toxicity reduction through chemical and biological modification of spent pulp bleaching liquors

EPA-600/2-80-039 January 1980

T O X I C I T Y REDUCTION THROUGH CHEMICAL AND BIOLOGICAL MODIFICATION OF SPENT PULP BLEACHING LIQUORS

Carl ton W. Dence, Chun-Juan Wang, and Pat r i ck R. Durkin State Un ivers i ty o f New York

Syracuse, New York 13210 College o f Environmental Science and Forestry

Grant R 804779

Pro ject Officers

Michael D. S t ru t z H. K i r k Wi l l a rd

Food and Wood Products Branch Indus t r i a l Environmental Research Laboratory

Cincinnat i , Ohio 45268

INDUSTRIAL ENVIRONMENTAL RESEARCH LABORATORY OFFICE OF RESEARCH AND DEVELOPMENT

U.S. ENVIRONMENTAL PROTECTION AGENCY CINCINNATI, O H I O 45268

0 I SCLAIMER

This repo r t has been reviewed by the Indus t r i a l Environmental Research Laboratory, U. S. Environmental Protect ion Agency, and approved for pub1 ica- t ion . Approval does not s i g n i f y t h a t the contents necessarily r e f l e c t the views and p o l i c i e s of the U.S. Environmental Protect ion Agency, nor does mention of t rade names o r commercial products cons t i t u te endorsement o r recomnendation for use.

ii

FOREWORD

When energy and material resources are extracted, processed, converted, and used, t h e related pol lut ional impacts on our environment and even on our health of ten require t h a t new and increasingly more e f f i c i en t pollution con- t r o l methods be used. The Indus t r ia l Environmental Research Laboratory-Cin- c innat i ( I E R L C i ) assists i n developing and demonstrating new and improved methodolcgiee t h a t will meet these need8 both e f f i c i en t ly and economically.

The report investigates t he reduction of t h e tox ic i ty of spent pulp bleaching l iquors through chemical and biological modification. was determined over a range of concentrations, before and aFter modification, t o determine t h e i r e f f ec t on t h e growth of fungi, a lga and duckweed, and on t h e survival of Daphnia Magna. vest igated i n t h i s study included treatment with chlorine, chlorine dioxide, hypochlorite, ozonation and hydrogen peroxide treatment, alum and lime addi- t i o n , carbon adsorption, and by modification of t h e conventional chlorination and caust ic extraction bleaching stages. For fur ther information on the sub- Jec t , contact t he Food and Wood Products Branch, IERL Cincinnati.

Toxicity

Methods of chemical treatment t h a t were in-

David G. Stephan Director

Indus t r ia l Environmental Research Laboratory Cincinnati

iii

ABSTRACT

Chlorophenols s im i la r t o or i den t i ca l w i th those detected i n spent ch lo r i na t i on and caust ic ext ract ion 1 iquors were synthesized and tes ted over a range of concentrations t o determine t h e i r ef fect on the growth of several fungi, an alga (Chlore l la renoidosa), and duckweed (Lemna e r u s i l l a ) and on the surv iva l m a magna.

genera l ly ind icated tha t growth repression and t o x i c i t y increased w i t h i n - creasing numbers of chloro substituents on the phenolic r ing ,

though differences -fesr;; were oun the responses of the i n P- v ual t e s t organisms t o the chlorophenols, the r e s u l t s

I The chlorophenols referred t o above were subjected t o a number of chem- i c a l and b io log i ca l treatments t o assess t h e i r s u s c e p t i b i l i t y t o degradation when present as components o f spent ch lo r i na t i on and caust ic ex t rac t ion 1 i- quors. Chlorophenol degradation was determined by gas chromatographic and/ o r u l t r a v i o l e t spectroscopic analysis f o r unreacted s t a r t i n g mater ia l Ozone and ch lo r i ne d iox ide e f f e c t i v e l y degraded the chlorophenol s, but com- p l e t e removal required the app l ica t ion o f excessively la rge amounts of chem- i c a l . I n the case of t he ozonization of tetrachloroguaiacol , decreases i n phenol breakdown were para l le led by reductions i n t o x i c i t y as ind icated by Da hnia magna. A l k a l i treatment o f the chlorocatechols a t room temperature h o v e d an ef fect ive means of reducing chlorophenol t ox i c1 ty.

B io log ica l treatment of the chlorophenols consisted of the app l i ca t i on of pure cu l tu res o f three d i f f e r e n t fungi and a mixed microbia l populat ion fo r periods ranging up t o 15 days. Degradation var ied widely among the va r i - ous phenols and f o r the same phenol t reated w i t h d i f f e r e n t fungi. s i m i l a r conditions, aerat ion i n the absence of any microorganisms was effec- t i v e i n varying degrees w i t h catechol (1.2-dihydroxybenzene) der iva t ives SUS- t a in ing the greatest amount o f degradation,

The spent l i q u o r s from the ch lo r i na t i on and caust ic ex t rac t ion bleach- i n g stages of a pine k ra f t pulp (Kappa No. 25.5) were f ract ionated by means of d ia l ys i s , gel permeation chromatography, and solvent (ether) extract ion; and the f ract ions bioassayed f o r t h e i r ef fect on a fungus, As e r i l l u s

6 m o l e k e * m a t e r i a l s displayed the greatest repression of fungal growth and were the most tox i c t o the Daphnia.

Spent ch lo r i na t i on and caust ic ex t rac t i on l i q u o r s were subjected t o a v a r i e t y of chemical treatments and the r e s u l t i n g ef fects on acute t o x i c i t y determined. Treatment w i t h elemental chlor ine, hypochlorous acid, hypo- ch lo r i t e , ozone and hydrogen peroxide produced increases i n the t o x i c i t y of the spent l i quo r . A modest reduct ion i n t o x i c i t y accompanied treatment of spent ch lo r i na t i on l i q u o r w i th ch lo r ine dioxide. T o x i c i t y reduct ion was

Under

fumi atus and Da hnia ma na. Without exception, the f rac t i on -Ep-? contain ng

i v

also achieved by the add i t ion of alum and l ime t o spent caust ic ex t rac t ion l i quo r . The concentration of t ox i c mater ia l expressed as t o t a l organic carbon (TOC) was, however, higher i n each instance. As i n the case of the chlorophenols, substant ia l reductions of t o x i c i t y were achieved by the addi- t i o n o f a l k a l i t o spent ch lo r i na t i on l i quo r . Tox i c i t y reduct ion i n t h i s instance para l le led decreases i n phenol and organ ica l l y bound ch lo r i ne contents.

B io log ica l treatment of spent ch lo r i na t i on and caust ic ex t rac t ion 1 i- quors involved the app l ica t ion o f a fungus (Candida u t i l i s ) , an un ident i f ied bacterium, and a mixed microbial p o p u l a t i o n , w e - supplemental carbon sources. The microbia l mixture and the fungus effected essen t ia l l y com- p l e t e e l im ina t ion of t o x i c i t y from spent ch lo r i na t i on l i q u o r and small reductions i n the t o x i c i t y o f spent caust ic ex t rac t ion l i quo r . The bacterium was, on the other hand, comparatively ine f fec t i ve i n achieving the same ob5 ec ti ve . and caust ic ex t rac t ion bleaching stages was a lso evaluated. The subst i tu - t i o n of ch lo r ine d iox ide f o r ch lo r ine i n the ch lo r i na t i on stage proved t o be d i s t i n c t l y benef ic ia l i n reducing t o x i c i t y as was the s u b s t i t u t i o n of oxygen for ch lo r i ne i n the same stage. Small reductions i n t o x i c i t y attended the i n t roduc t i on of hydrogen peroxide i n t o the f i r s t caust ic ex t rac t i on stage but add i t i on of hypochlor i te i n the same stage was less ef fect ive than a l k a l i alone i n t h i s regard.

T o x i c i t y reduct ion through modif icat ion of conventional ch lo r i na t i on

This repo r t was submitted i n f u l f i l l m e n t of Contract No. R804779010 by the Research Foundation of the State Un ivers i ty of N e w York under the sponsorship of t he U.S. Environmental Protect ion Agency. This r e p o r t covers a per iod from September 21, 1976, t o September 20, 1979 and work was com- p le ted as Of September 20, 1978.

V

CONTENTS

Foreword ................................................................ iii Abstract ................................................................ i v Figures ................................................................. v i i Tables .................................................................. v i i i Acknowledgment .......................................................... x i

1 . Int roduct ion .................................................... 2 . Conclusions ..................................................... 3 . Recomnendations ................................................. 4 . Mater ia ls and Methods ...........................................

Chemical s ..................................................... Bio log ica l t e s t organisms ..................................... Spent b l eachi ng 1 i quors ....................................... Fractionated spent bleaching 1 iquors .......................... Chemical treatment o f chlorophenols ........................... Chemical treatment o f spent c h l o r i n a t i o n and caust ic ext ract ion

1 iquors .................................................... Chlor inat ion and caust ic ex t rac t i on stage modif icat lons ....... Bio log ica l treatment o f phenol s and chlorophenol s .............. Bio log ica l treatment o f spent ch lo r i na t i on and caust ic

ex t rac t i on 1 iquors ........................................ Acute t o x i c i t y t es ts ......................................... Gas chromatographic analysis ................................. Analy t ica l procedures ........................................

5 . Results and Discussion ......................................... Acute t o x i c i t y o f phenols .................................... Acute t o x i c i t y o f whole and f ract ionated spent ch lo r i na t i on

(SC) and caust ic ex t rac t i on . (SCE) l i q u o r s ................. Degradation o f phenols by chemical treatment ................. Degradation o f chlorophenols by b io log i ca l treatment ......... Chemical treatment o f spent c h l o r i n a t i o n and caust ic ext ract ion

1 iquors ................................................... Bio log ica l treatment o f spent c h l o r i n a t i o n and caust ic

ex t rac t i on 1 iquors ........................................ Reduction i n SCL and SCEL t o x i c i t y through modif icat ions

o f the bleaching process .................................. References ............................................................. Amendi ces .............................................................

.

. . A . Jack Meyer's modif ied medium f o r Chlore l la ..................... B . Hutner's medium f o r duckweed ...................................

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13 14 16 17 19 19

29 48 60

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86 91 97 97 98

v i i

FIGURES

Number

1

2 Effect of creosol (VII) and three chloroguaiacols on the

3 Effect of age on the color of spent chlorination and caustic

Effect of 2,4,6-trichlorophenol ( V I ) and three chlorocatechol s on the growth of Aspergill us fumigatus ...............

growth of Aspergill us fumigatus ...........................

extraction 1 iquors stored a t room temperature .............. 4 Rate of loss of organically bound chlorine from spent

chlorination and caustic extraction 1 iquors ................. 5 Response of Aspergillus fumi atus t o whole spent chlorination

6 Gel permeation chromatograms of retentate ( I ) and dialysate

7 Relationship between the dry weight of fungus and the total

and caustic extraction + iquors ............................

( I I ) w i t h Sephadex 6-25 .....................................

organic carbon i n the fractions obtained by single d ia lys i s ....................................................

8 The ef fec t of the ether extracts of SCL ( A ) , SCEL ( B ) , and of ether-extracted SCL (C) on the growth of Aspergillus fumigatus ...................................................

9 A typical elution diagram for the fractionation of spent caustic extraction liquor ether extract on s i l i c a gel ...............

10 Degradation of selected phenols through reaction w i t h chlorine dioxide .....................................................

11 Effect of carbon treatment on the color and TOC of lime-

12 Detoxication ( A ) of spent chlorination liquors by alkaline

treated spent caustic extraction 1 iquor .....................

treatment ...................................................

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TABLES Number

1 Fungi Used i n the Invest igat ion .............................. 2 Phenolic Compounds Used i n the Invest igat ion ............... 3 Minimum Concentrations o f Phenols Required t o Prevent Growth

o f Various Fungi ........................................... 4 T o x i c i t y Ranges o f Selected Phenols f o r Chlore l la

pyrenoidosa ................................................ 5 E f f e c t o f Four Phenols on the Vegetative Growth o f Lemna

pe rpus i l l a ................................................. 6 Acute Tox ic i t y o f Various Phenols t o ........... 7 Acute T o x i c i t y o f the Whole Spent Chlor inat ion and Caustic

- Ext ract ion Liquors t o ........................ 8

9

Acute T o x i c i t y o f Spen Caustic Extract ion Liquor Fractions

Acute Tox ic i t y o f Ether Extract ives o f Spent Chlor inat ion and

t o Daphnia magna ...........................................

Caustic Ext ract ion Liquors t o ................ 10 T o x i c i t y o f the Sub-fractions o f the Spent Chlor inat ion Ether

Ext ract as Indicated by Daphnia magna and Aspergi l lus fumigatus ..................................................

11 T o x i c i t y Character ist ics o f Spent Chlor inat ion and Caustic Ext ract ion Liquors Ether Extracts Fractionated by Chromatog- raphy on S i l i c a Gel ........................................

12 Elemental Composition o f the Spent Chlor inat ion Liquor (A) and Spent Caustic Extract ion Liquor (B) Ether Extracts and Ether Ext ract Sub-fractions ............................

13 Acid ic Group and Total Hydroxyl Contents o f the Spent Chlor in- a t i o n Liquor (A) and Spent Caustic Extract ion Liquor (B) Ether Extracts and Ether Extract Sub-fractions .............

i x

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Number Page Ozonization Rate Constants o f Phenols ......................... 50

De tox i f i ca t i on o f Tetrachloroguaiacol ( X V I ) by Ozonization a t pH 10 .................................................... 53

Ozone Oxidation o f tetrachloroguaiacol-Spent Caustic Extract ion Liquor Combinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Chl or1 ne Dioxide Oxidation o f Tetrachl oroguai acol -Spent Caustic Ext ract ion Liquor Combinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

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Effect o f A l ka l i ne Treatment (pH 11) on the Tox ic i t y o f Some Chlorophenols t o Daphnia magna ..............................

Effect o f Aeration on Removal o f Chlorophenols ................ Rate o f Removal o f Phenols dur i t g Aeration . . . . . . . . . . . . . . . . . . . . Biodegradation o f Phenols by Fungi i n L iqu id Culture Media .... Effect o f Sodium Hypochlorite Treatment on the Acute Tox ic i t y

o f Spent Caustic Extract ion Liquor t o Daphnia magna .........

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Character is t ics o f Or ig ina l ar.d Ozonized Spent Chlor inat ion and Caustic Ext ract ion Liquors ..............................

Propert ies o f Alum-Treated Spent Caustic Extract ion Liquor . . . . Effect o f Lime Treatment on Spent Caustic Extract ion Liquor ... Propert ies o f Lime-Treated SCEL a f t e r Reaction with Ac t i -

Acute T o x i c i t y o f Spent Caustic Ext ract ion Liquor Treated

Acute T o x i c i t y o f Carbon-Treated Spent Caustic Ext ract ion

Effect o f Ozone Oxidation on the Propert ies o f Lime-Treated

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vated Carbon ................................................ 71

Sequent ia l ly w i t h Lime and Act ivated Carbon ................. 74

Liquor ...................................................... 74

Spent Caustic Ext ract ion Liquor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

X

Number

30 E f fec t o f Chlorine Dioxide on the Propert ies o f Lime-Treated

E f fec t o f Hydrogen Peroxide Oxidation on the Propert ies o f

Acute Tox ic i t y o f Lime-Treated Caustic Extract ion Liquor

Spent Caustic Extract ion Liquor ............................. 31

Lime-Treated Spent Caustic Ext ract ion Liquor ................ 32

Reacted w i th Ozone, Chlorine Dioxide, and Hydrogen Peroxide ....................................................

33 E f f e c t o f A l k a l i Treatment o f Spent Chlor inat ion Liquor on

Rate o f Removal o f Organical ly Bound Chlorine from Spent

E f f e c t o f A l k a l i on the T o x i c i t y o f Spent Chlor inat ion

E f f e c t o f Prolonged (two week) Neutra l izat ion on the T o x i c i t y

Phenol and Organical ly Bound Chlorine Contents .............. 34

Chlor inat ion Liquor w i t h Increasing pH ......................

Liquor t o Daphnia magna ..................................... 35

36 o f Spent Chlor inat ion Liquor t o ...............

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37 E f f e c t o f B io log ica l Treatments o f Spent Chlor inat ion Liquor

T o x i c i t y o f Ef f luents from Conventional and Process-Modified

and Spent Caustic Extract ion Liquor on Acute T o x i c i t y ........ 83

38 Bleaching Stages ............................................. 87

39 Characterization of Selected Conventjonal and Process- Modif ied Spent Bleaching Liquors .............................. 89

x i

ACKNOWLEDGMENT

The authors g r a t e f u l l y acknowledge the contr ibut ions o f Dr. Katu Sameshima, Dr. Brian Simson, Ms. Er ica Rowe, Mr. Richard Ztobro, M r . James C a i n a n d Mr. Joseph Fernandet i n the experimental phase o f the pro jec t .

x i i

SECTION 1

INTRODUCTION

It i s now a general ly accepted fact t h a t bleaching effluents are m i l d l y t o x i c t o f i s h and other aquatic organisms. The resu l t s of a recent i nves t i - ga t ion (1) have shown t h a t the compounds cont r ibu t ing t o t o x i c i t y are r e s i n acids, ch lor inated r e s i n acids, unsaturated f a t t y acids, f a t t y ac id deriva- t i v e s (epoxy s tea r i c and d ich lorostear ic acid), and chlorophenolics. The presence of r e s i n and f a t t y acids i n bleaching effluents can be traced t o i n e f f i c i e n t washing of the bleached pulp. During the ch lo r ina t ion and caus- t i c ex t rac t i on stages, these compounds are i n p a r t converted t o chloro and epoxy der i va t i ves .

The ch lor inated phenols are derived from the res idual l i g n i n i n the pulp and appear t o cons t i t u te the most t o x i c c lass of compounds comprising bleaching effluents. During ch lor inat ion, the chlorophenols are, t o a la rge degree, ox id ized and broken down t o ch lor ine-subst i tu ted a l i p h a t i c compounds. The impact of the l a t t e r compounds on the t o x i c i t y o f bleaching effluents has not been systemat ical ly evaluated.

A1 though the exact con t r ibu t ion of chlorophenols t o the t o t a l t o x i c i t y of bleaching effluents may be d i f f i c u l t to'assess w i t h any degree of conf i - dence a t present, there can be l i t t l e doubt bu t t h a t i t i s a t l e a s t a major and q u i t e poss ib ly dominant one. The cont inuing i d e n t i f i c a t i o n of chlorophenols i n spent ch lo r ina t ion and caust ic ex t rac t ion l'lquors serves t o emphasize the importance of learning how such compounds respond t o various chemical and b io log i ca l treatments i n order t h a t t h e i r removal from these spent l i q u o r s may be fac i l i t a ted .

I n low was

1.

2.

response t o the aforementioned need, the inves t iga t ion described be- undertaken w i t h the fo l lowing object ives i n mind:

To ascer ta in the r e l a t i v e degrees of t o x i c i t y displayed by phe- no ls and chlorophenols s im i la r to, o r i den t i ca l w i t h , those prev i - ously detected i n spent ch lo r i na t i on and caust ic ex t rac t ion liquors when contacted w i th a v a r i e t y o f aquatic l i f e forms.

To determine the f e a s i b i l i t y of using selected chemical and bio- l o g i c a l treatments o r combinations o f such treatments for the removal of those phenolic and chlorophenol i c residues previously demonstrated t o have a de leter ious e f f e c t on aquatic l i f e .

1

To pursue these object ives, we conducted experiments t h a t involved acute t o x i c i t y bioassays, and b io logica l and chemical treatments o f the indiv idual chlorophenols. These experiments were supplemented wi th s i m i l a r t e s t s t h a t used the spent l iquors from the ch lor ina t ion and caust ic ex t rac t ion o f a southern pine k r a f t pulp.

2

SECTION 2

CONCLU S I OPlS

i, an alga (Chlorel la renoidosa), and a vascu-

of phenols and chlorophenols. The s e n s i t i v i t y were found su i tab le as +-- p ant t e s t organisms fo r

acute t o x i c i t y bioassays ,e, o the phenols and chlorophenols used i n the inves-

due t o i t s greater sens i t + v ty.

of these organisms decreased i n the order alga > vascular p l a n t > fungi. An aquatic invertebrate (Da hnia magna) a lso proved sat is factory for use i n

t i g a t i o n . t i c ex t rac t i on l iquors, Da hnia magna was found t o be more useful than fungi

I n acute t o x i c i t y bioassays invo lv ing spent c h l o r i n a t i o n and caus-

Chlorophenols s i m i l a r t o o r i den t i ca l w i t h those present i n spent chlo- r i n a t i o n and caust ic ex t rac t i on l i quo rs can be r a p i d l y and extensively degraded with ozone and ch lor ine dioxide, However, t h e i r complete el imina- t i o n requires i no rd ina te l y large appl icat ions of chemicals p a r t i c u l a r l y when present w i t h other oxidizable mater ia ls as would be the s i t u a t i o n i n spent c h l o r i n a t i o n and caust ic ext ract ion l iquors.

the t o x i c i t y of so lu t ions o f the t e s t chlorophenols, Chlorocatechols can be most successfully detoxi f ied by such a treatment.

A l ka l i ne treatment (pH 11) i s e f f e c t i v e i n varying degrees i n reducing

Chlorophenols of the type included i n the i nves t i ga t i on can be exten- s i v e l y degraded by simple aerat ion ( in the absence of any microorganisms i n 1-week treatments). A s im i l a r effect can be achieved by b io log i ca l t r e a t - ments i nvo l v ing i nd i v idua l fungi o r a mixture o f microorganisms. p lay widely varying degrees of a b i l i t y t o break down a given chlorophenol.

Fungi d i s -

Some of the propert ies of spent ch lo r i na t i on and caust ic ex t rac t i on 11- quors were found t o change as the l i q u o r s aged, T0xicit.y t e s t s can be made on reasonably aged l iquors, however, wi thout the expectat ion of changes havi ng occurred.

Based on the r e s u l t s of f ract ionat ion treatments, the t o x i c i t y of spent c h l o r i n a t i o n and caust ic ext ract ion l i q u o r s gives evidence of being a t t r i - butable t o the lower molecular weight substances contained therein.

I n general, chemical treatment does not appear t o represent a very promising approach for reducing o r e l im ina t i ng the t o x i c i t y of spent c h l o r i - nat ion and caust ic ext ract ion l iquors. former l i q u o r through treatment w i th a l k a l i may have a future,provided the technical and economic f e a s i b i l i t y of such a treatment can be demonstrated

However, d e t o x i f i c a t i o n of the

3

by further experimentation. remove toxic material from spent caustic extraction liquor. However, the sludge that contains the toxic substances still has to be disposed of.

Lime and alum addition can be used to partially

Modifications of conventional bleaching treatments (in which chlorine dioxide and oxygen are substituted for chlorine in the chlorination stage, and hydrogen perioxide is added to the caustic extraction stage) are effective in substantially reducing the toxicity of the corresponding effluents.

4

SECTION 3

RECOMMENDATIONS

As described below i n the body of t h i s report, attempts t o reduce the t o x i c i t y of bleaching effluents through modif icat ion of conventional bleaching stages produced some very encouraging resul ts . We recommend, therefore, t h a t research along these l i n e s be continued and expanded so t h a t the po ten t i a l of t h i s approach may be f u l l y assessed. I n view of the general success the paper indust ry has had using b io log i ca l forms of waste t rea t - ment, we fur ther recommend that, i n conjunction w i t h the above bleaching stage modifications, the eff luents from such treatments a lso be subjected t o some form of b io log i ca l treatment i n order t o assess the effect of the modif icat ion on t h e i r s u s c e p t i b i l i t y t o biodegradation.

Improved ef f ic iency w i t h respect t o the b io log i ca l systems used i n the p u r i f i c a t i o n of paper m i l l wastes could r e s u l t i n an increased margin of safety w i t h respect t o the a b i l i t y of a p a r t i c u l a r treatment system t o con- s f s t e n t l y d e l i v e r t o x i c i t y - f r e e ef f luents dur ing m i l l upsets, o r as a r e s u l t of other operational problems. The wide range of a b i l i t i e s displayed by the various microorganisms used i n the present i nves t i ga t i on for breaking down chlorophenols suggests the p o s s i b i l i t y of manipulating the composition of the microbia l populat ion i n the biologica' l treatment system so as t o f a c i l i - t a t e t h i s objective. On t h i s basis,we recommend t h a t the search for addi- t i o n a l microorganisms having t h i s p a r t i c u l a r c a p a b i l i t y be continued and t h a t the effect of environmental t e s t condi t ions (i .e., pH, nutr ients, temp., etc.) be studied to ascertain how these should be contro l led t o achieve the maximum r a t e of degradation.

