document, 94. 0171X.

Blaise, C., Sergey, G., Wells, P., Bermingham, N., Van Coillie, N.,

1988. Biological testing-development, application and trends in

Canadian Environmental Protection laboratories. Toxicol. Assess.

3, 385406.

Boncz, M.A., Brunning, H., Rulkens, W.H., Sudh.olter, J.R., 1993.

Kinetic and mechanistic aspects of the oxidation of chlorophenols

by ozone. Water Sci. Technol. 35, 6572.

Canton, J.W., 1991. Catch-up operation on old pesticides: anintegration. RIVM Rep. 678 80 1002.

Chen, C-Y., Chen, J-N., Chen, S-D., 1999. Toxicity assessment of

industrial wastewater by microbial testing method. Water Sci.

Technol. 39, 139143.

Conover, W.J., 1971. Practical Non-Parametric Statistics. Wiley, New

York.

Cronin, M.T., Dearden, J.C., Dobbs, A.J., 1991. QSAR studies of

comparative toxicity in aquatic organisms. Sci. Total Environ. 109/

110, 431439.

Demirba-s, G., G.ok-cay, C.F., Dilek, F.B., 1999. Treatment of organic

chlorine in pulping effluents by activated sludge. Water Sci.

Technol. 40, 275279.

Dube, M.G., Culp, J.M., 1997. Growth responses of periphyton and

chronids exposed to biologically treated kraft pulp mill effluent.

Water Sci. Technol. 35, 339345.

Dugan, P.R., Lundgren, D.C., 1968. Appl. Microbiol. 8, 357361.

Dyer, J.C., Mignone, N.A., 1983. Handbook of Industrial Residues,

Vol. 1, Environmental Engineering Series. Noyes Publications,

Park Ridge, NJ, USA.

Ekama, G.A., Dold, P.L., Marais, G.R., 1986. Procedures for

determining influent COD fractions and the maximum specific

growth rate of heterotrophs in activated sludge systems. Water

Sci. Technol. 18, 91114.

Fracasso, M.E., Leone, R., Brunello, F., Monastra, C., Tezza, F.,

1992. Mutagenic activity in wastewater concentrates from dye

plants. Mutat. Res. 298, 9195.

Germirli, F., Orhon, D., Artan, N., 1991. Assessment of the initial

inert soluble COD in industrial wastewater. Water Sci. Technol. 23,

10771086.Haggblom, M., Salkinoja-Solonen, M., 1991. Biodegradability of

chlorinated organic compounds in pulp bleaching effluents. Water

Sci. Technol. 24, 161170.

Hayes, E., Woods, C., Lockett, P., 1999. Toxicity testing in the

textile industry: an evaluation of current methods. International

Conference on Water and Textile, London, UK, 1012 May,

pp. 4856.

Kingstad, K.P., Lindstrom, K., 1984. Spent liquors from pulp

bleaching. Environ. Sci. Technol. 18, 236247.

Kinnersley, D., 1990. Discharge consent and compliance policy:

a blueprint for the future. NRA Water Quality Series, No. 1.

Landner, L., 1994. Harmfull Chemicals, a Practical Guide for

Assessment of Chemicals. National Environmental Protection

Agency, Sweden.Minke, R., Rott, U., 1998. Anaerobic treatment of split flow

wastewater and concentrates from the textile processing industry.

Water Sci. Technol. 40, 169176.

Naru, M., Goto, M., Kato, H., Oshima, Y., 1993. Study on endocrine

disrupting chemicals in wastewater treatment plants. Water Sci.

Technol. 43, 101108.

Nirmalakhandan, N., Arulgnanendran, V., Mohsin, M., Sun, B.,

Cadena, F., 1993. Toxicity of mixtures of organic chemicals to

microorganisms. Water Res. 28, 543551.

Norussis, M.J., 1986. SPSS/PC for the IBM/P, XT/AT. SPSS Inc.,

Chicago.

NRA, 1993. The toxicity-based consent, a step towards more effective

control of complex effluents within the UK. NRA Information

Leaflet.



13/13

NRA, 1994. Direct toxicity assessment, a step towards better

environmental protection within the UK. NRA Information

Leaflet.

NRA, 1995. Aquatic toxicity, control and assessment, an update on

current research and development. NRA Information Leaflet.

Orhon, D., S.ozen, S., -Cokg .or Ubay, E., Genceli, E., 1998. The effect

of chemical settling on the kinetics and design of activated sludge

for tannery wastewaters. Water Sci. Technol. 38, 355362.Pelezar, M.T., Chan, E.C.S., 1972. Laboratory Experiences in

Microbiology, 3rd Edition. McGrawHill, New York.