I n the current invest igat ion, acute t o x i c i t y bioassays r e l i e d l a r g e l y on the response of a s ing le species, Da hnia ma na. This approach made i t possible t o screen a large number o f *+ p eno s an chlorophenols as wel l as spent c h l o r i n a t i o n and caustic ex t rac t i on 1 iquors before and a f te r various treatments. The relevance o f t h i s work could be subs tan t i a l l y increased by applying more sophist icated t e s t methods and by increasing the number of aquatic p lan t and animal species tested.

The f e a s i b i l i t y of using an alga and a vascular aquatic plant, t o x i c i t y bioassays of t i ons and the f indings (described i n the body of t h i s repo r t ) i nd i ca ted - the i r p o t e n t i a l u t i l i t y i n t h i s regard, p a r t i c u l a r l y from the po in t of view of t h e i r increased s e n s i t i v i t y as compared t o fungi. i nvo l v ing these organisms be continued and broadened t o include bleaching l i q u o r s before and a f t e r modification.

We propose t h a t the t e s t s

5

A1 though Daphnia represent an extremely important c lass of aquatic i n - vertebrates and are thus a highly relevant t e s t species, a sound program of hazard assessment should attempt t o evaluate effects on several organisms and diverse h a b i t a t s , phenols and spent chlorination and caustic extraction 1 iquors be performed using Gammarus, a genus of amphipods commonly found i n streams and brooks. Based on the comparative behavior of t h i s organism and Daph?ia when exposed t o other toxicants, the use of Gammarus i n acute toxicity bioassay m i g h t be expected t o indicate s i g n i f i c a n w e n c e s i n species susceptibil i ty rather than simply parallel the resul ts obtained using Daphnia.

For this reason,we recommend that bioassays of chloro-

6

E

w

SECTION 4

MATERIALS AND METHODS

CHEMICALS

2,4,6-Trichlorophenol , 2,4-dichlorophenol , catechol (1.2-di hydroxyben- zene) , guaiacol (2-methoxyphenol ), &-cresol and phenol were obtained commer- c i a l l y . A l l other phenols and chlorophenols used i n the inves t iga t ion were prepared as described by Gess and Dence (2). Cain (3 ) . Nonni (4), Oence e t a l . (5) . and W i l l s t a t t e r and M i l l e r (6).

Methyl dehydroabietate was prepared by methylation of the f ree ac id w i th diazomethane. This ester was not isolated, but gas chromatographic analys is of the methylated mater ia l i n the product mixture revealed the presence of only one peak.

BIOLOGICAL TEST ORGANISMS

Animals

Da hnia ma na were purchased from Wards' Natural Science Establishment,

a recons t i tu ted fresh water described by D'xgostino and P'f;bvasoli (7). The organisms were fed yeast and the photoperiod was kept on a 12-hour l igh t /12- hour dark cycle. Algal and protozoal contamination of the stock cu l tu res provided add i t iona l n u t r i e n t sources. F i r s t instar,or 1-3 day old Da hnia,

nongravid mature organisms t o 250 m l of fresh DM2 (17 + l0C, pH 7.0) and adding 5 m l of homogenized yeast (1 mg/ml) per day un tm eggs developed. A t t h i s time, feeding was terminated and young were harvested da i l y .

Inc. +f- Stoc cu tures were maintained a t 17 + loc, pH 7.0 + 0.1 u n i t s i n DM2,

for use i n the b io log i ca l assays were obtained by t ransferr ing groups +5 o

Br ine shrimp (Artemia sa l ina) eggs, d i s t r i bu ted by Metaframe Corpora- t ion, were p u r c h a s - n e s e organisms were hatched i n synthet ic sea water (8) 24 hours p r i o r t o test ing. The synthet ic sea water was a lso used as the d i l u t i o n water i n a l l Artemia sa l ina bioassays.

Plants

fungi ( l i s t e d below i n Table 1). an unident i f ied bac ter ia l cu l ture, an alga (Ch lore l la

The p lan t t e s t organisms used i n the inves t iga t ion consisted of several

renoidosa Chick), and a vascular p lan t (Lemna p e r p u s l l l a Torr.), comnonry Y-----T nown as uckweed.

7

TABLE 1, FUNGI USED I N THE INVESTIGATION

No. l-ungus name

1 Aspergil lus fumigatus Fres. (C-78)

2

3 Paecilomyces v a r i o t i Baiher (C-64)

4 Penic i l l ium va r iab i l e Sopp (C-78)

5 Trichoderma koningi i Oud. (C-65)

6 Aspergil lus n iger van Tiegh. (Wang 1152)

Cladosporium herbarum Link ex Fr ies (C-5)

7 Candida u t i l i s (Henneberg) Lodder & Kreger-van R i j

Fungi 1-5 were organisms most comnonly i so la ted from samples co l lected a t various s i t e s of a k ra f t paper m i l l waste treatment plant. The bacterium included i n the tes t ing was ident i f ied i n the same samples.

Candida u t i l i s i s an unicellar, yeast - l ike organism containing 50% edib le prater - SPENT BLEACHING LIQUORS

A p ine k ra f t pulp (Kappa No. 25.5) was chlor inated and the resu l t i ng pulp extracted w i th a1 k a l i using the conditions described below:

C hl o r i nat ion Caustic ext ract ion

Chlorine o r a l k a l i applied, X 5.25 3.6

Residual chlorine, %* 0,1-0.2 - Cons i s tency , % 3 10

Time, min, 60 60

Temperature, "C 22 70

F ina l pH 1.7 12.3-12.4

*After f i l t r a t i o n through a s intered glass Buchner funnel t o remove the pulp fines, the residual ch lo r ine was completely eliminated.

The ch lor inated pulp was washed w i th d i l u t e (0.01 N) HC1 p r i o r t o the a l k a - l i n e ex t rac t ion stage i n order t o maintain the same pH environment i n the pulp as existed during the ch lor inat ion.

8

FRACTIONATED SPENT BLEACHING LIQUORS

D i a 1 ys i s

Spent ch lo r ina t ion l i quo r was dialyzed i n a cellophane tube against d i s t i l l e d water for 24 hours a f te r f i r s t being concentrated t o 1/10 of i t s o r i g i n a l volume. The r a t i o of spent l i q u o r t o d i s t i l l e d water dur ing d ia ly - s i s was 1:4n. Spent caust ic ex t rac t ion l i q u o r was dialyzed d i r e c t l y under the same condit ions. I n the case of extensive d i a l y s i s of the spent caust ic ex t rac t ion l iquor , the d i a l y s i s was repeated a f te r the dialyzed l i q u o r was concentrated t o i t s o r i g ina l volume. When sodium i o n removal was required, the re ten ta te was fur ther dialyzed against 0.1 N HC1 and then against d is - t i 11 ed water,

Gel Permeation Chromatography

onto the top of a 20 mn x 1 mn column of Sephadex 6-25 (40 g) and developed by e l u t i o n w i th d i s t i l l e d water. The e l u t i o n o f mater ia l was followed by measuring the absorbance of the e luate a t 280 nm.

A concentrated neutral ized sample of spent l i q u o r (10 m l ) was charged

Ether Ex t rac t ion

Three l i q u i d - l i q u i d extractors (400 m l , 1-11 and 10-11 capaci ty) were used depending on the experimental requirements. Spent ch lo r ina t ion 1 iquor was extracted d i rec t l y ; spent caust ic ex t rac t ion 1 iquor was extracted a f te r ac id i fy ing t o pH 2 and without removing the p rec ip i t a te which thereupon formed . S i 1 i c a Gel Chromatography

Ether ext racts of the spent l i q u o r were fur ther f ract ionated on a column of 60-200 mesh s i l i c a gel (Grade 950, Fisher Chemical Company). A layer of anhydrous sodium sul fate was added t o the s i l i c a gel t o remove small amounts of water i n the solvent ext ract . The r a t i o s of sample t o s i l i c a gel and sodium sul fate were 1:150 and 1:25, respect ively. was e lu ted consecutively wi th petroleum ether, d ie thy l ether, and methanol.

CHEMICAL TREATMENT OF CHLOROPHENOLS

The column

Chlor ine Dioxide

Ten mi l l imo les of the phenols i n 30-40% aqueous ethanol were reacted i n the dark a t room temperature w i t h amounts of ch lo r ine d iox ide ranging from 1-15 equivalents/mole of phenol u n t i l the oxidant was completely exhausted. The product mixtures were exhaust ively extracted w i th chloroform t o remove any unreacted phenol and the ext racts were d r ied over anhydrous MgS04, concentrated to %3 m l , and subjected t o gas chromatographic analysis. P r io r t o gas chromatographic analysis, the ex t rac t containing the one catechol tested was s i l y l a t e d as described i n the sect ion Gas Chromatographic Analysis,

9

I n an experiment designed t o t e s t the eff iciency of phenol ox idat ion i n the presence of a bleaching l iquor , a so lut ion of tetrachloroguaiacol (24 mg) was d i l u t e d w i th varying amounts of spent caust ic ex t rac t i on l i q u o r and reacted w i t h 13 mg of ch lor ine dioxide a t room temperature u n t i l the l a t t e r was depleted. The residual phenol was recovered by ex t rac t i on w i t h chloroform and determined by gas chromatographic analysis as described above . Ozone

Ozone was generated w i t h a Welsbach Model LOA-1 PSI corona generator using oxygen as the gas source. Two l - l i t e r gas washing b o t t l e s connected i n ser ies were attached t o the generator. Both bo t t l es were equipped w i t h f r i t t e d glass di f fuser tubes t o provide a small bubble size. The r a t e of ozone generation was determined by placing a 5% K I so lu t i on i n one of the b o t t l e s and measuring the amount of iod ine l iberated i n a given t ime i n t e r - val fo l lowing i n t roduc t i on of the ozone stream.

The phenols (0,207 mnole) were each dissolved i n 500 m l of a 20% ethanol (v/v) s o l u t i o n containing 3 g of a buffer mixture comprised of 90 g of KH2P04 and 8 g of K HPO4. This buffer concentration produced an i n i t i a l

ac t i on period. The phenol solut ions were ozonized a t amhient temperature (22 2 2 O C ) for-30-second i n t e r v a l s i n i t i a l l y and for longer periods near the end of the treatment. After each react ion i n t e r v a l r the ozone was d i - verted t o the atmosphere through a t - j o i n t i n the assembly and a 3 4 sample was withdrawn from the reactor and analyzed by i on i za t i on dif ference spectroscopy (9).

As a check on the accuracy of the above method, the residual phenol content of a few ozonized solut ions was measured by gas chromatography. these tests , 200 ml of a 4.14 x 10-4 M so lu t i on of each model ( i n 20% ethanol) containing 1.2 g o f the same buffer mixture described above was div ided i n t o two equal port ions. One 100-ml por t ion was s e t aside as a con- t r o l and the 100-ml po r t i on was reacted w i t h ozone for one minute a t a flow r a t e of e i t h e r 2.64 o r 3.6 mg/min. A t the conclusion of the react ion, both the con t ro l and ozonized samples were extracted w i th 70 m l of chloroform. The solvent phase was d r ied over anhydrous MgS04, evaporated t o ~3 ml and s i 1 y l ated . used for each ozonizat ion react ion period. The ozonized product mixtures i n t h i s instance were extracted w i t h 250 m l of chloroform. The ex t rac ts were d r ied over anhydrous MgS04, concentrated t o ' ~ 3 ml, and analyzed by gas chromatography.

A ser ies of so lu t ions was prepared, each member of which contained a 0.1 mnole amount of t h i s phenol d issolved i n 100 m l of a buffer so lu t ion 0.08 N i n Na2C03 and 0.02 N i n NaHCO ranging from 0.8 t o 0.8 mg/min. A t the end of t h i s treatment, the product

-

pH of approximately 6. f 5 which stayed v i r t u a l l y constant throughout the re-

I n

I n the treatment of f u l l y e the r i f i ed phenols, a new 50Q-ml sample was

Tetrachloroguaiacol was ozonized i n an a l ka l i ne medium.

(pH 10.3). These so lut ions were reacted a t a flow r a t e

10

mixtures were acidified and extracted w i t h ether. The solvent layers were dried over anhydrous Na2S04, concentrated t o dryness, and the residues re- dissolved i n C H C 1 3 prior to gas chromatographic analysis.

CHEMICAL TREATMENT OF SPENT CHLORINATION AND CAUSTIC EXTRACTION LIQUORS

Chlorine-Containing Oxidants

Elemental chlorine ( i .e. , chlorine water), hypochlorous acid, and chlorine dioxide were applied to spent chlorination liquor and allowed to react t o complete exhaustion a t room temperature. chlorous acid, the reaction mixture was buffered to pH 4.75 by the addition of acetate. Sodium hypochlorite was applied to spent caustic extraction l i - quor a t 60°C and the reaction was continued u n t i l the oxidant was completely consumed.

In the application of hypo-

Ozone and Hydrogen Peroxide

perature u s i n g the ozone generator described above, A 250-ml sample of spent chlorination liquor (pH 2 ) was reacted w i t h ozone introduced a t a flow ra te of 4.65 mg/min. The first 9.3 mg of ozone applied were completely consumed by the liquor under the test conditions. The pH of a 250-1111 sample of spent chlorination liquor was buffered a t 8.0 by the addition of 4 g of KzHP04. The ozone flow ra t e was 4.75 mg/min. and the f irst 16.6 mg of ozone were completely absorbed by the liquor. A 150-1111 sample of spent caustic extraction liquor was reacted w i t h ozone (flow rate , 4.75 mg/min.) a f t e r the pH WAS f i rs t adjusted to 8.0 by the addition of 2 g of KHzP04. Absorp- t ion of ozone was complete up to the addition of 143 mg (1.30 min.) .

cate and magnesium sulfate added, and the mixture reacted w i t h hydrogen peroxide a t 70OC. Consumption of peroxide occurred slowly and not u n t i l a f t e r 24 hours was the applied dosage (4 meq/l50 ml of liquor) to ta l ly consumed.

All ozone treatments of the spent liquors were performed a t room tem-

Spent caustic extraction liquor was buffered (%pH 10.5). sodium s i l i -

Sodium Hydroxide

The pH of spent chlorination liquor was adjusted to various levels by the addition of sodium hydroxide and maintained a t the i n i t i a l level by the addition of more alkal i as required. After a predetermined reaction period a t room temperature, the phenol and organically bound chlorine contents and the acute toxici ty of . +e solutions were determined, as described i n l a t e r sections . Aluminum Sulfate, Lime and Activated Carbon

Aluminum sulfate (1.5 g ) was added to spent caustic extraction liquor

The supernate from the cen-

(220 m l ) and allowed to react for one hour a t room temperature (pH 5.5). The precipi ta te was removed by centrifugation, dissolved by addition of H C 1 , and subsequently dialyzed through cellophane.

11

t r i f uga t i on and the retentate from the d i a l y s i s were d i l u ted t o the volume of the o r i g i n a l t e s t sample, tested f o r phenol content, and bioassayed for acute t o x i c i t y .

Lime treatment of spent caust ic ex t rac t ion l i q u o r consisted of the addi- t i o n of a saturated so lut ion o f slaked l ime (Ca(0H)z) containing 2.2 g of the l a t t e r t o a 220-ml sample o f the l i q u o r (pH 11). The react ion mixture was s t i r r e d for several hours a t room temperature and the p rec ip i t a te re - moved by centr i fugat ion. Carbon dioxide was introduced i n t o the supernate t o p rec ip i t a te the calcium as CaCO . The combined prec ip i ta tes were red issolved by the add i t ion of HC1 and dialyzed through cellophane for two days w i t h frequent changes of water. The volumes of supernate and d i a l y s i s retentate were adjusted t o the o r ig ina l sample volume and the resu l t i ng solu- t ions analyzed fo r phenol content and acute t o x i c i t y .

I n those instances where spent caust ic ex t rac t ion l i q u o r was t reated sequent ia l ly w i t h l ime and act ivated carbon o r a chemical oxidant such as ch lo r ine dioxide, ozone o r hydrogen peroxide, a so lu t ion of slaked l ime (15 g / l ) was added t o the l iquor ; the mixture s t i r r e d for 20 minutes, and then allowed t o stand for one hour. The p rec ip i t a te was removed by f i l t r a - t i o n through No. 41 Whatman f i l t e r paper. Subsequent treatment of the fil- t r a t e was proceeded by pH adjustment t o 9.4 by the add i t ion of carbon diox- i d e and t o 5.0 w i t h HC1. The CaC03 formed a f te r CO2 add i t i on was removed by f i l t r a t i o n .

Treatments of spent caust ic ex t rac t ion 1 iquor w i t h act ivated carbon were performed using a sample of Nuchar S-A provided by Westvaco Corporation. Samples of the l i q u o r were t reated i n a batch process w i th the act ivated carbon fo r 18 hours w i th continuous s t i r r i n g . A t \ the end of t h i s period, the carbon was removed by f i l t r a t i o n through No. 41 Whatman paper.

CHLORINATION AND CAUSTIC EXTRACTION STAGE MODIFICATIONS

I n the sequential ClOZ/C12 treatment, 1.0% of C102 and 2.6% of C 1 were appl ied t o the k ra f t pulp a t room temperature and a consistency o f 3%. These combined amounts were equivalent t o 5.25% ch lo r i ne and the proport ions corresponded t o a 50% replacement of ch lo r ine w i t h ch lo r i ne dioxide. The bleaching was continued u n t i l the ch lo r ine was exhausted ( f i n a l pH, 2.1). Chlor ine d iox ide (2%) alone was applied t o the pulp under the same condi- t ions used fo r the sequential treatment ( f i n a l pH, 2.85).

Peroxide treatment of a chlor inated k r a f t pu lp consisted of apply ing 0.5% H202 (10% cons., 70°C, 3 h r ) a f te r f i r s t ad jus t ing the pH of the bleach t o 11.2. Hypochlori te treatment as a replacement of the conventional caus- t i c ex t rac t i on stage consisted of applying 2% of sodium hypochlor i te t o the pulp (10% cons., 45"C, 1.5 hr, i n i t i a l pH %12.5). dant was consumed t o complete exhaustion a t the conclusion of the react ion per i od .

I n each case, the ox i -

12

The alkal i /oxygen l i q u o r was prepared by treatment of a sample of southern pine k r a f t (Kappa No. 32.9) wi th 2.5% of oxidized white l i q u o r (as NaOH) a t 80 psig oxygen a t 27% consistency for 30 minutes a t 110OC. The l i q u o r used i n the acute t o x i c i t y bioassay was obtained by d i l u t i n g the 27% consistency pulp t o 4% and centr i fuging off the l i quo r . The t o t a l organic carbon (TOC) content of the l i q u o r was 405 ppm.

BIOLOGICAL TREATMENT OF PHENOLS AND CHLOROPHENOLS

Al iquots of stock solut ions of the various phenols ( 3 mg/ml i n 40% ethanol) were chosen, such tha t a f te r d i l u t i o n t o the f i n a l volume (100 m l ) , the concentrat ions of compounds having one o r l ess ch loro subst i tuents and those having two o r more chloro groups were 10 and 50 ppm, respect ively. One m l of each fungal spore suspension (10) was used as inoculum. For each of the phenols tested, a control sample containing no fungus was set up and inoculated w i t h s t e r i l e d i s t i l l e d water, Two rep l i ca tes were prepared a t each concentrat ion l e v e l and a lso for the control .

Flasks containing the solut ions described above were placed on a ho r i - Fol lowing the incubation zontal shaker and incubated a t 28°C f o r one week.

period, the contents of each f l a s k w e r e f i l t e r e d through a Sei tz f i l t e r and the f i l t r a t e s of r e p l i c a t e tests combined t o y i e l d approximately 200 m l of solut ion. I d e n t i c a l volumes of the f i l t r a t e s were extracted w i t h chloroform, and the l a t t e r was concentrated i n vacuo t o 3 m l and d r i e d over anhydrous magnesium sulfate. The dr ied s o F e n t e x t r a c t was subsequently analyzed fo r res idual phenol by gas chromatography as described i n a l a t e r section.

BIOLOGICAL TREATMENT OF SPENT CHLORINATION AND CAUSTIC EXTRACTION LIQUORS

The pH of both the spent ch lo r i na t i on l i q u o r (SCL) and spent caust ic ex t rac t i on 1 iquor (SCEL) were adjusted t o 7.0 w i t h sodium hydroxide and hydrochloric acid, respectively, over a two-hour period. Samples of the l i q u o r s were s t e r i l i z e d by f i l t r a t i o n through a Sei tz f i l t e r f i t t e d w i t h a #6 s t e r i l i z i n g pad. One hundred-ml a l i quo ts of the f i l t r a t e s were subsequently transferred t o s t e r i l e 250-ml Erlenmeyer flasks. One sample of each l i q u o r was inoculated wi th 5.0 m l o f sludge (obtained from a bleached k r a f t m i l l waste treatment plant), yeast o r bac te r ia l c e l l suspension. I n addi t ion, one f lask was l e f t as an uninoculated contro l . The f lasks were placed on a hor izonta l shaker and incubated a t 28OC for 7 days. Fol lowing the incubat ion period, the flasks were f i l t e r e d using a Sei tz f i l t e r f i t t e d w i t h a s t e r i l i z i n g pad. The f i l t r a t e s o f both the t e s t and con t ro l samples’ were then bioassayed for acute t o x i c i t y using Da hnia ma na as the t e s t

p o s i t i o n of the buffer and nu t r i en t solut ions are i n d i v i d u a l l y described below fo r the treatment w i t h sludge (mixed microbia l populat ion) yeast (Candida u t i 1 i s ) and bacterium.

organism. The preparation of the fungal and bacter ++ a1 nocula and the com-

13

S l u d g e (mixed microbial populat ion

was added a s a n i t rogen source and monobasic potassium phosphate a t a con- c e n t r a t i o n of 0.01 g/1, served a s a phosphorous source. A second experiment was conducted i n w h i c h no supplemental nutrients were added t o the l iquor s . In this experiment, monobasic potassium phosphate (0.10 g/1) was added t o the spent c a u s t i c e x t r a c t i o n l i quor (SCEL) and d i b a s i c potassium phosphate (0.10 g/1) was added t o the spent c h l o r i n a t i o n l i q u o r (SCL).

Yeast (Candida u t i l i s )

Monobasic potassium phosphate (0.10 g/1) was added t o the spent caus- t i c e x t r a c t i o n l i q u o r and d i b a s i c potassium phosphate (fl.19 g/1) was added t o the spen t c h l o r i n a t i o n l i quor . - C . u t i l i s could no t grow i n spent bleaching l i q u o r s i n w h i c h no supplemental n u t r i e n t s were added, added t o the l i q u o r s .

In the i n i t i a l experiment,asparaginer a t a concent ra t ion of 0.2 g / l ,

Prel iminary experiments showed t h a t

Therefore, glucose a t a concent ra t ion of 1.0 g/1 was

Inoculum was prepared according t o the fol lowing procedure: Several l oopfu l s of a 3-day-old culture of C. u t i l i s (NRRL Y-900) growing on y e a s t morphology s l a n t s were dispersed i n - 5 0 m ster i le d i s t i l l e d water i n a 125-1111 Erlenmeyer f l a s k . Enough y e a s t cel ls were added t o y i e l d a cell suspension having an absorbance reading of 0.32 a t 540 nm measured on a Spectronic-20 spectrophotometer.

A Bacterium

Mono and d i b a s i c potassium phosphate were added t o samples of the f i l t e r - s t e r i l i z e d SCEL and SCL, r e spec t ive ly , a t a concent ra t ion of 1 .O g/1. In a d d i t i o n , g lucose a t a concen t r a t ion o f 1.0 g/1 was added t o both l i quor s .

Several loops of a 4-day-old bac- terial culture (Ziobro, culture 2-R) growing on nutrient aga r slants were d ispersed i n 2@ ml of s ter i le d i s t i l l ed water. Enough cel ls were added t o y i e l d a cell suspension having an absorbance reading of 0.45 a t 540 nm, measured on a Spectronic-20 spectrophotometer.

Inoculum was prepared a s follows:

ACUTE TOXICITY TESTS

Daphnia magna

either f i r s t i n s t a r o r 1-3 day o ld animals . tests was DM2 prepared from doubly d i s t i l l ed water (5 ) . a pH of 7.n and was ae ra t ed t o s a t u r a t i o n p r i o r t o use. of the va r ious tes t compounds were prepared by d i s so lv ing the appropr i a t e amount of the ma te r i a l i n a s u i t a b l e s o l v e n t (DMSO) and adding the desired volume of DMSO/toxicant s o l u t i o n t o the d i l u t i o n water. were f i r s t ad jus t ed t o pH 7.0-7.5 through the a d d i t i o n o f NaOH o r HC1 imme- d i a t e l y p r i o r t o i n i t i a t i n g the test .

Acute t o x i c i t y tes ts were conducted on the var ious m a t e r i a l s using The d i l u t i o n water used i n the

This s o l u t i o n had Stock s o l u t i o n s

Spent l i q u o r samples

The t o x i c a n t s o l u t i o n s were then

14

s e r i a l l y d i l u t e d t o the required concentrat ion levels. Each t e s t consisted of 5-6 concentration l eve l s w i t h d i l u t i o n factors of 0.5-0.9 between each concentration and 10 organisms per concentration. A1 1 tes ts were conducted a t 17 + 1°C i n a constant temperature bath. lack of gross movement was used as the-cri t e r i o n for death. jected t o p r o b i t analysis (11) using the Bio-Med computer a t Syracuse Uni- v e r s i t y o r through the use of tables (12.13) based on the moving average method o f Thompson (14).

Concentration-response patterns were e i t h e r sub-

Brine Shrimp

Before i n i t i a t i n g each assay, the spent l i q u o r s were adjusted t o pH 7.4-7.6 w i t h e i t h e r NaOH or HC1 and the s a l i n i t y was adjusted t o t h a t of the standard sea water formulation (22 parts/thousand) by adding NaC1. The organisms were exposed t o t e s t volumes of 50 m l i n Carol ina cu l tu re dishes having dimensions of 3.5 x 1.5 inches. Each l i q u o r was tested a t f i v e concentrat ions w i t h 20 organisms per concentrat ion and a d i l u t i o n factor of 0.5 between concentrations. During the tests, the pH ranged from 7.2 t o 7.6 and temperature ranged from 20 t o 22°C. M o r t a l i t y was recorded a f te r 24 hours and mortality-response patterns were subjected t o p r o b i t analysis (11).

Fungi

The basal medium f o r the hioassy consisted of a s o l u t i o n of 3.00 g of glucose, 1.25 g of peptone, and 1.25 g of yeast e x t r a c t i n 1000 m l of d i s - ti-lled water (pH 6.5). Each t e s t compound was dissolved i n 40% ethanol and maintained a t a stock concentrat ion of 3.0 mg/l (3000 ppm). The stock solu- t i o n was s e r i a l l y d i l u t e d t o concentrat ion l e v e l s of e i t h e r 200, 100, 50, 10 and 0 ppm (contro l ) o r 50, 25, 10, 7.5 and 0 ppm (control),depending upon the number of ch lor ine atoms i n each compound. Af ter d i l u t i o n of the t e s t so lu t i on w i t h the basal medium and add i t i on of the spore suspension, the f i n a l volume was 21) m l and the concentrat ion of ethanol, 2%.

F ive r e p l i c a t e samples of e igh t phenols were prepared for assay of As e r i l l u s fumi atus. Two r e p l i c a t e samples o f 17 phenols were tested h h i o n a l species of fungi used i n the invest igat ion. The t e s t so lu t ions were placed on a hor izonta l shaker and incubated a t 28°C for 3 days. Growth was measured by v isual observation and by d ry weight deter- minat ion of the fungal mycelia.

Acute t o x i c i t y bioassays of spent c h l o r i n a t i o n and caust ic ex t rac t i on l i q u o r s were a lso performed,using s i x c m o n fungi as the t e s t organisms. P r i o r t o test ing, the pH of the two l i q u o r s was adjusted t o near n e u t r a l i t y by the add i t i on of NaOH o r HC1. Dupl icate samples o f each o f the whole liquors and samples d i l u t e d t o 1/2, 1/4 and 1/10 o f t h e i r i n i t i a l volumes were added t o a glucose-peptone-yeast medium and one m l o f the various spore suspensions added t o each. The samples were placed on a hor izontal shaker and incubated for 7 days a t 28°C. Growth o f the various cu l tures was e s t i - mated v i sua l l y .

15

A1 ga

The green alga, Chlore l la was grown i n modif ied Jack i n acid-clean f lasks on a ro ta ry Myer's medium for Chlore l la and

shaker (150 rpm) a t 18°C and under an i l l um ina t ion of 650 foot-candles of continuous l i g h t . "Algal Assay Procedur : B o t t l e Test" (15), except t h a t a l a rge r amount of

and the f i n a l concentrat ion of ethanol i n the c u l t u r e medium was 0.1%. The absorbance of the a1 ga i n the growth medium, measured spectrophotometrically a t 650 nm, was used as a measure of the amount of growth.

Inoculum was prepared as described i n the EPA publ icat ion,

inoculum was used (10 E cel ls/ml) . Test chemicals were dissolved i n ethanol

The i n i t i a l concentrations of the phenols tested were 100, 10, 1 , and 0.1 ppm o r 10, 1, 0.1, and 0.01 ppm depending on the ant ic ipated degree of t o x i c i t y . The range of t o x i c i t y was determined t o be from the l eve l of concentrat ion a t which no growth occurred t o t h a t where some growth occurred, even if i t was reduced from tha t of the control . T o x i c i t y l e v e l s were de- l ineated more prec ise ly by select ing and t e s t i n g a v a r i e t y of concentrations from w i t h i n the previously determined t o x i c i t y range. The absorbance of the alga i n the growth medium (measured spectrophotometrical ly a t 650 nm) was used as a measure of the amount of growth a t the end of 12 t o 14 days.

T o x i c i t y t e s t s of spent ch lo r i na t i on and caust ic ex t rac t i on 1 iquors were performed i n a manner s im i la r t o those described above for the phenols w i th the fo l lowing modif icat ions: The l i q u o r s were w i t h NaOH o r HC1, buffered w i t h phosphate and s t e r i l i z e d w i t h a Sei tz f i l t e r p r i o r t o being tested. The intense co lo r of the l i quo rs prevented ef fect ive use of spectrophotometry t o record the resul ts. As an a l t e r n a t i v e approach, d i r e c t counts from a hemocytometer were used. were tested.

Liquor d i l u t i o n s of 1:2, 1:4 and 1:8

Duckweed

Lemna e r u s i l l a (duckweed). The t e s t phenols were dissolved i n 4% DMSO.

of 20, 15, 10 and 5 ppm. 4,5-Dichlorocatechol and 2,4,6-trichlorophenol were tested a t concentrations o f 10, 5, 1, and 0.1 ppm, Each f lask con- t a i n i n g 50 m l of the c u l t u r e medium and the phenol so lu t i on was inoculated w i t h a 3-frond colony o f L. er u s i l l a . The f lasks were incubated a t 2 5 O C

of fronds was counted using a stereoscopic microscope.

One half. strength o f Hutner's medium plus sucrose was used for growing

--fe-- 2,4-Dich orophenol; and 4,5-dichloroguaiacol were tested a t concentrations

+ T - T 59 oot-candles for 10 days, The number under a constant i l luminaTion o

GAS CHROMATOGRAPHIC ANALYSIS

Dehydroa b i e t i c Ac i d

Pine k r a f t pulp (Kappa No. 25.5) was washed w i t h water, dewatered t o 36.5% consistency and extracted consecutively for 24-hour periods w i t h 8000-, 5000-, and 5000-ml amounts o f sodium hydroxide so lu t i on (pH 10.5) a t room temperature. A f t e r each treatment, the pulp was f i l t e r e d and washed

16

by performing the acety la t ion a t room temperature f o r 17 hours.

Molecular weights were determined using a Hewlett-Packard Model 302 vapor pressure osmometer. A non-aqueous probe thermostatted a t 6 5 O C was used and the samples were dissolved i n methyl cel losolve. Benzil was used as a ca l i b ra t i on standard.

a Chloride was measured by potentiometric t i t r a t i o n w i th standard AgNO solut lon as described by Shiner and Smith (19) and modified by Braddon an Dence (20). Organical ly bound ch lor ine was determined by applying an appro- p r i a te s ize sample (adjusted t o pH 8 if not i n i t i a l l y a lka l ine) on a 5.5 cm No. 1 Whatman f i l t e r paper whi le simultaneously evaporating the water w i th the a i d of a h a i r dryer. The dr ied f i l t e r paper was burned i n a Schoniger combustion f lask containing 50 m l of d i s t i l l e d water. Potentiometric ti t r a - t i o n of the resu l t i ng so lut ion w i th standard AgN03 so lu t ion provided the t o t a l ch lor ine (inorganic and organic) i n the sample. The organical ly bound ch lor ine was calculated by subtract ion o f the inorganic chlor ide con- ten t (see above) of the o r i g i n a l sample from t h i s value.

Chlorine, ch lor ine dioxide, ozone and hydrogen peroxide were determined iodometrical ly. lyzed by the addi t ion of ammonium molybdate.

I n the case of hydrogen peroxide, the reduction was cata-

Ion iza t ion di f ference spectra were recorded on a Unicam Model SP80OA recording spectrophotometer, a 1- o r 10-mn l i g h t path were used.

Semimicro matched quartz c e l l s having e i ther

18

SECTION 5

RESULTS AND DISCUSSION

ACUTE TOXICITY OF PHENOLS

The phenols and phenol ethers used i n the i n v e s t i g a t i o n a re l i s t e d i n Table 2, Thei r se lec t i on was based on a number o f c r i t e r i a , i nc lud ing main ly s t r u c t u r a l i d e n t i t y o r s i m i l a r i t y t o compounds prev ious ly i d e n t i f i e d i n spent c h l o r i n a t i o n and caust ic e x t r a c t i o n l i q u o r s and a des i re t o achieve a broader understanding of t h e s t r u c t u r e - t o x i c i ty r e l a t i o n s h i p s p e r t a i n i n g s p e c i f i c a l l y t o these types of phenols and chlorophenols. The phenols l i s t e d i n Table 2 thus cons is t p r i n c i p a l l y of t he var ious ch lo r i ne - s u b s t i t u t e d d e r i v a t i v e s o f mono- and d i h y d r i c (i ,e., catechol) r i n g types. F u l l y methylated s t ruc tu res were u t i l i z e d on ly t o a l i m i t e d degree and that s o l e l y f o r t he purpose of gauging the ef fect of a f ree phenol ic hydroxyl group i n a p a r t i c u l a r measurement o r treatment.

I n t h e i n i t i a l phase of t he p ro jec t , the phenol ic compounds studied contained a methyl subs t i t uen t a t the p o s i t i o n para t o a phenol ic hydroxyl group ( L e . , a t t h e s ide chain pos i t i on ) . Later, these compounds were abandoned i n favor of s t ructures conta in ing,a c h l o r o subs t i t uen t a t t h e para p o s i t i o n s ince the l a t t e r types were concur ren t ly being i d e n t i f i e d i n spent c h l o r i n a t i o n and caust ic e x t r a c t i o n l i q u o r s (21-23) . The general behavior of t he two aforementioned s t r u c t u r a l types w i t h respect t o reac- t i v i t y and acute t o x i c i t y probably do no t d i f f e r great ly , e s p e c i a l l y when a comparison i s made between compounds having I d e n t i c a l numbers of ch lo ro subs t i t uen ts on the same type of phenolic r ing .

Effect on Fungi

A d e t a i l e d study was made of the ef fect of e i g h t phenols on As e i l l u s

These p l o t s reveal t h a t i n h i b i t i o n of fungal growth increased I n p ropor t i on t o the number of ch lo ro subst i tuents attached t o t h e phenol ic r i n g . 2,4,6- Tr ichlorophenol ( V I , Table 2) had the greatest e f f e c t of t h e phenols tes ted i n reducing the growth o f the fungus.

fumi atus. The p l o t s shown i n Figures 1 and 2 i n d i c a t e the ef fect 4i4;1;---- o * cress ng concentrat ions of t he phenols on the amount of fungal growth.

Expansion of the t e s t i n g t o inc lude f i v e a d d i t i o n a l fungi and a t o t a l of 17 phenols produced the r e s u l t s shown i n Table 3. used i n t h e t e s t i n g had been prev ious ly found t o be present i n water sampled a t var ious s i t e s of a bleached k r a f t m i l l waste treatment p lan t , Although

Each of t he fungi

19

TABLE 2. PHENOLIC COMPOUNDS USED IN THE INVESTIGATION

f 3

OH

I R,=R,=R,=H I1 R,=R,=H; R,=CH, I1 RI=RS=H; R,=C1 IV R,=R,=Cl; R,=H V R,=R,=Cl; R,=CH,

VI R,=R,=R,-Cl

OH

VI1 R,=R,=R,=H VI11 R,=C1 ; R,=R,=H

IX R,=R,=H; R,=C1 X R,=R,=Cl; R,=H

XI R,=R,=Cl; R,=H XI1 R,=H; R,=R,=Cl

XI11 R,=R,=R,=Cl

OH

XVII R,=R,=R,=H XVIII R,=R,=H; R,=C1

XIX R,=R,=Cl; R,=H XX R,=R,=R,=Cl

XXI R,= R,uH; R2=R,=Cl XXII R,=R,=R,=Cl; R,=H

XXIII R,=R,=R,=R,=Cl

XIV R1=R,=H; R,=R,=Cl XV R,=R,=RS=C1 ; R,=H

XV I R , =R ,=R , =R , =C 1

XXIV R,=R,=R,=H XXV R,=Cl; R,=R,=H

XXVI R,=H; R,=R,=Cl XXVII RI=R,=R,=C1

20

a " T R A T I O N OF PHENOL, ppm

Figure 1 . Effect of 2B4B6-trichloropheno1 ( V I ) and three chlorocatechols on the growth of s e r i l l u s fumi atus. a re based on f ive Bfg----* rep cates. e r ca bars represent the standard er ror of the rep1 icates. )

(Values for dry weight

21

Figure 2.

J 0 e l

IO 50 I00 I50 200 CONCENTRATION OF PHENOL, ppm

Effect of creosol (VII) and three chloroguaiacols on the growth of As e r i l l u s fumi atus. (Values for dry weight are based on f ive -+-+ rep cates. e r t ca l bars represent the standard error of the repl icates. )

22

---

TAR1 F 3. -IN p p ~ ] OF PHFW s R E O U ~ F D TO. PRFVFNT GROMTH OF VARIOUS FUNGI

I I1 V V I V I1 VI11 I X X

Tr i c hoderma koningii > b200 25 25 >200 100 100 25

Aspergi 11 us n iaer > 200

N * w > 200 ,200 As e r i l l u s

Paeci lomyces v a r i o t i > 200 >200

50

25

10 >200

10 ,200

50 10 >200

100

100

100

200

200

200

25

25

50

qp&!!! > 200 >200 25 7.5 50 50 25 >200

Penici l l ium var iabi 1 e > 200 50 25 200 100 25

I n Ppm a

b>Indicates growth was evident a t the highest concentration tested.

(conti nued )

TABLE 3 (continued)

X I X I I XIII X V I I X V I I I X I X xx X X I V xxv ~ ~~~

25 >50 100 109 >50 50 >200 200 b > 50 T r i c hodenna

koningi i

Aspergillus n i ger > 50 25 25 >200 200 >50 50 >200 >200

> 50 25 25 >200 200 >50 25 >200 >200

N Aspergil lus

f mi ga tus

Paecilomyces var i o ti

Cladosporium herbarum

50

25

25

25

25

10

‘>200

100

200

100

> 50

25

25

7.5

>200

100

>200

100

> 50 50 >50 >200 100 >50 25 >200 >200 Peni c i 11 i um var ia b i 1 e

-

b >Indicates growth was evident a t the highest concentration tested.

some var iat ions occurred i n the response of the isolates, the f indings gen- e r a l l y duplicated those noted above for As e r i l l u s fumi atus w i th respect

sion of fungal growth. An exception t o t h i s general trend was noted i n the case of the veratrole der ivat ives ( X X I V - X X V I I , Table 2) where increasing subst i tu t ion of ch lor ine appeared t o exert no noticeable effect on fungal growth.

The data i n Table 3 once again underscore the strongly i n h i b t t o r y effect of 2,4,6-trichlorophenol. The potency of the l a t t e r compound as a toxicant i s well-documented and i t i s found on the EPA's l i s t of hazardous chemicals. A t lower leve ls of ch lor ine substi tut ion, the effect of the phenols on fungal growth was similar, provided the comparison was made between compounds having the same number of chloro substituents.

Among the s i x fungi tested, Clados orium appears t o be the l e a s t to le ran t t o the applied phenols. 7--e-T; n genera however, t h i s fungus and others are more res is tan t t o phenols o f the type tested than are fish, i n - vertebrates, and other aquatic organisms.

t o the effect of an increasing number 0 *+ c or0 su s t tuents on the repres-

Effect on the Alga Chlorel la pyrenoidosa

The t o x i c i t y range of selected phenols toward an alga, Chlorel la pyrenoidosa, was determined and the resu l ts obtained are presented i n Table 4.

TABLE 4. TOXICITY RANGES OF SELECTED PHENOLS FOR CHLORELLA PYRENOIDOSA

Phenol Concentration, ppn Growth Presenta Growth Absent

Catechol (lS2-dihydroxybenzene) 20 40 2,4-Dichlorophenol ( I V ) 8 10 4.5-Dic h l orocatechol ( X X I ) 2 4 4.5-Dic h? orogua iacol ( X I V ) 1 10 Trichlorocatechol ( X X I I ) 0.1 1

'Determined w i t h a Spectronfc-20 spectrophotometer and checked by c e l l count using a hemocytometer.

As i n the previous tests w i t h fungi, the enhancement of t o x i c i t y re- s u l t i n g from the in t roduct ion o f chloro substi tuents on the phenolic r i n g i s c l e a r l y evident. On the basis of the l i m i t e d data i n Table 4, the phenol concentration leve ls a t which the Chlorel la ceased t o grow are consider- ab ly lower than those f o r s imi lar c o m p m t e d against fungi. For ex- ample, a concentration o f 1 ppm tr ichlorocatechol completely inh ib i ted the growth o f Chlorel la whereas i n the case of the s i x fungi tested (cf. Table

25

3), the corresponding range was 7.5-50 ppm. t i v i t y , Ch lo re l l a i s t he re fo re more su i tab le f o r use as a bioassay microorganism i n the t e s t i n g o f low concentrat ions of phenols.

Effect on the Vascular P lan t -- Lema p e r p u s i l l a (Duckweed)

Because o f i t s g rea ter sensi-

Duckweed i s a small aquat ic p l a n t t h a t i s wide ly d i s t r i b u t e d throughout the Uni ted States. bioassays was examined by determining the e f f e c t o f various concentrat ions of four phenols on i t s growth. Table 5.

I t s u t i l i t y as a t e s t organism i n acute t o x i c i t y

The r e s u l t s o f these t e s t s are shown i n

2,4,6-Trichlophenol ( V I ) was again found t o cause the grea tes t suppression o f growth i n the l i m i t e d ser ies o f phenols tes ted and, a t a concen- bfaCippmgfa6dgpm, i t t o t a l l y i n h i b i t e d growth. The suppression o f growth the rea f te r decreased i n the order o f 4,5-dichlorocatechol (XXI) , 4,5-dichloroguaiacol ( X I V ) , and 2,4-dichlorophenol ( I V ) .

Based on a comparison o f a l i m i t e d number o f phenols, therefore, Lemna 1

appears t o be more s e n s i t i v e t o such compounds than fungi , bu t l e s s sensi- t i v e than Ch lore l la . However, duckweed i s much eas ie r t o work w i t h i n the labora tory and as a b i o l o g i c a l t e s t organism, i t i s l ess expensive t o use.

E f fec t on Daphnia magna

The response o f D?phnia ma na t o chlorophenols s i m i l a r t o o r i d e n t i c a l w i t h those i d e n t i f i e d i n spent 4m c o r i n a t i o n and caus t ic e x t r a c t i o n l i q u o r s i s shown by t h e data compiled i n Table 6. Durkin (24) has found t h a t several o f t he phenols l i s t e d i n t h i s t a b l e i n t e r a c t w i t h themselves, w i t h c e r t a i n u n i d e n t i f i e d components o f spent c h l o r i n a t i o n l i q u o r , and w i t h sodium c h l o r i d e i n low concentrat ions, thereby a l t e r i n g somewhat the magnitude o f t he values repor ted i n t h e tab le . This f i nd ing has impor tant imp l i ca t i ons regarding the v a l i d i t y o f attempts t o account f o r t h e t o t a l t o x i c i t y o f a p a r t i c u l a r l i q u o r through the sum o f e f fec ts o f i n d i v i d u a l t o x i c components; nevertheless, t he f i nd ing does no t i n v a l i d a t e the more pronounced trends ind ica ted by the data i n Table 6.

t he acute t o x i c i t y o f the various chlorophenols increased i n p ropor t i on t o the number o f ch lo ro subs t i tuents on the phenolic r i ng . a l so noted above i n the d iscuss ion o f t he response o f p l a n t t e s t organisms t o many o f t h e same phenols. the ch lorocatechols ( X V I I I , X X - X X I I I , Table 6) seem t o have the grea tes t e f f e c t on t h e Da hnia. Somewhat su rp r i s ing l y , 2,4,6-trichlorophenol (V I ) ,

proved t o be somewhat l ess t o x i c than 3,4-dihydroxy-2,5,6-trichlorotoluene (XX) and 3,4,5-trichlorocatechols (XXI I ) i n t h e t e s t s w i t h Daphnia. catechols d id , however, have a pronounced e f f e c t on r e t a r d i n g the growth o f the p l a n t s exposed t o them (see Tables 3-5).

I n a recent i nves t i ga t i on ,

One of t h e more cons is ten t trends shown by the data i n Table 6 i s t h a t

Th is same t rend was

As a class, and w i t h respect t o acute t o x i c i t y ,

which ranked as + aving a very t o x i c e f f e c t on the p l a n t t e s t organisms,

Chloro-

26

TABLE 5. EFFECT OF FOUR PHENOLS ON THE’ VEGETATIVE GROWTH OF LEMNA PERPUSILLA~ - Cone en t r a -

t ion , PW Number of Fronds, 4; of Control

0.1 I ’ 5 10 15 20 Phenol 0.0 (control ) CI

C I Q 70 29 10 7 OC b I V 100 -

X I V 1 00 77 55 20 10 7

xx I 100 95 95 20 0

V I 100 94 31 0 0 - - CI

’Ten days of incubation bNot tested ‘No growth

TABLE 6. ACUTE TOXICITY OF VARIOUS PHENOLS TO pAPH"AGNA 24 hr LC50 or EC59

(95% confidence interval), a Compound IJ moles/l

2.4-Dic hlorophenol IV 2,4,6-Tric hl orophenol VI 4-Methyl guaiacol VI I 6-C hl oro-4-methyl gua i acol VI I I 5-C hloro-4-methyl guaiacol IX 5.6-Di c hl oro-4-met hyl gua i acol X 2,5-Dichloro-4-methylguaiacol XI 2,6-Dichl oro-4-methyl guaiacol XI I 2 , 5, 6-Tri c hl oro-4-methyl guaiacol XI I I 4.5-Dichloroguaiacol XIV 4,5,6-Tric hloroguaiacol XV Tetrac hl oroguaiacol XVI 3,4-Dihydroxytoluene XVII 3,4-Di hydroxy-6-chlorotoluene XVI I I 3,441 hydroxy-2.5.6-tric hl orotol uene XX 4,5-Dic h l orocatec hol XXI 3,4,5-Tric hl oroca tec hol XXI I Tetrachlorocatechol XXIII 4-Methylveratrole XXIV 5-C hloro-4-methyl vera trol e XXV 2.6-Dic hl oro-4-methyl veratrol e XXVI 2,5,6-Trichl oro-4-methylvera trol e XVI I

51.9 (40.2-90.1)b 13.7 (11 .4-16.0)b

49.9 (16.4-xc) 177 (1 9. 5-xc)

150 (76.1-1470)

13.0 (6.77-75.4) 25.5 (13.4-212) 14.6 (8.79-46.0) 7.45 (2.74-xc) 98.3 (79.0-1 35)

b

4.96 (4.19-6.21 )b

22.0 (1 5.6-25.5)

27.6 (21,O-36.5) 13.8 ( 7.6-25.1) 4.90 (4.46-5.30)

' 6.64 (4.76-7.81)b 3.39 (2.92-3.86) 2.23 (l.25-2.80)b 1200 ( 557-2640) 46.3 (20-xC) 2n.7 (6.89-x') 2.36 (1.08-3.53)

0.424 I). 206 0.403 0.905 2.63 0.477 0.438 0.290 1.16 0.309 0.261 0.230

d d d

- - -

0.240 0.222 0.319 0.472 0.520 0.873 0.262

'As discussed by Finney (11). the "g" value can be used as an index of the

In general, "g" values quality of the bioassay, i.e., how well the observed mortality pattern cor- responds to the best estimate of the probit line. of 0.4 or less indicate good agreement. bLC50 values; all others, EC5 . The LC5 values are based on cardiac arrest

'Meaningful upper limit not available due to low mortality at highest con- d

whereas the EC50 values rela ? e to immob 0 lity as response criteria. centration tested. Not determined.