Redenbach, A.E., 1997. Sensory evaluation of fish exposed to pulp and

paper mill effluent: a case study of method used for environmental

effects monitoring. Water Sci. Technol. 35, 357363.

Schowanek, D., Fox, K., Holt, M., Schroeder, F., Koch, V., 2001.

GREAT-ER: a new tool for management and risk assessment of

chemicals in river basin. Water Sci. Technol. 43, 179185.

Siegel, S., 1956. Non-Parametric Statistics for the Behavioral Scientist.

McGrawHill, New York.

Slabbert, J.L., 1996. Guidelines for toxicity bioassaying of water and

effluents in South Africa. Contact Report for the Water Research

Commission. Project No. K5/358/0/1. Division of Water Technol-

ogy, Pretoria, South Africa.

Slabbert, J.L., Venter, E.A., 1999. Biological assay for aquatic toxicitytesting. Water Sci. Technol. 39, 367373.

Solomon, K., Bergman, H., Huggett, R., 1996. A review and

assessment of the ecological risk associated with the use of chlorine

dioxide for the bleaching of pulp. Pulp Paper Can. 97, 3544.

Sponza, D., 1999. Assessment of biotoxicity analysis for different

industrial effluent. Priorities in the Environmental Pollution,

Vol. 1. Gebze, Kocaeli, Istanbul, 1819 November, pp. 522533.

Sponza, D., 2002a. Incorporation of toxicity tests into the Turkish

industrial discharges monitoring systems. Arch. Environ. Contam.

Toxicol. 43, 186197.

Sponza, D., 2002b. Necessity of toxicity assessment in Turkish

industrial discharges. Examples from metal and textile industry

effluents. Environ. Monit. Assess. 73, 4166.

Sun, Y.B., Chang, H.M., Joyce, T.W., 1989. Dechlorination

and decolorization of high molecular-weight chloroligninfrom bleach plant effluents by an oxidation process. TAPPI J. 72,

209213.

Tchobanoglous, G., Burton, F.L., 1991. Wastewater Engineering

Treatment Disposal Reuse, 3rd Edition. Metcalf & Eddy, USA.

Tirsch, F., 1989. Pulp and paper effluent management. JWPCF 61,

876878.

Tonkes, M., de Graaf, J.J., Graaansma, J., 1999. Assessment of

complex industrial effluents in The Netherlands using a whole

effluent toxicity approach. Water Sci. Technol. 39, 5561.

Trevizo, C., Nirmalakhandan, N., 1999. Prediction of microbial

toxicity of industrial organic chemicals. Water Sci. Technol. 39

1011, 6369.Turkish Environment Regulation, 1992. Official Gazette No. 19880,

21 September, V. 2, Ankara.

Turkish Water Pollution Control Regulation, 1998. Official Gazette

No. 19919, 4 September.

U.S. Environmental Protection Agency (USEPA), 1981. Technical

Support Document for Water Quality Based Toxic Control. EPA/

505/2-90-001.

U.S. Environmental Protection Agency (USEPA), 1989. Generalized

Methodology for Conducting Industrial Toxicity Reduction

Evaluations TREs. EPA 600/2-88/070.

U.S. Environmental Protection Agency (USEPA), 1991. Technical

Support Document for Water Quality Based Toxic Control. EPA/

505/2-90-001. Office of water, Washington, DC.

U.S. Environmental Protection Agency (USEPA), 1996. The Applica-

tion of Toxicity Based Criteria for the Regulatory Control ofWastewater Discharges. Washington, DC, pp. 234287.

Volskay, V.T., Grady, C.P.L., 1988. Toxicity of selected RCRA

compounds to activated sludge microorganisms. JWPCF 60,

18501856.

Vyryan, T., Coombe, S., Keith, M., Matthew, J., 1999. An abbreviated

guide to dealing with toxicity. Water Sci. Technol. 39, 9197.

Wharfe, J.R., Tinsley, D., 1995. The toxicity based and the wider

application of direct toxicity assessment to protect aquatic life.

J. Chart. Inst. Water Environ. Manage. 9, 526530.

Whitehouse, P., Dijk, P., 1996. The precision of aquatic toxicity

tests: the implications for the control of effluents by direct

toxicity assessment. In: Tapp, J.F., Wharfe, J.R., Hunt, S.M.

(Eds.), Toxic Impacts of Wastes on the Aquatic Environment.

Cambridge.

Zar, J.H., 1984. Biostatistical Analysis, 2nd Edition. PrenticeHall,Englewood Cliffs, NJ.

Zaror, C., Varrosco, V., Perez, L., Soto, G., 2001. Kinetics and toxicity

of direct reaction between ozone and 1,2-dihydrobenzene in dilute

aqueous solution. Water Sci. Technol. 43, 321326.


application of toxicity tests into discharges of the pulp-paper

Documents