28

The isomeric dichloro-4-methylguaiacols (X-XII) show s t a t i s t i c a l l y i n - s i g n i f i c a n t d i f fe rences i n t h e i r LC5ovalues, a f ind ing genera l ly dupl icated when fung i were used as t e s t organisms (see Table 3 ) . On the other hand, the e f f e c t o f increas ing numbers of ch lo ro subst i tuents t h a t was apparent i n increas ing the t o x i c i t y of the vera t ro les (XXIV-XXVII) t o Daphnia, was n o t observed i n the treatment o f the fungi w i t h representat ives o f t h i s c lass. Vera t ro le der iva t ives are a typ ica l o f the phenols detected i n spent c h l o r i n a t i o n and caust ic e x t r a c t i o n l iquors ; the observed d i f fe rence i n the response o f the fung i and Daphnia toward vera t ro le d e r i v a t i v e s probably has l i t t l e s ign i f i cance f o r our purposes i n the present inves t iga t ion .

ACUTE TOXICITY OF WHOLE AND FRACTIONATED SPENT CHLORINATION LIQUOR (SCL) AND CAUSTIC EXTRACTION LIQUOR (SCEL)

I n t h i s segment o f the inves t iga t ion , the t o x i c i t y o f the whole spent c h l o r i n a t i o n and caus t ic e x t r a c t i o n l i q u o r s was examined us ing a fungus

been c r i t i c a l l y discussed by Windaus and Petermann (25). Da hnia magna

ca t ions and because i t has been shown t o g ive r e s u l t s t h a t have a good cor- r e l a t i o n w i t h bioassay data t h a t i s based on the u t i l i z a t i o n of f i s h (26).

As e r i l l u s fumi atus) and an aquat ic inver tebra te (Daphnia ma na) as t e s t *T&ty o f the use o f fungi i n acute t o x i c i t y 4- ioassay has

was se lected on the bas is o f i t s comparative s i m p l i c i t y i n -8- ioassay a p p l i -

I n an e f f o r t t o i d e n t i f y and de f ine more thoroughly the chemical and phys ica l c h a r a c t e r i s t i c s responsible f o r t o x i c i t y , the whole 1 iquors were subsequently f ract ionated by var ious means and the i n d i v i d u a l f r a c t i o n s sub- j e c t e d t o acute t o x i c i t y bioassay and, t o a lesser degree, chemical analy- s i s . I n t h i s phase o f the inves t iga t ion , the cont r ibu t ions o f the var ious f r a c t i o n s t o the t o x i c i t y o f the whole l i q u o r s were ca lcu la ted us ing the " t o x i c u n i t " method described by Sprague (27). app l ied throughout the course o f t h i s work, the t o x i c u n i t content was c a l - cu la ted as fo l lows:

As def ined by Sprague and

Total Organic Carbon (TOC) o f Test Sample (ppm) expressed as TOC ( PPm 1 Toxic U n i t s (T.U.) =

LC50

S t a b i l i t y o f SpentChlor inat ion L iquor (SCL) and Caustic E x t r a c t i o n L iquor [ SCEL 1

subject ing the spent c h l o r i n a t i o n and caus t ic e x t r a c t i o n 1 iquors t o var ious f r a c t i o n a t i o n s , the s t a b i l i t y o f those l i q u o r s w i t h respect t o c o l o r , BOD5, o r g a n i c a l l y bound ch lor ine, and acute t o x i c i t y on storage was determined i n order t o evaluate the changes t h a t would occur s o l e l y as a r e s u l t o f a p a r t i c u l a r form o f treatment.

BOD t e s t s were performed on samples o f the spent c h l o r i n a t i o n and caus- t i c e x t r k t i o n l i q u o r s t h a t ranged i n age from 1 t o 26 days. No s i g n i f i c a n t t rends were observed i n e i t h e r case, and the average values f o r spent c h l o r - i n a t i o n and caus t ic e x t r a c t i o n l i q u o r s were 125 and 437 mg 02/1, respect ive ly . As shown by the p l o t s i n Figure 3, the c o l o r o f both caus t ic e x t r a c t i o n l i q u o r s decreased a t a greater r a t e i n i t i a l l y than i t d i d a f t e r prolonged standing. The d i f f e r e n c e i n t h e c o l o r o f the two e x t r a c t i o n l i q u o r s

29

c C

SCEL - (CHLDRINATED PULP PRE-WASHED WITH 0.01 N HCI) r\ v

WASHED WITH TAP WATER)

I 1 I I

I I

I I

I I

0 4 8 12 16 20 24 DAYS

Figure 3. Effect of age on the color of spent chlor inat ion and caustic extract ion l iquors stored a t room temperature.

is a t t r ibuted t o the greater retention of solubilized material i n the chlorinated p u l p washed w i t h d i lu te acid as compared to the one washed w i t h tap water. vir tual ly constant throughout the t e s t period.

In contrast , the color of the spent chlorination l iquor remained

The s t a b i l i t y of the organically bound chlorine i n the two liquors i s shown by the p l o t s i n Figure 4 . The i n i t i a l loss of chlorine i n the case of the spent caustic extraction l iquor i s seen t o be substantial a t both 4" and 20°C. The spent chlorination l iquor a lso lo s t organically bound chlorine on storage a t room temperature b u t a t approximately only 1/3 the r a t e observed for the caustic extraction liquor. was reduced as the pH of the liquor was progressively decreased.

The loss of organically bound chlorine

In the acute toxici ty determinations, samples of spent chlorination l iquor (pH 2 ) were stored i n the dark a t 5°C and periodically tested against Daphnia magna. 24-hr 24 LC50 values were not s t a t i s t i c a l l y s ignif icant . When allowed t o stand a t room temperature for 37 days, the same liquor likewise showed l i t t l e s ignif icant change i n acute toxicity. After 80 days' storage, however, a s l i g h t decrease i n acute toxicity was observed, b u t fur ther testing a f t e r more prolonged standing i s required to confirm this trend. i n g t o note, however, tha t should the trend toward decreased toxici ty on standing be confirmed, i t would parallel the decrease i n organically bound chlorine content for the liquor noted above and m i g h t indicate a cause and effect re1 a t i ons h i p .

For periods of storage u p to 39 days, the variations i n

I t i s interest-

Spent caustic extraction liquor was also stored i n the dark a t room temperature and intermittently monitored for acute toxici ty . After 80 days, no s ignif icant trend w i t h respect t o toxici ty change was observed. In this s i tuat ion, loss of organically bound chlorine appears to have had no ef fec t on acute toxicity.

The apparent s t a b i l i t y of the spent chlorination liquor w i t h respect t o acute toxici ty i s probably due, t o a large extent, t o i t s h i g h acidity. As will be shown l a t e r , a lkal i treatment of this l iquor had the effect of greatly decreasing i t s toxicity i n a re la t ive ly short period of time. Smaller increases i n the pH of spent chlorination liquor of the order of magnitude produced by d i l u t i n g i t w i t h chlorinated p u l p washings would un- doubtedly have produced a similar e f f ec t , a l b e i t over a greater period of time,

Collectively, the above f i n d i n g s indicate tha t although some of the properties of spent chlorination and caustic extraction 1 iquors change on standing, acute toxici ty testing can be performed on reasonably aged samples of such liquors w i t h o u t changes occurring i n the course of the tes t .

31

w ru

SCEL, 2OoC /

I I I I I

20 f I 4 8 12 16

I I

I I

I I

I I

I I

20 f I 4 8 12 16

SCEL, 4 O C

2 0 ° C

DAYS

+SCEL, 2OoC

+ SCEL, 4 O C

I I

I I f54 58

Figure 4. The rate of loss of organically bound chlorine for spent chlorination and caustic extraction liquors.

Responses of the Test Organisms t o the Whole Spent Ch lor ina t ion L iquor (SCL) and Spent Caustic Ex t rac t i on L iquor (SCEL)

A t t h e i r o r i g i n a l concentrations, both the spent c h l o r i n a t i o n l i q u o r and spent caus t i c e x t r a c t i o n l i q u o r s were d i s t i n c t l y t o x i c t o Da hnia magna. As seen i n Table 7, t he 24-hr LC

l e s s t o x i c than the SCL when compared on the same (TOC) basis r e s u l t has been r e c e n t l y repor ted by P f i s t e r and Sjostrom (28). u n i t s (TOC/LC 0) were ca lcu la ted as 3.21 and 4.12 fo r SCL and SCEL, respec-

u n i t s by t h e t o t a l l i q u o r volumes which are regulated by the pu lp consis- tenc ies of 3% and 10% f o r c h l o r i n a t i o n and caus t ic ext ract ion, respec t ive ly . The t o x i c i t y loads a r e about 104 (SCL) and 37 (SCEL) t o x i c u n i t s per kg of

value of the SCL i s about s i x +- t mes smal ler than t h a t of t he SCEL, w io i c h means t h a t the SCEL i s considerably

t i v e l y . The $ o t a l t o x i c i t y loads were ca lcu la ted by m u l t i p l y i n g the t o x i c

A s i m i l a r The t o x i c

P u l p .

Table 7. ACUTE TOXICITY OF THE WHOLE SCL AND SCEL TO DAPHNIA MAGNA - scLa S C E L ~

Test 24-hr LCs0, 24- h r LC50 No. TOC, Ppn ppm of TOC TOC, ppm ppm of TOC

1 222 49 1492 41 9

- 2 249 67 1900 301

3 21 5 94 1577 472 4 249 96 1553 420

5 248 44 1621 440 6 230 43 1845 4 24 7 223 91 1624 41 2

8 21 4 90 1480 285 xc 231 72 1637 397 15 24 155 67

d 0

Se 6 33 9 17

aspent c h l o r i n a t i o n l i q u o r bspent caus t i c ex t rac t i on I i quor

= standard dev ia t i on es = percent sca t te r i ng

c, x = mean va lue

The pu lp used i n t h e present i n v e s t i g a t i o n was southern p ine k ra f t . many papers i t i s repor ted t h a t t he wood ex t rac t ives , espec ia l l y r e s i n acids, p l a y an impor tant r o l e i n the t o x i c i t y o f t he spent pu lp ing l i q u o r s t o f i s h (21.29).

I n

To evaluate t h e poss ib le c o n t r i b u t i o n of the wood e x t r a c t i v e s t o

33

the SC and SCE l i q u o r s , the k r a f t pulp was thoroughly washed w i t h water and then ext racted successively w i t h several por t ions of aqueous sodium hydrox- i d e w i t h in termediate washes under both ambient and elevated temperature condi t ions. Without making any attempt t o i d e n t i f y and quant i fy a l l of the r e s i n and f a t t y acids i n the a l k a l i n e ext racts , dehydroabiet ic a c i d was iden- t i f i e d as i t s methyl es te r i n about 0.01% y i e l d i n the pr imary ex t rac t . The y i e l d s of t o t a l ext racted mater ia l and methyl dehydroabietate decreased pro- g ress ive ly w i th each e x t r a c t i o n treatment. and f a t t y ac ids ( e x t r a c t i v e s ) from brown stock would thus appear t o be un- a t t a i n a b l e under o rd inary technica l washing condi t ions.

i n t o the spent c h l o r i n a t i o n and caust ic e x t r a c t i o n l i q u o r s on t o x i c i t y , the pulp was f i r s t ext racted w i t h ethanol-benzene s o l u t i o n (1:2) and then bleached under t h e same condi t ions as described i n the Mater ia ls and Methods section. LC50, 60 ppm o f TOC) which was comparable t o the value found f o r SCL corresponding t o t h e unextracted k r a f t pu lp (see Table 7) . i n g t o t h e pre-extracted pu lp showed, on the o ther hand, a s l i g h t l y reduced t o x i c i t y (24-hr LC50, 600 ppm of TOC) as compared t o t h a t found fo r the liquor corresponding t o the unextracted pulp. A1 though the t o x i c i t y reduct ion was about 30% when ca lcu la ted as t o x i c i t y u n i t s , the c o n t r i b u t i o n t o the to - t a l t o x i c i t y l o a d (SCL + SCEL) was ca lcu la ted t o be l e s s than lo%, thereby support ing t h e conclusion t h a t the wood e x t r a c t i v e s and t h e i r ch lo r ina ted d e r i v a t i v e s a r e minor cont r ibu tors t o the combined t o x i c i t y of SCL and SCEL.

The response o f A. fumigatus t o the o r i g i n a l spent l i q u o r s i s shown by the p l o t s i n F igure 5. The d r y weight of fungal, mycelium increased i n the case of spent c h l o r i n a t i o n l i q u o r . Because of t h e low concentrat ion of s o l i d s i n the o r i g i n a l SCL, a sample of the same l i q u o r concentrated t o one- f i f th of i t s o r i g i n a l volume was a l s o tested and a c l e a r i n d i c a t i o n of growth s t i m u l a t i o n was observed (Figure 5) . On t h e o ther hand, the SCEL showed o n l y a s l i g h t growth i n h i b i t i o n . S u p e r f i c i a l l y , t h e r e s u l t s obta ined w i t h !. fumigatus seem t o c o n t r a d i c t those garnered by the use of Da hnia magna; i.e., n e i t h e r of the two spent l i q u o r s appears harmful t o h g u s , which seems t o support the view which c la ims the i n v a l i d i t y of fungi as t e s t organisms (25). dif ference i s more apparent than r e a l and i s a r e f l e c t i o n of s e n s i t i v i t y d i f fe rences i n the t e s t organisms.

The complete removal of r e s i n

TO est imate the e f f e c t o f ex t rac t i ves c a r r i e d over from the brown stock

The r e s u l t i n g spent c h l o r i n a t i o n 1 iquor showed a t o x i c i t y (24-hr

The SCEL correspond-

However, as w i l l be demonstrated below, t h i s

Frac t ionat ion of the Spent Bleaching Liquors According t o Molecular Size

Of t h e var ious separat ion methods ava i lab le , those based mainly on mo- l e c u l a r s i z e differences, i .e., d i a l y s i s and gel permeation chromatography, were examined i n order t o determine the molecular s i z e d i s t r i b u t i o n of the tox icants i n t h e spent bleaching l i q u o r s . Although i t i s e s s e n t i a l l y impos- s i b l e t o demonstrate a r e l a t i o n s h i p between b i o l o g i c a l a c t i v i t y and i n d i - v idual chemical components wi thout i s o l a t i o n and p u r i f i c a t i o n , attempts were made i n the present i n v e s t i g a t i o n t o ef fect a gross molecular s i z e d i s t r i - bu t ion o f the a c t i v e substances i n spent bleaching l i q u o r through a f rac- t i o n a t i o n approach.

34

- ORIGINAL SCL

-0-0-0-SCL CONCENTRATED 5-FOLD

Figure 5. The response of Aspergillus fumigatus to the whole SCL and SCEL.

35

Dialysis of SCL gave two fractions. The maximum amount of material i n the SCL passing through the cellophane tube (dialpate) i n a 24-hr period corresponded t o 95% of the whole liquor. The corresponding value for the SCEL was 30%. SCEL contained a greater percentage of higher molecular weight material t h a n the SCL. the SCL and the SCEL was also evident from the patterns of their gel permea- t i on chroma tograms.

Failure o f the cellophane membrane t o retain more t h a n 5% of the TOC i n SCL negated the use of dialysis as a useful technique for the fract ionat ion of solids i n this particular l iquor, SCEL was, however, fractionated using b o t h dialysis and gel permeation chromatography w i t h the results shown i n Table 8.

T h i s large difference may be taken as an indication t h a t the

The large difference i n the distribution of molecular sizes between

The retentate designated as “A” indicates a retentate obtained after a single 24-hr dialysis while the retentate designated as “B” refers t o a retentate obtained after extensive and repetitive dialysis. The number average molecular weights of these retentates as measured by vapor pressure osmom- etry were about 3100 and 7000, respectively. These numbers are close t o the value (4000) reported for the acid precipitate of a SCEL (30) b u t larger t h a n the number (500) reported for the precipitate formed by lime treatment of SCEL (31). In the latter case, small amounts of ash or low molecular weight organics may have contributed to the low results.

Both retentate A and dialyzate A were further fractionated by means of gel permeation chromatography in to five sub-fractions as shown i n Figure 6. The order of acute toxic i ty among the fractions listed i n Table 8 indicates t h a t the smaller the average molecular size of ‘the acute material i n the whole liquor o r i t s fractions, the greater the acute toxici ty .

The combined toxic u n i t contents of the dialyzates and retentates corresponding t o the A and B treatment series revealed t h a t 40 and 59%, respectively, of the number or ig ina l ly present i n the whole liquor were unaccounted for. These losses may have resulted from the increased a lka l in i ty i n the dialysates as a consequence of the residua bei ng concentrated.

Retentate A and dialyzate A were a l s o fungus As er illus fumigatus. As shown by

large amount of scatter i n the da ta may be r a t i o n of materials i n t o the dialvzate and

this fungus + ecreased when incubated w i t h

liquor i n the cellophane tubes

subjected t o bioassay using the Figure 7, the oven dry weight of he concentrated dialysate. The taken as an indication t h a t sepa- retentate fractions was incom-

plete. The gel permeation chroma‘tograms i n Figure 6 appear t o support this contention. Regardless, the sense of the data i n Figure 7 i s t h a t certain growth inh ib i tors are present i n the dialyzate of the SCEL; the effect of w h i c h becomes more pronounced as the concentration of materials i n the tes t solution i s progressively increased.

36

w v

TABLE 8. ACUTE TOXICITY OF SCEL FRACTIONS TO M P H N I A MAGNA

Tox ic i t y Uni ts %f

Met hod L i quor F rac t i on Ppm Or ig ina l TOC, ppm T.U. 0 r i g i nal

T O C LC50 Separation % of as

1637 100 3 97 4.12 100 Whole SCEL -

Retentate Aa 1065 65 1395 0.76 18

Dialyzate A" 572 35 330 1.73 42 D i a 1 ys i s SCEL

Retentate Ba 525 32 5000 0.11 11

Dialyzate 6' 1112 68 ago 1.25 30

R1 862 53 >1593 <O. 54 4 3 Retentate A

R2D 203 12 > 496 <O. 41 <lo Gel Permeation Chromatography

108 7 N O ~ -- -- Dialyzate A D: 24 1 15 78 1 0.31 a

D1

D 3 b 223 13 23 5 0.95 23

'A and B re fer t o s ing le and mu l t i p le d i a l y s i s treatments, respect ively.

bSee Figure 6.

'Not determined

(I) RETENTATE A n ELUATE VOLUME, ml.

0 (II) DiALYZATE A 20 8

6

9

8

W

a IO m 0

a IO0

ELUATE VOLUME, ml.

Figure 6. Gel permeation chromatogramsof retentate ( I ) and dialysate (11) with Sephadex 6-25,

38

40

>. 0 a

C

(e) RETENTATE A

\ 0) DIALYZATE A

e

e 0

8 I I 1

lobo 2doo 30m Toc OF LIQUOR SAMPLE, ppm

Figure 7. Relationship between the dry weight of fungus and the TOC of the fractions obtained by single dialysis.

39

Toxicity Characterist ics of SCL and SCEL Ether Extracts

Solvent extraction and adsorption on activated carbon have been among

Since desorption of materials adsorbed on activated carbon i s the most frequently adopted techniques fo r the extraction of organic matter from water. sometimes d i f f i c u l t t o achieve, the solvent extraction approach seemed pre- ferable as a means of recovering the toxic materials from the spent liquors. Moreover, the r e su l t s presented i n the above section suggested tha t the toxic substances present i n both spent chlorination and caustic extraction l iquors were mainly low molecular weight compounds. solvent extraction’appeared t o be a logical approach for removing the toxic compounds from the spent liquors. Although i t has been shown t h a t ethyl ace- t a t e i s superior t o diethyl ether a s an extractant for SCL and SCEL (32,331, the l a t t e r solvent was selected for use since the dissolved sol ids can be more safely recovered from i t .

long periods i n 1 i q u i d - l i q u i d extractors, progressively w i t h increasing extraction time. T h u s , a 5 - l i t e r sample of the SCL was extracted f o r up to l Q 6 hours and the yield of extractives a t the end of this period was found to be 37% based on the TOC of the original liquor. Of the TOC extracted i n 196 hours, about 80% was removed i n the f i r s t 96 hours. Fractions collected a t the end of consecutive 96- and 100-hr extrac- t ion periods were designated A1 and A28 respectively.

for 184 hours w i t h ether. The percentage of the original TOC recovered a t the end of this extraction was about 20%. the ether ex t rac ts were collected a f t e r 62 and 184 hours of consecutive ex- t ract ion and designated as B and 0 , respectively. As i n the case of the SCL, the majority (about 70%] of TO8 was extracted from the whole liquor w i t h i n the f i r s t 62 hours.

In such a case, conventional

Ether extraction of the spent l iquors was carried o u t for comparatively T h e yields of extractives increased

A 750-ml sample of the SCEL was acidif ied to pH 2 and similarly extracted

For <the purpose of fractionation,

As shown by the d a t a i n Table Q, most of the acute toxici ty a s shown by Da hnia ma na was found i n the i n i t i a l l y collected fract ion of each liquor ++ i . e . , i n 1 and 61). The extracted SCL was a l so subjected to acute toxici ty bioassay a f t e r f i r s t removing trace residues of ether by means of a f lash evaporator. The residual organics i n extracted liquor represented 49% of the original TOC and showed an LC50 of 465 ppm of TOC. t h a t most of the substances toxic to Da hnia ma na a r e extractable w i t h ether. or fract ion B1 (Table 9) indicates t h a t e ther extraction i s more effect ive than the methods adopted previously ( i .e . toxic materials of the SCEL.

These results indicate

The LC50 value of 71 ppm of T C

d ia lys i s and gel permeation chromatography) for concentrating the

The response of Aspergillus fumigatus to the ether extracts of the SCL ( A ) and SCEL (€3) and t o the ether-extracted SCL ( C ) i s shown bv the plots i n Figure 8. stance, 31% and 25%. respectively. As i s c lear ly demonstrated by plots A and B, both ether extracts showed growth stimulation charac te r i s t ics a t lower concentrations of the extracts b u t d i s t i n c t growth inhibit ion a t higher concentrat ions,

The yields of ether extracts for SCL and SCEL were,-in t h i s i n -

40

TABLE 9, ACUTE T O X I C EFFECTS ON DAPHNIA MAGNA OF ETHER EXTRACTIVES OF SCL AND S C C

T O C 24-hr LCso Toxic u n i t s

% of PPm of 'x: of

SCLa 231 100 72 3.21 100

Frac t ion ppm o r i g i n a l TOC T.U. o r i g i nal

68 30 23 2.96 92

15 7 28 0.54 17

S C E L ~ 1637 100 397 4.12 100

21 8 14 71 3.07 75

92 6 204 0.45 11

'Whole l i q u o r (see Table 7 )

bA1 and A2 correspond t o mater ia l removed from SCL a f t e r consecutive 96- and 1 00- h r e x t r a c t i o n per iods , respec ti ve l y .

'B and B2 correspond t o mater ia l removed from a c i d i f i e d SCEL a f te r consecu- t f ve 62- and 122-hr e x t r a c t i o n periods, respec t ive ly .

Thus, there i s an apparent p a r a l l e l between the g rowth - inh ib i t i ng ef fect of the e ther ex t rac ts on played by the same conclusion t h a t t he t o x i c components o The mutagenic compounds i n SCL have l i k e w i s e been found t o be ether ex t rac t - ab le (34). The f i nd ing t h a t t he e ther ex t rac ts of SCL and SCEL d i sp lay acute t o x i c i t y toward Da hnia ma na a t a concentrat ion l e v e l where growth

SCL supported fungal growth may have been the d i r e c t r e s u l t o f the se lec t i ve removal of a l a r g e percentage o f the tox icants by ether e x t r a c t i o n as already demonstrated.

t he acute t o x i c i t y d i s - h i c h i s support ive of the

are e ther ext ractable.

of As e r i l l u s fumi a tus +Pr s s t mu ated probably i s ascr ibab le t o a di f ference i n 7ee7T-hi t e s e n s i t v ty o t e two organisms. The f a c t t h a t t he ether-extracted

T o x i c i t y Charac ter is t i cs o f SCL Ether Ex t rac t Sub-fract ionated on the Basis of A c i d i t y

An e ther s o l u t i o n o f the e x t r a c t from a f r e s h l y prepared sample of SCL ( y i e l d , 31%) was fu r the r separated i n t o 1% NaHC03 soluble, 1% NaOH soluble, and neut ra l f r a c t i o n s (designated as A , Ab, and A,, respec t ive ly ) . sponse of Daphnia magna t o these f rac tvons a re recorded i n Table 10.

The re -

41

CONCENTRATION AS TOC ppm

Figure 8. The effect of the ether extracts of S C L ( A ) , S C E L ( B ) , and of ether-extracted SCL(C) on the growth of Asper- 91 11 us fumigatus .

42

TABLE 70. T O X I C I T Y OF THE SUB-FRACTIONS OF THE SCL ETHER EXTRACT AS INDICATED BY DAPHNIA MAGNA AND A S P E R G I U S FUMIGATUS

TOCa Toxic u n i t s a Dry weightb 24- h r LC 50 of

A. fumigatus % of Ppm of % of c1 - Ppm o r i g i n a l TOC i n mg

Symbol ' F rac t i on

. T.U. o r i g i n a l w/w, %

A whole e ther e x t r a c t 72 31 29 2.48 77 25.0 1.5

weak ac ids 60 26 1500 0.04 1 16.0 43.5 P W

very weak ac ids 7 3 23 0.30 9 6.8 39.7 Ab

A, neu t ra l s 3 1 18 0.17 5 6.6 50.7

a13ased on data f o r t he whole SCL l i s t e d i n Table 9

bCompared a t a TOC concentrat ion of 2800 ppm

As I s apparent from the data i n t h i s table, most of the material i n the whole ether ext ract was col lected i n the A (weak acid) sub-fraction. The compounds comprisfng t h i s group are, i n a17 1 i k e l i hood, la rge ly carboxyl ic acids. The acute t o x i c i t y o f t h i s sub-fraction i s considerably lower than those o f the whole ether ext ract and the very weakly ac id ic and neutral frac- t ions. Although smal l i n quanti ty, these l a t t e r f ract ions are highly toxic.

The compounds i n sub-fraction A are probably phenolic i n nature and

i n fact , been ident i f ied by L i n d s t r h and Nordin (23) i n SCL. Several of these compounds have been found t o be acutely tox ic to f i s h (21). L i n d s t r h and Nordin (35) have a lso ident i f ied a substantial number of neutral compounds (several sulfur- and chloro-containlng), the t o x i c i t i e s of which have not been established i n a l l cases. Presumably, sub-fraction & consists of compounds of the type i den t i f i ed by the above Swedish researchers.

lower compared t o tha t of the whole ether ex t rac t (Table 10). The loss i n tox ic u n i t content can be ascribed t o reactions (other than neutra l izat ion) of cer ta in components of the whole ether ex t rac t w i th the a lka l ine solut ions (NaOH and NaHCO ) used i n the subf rac t ionat ion treatments. Simflar reduc-

kal ine and subjected t o bioassay using f i s h (36) and sections) as the t e s t organisms. The data i n Table 1 three subkfractions as being bet ter able t o support fumi atus than the whole ex t rac t support these f i n d i

(34) i s also important i n h i s connection,

Although t o x i c i t y I s f ten d i r e c t l y re la ted t o the ch lor ine content of a material, the data i n Table 10 ind icate t h a t t he sub-fraction with the highest ch lor ine content (A ) also shows the lowest acute tox i c i t y , I n t h i s instance, the type of-3truc!ure(s) t o which the ch lor ine i s bound may have a greater i nf 1 uence on t o x i i t y than ch lor ine content.

Tox ic i ty and Chemical Cha ac te r i s t i cs of SCL and SCEL Ether Extracts Sub- fract ionated by Adsorption Chromatoqraphy

The ether extracts designated A i , A2, B i , and B were sub-fractionated by means of adsorption chromat A t yp ica l e lu t i on diagram i s shown i n Figure 9. The ma na t o the various f ract ions obtained by t h i s type

previously by the data reported i n Table 1.1, namely, t ha t the tox ic substances i n SCL and SCEL are pre feren t ia l l y extracted by ether i n the i n i t i a l stages of the treatment. As i s also apparent from the data i n Table 11, most of the t o x i c i t y i n A1 and B1 was recovered when these fract ions were chroma- tographed on a s i l i c a gel column using ether as the elutant. Because these sub-fractions (A1E and B1E) represent a major f rac t ion of the t o x i c i t y found i n the whole SCL and SCEL, respectively, they appear t o be excel lent s ta r t i ng m a t e r i a l s for fur ther t o x i c i ty character1 zation.

several monohydric chlorophenols, ch v orocatechol s, and chloroguaiacols have,

The tox ic u n i t contents of the three sub-fractions are substant ia l ly

t ions i n acute 2 o x i c i t y have been observed when whole SCL has been made a l -

+ t e mutagenic propert ies of SCL are grea t ly reduced

rs- n able 11. The resu l t s I n t h i s tab le dupl icate t h

44

2- 0 -

loo--

ELUTION VOLUME, ml.

+ 75- 8 E 50- LL 0

I: (3 W

+ 25- -

Figure 9. A typical elution diagram for the fractionation of SCEL ether extract on s i l i ca gel .

B E I

2

45

TABLE 11 . TOXICITY CHARACTERISTICS OF SCL AND SCEL ETHER EXTRACTS FRACTIONATED BY CHROMATOGRAPHY ON SILICA GEL

Fractiona T O C

% of 24- hr LC50

PPm of Toxic u n i t s

% of Ppn o r i g i na 1 . TOC T.U. O r i g i na 1

SCLb 231 lor) 72 3.21 100

SCEL 1637 locl 3 97 4.12 100

50 22 15 3.33 104

18 8 77 0.23 7 A1 E

AIM A2E 10 4 31 0.32 10

A2M 4 2 38 0.11 3

107 7 39 2.74 67

111 7 138 0.80 20

61 4 164 0.37 0

B2M 31 2 47 0.66 16

E

aA1 and A2 correspond t o ether ext racts of SCLtobtained i n two consecutive ex t rac t i on treatments (see Table 9).

B1 and B2 correspond t o ether ext racts of ac id i f i ed SCEL obtained i n two consecutive ex t rac t ion treatments (see Table 9).

E and M re fe r t o e l u t i o n of mater ia l from s l l i c a gel column w i t h d ie thy l ether and methanol , respect ively.

bData correspond t o the mean values shown i n Table 7.

I n comparison w i th the sub-fractions of SCL ether ex t rac t i ves e lu ted from a s i l i c a gel column w i t h methanol (A M), the corresponding sub-fract ion

s u l t s discussed i n a previous section, the difference may b the r e s u l t of a greater con t r i bu t i on t o t o t a l t o x i c i t y ( in the case of SCE 5 by wood extrac- t i ves .

from SCEL (RIM) displayed a high tox i c un 1 t content. As suggested by re-

Elemental and funct ional group analyses were performed on the ether ext racts o f SCL and SCEL ( ~ ~ 2 6 0 hours of ex t rac t ion) and on sub-fractions obta ined by chromatography on s i l i c a gel w i t h the r e s u l t s shown i n Tables 12 and 13. The only s i g n i f i c a n t differences among the f ract ions are found i n

46

TABLE 12. ELEMENTAL COMPOSITION OF THE SCL(A) AND SCEL(B) ETHER EXTRACTS AND ETHER EXTRACT SUB-FRACTIONS

Weight, % Number/h - Frat t i o na Y i e 1 d , 4: Mn C H c1 0 C H c1 0

A 58.6 292 38.7 3.4 23.0 34.9 9.4 9.7 1.9 6.4 AE 27.5 307 39.1 3.6 23.2 34.1 10.0 11.0 2.0 6.6 AM 31.1 793 36.6 3.4 15.3 44.7 24.2 26.9 3.4 22.1

B 21.8 225 46.4 4.4 7.4 41.8 8.7 9.8 0.5 5.9 BE 6.7 256 50.1 5.0 6.1 38.8 10.7 12.7 0.4 6.2 BM 15.1 746 44.0 3.6 7.6 44.8 27.3 26.8 1.6 20.9

'A and B = ether ext racts of SCL and SCEL, resp.; E and M = ether and methanol, resp., solvents appl ied consecutively t o eluted mater ia l from s i l i c a gel column.

bBased on TOC of the whole l iquors.

TABLE 13. A C I D I C GROUP AND TOTAL HYDROXYL CONTENTS OF THE SCL(A) AND SCEL (B) ETHER EXTRACTS AND ETHER EXTRACT SUB-FRACTIONS

Weak acids Very weak acids Total hydroxyl

Fractiona meq./g No. /An meq . /g ' No. /h meq./g No. /Rn

A 3.79 1.11 2.60 0.76 4.82 1.41 AE 4.07 1.25 2.04 0.63 5.00 1.54

AM 3.91 3.10 2.50 1 .!XI 5.18 4.11

B 6.70 1.51 1.99 0.45 5.67 1.28

BE 7.58 1.94 2.00 0.51 6.27 1.61

BM 2.77 2.07 1.94 1.45 5.31 3.96

aSymbols i d e n t i f i e d i n footnote a, Table 12.

the ch lo r i ne content and number average molecular weight values. The higher ch lo r i ne content of the SCL ether e x t r a c t as compared t o t h a t of the SCEL ether e x t r a c t may expla in the greater t o x i c i t y o f the former. Comparison of the number average molecular weight values l i s t e d i n Table 12 for the E and M sub-fractions w i t h the appropriate acute t o x i c i t y data i n Table 11 shows t h a t greater t o x i c i t y i s associated w i t h the sub-fract ions composed of lower

47

average molecular weight material as was speculatively assumed previously i n this paper on the basis of less firm data.

The resu l t s presented i n th is section have provided evidence indicating tha t a l l or most of those compounds i n SC and SCE liquors contributing to toxici ty a re of low molecular weight and as such may be conveniently separated from the non-toxic substances i n these liquors using separation techniques based on molecular s ize , Of the fractionation approaches attempted, ether extraction proved to be one of the more satisfactory for effecting the non-destructive recovery of low molecular weight ( i .e . , toxic) substances i n SCL and SCEL. T h i s achievement i s particularly important i n the case of SCL which is especially prone to undergo changes which affect i t s toxicity. Ether extracts of SCL and SCEL therefore represent excellent s tar t ing mater- i a l s for further work relating t o toxicity.

DEGRADATION OF PHENOLS BY CHEMICAL TREATMENT

Treatment w i t h Ozone

W i t h respect t o i t s reactions w i t h organic materials, ozone i s best known fo r i t s a b i l i t y to react w i t h and ultimately cleave al iphat ic carbon- carbon double bonds (37.38). When applied t o spent bleaching liquors, ozone could therefore be expected to react w i t h f a t ty acids and l i g n i n fragments containing such groups, Although less readily attacked than al iphat ic carbon-carbon double bonds, phenols as a c lass of compounds a re reactive toward ozone (39). ozone functions by breaking down the aromatic rings of l i g n i n and simple phenols forming3 u l timately,al iphatic carboxylic acid fragments.

Publ ished f i n d i n g s (39-46) consistently indicate that

An inspection of relevant l i t e ra ture reveals that of the phenols reacted w i t h ozone, re la t ively few possessed aromatically bound chloro substi t u - ents and even these were atypical of chlorophenol types identified t h u s f a r i n spent chlorination and caustic extraction liquors (21-23). obtain information more relevant t o the s i tuat ion actually encountered i n spent chlorination and caustic extraction 1 iquors, chlorophenols representa- tive of the phenolic types identified i n such liquors were ozonized i n a s l igh t ly acidic aqueous medium and the i r ra tes of disappearance individually determined.

In order to

Sufficient amounts of the aforementioned phenols were dissolved i n ethanol/water solutions to provide 0.414 mM solutions. After the flow r a t e had been separately determined, the ozone was introduced into the solution of phenol through a f r i t t e d glass diffuser tube. After varying amounts of ozone had been applied, the residual phenol was determined by means of gas chromatography and ionization difference spectroscopy. A detailed descrip- t ion of the apparatus and procedures i s provided i n the EXPERIMENTAL section.

48

Calcu lat ion of Otoniza t i on Rate Constants--

The procedure used t o obta in r a t e constants for the reac t ion o f ozone w i t h the various phenols was the same as tha t developed by Gould and Webber (45).

Phenol + O3 ka Products + O2 The r a t e expression the authors derived fo r t h i s process i s as fol lows:

i n (Pho/Ph) = ka D t where Pho = i n i t i a l concentrat ion of phenol (moles/l)

Ph = concentrat ion of remaining phenol (moles/l) ka = r a t e constant (moles of phenol removed/mole of ozone)

The system i s assumed t o be a simple one:

-+

0 = dose r a t e (moles of ozone fed per minute/mole of phenol per

t = t ime (minutes) l i t e r i n i t i a l l y present)

The p l o t of- the natura l l o g of Pho/Ph versus D t y i e l d s a s t r a i g h t l i n e w i t h a slope equal t o ka. The r a t e constants were ca lcu lated by tak ing a l l the values o f Ph /Ph and D t f o r each sample withdrawn from the reac t i on vessel, up t o the po?nt where 60% of the s t a r t i n g compound had reacted. This value was chosen as the upper l i m i t t o avoid interference i n the u l t r a v i o l e t spec- t roscopic measurements of res idual phenol by accumulating degradation products. The slope (ka) was then determined using an HP-55 ca l cu la to r w i t h automatic l i n e a r regression, i n order t o e l iminate the er ro rs inherent i n a graphical method. those po in ts corresponding t o the f i r s t 20% of phenol removal, bu t i n many instances the i n i t i a l react ions were so rap id t h a t i nsu f f i c i en t data were ava i l ab le t o a l low the slope t o be determined w i t h any degree of accuracy.

Idea l l y , i t would have been des i rab le t o use only

Ef fect of St ructure on Ozonization Rate--

The r a t e constants fo r the reac t ion o f ozone w i t h var ious model phenols are compiled i n Table 14. I n i t i a l l y , the res idual phenol concentrat ions were measured gas chromatographically. This approach proved t o be t ime con- suming, however, because of the l a rge number of samples involved and the time needed t o prepare each sample fo r analysis. U1 t r a v i o l e t spectroscopy based on measurement of i on i za t i on di f ference spectra appeared t o represent a much simpler and more convenient a1 te rna t i ve approach. Analyses subsequent ly performed on the ozonizat ion product mix ture o f t r i ch lo rocreoso l ( X I I I ) i nd ica ted t h a t the y ie lds of res idual phenol, as determined by the spectroscopic and chromatographic methods, agreed t o w i t h i n 2%. Accordingly, the UV spectroscopic method was therea f te r adopted f o r use i n determining the res idua l phenolic content of phenol/ozone product mixtures. The gas chromatographic method continued t o be used f o r the determination of vera- t r o l e der iva t ives ( X X I V - X X V I I ) since they have no ion izab le groups.

49

TABLE 14. OZONIZATION RATE CONSTANTS OF PHENOLS

Moles of ka phengl Compou nda I n i t i a l pH Fina l pH removed/mol e 01 -

I I1 V

VI I VI11 IX X XI XI1 XI11 xv I XVII XVIII XIX - xx

XXIV xxv

6.15 6.15 6.15 6.15 6.15 6.15 6.15 6.15 6.15 6.15 10.3 6.15 6.15 6.10 6.15 6.15 6.15

6.05 5.85 5.83 5.85 5.45 5.90 5.42 5.68 5.30 5.20 > 10 5.65 5.29 6.05 6.10 5.65 ,

6.15

0.91) 0.83 2.44 2.43 1.62 1.11 2.02 2.04 2.56 2.61

~ 1 3 . 6 3.72 2.53 5.25 4.42 1.77 0.20

xxv I 6.14 6.14 0.02 XXVII 6.15 6.15 - C

aSee Table 2 for structures.

bCompound XVI: i n i t i a l conc., 1.01 mM; 03 flow rate, 0.72 mg/min.

'No reac t i on a f te r app l i ca t i on o f 27 mg o f 03.

A s was stated previously, on ly data corresponding t o the removal of up t o 60% of the o r i g i n a l phenol were used t o ca l cu la te the r a t e of constants i n order t o reduce the p o s s i b i l i t y of phenol ox ida t ion products i n te r fe r i ng w i t h the measurements. I n a number of instances, d i f f e r e n t i a l absorbance was noted a t wavelengths s l i g h t l y higher than the absorpt ion maxima found for the phenols (297-313 nm). This d i f f e r e n t i a l absorbance increased w i th increasing ozone input, thereby i nd i ca t i ng the res is tance of the cont r ibu t - i ng s t ruc tu re(s ) t o breakdown by the oxidant,

A l l others: i n i t i a l conc. 0.414 mM; 03 flow rate, 2.5 mg/min.

50

I n general, the data i n Table 14 i nd i ca te t h a t the r e a c t i v i t y o f the phenols toward ozone decreases i n the order: catechols (XVII-XX) , guaiacols ( V I I - X I I I ) , cresols (I1 and V), and verat ro les ( X X I V - X X V I I ) . This sequence i s cons is tent w i t h the order genera l ly observed fo r the r e a c t i v i t y of oxidants, inc lud ing a sparse amount of information r e l a t i n g s p e c i f i c a l l y t o ozone (47), toward mono- and d ihyd r i c phenols and t h e i r ethers,

The ra tes of react ion of phenols of the guaiacol ( V I I - X I I I ) and catechol (XVII-XX) types w i th ozone decrease w i th the i n t roduc t i on of one ch loro subst i tuent onto the r i ng . This f ind ing i s predic tab le s ince ozone func- t i ons as an e lec t roph i l e (see ref . 48) and the chloro subst i tuent reduces the e lec t ron densi ty of the r ing . However, as the number of ch loro subst i - tuents increased, the r a t e constants (cf. Table 14) a c t u a l l y showed a pro- gressive, a l b e i t a somewhat e r r a t i c , increase ra ther than the expected decrease. An explanat ion fo r t h i s apparent anomaly i s not r e a d i l y discerned from the data a t hand. based on the statements of other inves t iga tors (39,48,49) i nd i ca t i ng t h a t the intermediate ox ida t ion products of phenols w i t h ozone (e.g. , o-benzoquinones and muconic ac ids) are themselves r e a d i l y attacked and degraded by ozone, Thus, there ex i s t s a compet i t ion fo r ozone between res idual phenol and i t s breakdown products. the ef fect of ch lo ro subst i tuents on reducing the r a t e of ozone a t tack i s greater when they are present on phenol breakdown products than when they are s i tua ted on phenolic r ings. present s i tua t ion , i t has t o be assumed t h a t the number and poss ib ly the p o s i t i o n of the ch loro subst i tuents on both the phenol and i t s breakdown products are c r i t i c a l var iables.

However, one working hypothesis can be evolved

I n the case a t hand, i t i s conceivable t h a t

I n apply ing t h i s i n t e r p r e t a t i o n t o the

Comparison of the r a t e constants (Table 14) fo r the reac t i on of phenol i t s e l f (I) and K c r e s o l (11) reveals t h a t the e f f e c t of H and CH3 subst i tuents a t the p o s i t i o n para t o the phenolic hydroxyl i s not pronouncedly d i f - ferent. Contrary t o the behavior displayed by phenols V I I - X I 1 1 and X V I I - X X , i n t roduc t i on of one ch loro subst i tuent onto the cresol r i n g d i d not r e s u l t i n a reduct ion of the reac t ion r a t e w i t h ozone, as was observed w i t h phenols of the guaiacol and catechol types. The reac t i on r a t e did, however, increase systemat ica l ly as the number of ch lo ro subst i tuents increased from 0 t o 2. The reac t i on sequence proposed (42,45,48,50) fo r the ox ida t i ve breakdown of monohydric phenols w i t h ozone involves a p r i o r conversion t o a catechol through hydroxy lat ion a t a pos i t i on or tho t o the phenolic hydroxyl group. Whether t h i s add i t iona l step i s su f f i c i en t t o expla in the s l i g h t l y d i f f e r - ent ef fect of ch loro subst i tuents on the observed rates of mono- and dihy- d r i c phenol ozonizat ion cannot be determined w i t h the evidence a t hand. The problem i s fu r ther complicated by the fac t t h a t conversion of monohydric phenols t o catechols may be prevented by the presence o f ch lo ro subst i tuents a t the pos i t ions or tho t o the phenolic hydroxyl group,

i d e n t i f i e d thus f a r i n spent ch lo r i na t i on and caust ic ex t rac t i on l i q u o r s (21-23) but were included i n the study fo r purposes o f comparison w i t h other phenolic types and w i t h the hope of obta in ing in format ion regarding the mechanism of ox ida t ive breakdown w i th ozone,

The f u l l y e the r i f i ed phenols ( X X I V - X X V I I ) a re a typ i ca l of the phenols

The r a t e data i n Table 14

51

c l e a r l y demonstrate the r e l a t i v e unreac t iv i t y of these compounds toward ozone.

The r a t e constants i n t h i s series of compounds decrease progressively w i t h increas ing s u b s t i t u t i o n o f ch lo r i ne as would be predic ted assuming an e l e c t r o p h i l i c a t tack o f ozone and no other complicating s ide effects.

The observed s t a b i l i t y o f the t r i ch lo rove ra t ro le d e r i v a t i v e (XXVII) toward ozone contrasts sharply w i t h the behavior of t he corresponding guaia- co l and catechol compounds XI11 and XX, respectively, both of which were extens i ve l y degraded by ozone under the same condit ions. Other evidence i n d i - ca t i ng t h a t a d i f f e r e n t mode of aromatic r i n g degradation i s operat ive i n the ozonizat ion o f ve ra t ro le der iva t ives l i e s i n the observation t h a t the ozonizat ion product mixtures of such compounds were co lo r l ess whereas those of the guaiacols and catechols were i n i t i a l l y h igh l y colored. This f ind ing suggests t h a t aromatic r i n g s i n veratrole-type s t ruc tu res are no t degraded through an o-benzoquinone intermediate.

I n o ther studies (41,46) of t he reactions of s i m i l a r (vera t ry l - type) compounds w i t h ozone, the product analys is showed that , s i m i l a r t o the be- hav ior of t he guaiacol and catechol types, r i n g sc iss ion occurred mainly between the oxygen-bearing carbons. It i s important t o note, however, t h a t t h i s i s no t exc lus ive ly the case since Kra tz l e t a l . (46) have detected dimethyl oxalate among the ozone ox ida t ion products of v e r a t r o l e and 4- methylveratrole ( X X I V ) . Th is f ind ing ind icates a rup ture of t he bonds between carbon atoms 2 and 3 and 4 and 5 and represents a complicating factor i n attempting t o r a t i o n a l i z e the effect of s t ruc tu re on ozonizat ion ra te .

I n con t ras t t o the o ther compounds l i s t e d i n Table 14, te t rach lo ro- guaiacol ( X V I ) was ozonized i n an a l k a l i n e (pH 10.3) medium and under s l i g h t l y d i f f e ren t condi t ions w i t h respect t o i n i t i a l concentrat ion and ozbne flow r a t e than those employed w i th the former set, Although the ozone treatment of tetrachloroguaiacol was no t performed under the r i g o r - ously c o n t r o l l e d condi t ions required fo r the acqu is i t i on of re1 i a b l e k i n e t i c data, a r a t e constant fo r t he reac t ion was nevertheless ca lcu la ted (Table 14). A f te r making ample allowance for the po ten t i a l inaccuracies of the measurement, the value reported f o r the r a t e constant i s s t i l l s u f f i c i e n t l y higher than the r a t e constants reported fo r the other phenols t o j u s t i f y the conclusion t h a t the reac t ion of tetrachloroguaiacol w i t h ozone i s enhanced i n an a l k a l i n e medium, documented i n the l i t e r a t u r e where several inves t iga tors (40,45,49,50) concluded t h a t phenols a re ox id ized i n preference t o other substances i n a l - k a l i n e media. The effect of the a l k a l i may be ascribed t o an enhancement o f t he e lec t ron dens i ty on the r i n g as a consequence of forming a phenolate anion, thereby promoting the e l e c t r o p h i l i c a t tack of ozone. An a l t e r n a t i v e o r complementary explanation i s that , i n aqueous, a l k a l i n e media, ozone i s decomposed i n t o hydroxyl rad i ca l s (51) which are more reac t i ve toward phe- no ls than ozone i t s e l f .

Support fo r t h i s i n t e r p r e t a t i o n i s we l l

The r e l a t i o n s h i p between the extent of removal of tetrachloroquaiacol and the acute t o x i c i t y of the product so lut ions toward Daphnia magna i s shown by the data i n Table 15.

52

TABLE 15. DETOXIFICATION OF TETRACHLOROGUAIACOL ( X V I ) BY OZONIZATION AT pH 1 r)

Ozone consumed/ a 24-hr Lc50

(mnoles/mmole) % o r i g i n a l Phenol ppm of carbon i n i t i a l phenol onc. Phenol loss, ppm based on

0 0 0.22 0.06

1.57 50 4.1 1.31

5.13 100 >lo >3 , 21

' I n i t i a l conc. of phenol, 259 mg/1; ozone flow rate, 0.60 mg/min.

I t i s apparent from the values l i s t e d t h a t tetrachloroguaiacol can be to- t a l l y removed from so lu t i on by treatment w i t h ozone i n an a l k a l i n e medium. The decrease i n phenol content was roughly para l le led by a decrease i n acute t o x i c i t y as indicated by the response of Da hnia ma na t o the ozonized solut ions, It i s also important t o note t h a t w *le en p eno removal was cam- plete, the product mixtures were e s s e n t i a l l y non-toxic. A s i m i l a r f ind ing has been reported by Niegowski (40).

Extent of Phenol Removal--

Continued ozonization of the phenols and phenol ethers resul ted i n extensive breakdown of these compounds. A1 though ozonization was terminated shor t of 100% removal of the phenol, evidence was obtained suggesting t h a t t h i s goal was at ta inable i n a l l but a few instances. The f u l l y e the r i f i ed models ( X X I V - X X V I I ) proved considerably more r e s i s t a n t t o ox idat ive breakdown than the other classes of phenols tested, and t h e i r s t a b i l i t y increased w i t h increasing subs t i t u t i on of ch lo r i ne on the r i ng . 4-methyl - t r i ch lo rove ra t ro le ( X X V I I) was recovered essen t ia l l y i n t a c t a f te r the app l i ca t i on of a considerable excess of ozone.

I n the extreme case,

The ef f ic iency of phenol removal appeared t o decrease w i t h increasing app l i ca t i on of ozone, This s i t u a t i o n was seemingly the r e s u l t of the formation of increasing quan t i t i es of phenol ox idat ion products which competed successful ly w i t h residual phenol for the ozone. Because of such complexi- t i e s , few if any consistent trends were apparent i n the res idual phenol/ ozone consumption data, and attempts t o draw any r e l i a b l e conclusions regarding the re la t i onsh ip between the ozone required t o effect complete removal of the phenol s and t h e i r s t ruc tu re were unsuccessful.

The primary intermediate breakdown products o f phenol ox idat ion by ozone have not been extensively investigated. The sequence shown below i s a composite which combines experimental f indings w i t h the speculations of a number of i nves t i ga t o r s .

53

'OR,

CY

C

OH A

D E

Monohydric phenols (represented by A ) are bel ieved t o f i r s t undergo hydroxy- l a t i o n t o a catechol (B;R,=R,=H). The aromatic r i n g of B st ructures subsequent ly may undergo fu r the r a t tack by ozone d i r e c t l y forming a muconic ac ld de r i va t i ve (C) through rupture between the oxygen-substi tu ted carbon atoms. I n the case of 4-methylveratrole (B;R,=R,=CH,), Kratz l e t a l . (46) have ac tua l l y i d e n t i f i e d dimethyl cis,cis-B-methylmuconate among the reac t ion products. A1 ternat ive ly , c i s x s T m e t h y l m u c o n i c ac id (E) may be formed - v i a an o-benzoquinone i n t e m e (D). Sinte compounds of the l a t t e r type are colored, the formation o f colored ozonization reac t ion mixtures could be taken as evidence for the r e a l i t y of t h i s por t ion of the above sequence.

I n the present invest igat ion, ozonizat ion of a l l compounds except V, X X I V , X X V I and X X V I I resu l ted i n the formation o f colored product mixtures, thus supporting the concept t h a t a t l e a s t a po r t i on of the ove ra l l degrada- t i o n of the ma jo r i t y o f phenols tested occurred mediate. The co lo r i n i t i a l l y generated by the in t roduc t ion of ozone i n t o the phenol so lu t ions was discharged when 0.8 t o 2.0 moles of Og/mole of phenol was consumed. One group of inves t iga tors (49) has suggested tha t oxygen as a decomposition product of ozone plays a r o l e i n the ove ra l l process of phenol degradation.

an o-benzoquinone i n t e r -

Another pathway i n the degradation of phenols w i th ozone consists of the breaking of carbon-carbon bonds other than those between the oxygen- bearing carbons. Thus, Kratz l e t a l , (46) i d e n t i f i e d dimethyl oxalate (F;R,=R,=CH ) i n the product mixture from the ozonization of 4-methylvera- t r o l e (XXIV ! i n aqueous acet ic acid. l a ted ozone at tack on a phenol r i n g a t s i t e s other than the oxygen-substi- tu ted carbons.

Bernatek e t a l . (47) have a lso postu-

54

The r e a c t i v i t y of o-benzoquinones, muconic acids, and other possible phenol breakdown products toward ozone i n comparison w i t h t h a t o f phenols toward the same oxidant has not been extensively investigated. Generally, app l i ca t ion of the t h e o r e t i c a l l y required amount of ozone t o e l iminate com- p l e t e l y a given quant i t y of phenol has been found t o be insu f f i c ien t f o r t h i s purpose (42.44.45.47). This f inding has been in te rpre ted as i n d i c a t i n g a greater r e a c t i v i t y of ozone toward the primary ozonizat ion products than toward the s t a r t i n g mater ia l (47).

A s i m i l a r competit ion e x i s t s i n the spent bleaching l i q u o r s where the simple phenols are mixed w i t h phenolic breakdown products, l a r g e r l i g n i n fragments, and carbohydrates. The s p e c i f i c i t y of ozone for a monomeric phenol was tested by d i l u t i n g an aqueous so lu t ion of tetrachloroguaiacol ( X V I , Table 2) containing 0.1 mnole of the l a t t e r w i t h increasing amounts of spent caust ic ex t rac t ion l i q u o r and reac t ing the mixtures w i t h a f ixed amount of ozone. The r e s u l t s of t h i s experiment are compiled i n Table 16.

TABLE 16. OZONE OXIDATIONa OF TETRACHLOROGUAIACOL-SPENT CAUSTIC EXTRACTION LIQUOR COMB1 NATIONS

Carbon contr ibuted by Tetrac h l orogua iaco l te t rac h l o rowa iaco l % of TOC removed, %

30.7 69

12.8 44

4.2 14 ,

'Ozone applied, 5.28 mg; ozone flow rate, 0.66 mghin.; pH 10.3

These r e s u l t s reveal t h a t the organic compounds present i n the spent caust ic e x t r a c t i o n l i q u o r do indeed compete e f f e c t i v e l y w i t h the added t e t r a - chloroguaiacol fo r ozone. The bottom values i n each of the two columns probably come c losest t o approximating the s i t u a t i o n e x i s t i n g i n spent caust ic e x t r a c t i o n l i q u o r w i t h respect t o the f rac t ion of t o t a l organic carbon (TOC) contr ibuted by the phenolic component. tetrachloroguaiacol was far from quant f ta t ive. However, comparison of the corresponding values i n the two columns reveals t h a t the removal of t e t r a - chloroguaiacol (14%) was nevertheless greater than would have been expected on the basis of i t s con t r ibu t ion t o the t o t a l carbon of the system (4.2%) and some s p e c i f i c i t y of the ozone for the phenol i s indicated. preference i s moderated and contro l led by the r e l a t i v e concentrat ion of the phenol and ozone, and the r e a c t f v i t y o f the other 1 iquor components toward ozone.

I n t h i s s i tua t ion , t h e removal of

However, t h i s

55

Another parameter r e f l e c t i n g the character and extent of the ozonization treatment i s the formation o f ch lo r i de through ox ida t ive breakdown of phenols containing o rgan ica l l y bound ch lor ine. t i ons of c h l o r i d e i o n i n the ozonized so lu t ions of the chlorophenols were determined as a funct ion o f the amount of appl ied ozone. these analyses revealed t h a t ch lo r ide i o n was produced r a p i d l y and extensively a f t e r the a d d i t i o n o f on ly a small amount of ozone t o phenols of the guaiacol, catechol, and cresol types. Low y i e l d s of ch lo r i de from the vera t ro le deriva- t i v e s again ind ica ted t h e i r poor r e a c t i v i t y toward ozone. I n the extreme case, no ch lo r i de was detected when 3,4-dimethoxytrichlorotoluene ( X X V I I ) was ozonized.

I n the present study, the concentra-

The r e s u l t s of

The percent recovery o f ch lo r ide based on theore t ica l y i e l d was greatest (over 60%) fo r the monochloro compounds V I I I , I X , X V I I I , and X X V . ch lo ro and t r i c h l o r o der iva t ives a l l produced ch lo r i de i o n y i e l d s i n the range 30 t o 50% o f theore t ica l ; i nd i v idua l d i f ferences were too small t o r e l a t e t o s t r u c t u r a l features w i th any degree o f confidence. the y i e l d of c h l o r i d e i o n approach the theo re t i ca l value fo r 100% removal, and i t must be presumed t h a t chlorine-containing, ozone-resistant breakdown products were formed, This conclusion i s supported by the r e s u l t s of a study by G i l b e r t (44),who ozonized several monohydric chlorophenols and recorded ch lo r i de y i e l d s ranging from 60-97% when approximately 4 moles of ozone/mole of phenol were applied.

The d i -

I n no case d i d

Treatment w i t h Chlor ine Dioxide

The a b i l i t y of ch lo r i ne d iox ide t o r e a c t w i th and degrade phenolic l i g n i n model compounds i s we l l documented i n the l i t e r a t u r e (4, 52-55). Th is fact, coupled w i t h the current i ndus t r y t rknd toward greater u t i l i z a t i o n of c h l o r i n e d iox ide i n the f i r s t bleaching stage provided the impetus fo r performing a few experiments i nvo l v ing the react ions of ch lo r i ne d iox ide on phenols s i m i l a r t o o r i d e n t i c a l w i t h those detected i n spent ch lo r i na t i on and caus t ic ex t rac t i on 1 iquors.

I n these experiments, selected phenols (conc,, 10 mM) were reacted w i t h vary ing amounts of ch lo r i ne d iox ide i n the dark a t ambient temperature u n t i l the oxidant was completely consumed. ex t rac t i on of t he product mixture w i t h chloroform and the amount determined by gas chromatography. The r e s u l t s a re shown by the p l o t s i n Figure 10, The p l o t s i n d i c a t e the l i k e l i h o o d of completely removing the phenols tested, provided s u f f i c i e n t amounts of ch lo r i ne d iox ide are appl ied. exception of 2,4-dichlorophenol ( I V ) , the responses of t he phenols show no pronounced differences. o r e t i c a l l y requ i red t o convert the var ious phenolic r i n g s i n question t o non-aromatic moiet ies, i t i s apparent t h a t the removal of the phenols tested i s a very i n e f f i c i e n t process.

The res idua l phenol was recovered by

With the poss ib le

Since e i t h e r 2 o r 4 ox id i z ing equivalents are the-

The e f f i c iency of phenolic r i n g breakdown by ch lo r i ne d iox ide was fu r ther tes ted by applying a f ixed amount of ch lo r i ne d iox ide t o a so lu t i on of tetrachloroguaiacol ( X V I ) d i l u t e d w i t h increasing amounts of spent caus- t i c e x t r a c t i o n l i q u o r . The r e s u l t s (see Table 17 reveal a decrease i n the d e t o x i f i c a t i o n fac to rs ranging from 0.701 t o 0.82 b .

56

80

70

IC

0.

-@ CI

--*-e-*- c@" \ \ \\\

+ I 4 6 8 IO I2 14 16

EQUIVALENTS OF CIOz/MOLE OF PHENOL

Figure 10. Degradation o f selected phenols through reaction with chlorine dioxide.

57

TABLE 17. CHLORINE DIOXIDE OXIDATIONa OF TETRACHLOROGUAIACOL-SPENT CAUSTIC EXTRACT I ON LIQUOR COMB I NAT I ONS

Carbon contr ibuted by Tetrachloroguaiacol tetrachloroguaiacol, % o f TOC removed, X

100

30

10

5

95

81

58

19

'Applied 17 mg o f C102 t o 24 mg of te t rachloroguaiacol a t pH 6.

e f f ic iency of phenol removal w i t h increasing d i l u t i o n by the spent l i quo r . A s i m i l a r f ind ing was previously noted i n the case of t he tetrachloroguaiacol/ spent caus t ic ex t rac t ion l i q u o r combinations w i t h ozone (Table 16). Compari- son of the data i n the two tables leads t o the conclusion that, i n the presence of spent caust ic ex t rac t ion l iquor , the s e l e c t i v i t y of ch lo r i ne d iox ide fo r tetrachloroguaiacol i s greater than t h a t of ozone.

Treatment w i t h A1 kal i

Data presented below and elsewhere (24.36) have demonstrated the detox i - f i c a t i o n ef fect of a l k a l i on spent c h l o r i n a t i o n l i quo r . I n an attempt t o re- l a t e the de tox f f i ca t ion of the l i q u o r t o spec i f i c phenol o r a t l e a s t phenolic types, aqueous solut ions of several phenols, prev ious ly i d e n t i f i e d i n spent c h l o r i n a t i o n l i q u o r (23) were prepared and adjusted t o pH 11 by add i t i on of a l k a l i and subsequently stored i n the dark a t 5OC f o r 24 hours. The appro- p r i a t e concentrations of the phenols tested were f i r s t estab l ished by range- f ind ing t o x i c i t y studies. A con t ro l sample was a l so prepared and allowed t o stand a t neut ra l pH under otherwise i d e n t i c a l condit ions. During the stand- i n g period, the pH of the a l ka l i - t rea ted guaiacols ( X X I V and XXV, Table 2). 2,4-dichlorophenol ( I V ) , and 2,4,6-trichlorophenol ( V I ) remained constant whereas the pH of the a l ka l i - t rea ted catechols ( X X I , X X I I , and X X I I I , Table 2) dropped from 11 t o 9.9. A t the end of 24 hours, a l k a l i - t r e a t e d samples were readjusted t o pH 7 w i t h 1 N HC1. Standard d i l u t i o n water was added t o each untreated tox i can t so lu t i on i n a volume equal t o the t o t a l volume of NaOH and HC1 added t o the corresponding t reated sample. A l l phenol so lu t ions were bioassayed a t 14OC using cardiac a r r e s t as the response c r i t e r i o n .

The r e s u l t s of these bioassays are summarized i n Table 18. A l l of the

o f untreated chlorophenol, chlorocatechol s evidenced pronounced d e t o x i f i c a t i o n on a1 kal i n e treatment. A t concentrat ions greater than 20 times the LC

For the chloroguaiacols, 2,4-dichlorophenol and 2,4,6-trichloropheno , no s i g n i f i c a n t differences wereapparent i n the t o x i c i t i e s of t he t rea ted and untreated samples. However, w i t h each of these compounds, the t o x i c i t y of the t rea ted sample was consls tent ly lower than the untreated sample, w i t h d e t o x i f i c a t i o n factors ranging from 0.701 t o 0.826.

-e- a1 ka l i - t rea ted chlorocatechols caused no s i g n i ?? cant m o r t a l i t y i n Da hnia.

58

TABLE 18. EFFECT OF ALKALINE TREATMENT (pH 11) ON THE TOXICITY OF SOME CHLOROPHENOLS TO O A P U MAGNA

Chlorophenol i n p moles / l i te r 9 Ab 9 24 hr LC5

(95% confidence n terva l ) a

4.5-Dichl oroca tec hol ( X X I ) u n t rea t p t reated

3.4.5-Trichl orocatec hol (XXI I ) untreated t reated

3.4.5.6-Tetrachl orocatechol ( X X I I I ) untreated t reated

4.5-Dichloroguaiacol ( X I V ) untreated t reated

4.5.6-Tric h l orogua iaco l (XV ) untreated t reated

2.4-Dichlorophenol ( I V ) untreated t reated

2.4.6-Trichlorophenol ( V I ) untreated t reated

6.64 (4.P6-7.81) >i78

3.39 (2.92-3.86) >62.0 C O X ]

2.23 (1.25-2.89) >57.5 Cl0X-J

98.3. (79.0-135) 119 (96.6-189)

22.0 (15.6-26.5) 31.4 \(26.3-40.8)

51.9 (40.2-90.1) 68.2 (51.2-175)

13.2 (11.4-16.0) 16.9 (14.8-21.5)

n.240 0 .937 -

0.222 ~0.055 -

0*309 9.826 0.299

0.242 9*261 0.701

Q*424 0.761 q. 408

Oo2O6 0.811 0.276

L . "As discussed by Finney (11). the "g" value can be used as an index of the q u a l i t y o f the bioassay, i.e., how wel l the observed m o r t a l i t y pa t te rn cor- responds t o the best estimate o f the p r o b i t l i n e . 0.4 o r less ind ica te good agreement

I n general, "g" values of

bLC5~ o f untreated compound 'Each treatment consisted o f addi t ion o f NaOH t o b r ing pH t o 11.0 dPercent m o r t a l i t y a t spec i f ied concentration

LC50 o f t reated compound

The de tox i f i ca t i on of the chlorocatechols (XXI-XXIII) poss ib ly may be associated w i t h loss of o rgan ica l l y bound ch lo r ine since s t ructures of t h i s k ind have prev ious ly been shown t o undergo a1 ka l i -catalyzed hydro lys is of

59

chloro subst i tuents (19). Conversely, 4,5-dichloroguaiacol ( X I V ) t ha t sus- ta ined a comparatively small loss o f o rgan ica l l y bound ch lo r ine i n the same study, showed a s t a t i s t i c a l l y i n s i g n i f i c a n t decrease i n acute t o x i c i t y (Table 18). involvement o f other, as y e t un ident i f ied , factors .

This observed re la t i onsh ip does not, however, preclude the

DEGRADATION OF CHLOROPHENOLS BY BIOLOGICAL TREATMENT

The biodegradation t e s t s involved add i t i on o f i nd i v idua l microorganisms ( fungi) and a mixed microbia l populat ion t o so lu t ions o f various phenols; the phenols were i d e n t i c a l t o those detected i n spent ch lo r i na t i on and caust ic ex t rac t ion l iquors . Since, i n the course o f performing the b io log ica l t rea t - ments, the aqueous phenol so lu t ions were exposed t o a i r throughout the t e s t period, con t ro l experiments were conducted i n the absence o f microorganisms i n order t o evaluate the e f f e c t o f aerat ion alone on the removal o f the phenols. I n these tests, so lu t ions o f the various chlorophenols (conc. , 20 ppm) were f i l t e r e d through a Sei tz f i l t e r t o remove any microorganisms, then shaken on a hor izon ta l shaker f o r one week a t 28OC. The residual phenol was removed by so l vent ex t rac t i on and determined by gas chromatography.

The r e s u l t s o f the aerat ion treatments are shown i n Table 19. The ex- t e n t o f phenol removal appears t o be re la ted t o the s t ruc tu re o f the phenolic r ing . h ighest y i e l d (70-75 percent), 50 percent o f the d i - and tr ichlorophenols ( IV and V I , resp.) remained a f t e r one week, wh i le less than 5 percent o.f the catechols survived a f t e r the same period. No attempt was made t o character- i z e the phenolic breakdown products, but i t was speculated t h a t ox idat ion of the phenolic r i ngs t o quinonoid and acyc l i c moiet ies probably occurred, p a r t i c u l a r l y i n the case o f the catechols (XXI'and X X I I I ) . The co lo r developed i n the aerated so lu t ions i s support ive o f the proposed formation of quinonoid un i t s . s t i t u e n t s i s a l i k e l y p o s s i b i l i t y .

o f several phenols f o r vary ing periods o f up t o 10 days a t ambient temperature. phenol measured by i o n i z a t i o n d i f fe rence spectroscopy. The resu l t s of t h i s analys is a re shown i n Table 20. I n agreement w i t h the f ind ings reported i n Table 19, the catechol der iva t ives ( X V I I , X V I I I , XX) were degraded more extens ive ly than the guaiacols ( I X , X I I I ) . Among the catechols, degradation decreased s l i g h t l y w i th increasing numbers of ch lo ro subst i tuents on the phenolic r i ng . I n a l l instances, the ra tes o f phenol removal were low.

Thus, both guaiacol der iva t ives ( X I V and X V I ) were recovered i n the

I n add i t i on t o ox idat ion, some hydro lys is o f ch loro sub-

I n a somewhat s i m i l a r inves t iga t ion , Cain (3 ) aerated aqueous so lut ions

Samples were withdrawn a t various i n t e r v a l s and the amount o f res idual

The extents o f phenol removal found i n the present inves t iga t ion were s l i g h t l y greater i n the case o f the guaiacols and very much greater f o r the catechols than those ind ica ted by Cain's r e s u l t s a f t e r comparable aerat ion periods. The explanation f o r t h i s d i f f e rence may be re la ted t o the roughly 4 - fo ld higher phenol concentrat ions and a s l i g h t l y lower react ion temperature t h a t Cain used. The ana ly t i ca l methods employed i n the two invest igat ions, gas chromatography and ion i za t i on d i f f e rence spectroscopy, may a lso have contr ibuted t o some o f the di f ference.

60

T-9. FFFFCT OF A F W ON RFWVAI OF -1 Sa

Residual Chlorophenol . (Xlb

Ae a t i o Chlorophenol s a1 one + ungus J t

Aeration

4.5-Dichl oroguaiacol ( X I V ) Tetrac h l oroguaiacol ( X V I )

2.4-Dichlorophenol ( IV) 2.4.6-Tric hlorophenol ( V I )

70-75

50

55 16

15 40

-d <5 - 4.5-Dic h l orocatec hol ( X X I

Tetrachlorocatechol ( X X I I

aConc.: 20 ppm; aerat ion (shaking) time: 1 week; temp., 28OC bCorrected for incomplete recovery by solvent ex t rac t ion 'Candida u t i l i s (yeast) dNot determined

TABLE 20. RATE OF REMOVAL OF PHENOLS DURING AERATION

Residual ,phenol. X

Phenol

Time, Days

I X XI11 X V I I X V I I I xx 1 99 - 98 100 98 2 98 - 96 94 93 3 98 - 88 88 92 4 97 - 81 85 92 5 96 95 75 83 88

10 - 91 - - -

61

The expectat ion i s t h a t the various phenolic types l i s t e d i n Tables 19 and 20 w i l l behave i n a q u a l i t a t i v e manner when subjected t o aerat ion a s components of spent c h l o r i n a t i o n and caust ic ex t rac t ion 1 iquors. However, the r a t e and degree of t h e i r removal i n the technical aerat ion treatment cannot be accurately judged from the data i n Tables 19 and 20.

I n the few instances where aerat ion was performed i n the presence of a fungus (Candida u t i l i s ) , the amount of phenol removed was increased over t h a t resul t i n m a e r a t i o n alone. Under the t e s t condit ions, however, chlorophenol removal was incomplete i n every instance. Mueller, and Walden (56) found t h a t tetrachloroguaiacol could be biodegraded by an inoculum o f acclimated act ivated sludge a f te r an induc t ion per iod of approximately 5 days. The guaiacol was completely consumed a f t e r approximately 10 days' fermentation treatment. I n other biodegradation tests , three fungi, Paeci lom ces v a r i o t i , P e n i c i l l ium var iab i le , and Trichoderma konin i i , w e r h o - ab i 1 i ty t o remove phenol i c compounds from d e - y e a s t extract-peptone l i q u i d medium i n a one-week incubation period. The amount of res idua l phenolic compound i n the medium was determined by gas chromatography w i t h the r e s u l t s shown i n Table 21. The data i n t h i s tab le represent the combined effects of aera t ion and microorganism a t tack on phenol breakdown.

I n e a r l i e r work, Leach,

The most extensively degraded phenol was 4-methylcatechol (see Table 21 ) and a l l th ree fungi t o t a l l y removed t h i s compound from the media a f t e r one week of incubation. A t the other end of the r e a c t i v i t y scale, the ve ra t ro le de r i va t i ves ( X X I V and X X V ) proved t o be except ional ly r e s i s t a n t t o fungal a t tack. I n between these extremes, the fungi evidenced considerable d i f f e r - ences i n t h e i r a b i l i t y t o degrade the same compound,

NO cons is ten t co r re la t i ons were found between t o x i c i t y (see Table 3 ) and s u s c e p t i b i l i t y t o biodegradation. Thus, t he t o x i c effect of 2,4,6-trichlorophenol ( V I ) on Trichoderma kon ing i i and Pen ic i l l i um v a r i a b i l e were s i m i l a r whereas i n the biodesradation tests, the former f u n d t o t a l l v removed t h i s phenol from the mediim but the l a t t e r on l y t o the e i t e n t of io%.

Gas chromatographic analysis of the ethanol ex t rac t of the mycelia Paecilomyces v a r i o t i fo l lowing incubation ind icated t h a t the fungal mycelia had not r e m o v e d p h e n o l s from the media by physical adsorption. The removal of phenolic compounds from the media can therefore be a t t r i b u t e d t o fungal a t tack, coupled w i t h aerat ion effects.

Cain (3 ) examined the biodegradation of many of the phenols l i s t e d i n Table 2 by the mixed microbia l population present i n the sludge of a paper m i l l waste treatment p lan t . I n these tests , so lu t ions of t he phenols were inoculated w i t h d i l u t e d samples of the sludge and incubated a t ambient temperatures fo r periods up t o 15 days. a t selected reac t i on i n t e r v a l s using gas chromatography.

Residual phenol content was determined

62

TABLE 21. BIODEGRADATION OF PHENOLS BY FUNGI IN LIQUID CULTURE MEDIA Residual phenol, X

Initial Paecilomvces m d e m Penicillim Phenol cox. . ppn vari o t i koningii variabi 1

Creosol VI I 50 70 62 1.4 6-C h 1 orocreo so 1 VI11 50 82 70 79 5-Chl orocreosol IX 50 75 68 a3 5.6-Dichlorocreosol X 10 94 75 62 3,6-Dichlorocreosol XI 10 89 6.8 6.6 3.5-Dichlorocreosol XI I 10 70 34 16

Q, Trichl orocreosol XI11 10 77 0 0 4-Methyl catechol XVI I 50 0 0 0

4-Methyl -5,6-d ic hl oroca tec hol XI X 10 87 87 1 1 4-Methyl -3.5-6- tric hl orocatechol XX lb 40 50 44 4-Methylveratrol e XXIV 50 81 89 94 4-Methyl-6-chloroveratrole xxv 50 83 95 100

w

4-Methyl -5-chlorocatechol XVIII 50 70 44 61

p-Cresol - I 50 90 60 66 4-Met hyl - 2.6-di c hl orop henol V 10 53 6.0 3.0 2.4,6-Trichlorophenol VI 10 67 0 52

All of the phenols examined by Cain were a t l eas t par t ia l ly degraded under the t e s t conditions, The ra te and extent of phenol removal was found t o vary widely from compound t o compound and consistent trends correlating structure w i t h these two parameters were not apparent. As i n the previously described t e s t s employing individual f u n g i , the biodegradation w i t h microorganisms i n the sludge was performed il l concert w i t h aeration. sured decrease i n phenol content was therefore, in a l l probability, a compo- s i t e of the effects of biological and chemical processes. clear-cut trends i n Cain's data may therefore he due to the i r being masked as a resu l t of interference on the part of one or the other type of process.

The mea-

Failure to perceive

In view of the widespread use of biological processes i n the treatment of pulping and bleaching wastes, additional research aimed a t optimization of the operating parameters affecting phenol and chlorophenol breakdown would appear t o be highly desirable.

CHEMICAL TREATMENT OF SPENT CHLORINATION AND CAUSTIC EXTRACTION LIQUORS

I n these treatments, the chemical was generally applied to either of the two spent liquors b u t rarely to both. The choice of liquor to be used i n any given treatment was dictated by pH considerations; t h u s , those chemicals known t o be effect ive under alkaline conditions were applied t o spent caustic extraction liquor whereas those functioning i n the desired manner i n acidic media were added to the spent chlorination liquor.

In the application of those chemicals (oxidants) used as p u l p bleaching agents, the treatment temperatures selected coincided w i t h those used i n the bleaching process, pletely consumed i n order to avoid i t s contributing to acute toxici ty in any subsequent bioassays,

The treatments were continued u n t i l the chemical was com-

Chlorination Treatment

Spent chlorination liquor was reacted w i t h solutions of chlorine water a t room temperature without pH adjustment. In one treatment, 0.2 mg C12/ml spent liquor was consumed w i t h a corresponding 35% reduction of phenol content as indicated by ionization difference spectra measurement. In a second treatment, 0.8 mg C12/ml spent liquor was consumed w i t h an accompanying loss of 70% of the phenol content. The reacted solutions were subjected to bioassay u s i n g Daphnia magna as test organism, crease i n toxici ty was observed over that found for the untreated liquor. The toxici ty of the two treated liquors was no t s ignif icant ly different.

Results reported for the phenol contents of spent chlorination and caus- t i c extraction liquors here and elsewhere i n this report must be regarded as only approximate. This stems pa r t ly from the f ac t that the absorptivity v a l - ues of the component phenols of the liquor vary substantially among themselves. T h u s , any attempt t o calculate phenol concentration must involve the use of an "average" absorptivity value which cannot be determined accurately. Hence, the values for phenol content l i s t ed throughout this report indicate "phenol" content changes resulting from a specified treatment. usage i s suspect since the alkal i used to effect ionization may induce other

64

In both cases, a d i s t i nc t i n -

Even this

chemical changes i n the molecule t h a t affect i t s spect ra l charac ter is t i cs ,

. . -i.

:.a. . I .

Hypochlorous Acid Treatment

The pH of samples of ch lo r i na t i on l i q u o r was adjusted t o 4.75 and buffered w i t h acetate t o ob ta in a high concentrat ion of hypochlorous ac id a f t e r the a d d i t i o n of ch lo r ine . I n two treatments, 0.2 and 0.37 mg of H O C l / m l o f spent l i q u o r were consumed, which resu l ted i n the loss of 35 and 82% of t he phenols, respect ively, as ind icated by i o n i z a t i o n u l t r a v i o l e t spectroscopy. As i n the above instance, t he bioassay t e s t r e s u l t s ind ica ted an increase i n t o x i c i t y as a r e s u l t of the ox idat ion w i th hypochlorous ac id.

Sodium Hypochlor i te Treatment

A sample of SCEL was reacted w i t h 0.7 mg o f N a O C l / m l of l i q u o r a t 60OC.

With a la rger app l i ca t i on of hypochlor i te (2.0 mg/ml of Af ter three hours, the oxidant was completely consumed and the phenol content was reduced t o 49%. l i quo r ) , consumption a t t he same temperature was complete i n n ine hours and phenol removal was 95% complete.

Af ter these t e s t s had been completed, i t was discovered t h a t the commer- c i a l sample of sodium hypochlor i te used had a h igh ch lo r i de concentrat ion t h a t which could have affected the LC50 values of t he bioassay. Accordingly, another experiment was performed w i t h laboratory-prepared hypochlor i te (7.8 mg N a K l / m l l i quo r , 60°C, reacted t o exhaustion (%24 hrs)) having an accept- ab ly low c h l o r i d e content. The acute t o x i c i t y o f t he hypochlor i te- t reated l i q u o r s was determined using Daphnia magna and the r e s u l t s a re reported i n Table 22. As i n the previous instances where ch lo r i ne and hypochlorous ac id were applied, the t rend shown by the data i s t h a t hypochlor i te treatment ac- t u a l l y resu l ted i n an increase i n t o x i c i t y .

TABLE 22. EFFECT TO DAPAlPfA MAGNA OF SODIUM HYPOCHLORITE TREATMENT ON THE ACUTE T O X I C I T Y ~ P E N T CAUSTIC EXTRACTION LIQUOR

Exposure time, L iquor hrs. LC50 i n ppm TOC (95% C.I.)

Or ig ina l 24

96

NaOC1 -Treated 0.7 mg/ml l i q u o r 24

96

440 (41 2-469)

367 (286-490)

442 (400-489) 271 (1 10-668)

2.0 mg/ml l i q u o r 24 250 (2r)c)-314)

96 155 (122-196) a 294 - 7.8 mg/ml l i q u o r 24 a 28 1 - 48

65 aNot determined

The explanat ion f o r the increase i n t o x i c i t y of the spent l i quo rs as a consequence of the three aforementioned treatments i s not read i l y apparent. One p o s s i b i l i t y i s t h a t the treatments resu l ted i n an increase i n the amount of o rgan ica l l y bound chlor ine, p a r t i c u l a r l y t h a t subst i tu ted on the aromatic r i n g . The formation o f non-phenolic ox ida t ion products more tox ic than the phenols themselves i s also feasible, espec ia l l y i n the case of react ion w i th hypochlor i te. organic compounds t o smaller fragments u l t i m a t e l y would be expected t o have a benef ic ia l ef fect w i th respect t o t o x i c i t y reduct ion since organica l ly bound ch lo r i ne would be converted t o ch lo r i de i n the process.

Generally speaking, however, continued ox idat ion of chloro-

Treatment of Spent Chlor inat ion Liquor w i t h Chlor ine Dioxide

of l i q u o r ) a t room temperature u n t i l the oxidant was exhausted (-5 hours). The pH o f the reac t ion mixture remained a t 2 o r lower dur ing the treatment.

t o the value fo r the t h a t a modest amount of d e t o x i f i c a t i o n had resul ted.

Spent ch lo r i na t i on l i q u o r was reacted w i th ch lo r i ne d iox ide (0.27 meq/ml

The 24- and 48-hr LC5 l i q u o r were 153 and 75 ppm TOC, respect ive ly , us organism. When compared

the r e s u l t s ind ica te

Treatment of Spent Caustic Ext ract ion Liquor w i t h Hydrogen Peroxide

w i t h sodium s i l i c a t e and magnesium su l fa te , respect ively, and reacted w i th hydrogen peroxide ( 4 meq o f H 02/15r) m l of l i q u o r ) a t 7 O O C .

50% complete a f te r three hours' react ion. p o s i t i o n of peroxide was achieved on ly a f t e r ~ 2 4 hours of heating.

The 24- and 48-hr LC50 values f o r the peroxide-treated l i q u o r were 202 and 133 (ppm of TOC), respect ively, when tested w i t h Da hnia ma na, Compari- son of these values w i th those o f the untreated SCEL *?- ab e 7 reveals t h a t the peroxide treatment ac tua l l y produced an increase i n acute t o x i c i t y . This f ind ing agrees w i t h r e s u l t s reported by Bet ts and Wilson (57). Although add i t i ona l t e s t s should be performed t o conf i rm t h i s trend, the comparative un reac t i v i t y of the peroxide toward the spent caust ic ex t rac t ion 1 iquor, even a t elevated temperature, prompts the conclusion t h a t such a process would no t be techn ica l l y feasible i n any event.

Spent caust ic ex t rac t ion l i q u o r was buffered (%pH 10.5) and s tab i l i zed

Consumption of peroxide under these r e l a t i v e ? y severe condi t ions was very slow and was only

Complete consumption and/or decom-

Treatment of Spent Ch lor ina t ion and Caustic Ex t rac t ion Liquors w i th Ozone

a t pH 8, wh i l e spent caust ic ex t rac t ion l i q u o r was reacted w i th ozone only a t pH 8. Acute t o x i c i t y t e s t s were performed on the ozonized l i quo rs w i t h Da hnia ma na and br ine shrimp (Artemia sa l ina) . h e +

Spent ch lo r i na t i on l i q u o r was ozonized, both a t i t s ambient pH ( ~ 2 ) and

The r e s u l t s are sumnarized

The ozonizat ion consumption pat terns (cf. Table 23) f o r the three l i q u o r samples are s i m i l a r and consis t o f an essen t ia l l y quan t i t a t i ve uptake of ozone i n the i n i t i a l stage o f the treatment, followed by an ever-diminishing

66

TABLE 23. CHARACTERISTICS OF ORIGINAL AND OZONIZED SPENT CHLORINATION AND CAUSTIC EXTRACTION LIQUORS

Ozone, mg/ml .of l i q u o r "Phenol", Org. bound Acute t o x i c i t y TOC Color removals ch lor ine,

Appl i ed Consumed Ppm (co lo r u n i t s ) % ppm 24-Hr L C E ; ~ - i n ppm TOC (95% C . I . )

0 0.052 0.208 0,521

3 0

0.053 0.213 0.532

0 0.034 0.121 0.145

0 0.053 0.193 0.296

0 0 0.087 0.086 0.232 0.175

268 2 54 21 4 204

268 261 237 195

1640 1480 1360

Spent Ch lor ina t ion Liquor (pH 2) Br ine Shrimp

1,383 0 91 342 (126-a) 91 7 ( 4 4 69 - 57 5 7 67 - 429 55 16 284 ( 76-a)

Spent Ch lor ina t ion L iquor (pH 8 1

1,383 933 370 200

0 91 - 17 61 -

- 18 31 - 34 34 26 (19-33)

Spent Caustic Ex t rac t ion L iquor (pH 8)

18,290 0 209 1323 (466-a) 9,458 36 133 - 2,790 36 47 304 (238-434)

Daphnia magna 107 (81.2-212 )

-

53.2 (40.3-72.3)

'No meaningful est imate due t o low m o r t a l i t y

consumption w i t h cont inuing in t roduct ion o f ozone a t a constant f l o w ra te . Under the same condit ions, the t o t a l organic carbon (TOC) decreased i n each instance, but the t o t a l decrease observed w i t h i n the t e s t l i m i t s was only i n the range 17-27%. Thus, a s izable por t ion o f the spent l i q u o r const i tuents res is ted degradation t o v o l a t i l e carbon-containing compounds even i n the presence of excess ozone.

A decrease i n the co lo r o f the spent l i q u o r s accompanied the ozone treatment as has been reported and commented on prev ious ly (58,59). overa l l decrease i n co lo r was greater when the ozonizat ion was performed i n an a l k a l i n e medium.

The

The i n i t i a l and res idual phenol contents of the spent l i quo rs were mea- Because of the e r ro rs sured by means of i on i za t i on difference spectroscopy.

inherent i n t h i s method when applied t o complex mixtures, the data fo r r e - s idual phenol content i n Table 23 have t o be viewed w i t h some degree of skepticism. increase i n "phenol" content observed i n the case o f SCL (pH 2) i n the i n i - t i a l stages of ozonization, The obvious imp l i ca t i on of t h i s f inding, which has been substant iated i n rep l i ca te tests , i s t h a t e i t h e r add i t iona l ion izab le groups ( i nc lud ing phenolic hydroxyls) were created as a consequence of the ozonizat ion treatment o r t h a t pre-ex is t ing s t ructures were chemical ly modi- f i ed i n such a way t h a t t h e i r b absorbance values were increased. Although the exact extent of phenol removal i s open t o question, the ove ra l l sense of the data i n Table 23 i s t h a t i t decreases subs tan t i a l l y i n each instance.

Of p a r t i c u l a r i n t e r e s t w i th regard t o these data i s the apparent

An increase i n ch lo r ide ion content o r a decrease i n o rgan ica l l y bound ch lo r i ne content of ozonized chloro-organic mater ia l can be considered i n - d i c a t i v e of the extent o f ox ida t ive breakdown. I n the case of the spent l iquors , the organ ica l l y bound ch lo r ine content (Table 23) i s seen t o decrease w i t h increasing consumption of ozone i n a l l but one instance. I n a l l cases, however, some chlor ine-containing residues remain a t the conclusion of the treatment. The p o s s i b i l i t y t ha t extended ozone treatment of the l i q u o r s would r e s u l t i n complete removal of o rgan ica l l y bound ch lo r i ne seems u n l i k e l y i n view of the observed diminished r e a c t i v i t y o f the res idua l mater ia l toward ozone. Thus, spent l i quo rs i n i t i a l l y e i t h e r conta in ch lo r ine-subs t i tu ted s t ructures r e s i s t a n t t o ozone or are transformed t o such dur ing breakdown by ozone.

The r e s u l t s of the acute t o x i c i t y t e s t f o r the spent c h l o r i n a t i o n l i q u o r (pH 2) using Daphnia magna and br ine shrimp as the t e s t organisms are somewhat surpr is ing i n t h a t the ozonized l i q u o r proved t o be equal ly o r s l i g h t l y more t o x i c than the untreated sample. An e a r l i e r study of Bet ts and Wilson (57) showed t h a t ozonization of spent ch lo r i na t i on 1 iquor was ine f fec t i ve fo r re - ducing the acute t o x i c i t y toward salmon, Other data, co l l ec ted i n the cur- r e n t i nves t i ga t i on but no t included on Table 23, p o i n t t o a t rend i n which a small decrease i n acute t o x i c i t y appears w i th small app l i ca t ions of ozone bu t thereaf ter increases w i th increasing ozone dosages. These data a r e no t sta- t i s t i c a l l y re l i ab le , however, and hence cannot be in te rpre ted w i t h any degree of confidence.

68

Because of buffer s a l t s used t o cont ro l pH i n the a l k a l i n e ozonization treatments, Daphnia magna was replaced by b r i n e shrimp as the t e s t organism i n these cases. The LC50 values l i s t e d i n Table 23 show t h a t ozonization of spent c h l o r i n a t i o n and caust ic ex t rac t ion l i q u o r s i n an a l k a l i n e (pH 8) con- d i t i o n resul ted i n a decided increase i n t o x i c i t y under the appl ied treatment condi t ions. (Although no cont ro l data a re a v a i l a b l e f o r a sample of spent c h l o r i n a t i o n l i q u o r adjusted t o pH 8, r e s u l t s c i t e d below show t h a t a l k a l i n e treatment of such l i q u o r s a c t u a l l y causes a substant ia l reduct ion i n acute t o x i c i t y . )

Viewed c o l l e c t i v e l y , the r e s u l t s i n Table 23 r e l a t i n g t o acute t o x i c i t y lead t o the conclusion t h a t i n no instance does ozonizat ion cause a substan- t i a l reduct ion i n the t o x i c i t y o f spent c h l o r i n a t i o n and caust ic ex t rac t ion l i q u o r s a n d under a l k a l i n e ozonizat ion condi t ions, a decided increase i s produced. The reason fo r the f a i l u r e of ozone t o produce the expected benefi- c i a l ef fect i s no t obvious, p a r t i c u l a r l y i n view of the r e s u l t s reported previously; showing phenols of the types found i n spent c h l o r i n a t i o n and caust ic e x t r a c t i o n l i q u o r s t o be extens ive ly degraded by t h i s oxidant. However, the complexity of the spent bleaching l i q u o r s w i t h respect t o component composi- t i o n i s a factor t o be reckoned w i th i n at tempt ing t o r a t i o n a l i z e the effect of ozonizat ion on t h e i r t o x i c i t y . I t i s conceivable, therefore, t h a t the gain i n t o x i c i t y reduct ion r e s u l t i n g from the breakdown of phenolic substances by ozone could be o f f s e t by the formation of t o x i c mater ia ls o r as a r e s u l t of in te rac t ions between the l i q u o r components as described by Durkin (24 )

Treatment of Spent Caustic Ext ract ion Liquor (SCEL) w i t h Aluminum Sulfate

fo r one hour a t room temperature (pH 5.5). The p r e c i p i t a t e was removed hy centr i fugat ion, d issolved by add i t ion of HC1, and subsequently p u r i f i e d by d i a l y s i s through cellophane. r e t e n t a t e from the d i a l y s i s were adjusted t o the volume of the o r i g i n a l t e s t sample, tested f o r phenol content, and bioassayed for acute t o x i c i t y w i t h the r e s u l t s shown i n Table 24.

Aluminum sul fa te (1.5 g) was added t o SCEL ‘(220 m l ) and allowed t o reac t

The supernate from the cent r i fugat ion and the

The r e s u l t s show that,although the treatment w i t h alum removed a con- s iderable amount of the mater ia l i n the l i q u o r , a major p a r t of the t o x i c i t y i n i t i a l l y present was s t i l l re ta ined i n the supernate. This f ind ing accords w i t h the concept t h a t the aluminum coagulates the higher molecular weight components of the l i q u o r t h a t appear t o be less t o x i c than the lower molecu- l a r weight species.

Treatment of Spent Caustic Ext ract ion Liquor (SCEL) w i t h Lime

v ide a f i n a l concentrat ion o f 15 g/1. t a t e was removed by f i l t r a t i o n and the pH o f the f i l t r a t e was adjusted t o 9.4 by the i n t r o d u c t i o n o f C02. The l ime-treated l i q u o r , a f t e r removal of CaCC3, had the c h a r a c t e r i s t i c s recorded i n Table 25.

Suf f ic ient Ca(OH)2 was added t o spent caus t ic e x t r a c t i o n l i q u o r t o pro- Af ter standing one hour, the prec ip i -

69

,

P - - -

U U

TABLE 32. ACUTE TOXICITY OF LIME-TREATED CAUSTIC EXTRACTION LIQUOR REACTED WITH OZONE, CHLORINE DIOXIDE, AND HYDROGEN PEROXIDE

24-hr LC50 cone. of - Test oxidant, TOC, TOC, Tox i c Residual No. Oxidant meq/l I n i t i a l pH Ppn Ppn % (v/v) Uni ts t o x i c un i t s , %

0 - (SCEL) 1630 350 21.5 4.7 100

10 03

0

50

1 1 c10; 50

(Lime-Treated SCEL) 493 199 40.4 2 .5 53

5.0 370 204 55.1 1.8 39

5.0 409 61 14.9 6.7 143

50 9.4 434 281 64.8 1.5 32 12 H202b

'Heated a t 78OC for 12 hrs a f te r treatment bHeated a t 78OC f o r 4.5 hrs a f te r treatment

The amounts o f ch lo r ine dioxide, ozone and peroxide appl ied t o the lime- t reated l i q u o r were equivalent t o the addi t ion of 6.1, lfl.8, and 7.7 g/kg of pulp, respectively, i n a hypothetical bleaching stage i n which pulp consistency was 10%. Thus, even i n those instances where modest reductions i n acute t o x i c i t y were achieved, the cost i n terms of expenditure of chemical would have t o be considered economically p roh ib i t i ve and i t i s concluded tha t none of the above-described combination processes i s l i k e l y t o have much of a fu ture as a technical approach t o waste treatment.

Treatment of Spent Chlor inat ion Liquor w i th Sodium Hydroxide

leve ls throughout the t e s t by the add i t ion of aqueous sodium hydroxide. After a predetermined reac t ion per iod a t room temperature, the phenol and organica l ly bound ch lo r ine contents of the solut ions were monitored. The resu l t s are shown i n Tables 33 and 34.

EFFECT OF ALKALI TREATMENT OF SPENT CHLORINATION LIQUOR ON

The pH o f SCL was adjusted t o various pH leve ls and maintained a t those

TABLE 33. PHENOL AND ORGANICALLY BOUND CHLORINE CONTENTS

Loss o f organica l ly bound chlorine, X pH Phenol loss, % 16 hours 12 days

2 0 0 18 0 7

33 42 51

11 -

14

19

' -

36 -

44 -

55 10 69 38 74 11 80 65 75 12 73 - - 13 167 - -

The phenol loss was d i f f i c u l t t o quantify accurately due t o a s h i f t i n the absorpt ion maximum from 290 t o 310-320 nm i n the i on i za t i on difference spectrum. Nevertheless, the resu l t s c l e a r l y show a la rge decrease i n phenol content, p a r t i c u l a r l y i n the pH 7-10 range. The loss i n phenol content was para l le led by a decrease i n organica l ly bound chlor ine, which was time de- pendent. The maximum values for the loss of phenol and organica l ly bound ch lo r ine were i den t i ca l ( ~ 7 5 % ) and might be re la ted although t h i s was not esta b l i s hed.

78

k

TABLE 34. RATE OF REMOVAL OF ORGANICALLY BOUND CHLORINE FROM SPENT CHLORINATION LIQUOR WITH INCREASZNG pH

Loss o f o rgan ica l l y bound chlor ine, % PH ay 1 aY 9 Day 12

2 15 18 5 11 - 36 7 14 - 44 9 19 53 55 10 38 72 74 11 65 75 75

The ef fect of pH on the r a t e o f removal o f o rgan ica l l y bound ch lo r ine from spent c h l o r i n a t i o n l i q u o r i s revealed by t h e data i n Table 34. The trend for enhancement of the r a t e w i t h increase i n pH i s c l e a r l y evident, espec ia l l y i n the pH 9-10 i n t e r v a l . The maximum loss of ch lo r ine appears t o be reached i n shorter periods as the pH i s raised.

I n t e s t s designed t o determine the e f f e c t o f an a l k a l i n e treatment on acute t o x i c i t y , the pH of samples of spent c h l o r i n a t i o n l i q u o r (SCL) was adjusted t o 7, 9, 10 and 11 by the add i t ion o f sodium hydroxide so lu t ion and allowed t o stand i n the dark f o r 24 hours a t 5OC. The above samples and an untreated pH 2 SCL contro l were bioassayed a f te r the 24-hour per iod using Daphnia magna as the t e s t organism.

Table 35, was t o cause a reduct ion i n the acute t o x i c i t y of SCL. The amount of t o x i c i t y reduct ion increased w i t h increasing treatment pH, The benefi- c i a l ef fect of a l k a l i w i t h respect t o t o x i c i t y reduct ion i s i n agreement w i t h the r e s u l t s of an e a r l i e r inves t iga t ion reported by Leach e t a l . (36) who de tox i f ied spent c h l o r i n a t i o n l i q u o r w i t h combined aerat ion and a1 k a l i n e treatment over a six-day period.

Several o f the bioassays reported i n Table 35, espec ia l l y corresponding t o the higher pH treatment levels, gave u n s a t i s f a c t o r i l y h igh g values. a l l cases, t h i s i s a t t r i b u t a b l e t o low response ra tes even a t the highest concentrat ion tested (100%. 218 ppm TOC) and i s not t h e r e s u l t of hetero- geneous concentration-response patterns . ( i m n o b i l i t y as the response c r i t e r i o n ) a t pH 2 fo r a given exposure per iod d iv ided by the EC 0 of the a l k a l i n e t reated sample for the same exposure

creasing pH as i s i l l u s t r a t e d i n Figure 12. Although good m u l t i p l e estimates of potency over a wide pH range are ava i lab le on ly f o r 48-hour exposure periods, these data suggest t h a t the r e l a t i o n s h i p between d e t o x i f i c a t i o n and treatment pH i s l inear .

The ef fect of these a l k a l i n e treatments, as ind icated by the data i n

I n

Detox i f i ca t ion values, A , were ca lcu lated as the r a t i o of t h e EC50

period. These va 5 ues suggest I consistent decrease I n potency w i t h i n -

79

Since nei ther asparagine, which serves as a nu t r ien t , nor phosphate, which funct ions both as a buf fer and a nu t r ien t , are requi red f o r adequate growth o f the microorganisms comprising the seed, t h e i r e l im ina t ion i n d i - v i d u a l l y and i n combination Sn a r e p e t i t i o n of the aforegoing t e s t s could he lp i n i d e n t i f y i n g the cause o f the high t o x i c i t y often found i n the con- t r o l tests .

When proper ly performed, b i o l o g i c a l treatment general ly has been ef fec- t i v e i n reducing o r e l im ina t ing the t o x i c i t y of bleached k r a f t m i l l wastes (59-63). I n t h i s connection, the ava i lab le t o x i c i t y data, as can best be determined, appl ies exc lus ive ly t o the combined c h l o r i n a t i o n and caust ic ex t rac t ion e f f l u e n t ra ther than t o ind iv idua l bleach streams, and thus represents the composite property. The r e s u l t s i n Table 37 i n d i c a t e t h a t b i o l o g i c a l treatment o f the segregated streams (SCL and SCEL i n t h i s case) does n o t necessar i ly e f f e c t f u l l o r equivalent t o x i c i t y reduct ion since, i n t h i s p a r t i c u l a r treatment, SCL was d e t o x i f i e d t o a greater extent than SCEL.

Unl ike the previous s i tuat ion, invo lv ing a mixed microbia l populat ion ("seed") from a sludge, an external n u t r i e n t source (glucose was used) was required t o produce a respectable amount o f microorganism growth i n the cases of the experiments w i t h Candida u t i l i s (yeast) and the bacterium. With respect t o the former microorganism, SCL was successful ly de tox i f ied i n the presence of phosphate and glucose based on the r e s u l t from the 24- hour tes t . A f t e r 48-hour exposure t o the l iquor , however, the Da hnia magna displayed an except ional ly h igh incidence o f death ( t e s t 12). h t h e r previous instances, the contro l sample was considerably more t o x i c than the untreated 1 iquor ( c f . tests 1 and 11 ) .

t o degree o f d e t o x i f i c a t i o n general ly para l le led those obtained when a mixed pop- u l a t i o n of microorganisms (seed) was appl ied (compare tes ts 13 and 14 w i t h 7 and 8, Table 37).

e f f e c t i n g the biodegradation of SCL and SCEL, Candida u t i l i s was a lso se- lec ted by reason o f i t s being an ed ib le p r o t e i n source. extent t h a t i t would be able t o use SCL and SCEL as carbon sources f o r pro- t e i n production, t h i s fungus of fered some i n t e r e s t i n g p o s s i b i l i t i e s . Unfor- tunately, the carbon concentrat ion provided by the organic s o l i d s i n the two l iquors was i n s u f f i c i e n t t o provide the amount o f growth required t o make such a process f u l l y pract icable. Thus, although the f u t u r e of Candida u t i l i s does no t appear t o be p a r t i c u l a r l y b r i g h t w i t h respect t o the app l i - c a t i o n described above, addi t ional work should be performed before t h i s approach i s abandoned.

The r e s u l t s from the treatment o f SCEL with yeast i n regard

I n a d d i t i o n t o i t s po ten t ia l u t i l i t y as a microorganism capable of

Therefore, t o the

General ly speaking, the bacterium was i n e f f e c t u a l i n reducing the t o x i c i t y o f e i t h e r SCL o r SCEL ( tes ts 15-18, Table 37), and the death pat- t e r n of the Daphnia magna was very e r r a t i c i n the tes ts employing the former 1 i quor .

85

REDUCTION I N SCL AND SCEL TOXICITY THROUGH MODIFICATIONS OF THE BLEACHING PROCESS

An a l t e r n a t i v e t o the approach o f e l im ina t ing bleaching l i q u o r t o x i c i t y by chemical o r b io log i ca l treatment o f the bleaching ef f luent i s t o modify conventional bleaching stages i n such a way t h a t the offending chemicals are e i the r no t produced o r are formed i n smaller amounts. The l a t t e r approach has been adopted by several invest igators fo r the purpose of achieving not on ly t o x i c i t y reduct ion but color, COD,and BOD improvements as wel l . Notable i n t h i s connection are modif icat ions i nvo l v ing the p a r t i a l replacement of ch lo r ine by ch lo r i ne d iox ide i n the f i r s t stage (57, 64, 65) replacement of ch lo r ine w i t h hypochlor i te ( 6 6 ) , subs t i t u t i on of the f i r s t caust ic extrac- t i o n stage by a hypochlor inat ion treatment (64). and the add i t i on of hydrogen peroxide t o caust ic ex t rac t ion stages (67). (65), novel bleaching sequences invo lv ing combinations of gaseous ch lo r ine and ch lo r i ne dioxide, oxygen and hydrogen peroxide have been explored as possible means of reducing e f f l u e n t t o x i c i t y .

I n the present invest igat ion, several combinations o f the modi f icat ion treatments c i t e d above were evaluated for t h e i r e f fect on t o x i c i t y . Except as otherwise noted, the pulp used i n the bleaching t e s t s was a commercial sample of southern p ine k r a f t taken from the same batch used fo r the prepa- r a t i o n of the spent l iquors , The “conventional“ and process-modified 1 i- quors were-subjected t o acute t o x i c i t y bioassays using Da hnia ma na as the

exposure appear i n Table 38. are a lso expressed as tox i c un i ts .

I n another i nves t i ga t i on

t e s t organism. The t e s t condi t ions and LC5 values a t ++ var ous per ods of I n the case o P the 24-hour tests , the r e s u l t s

When the caus t ic ex t rac t ion stage was replaced by hypochlor i te and alka- l i n e hydrogen peroxide treatments (Table 38, t e s t s 3 and 4, resp.), the acute t o x i c i t y was no t subs tan t ia l l y d i f f e ren t from t h a t found when a con- vent ional ex t rac t i on stage was employed. The finding, w i t h respect t o hypochlor i te treatment, i s cont rary t o t h a t reported by Ga l l and Thompson (64); who determined t h a t in t roduc t ion of hypochlor i te i n t o the second stage ex t rac t i on had the effect of reducing the acute t o x i c i t y of the r e s u l t i n g eff 1 uent .

Sequential C102/C12 treatment (Table 38, t e s t 5 ) caused a pronounced reduct ion i n e f f l u e n t t o x i c i t y as compared t o the s i t u a t i o n where an equiva- l e n t amount o f ch lo r i ne alone was appl ied ( t e s t 1) . t r a c t i o n of the ClO2/Clz-treated pulp produced an ef f luent w i t h a somewhat lower t o x i c i t y than t h a t resu l t i ng from caus t ic ex t rac t i on of the pulp re - acted w i t h ch lo r i ne alone (cf. tes ts 2 and 6). i n t o the ex t rac t i on stage of the pulp from a C102/C12 sequential treatment a lso proved advantageous, On the other hand, the use of an equivalent amount o f hypochlor i te i n the caust ic ex t rac t i on of the same pulp a t the very best produced no advantage and may even have had a detr imental ef fect w i t h respect t o acute t o x i c i t y (cf . t e s t s 2 and 8).

Subsequent caust ic ex-

The in t roduc t i on of peroxide

86

TABLE 38. TOXICITY OF EFFLUENTS FROM CONVENTIONAL AND PROCESS-MODIFIED BLEACHING STAGES

Treatment o r ~ ~ 5 0 , PW T O C ~ . 24-hr t o x i c u n i t s Test no. treatment sequencea 24-hr 48-hr 72-hr 96-hr ( T O C / ~ ~ - L C ~ Q TOC)

1 2 3 4 5 6 7 8 9

10 11

94 480 430 524 186 660 789 350 380

690 43rj

51 41 0 28 0 467

55 421 47 6 190 150 423 265

- 380 220

- 100 340 180

- 160

70 31 0 140

2.4 2.9 3.0 2.9 1.4 2.3 1.8 3.6 0.6 2.0 2.6

400 3 50 280 - 12 A/ 0 560

'C = ch lo r ine (5.25%); E = caust ic ex t rac t ion (3.6% NaOH); P = hydrogen peroxide (0.5%); H = sodium hypochlor i te (2.0%); D = ch lo r ine d iox ide (2.0%); D + C = sequential ch lor ine d iox ide (1.9% C102); A/O = alkaline-oxygen (80 ps ig oxygen).

bValues corresponding t o second-stage ef f luents i n two-stage treatments.

When the f i r s t bleaching stage consisted of the sole application of chlorine dioxide ( t e s t 9), toxicity reduction was further enhanced as compared t o the cases where an equivalent amount of chlorine was used alone or sequentially i n combination w i t h chlorine dioxide ( t e s t s 1 and 5, resp.). Subsequent extraction of the dioxide-treated pulp w i t h a lkal i ( t e s t 10) generated a liquor having an acute toxicity less t h a n t h a t shown by the caustic extraction liquor derived from the chlorinated p u l p ( c f . t e s t s 10 and 2 ) b u t i n the same range as the liquor obtained by extracting the ClOZ/C12-treated pulp. Extraction of the dioxide-treated p u l p w i t h hypochlorite ( t e s t 11) appeared, on the other hand, t o enhance s l ight ly the toxici ty of the result- i n g liquor as compared t o the case where a conventional a lka l i extraction was employed (cf . t e s t s 70 and 11).

The final test recorded i n Table 38 ( t e s t 12) was performed in an i n - dustrial laboratory on a sample o f southern pine kraft (Kappa No. 39.2) and a portion of the spent l iquor was provided for acute toxici ty bioassay. The effectiveness of the alkaliloxygen treatment i s probably best judged by comparison w i t h the toxicity of other alkaline spent liquors l i s ted i n Table 38. i n t h i s case, is i n the same range as those for the other alkaline liquors l i s ted i n the table w i t h the exception of the s i tuat ions where hypochlorite was employed. However, the advantage o f the A/O treatment l i e s i n the fac t that i t requires no prior bleaching stage as the other modification sequences i n Table 38 do, thus eliminating a source of material having a relatively h i g h toxicity.

Since the foregoing bioassay results indicated the value of replacing a l l or part of the chlorine by chlorine dioxide i n the f i r s t stage as a means of reducing the acute toxicity of f irst- ahd second-stage effluents, these spent liquors were subjected to additional characterization t e s t s i n an e f for t t o understand the reason for this effect . The resul ts of these analyses a re shown i n Table 39.

Such a comparison leads t o the conclusion tha t the acute toxicity level ,

The par t ia l or total replacement not to have had d particularly s i g n i f 1 eve1 s of the f i r s t-s tage ef f 1 uents . effluents from the subsequent caustic f icant ly w i t h increasing substi tution f i r s t stage. The TOC contents of the cantly only as a resul t of a 109% rep i n the f i r s t stage.

of chlorine by chlorine dioxide i s seen cant effect on e i ther the color or TOC On the other hand, the color of the extraction treatments decreased s igni- of chlorine dioxide for chlorine i n the extraction liquors decreased s igni f i - acement of chlorine by chlorine dioxide

As expected, the chloride ion concentrations of a l l the liquors included i n Table 39 decreased as the proportion of chlorine dioxide used i n the f i r s t stage increased. washing chlorine- and/or chlorine dioxide-treated pulps w i t h d i lu t e hydrochloric acid rather t h a n w i t h tap water, the chloride ion contents of the caustic extraction liquors corresponding to pulp washed i n this manner are somewhat higher t h a n normal. by comparing the f i n a l two values under the heading "chloride" in Table 39.

Because of the practice followed i n this investigation of

The magnitude o f this difference can be judged

88

TABLE 39. CHARACTERISTICS OF SELECTED CONVENTIONAL AND PROCESS-MODIFIED SPENT BLEACHING LIQUORS

Treatment o r Org . -bound treatment Color TOC Chl o r i ge chlor ine, OrgeTc$lorine h,,c 1,,-3 s equencea (D t-c.0 u n i t s ) (ppm~b (PPm) DPm

C 1290 228 97 3 90 0,394 0.53 2.3

D + C 1360 260 600 97 0.373 0.61 2.3

D 1120 226 179 107 0.474 0.39 1.7

C,E 17,930 1410 840 21 0 0.148 0.28 0.20

D + C,E 13,100 1515 380 124 0,082 0.23 0.15 00 rD

D,E 71 90 1220 105 5 0.004 0.10 0.08

D ~ , E - - 45 10 - - - 'C = chlorine; D = ch lo r ine dioxide; E = caust ic extract ion; D + C = sequential ch lo r ine dioxide-

b\lalues correspond t o second-stage e f f l uen ts i n two-stage treatments. 'Measurement a t 290 m f o r ac id i c l i quo rs and 315 an for a l k a l i n e l iquors. dDioxide-treated pulp washed w i t h 0.01 N H2S04.

ch lo r ine treatment.

As expected, the o rgan ica l l y bound ch lo r i ne contents (Table 39) of the f i rs t -s tage 1 iquors were considerably higher than those of the corresponding caust ic ex t rac t i on 1 iquors. The o rgan ica l l y bound ch lo r i ne content of the l a t t e r decreased sharply as ch lo r ine was replaced by ch lo r i ne dioxide, and a t 100% replacement, the organ ica l l y bound ch lo r i ne content of t he l i q u o r was essen t ia l l y n i l . This f i n d i n g i s i n general agreement w i t h r e s u l t s reported by Rapson and Anderson (68). trend i s no t as c l e a r cut, bu t the organ ica l l y bound ch lo r i ne content of t he 1 iquor from the 100% d iox ide treatment i s subs tan t i a l l y higher than fo r the l i quo rs from the C and D + C stages. eff luent was the lowest o f the f i r s t - s t a g e l i q u o r s (see Table 38). t he expected d i r e c t re la t i onsh ip between organ ica l l y bound ch lo r i ne and t o x i c i t y was not demonstrated, cludes both a l i p h a t i c - and aromatic-bound ch lor ine, and i t i s t he l a t t e r which i s genera l l y thought t o be the more important con t r i bu to r t o t o x i c i t y .

I n the case of t he f i r s t - s t a g e l iquors , the

Inasmuch as the t o x i c i t y of t he D stage

However, the organ ica l l y bound ch lo r i ne analysis i n -

The A-absorbance (Aa) values i n Table 39 a re assumed t o represent p r i n - c i p a l l y changes i n the phenolic content of the l iquors . The problems associated w i t h the use of i o n i z a t i o n d i f fe rence spectroscopy fo r such a purpose have been discussed previously, and i t suffices t o emphasize here t h a t the measured changes i n Aa values a t best merely r e f l e c t oss ib le changes I n

s ign i f i can t decrease i n ''phenol" content was effected on ly i n the case where the c h l o r i n e was completely replaced by c h l o r i n e dioxide. The corresponding caustic- e x t r a c t i o n 1 iquor showed a systematic decrease i n "phenol" content w i t h increas ing replacement of ch lo r i ne by d iox ide i n the f i r s t stage, and t h i s change pa ra l l e led the decrease i n o rgan ica l l y bound ch lo r i ne content of the same l iquors . I n none of the t e s t s performed i n t h i s i nves t i ga t i on was the "phenol" content reduced t o zero, and j t may be speculated t h a t such a cond i t ion could no t be achieved without a p r o h i b i t i v e expenditure of chemical.

phenol content. With t h i s i n mind, the Aa data I n Ta h e reveal t h a t a

90

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67. DeLattre, M.G, and G. Papageorges. In t roduc t ion of Hydrogen Peroxide i n the A lka l i ne Ext ract ion Stages During the Bleaching of K r a f t Pulps. (Trans lat ion o f a l ec tu re given a t the 6 th Annual Convention of the Technical B r a s i l i a n Associat ion o f Cel lu lose and Paper, S5o Paulo, Bras i l , 19-23 November 1973) In te rox Coordination. Solvay & CIE, Brussels, Belgium, 1974. 21 pp.

68. Rapson, W.H, and Anderson, C.B. Mixtures o f Chlor ine Dioxide and Chlo- r i n e i n the Chlor inat ion Stage o f Pulp Bleaching. Pulp Paper Mag. Can., 67 ( 1 ) : T47-T55, 1 966,

96

APPENDIX' A

Stock Solut ions

1 M KN03 1 M MgSOq . 7H20 1 M KH2P04 Fe Versenola Micronutr ients 1 M Ca(N03)2 NaHC03 (15 g/1) Double d i s t i l l e d water

b

m l / l

12 10 9 1 1 0.1 1

966 1000 m l

aFe Versenol: Dissolve 26.1 g Versenol i h 276 m l 1 M KOH. Add double d i s t i l l e d water t o 500 m l . Dissolve 24.9 g

7H 0 i n 500 m l double d i s t i l l e d water. Mix the FeSO? wo * so ? ut ions s t i r r i n g r a p i d l y and aerate over- n ight. Store i n a foi l covered container under r e f r i g e r a t i o n because the r o t a t i o n i s l i g h t and temperature sensi t ive.

bM i cro nu tr i en t s : Ingredients Conc. i n Stock Solut ion g/1

C0C12 . 6H20

H 803 MnC12 . 4 H20 ZnS04 . 7 ~~0 CuS04 . 5 H20 Moo3 (97.5%)

97

0.040 2.86 1.81 0.222 0.079 0.015

APPENDIX B

Hutner's Medium f o r Duckweed

Solut ion 1:

Solut ion 2:

Solut ion 3A: 3.295 g ZnS04 . 7H20

17.7 g of Ca(N03)2 . 4H20 and 10.0 g o f NH4N03/liter

20 g of K;IHP04/l

d issolved i n 200 m l o f water

0.710 g H3B03 1.260 g Nap Moo4 . 2H20

Add HC1 t o disperse cloudiness 0.190 9 cUso4 . 5H20

0.010 g Co(NO3)2 0.897 MnC12 . 4H20

Solut ion 38: 25.09 of EDTA, add s u f f i c i e n t KOH t o d isso lve and a 4 0 0 m l water

Solut ion 3C: 1.25 g FeS04 . 7H20 dissolved i n 200 m l water

Solut ion C was prepared by adding 3B t o 3C and then adding 3A. The so lu t i on was d i l u t e d t o one l i t e r and the pH adjusted t o 6.

Solut ion 4: 25.0 g o f %SO4 . 7HO/l

Hutner's medium (1/2 strength) consists of 5 m l each o f so lu t ions 1-4 and 580 m l of d i s t i l l e d H 0. Ten g of glucose i s added, the pH adjusted t o 6.1-6.4 and td so lu t i on i s autoclaved.

98

1 1 TECHNICAL REPORT DATA

(Please read Insfmettons on the reveme before completing) I. REPORT NO. 12. 13. RECIPIENT'S ACCESSION NO.

EPA-600/2-80-039 I LTITLE ANDSUBTITLE ~ ~ ~ i c i t y Reduction Through Chemical And B io log ica l Modi f icat ion o f Spent Pulp Bleaching Liquors

I 8. PERFORMING ORGANIZATION REPORT NO.

Carl ton Dence, Chun-Juan Wang, and f. AUTHOR(S)

5 . R E P O R T D A T E January 1980 issu ing date

6. PERFORMING ORGANIZATION CODE

Pat r ick Durkin 10. PROGRAM ELEMENT NO. I . PERFORMING ORGANIZATION NAME A N D ADDRESS

1. DESCRIPTORS b.lDENTIFIERS/OPEN E N D E D TERMS

Pulping, I n d u s t r i a l Wastes, Phenols, Bio- Spent Chlor inat ion assay, chemical treatment, t o x i c i t y , b io- l iquors , chlorophenols, 1 og i ca l treatment Total organic carbon

State Un ivers i ty o f New York College of Environmental Science And Forestry Syracuse, N.Y. 13210

I n d u s t r i a l Environmental Research Lab. - C in t i . , OH Office o f Research and Development U.S. Environmental Protect ion Agency Cincinnat i , Ohio 45268

12. SPONSORING AGENCY NAME A N D ADDRESS

15. SUPPLEMENTARY NOTES

C. COSATl Field/Group

13/B

1 BB610 11. CONTRACT/GRANT NO.

18. DISTRIBUTION STATEMENT

RELEASE TO PUBLIC

R-804779 13. TYPE O F REPORT A N D PERIOD COVERED

14. SPONSORIN6 AG NCY C D E F i n a l : 9I7-Q

19. SECURITY CLASS (ThisRcport). 21. NO. OF PAGES

111 22. PRICE

UNCLASSIFIED UNCLASSIFIED

20. SECURITY CLASS (ThiJpge)

EPA/600/12

16. ABSTRACT mlTFi7p nenois s i m i l a r t o o r i d e n t i c a l w i t h those detected i n spent c h l o r i n a t i o n and caust ic ex t rac t ion l iquors were synthesized and tested over a range of concentrat ions t o determine t h e i r e f f e c t on the growth o f several fungi, an alga (Ch lore l la pyrenoidosa) and dirckweed JLemna p e r p u s i l l a ) and on the surv iva l o f Daphniamagna. -

B io log ica l treatment of the chlorophenols consisted of the a p p l i c a t i o n o f pure cu l tures o f three d i f fe ren t fungi and a mixed microbia l populat ion f o r periods rang- i n g up t o 15 days. Degradation var ied widely among the various phenols and for the same phenol t rea ted w i t h d i f ferent fungi.

o f chemical treatments and the r e s u l t i n g effects on acute t o x i c i t y determined. Treatment w i t h elemental chlor ine, hypochlorous acid, hypochlor i te, ozone and hydrogen peroxide produced increases i n the t o x i c i t y of the spent l i q u o r .

volved the a p p l i c a t i o n of a fungus (Candida u t i l i s ) , an u n i d e n t i f i e d bacterium, and a mixed microbia l population, together w i t h +mental carbon sources.

caust ic e x t r a c t i o n bleaching stages was a lso evaluated.

Spent c h l o r i n a t i o n and caust ic ex t rac t ion 1 iquors were subjected t o a v a r i e t y

B io log ica l treatment of spent ch lo r ina t ion and caust ic e x t r a c t i o n l i q u o r s i n -

T o x i c i t y reduct ion through modi f icat ion o f conventional c h l o r i n a t i o n and

1 I

E P A Form 2220-1 (Rov. 4-77) P R E V I O U S E D I T I O N I S OBSOLETE

0 U 5 GOVtRNUfNI PI1INIIN(IOflICt l W . b 5 7 - L l b / 5 5 7 9 99

toxicity reduction through chemical and biological modification of spent pulp bleaching liquors

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