The toxicity of pulp and paper mill effluents and corresponding measurement procedures

~4arer Research Vol. H), pp. 6."9 to 664 Pergamon Press 19"6. Printed in Great Britain.

REVIEW PAPER THE TOXICITY OF PULP A N D PAPER MILL EFFLUENTS A N D C O R R E S P O N D I N G MEASUREMENT PROCEDURES C. C. WALDEN B. C. Research. 3650 Wesbrook Crescent, Vancouver, B.C. V6S 2L2, Canada (Receiced 18 March 1976)

INTRODUCTION The impact of pulp and paper discharges on receiving waters results from the integrated action of oxygen demand, suspended solids, pH, color, and toxicity. In this presentation, effects due to toxicity only are examined, even though it is frequently impossible in natural situations to segregate effects due to toxicity from those due to other effluent characteristics. Most of the information comes from laboratory studies carried out under controlled conditions, with Iimited data available concerning the impact of toxicity under conditions in natural ecosystems. The toxicity of any complex effluent results from the combined action of a number of chemical constituents in the waste. Because the toxic chemicals in pulp and paper effluents are not completely identified, their effects must be assessed by a biological measurement. The bioassay is thus used as a quantitative measure of the integrated effect of the toxic materials in pulp and paper discharges. An important part of this study has been to define reproducible bioassay procedures to measure this biological response. It is anticipated that the reader will possess background knowledge concerning pulp and paper processing to appreciate the terminology used to describe the various processes and processing stages relating to effluent origins. Where necessary, the reader is advised to consult standard reference texts (Casey, 1961; Macdonald and Franklin, 1965). WATER

USAGE

IN

RELATION

TO

TOXICITY

Although the acute toxicity of various pulp and paper mill effluents is documented subsequently, almost invariably their toxicity is extremely limited. Despite their low toxicity, pulp and paper discharges may have a toxic impact on receiving waters because of the tremendous volumes discharged. Data relating to water usage for various types of pulping are given in Table 1. Even small groundwood mills, processing 50 tons d a y - t discharge 300,000-500,000 gal day-x. The bleached kraft pulp mill of 750 tons day- t capacity discharges 20 to 30 million gal of effluent day- x. 639

For the larger forest product complexes, involving mechanical and chemical pulping, production of bleached pulp and formulation of various papers, discharges in excess of 50 million gal day-1 are common. Thus, the toxic impact of pulp and paper discharges on the environment relates to the total toxic loading, involving tremendous volumes of wastewaters of limited toxicity, from a single point of discharge. Although control regulations usually limit both the concentration and the total weights of materials discharged, it is less common to do so with toxicity. One instance of such a procedure is the California State Water Quality Control Plan for Ocean Waters (1972), which limits the total discharge of toxic materials according to a formula representing the product of the discharge volumes times a measurement of toxicity. In recent years the pulp and paper industry has emphasized reuse of process waters, reducing considerably the volumes to be discharged. Currently, attempts are being made to achieve even greater reductions. However, reuse of process water does not necessarily reduce the release of toxic materials. Unless the changes result in destruction or diminished production of toxic substances, the net result is the same toxic loading, although in lesser volumes of water. Except to reduce the hydraulic loading on treatment systems, recycling may not affect toxicity unless zero discharge is achieved, as per Rapson (1967) and Histed and Nicolle (1973). Table 1. Effluent volumes of some pulping discharges ua=*: vot~a

s~dra~lle d t b a r ~ n s

~00 - I0.000

Cro~dwQ~

6.5~

(vtth rac:~ry)

3,000 - 20.000

g : s f : pulp~.~$

6,C00

- lo.O00

-

20.000

SulZ~t* p u / p i ~ (:o =ecovery)

20.0O0 - 3o,0o0

(,~t~ :*~vtry)

20,0~

el**=~t

20,000 - t o , G o o

- 30,000

640

C . C . WALDEN BIOASSAYS FOR MEASLRING ACUTE TOXICITY

Toxicity is a biological response, which when quantified in terms of the concentration of the toxicant. can constitute the basis for a bioassay procedure. In its broadest terms, the bioassay is "'the measurement of the potency of any stimulus, physical, chemical or biological, physical or physiological by means of the reactions it produces in living matter" (Finney. 1941}. To produce meaningful results, bioassay procedures require carefully controlled test conditions. For pulp and paper effluents, bioassays usually use an indigenous species of fish. with death as the response criterion. Chemical assays are not yet feasible as a technique for assessing the toxicity of pulp and paper effluents. Some of the toxicants have not been identified and, consequently, cannot be determined chemically. Moreover, chemical assays of toxicity are valid only when results can be related to biological responses by a previously established relationship. Where the toxic constituents of a waste are unknown, or only partially known, bioassays are the only method of measuring toxicity. The bioassay of pulp and paper effluents poses special problems. The low concentration of toxicants requires high concentrations of the effluents (v v - t ) in bioassay test solutions. The high oxygen demand of these test solutions necessitates oxygenation to maintain adequate oxygen levels for fish respiration during the test. These conditions are highly conducive to depletion of the unstable toxic constituents. Compounding these problems is the depletion of toxicants due to their uptake by test fish, as occurs even in bioassays of stable and highly toxic materials (Spracue, 1969, 1970, 1971; APHA, 1971}. Early workers examined some of the specific problems in the bioassay of pulp and paper effluents.. Warren and Doudoroff (1958) suggested guppies (Lebistes reticulatus) as test organisms and indicated a requirement for oxygenation during bioassay, Betts, Beak and Wilson (1967) devised a flow-through apparatus for replenishing test solutions, which required only small volumes of effluent and was applicable to pulp and paper effluents. Blosser and Owens (1970}, and Howard and Walden (1973), examined available bioassay procedures. More recently, Walden and McLeay (1974), and Walden, McLeay and Monteith (1975) have undertaken a detailed study of the problems specific to the bioassay of pulp and paper effluents.

Basic procedures for acute bioassays The basic procedure for any bioassay involves establishing the combination of concentration and exposure time which permits partial survival of test fish. The percentage survival usually chosen for * Synonymous terms are ZTso--effective time to death, and MST--median survival time. + Synonymous with TL.--median tolerance limit.

measurement is 50°o, since this value is the most accurate and permits bioassay data to be examined by probit analysis (Finney, 19411 and manipulated by analytical mathematical techniques ILitchfietd and Wi!coxon, 19531. Since 0 and 100°i~ survivals ha~e no meaning when data are processed by these procedures, these limiting survivals are seldom used in toxicity estimates. The ingestion of a lethal dose by test fish is a t\mction of concentration of the bioassay solution and the time of exposure. That is. the lower the concentration, the longer the exposure time must be to effect fish kills. The plot of paired concentrations and exposure times corresponding to fish survivals such as 50°, normally produces a hyperbolic curve, with the log-log plot producing a straight line. Bioassay procedures involve measurement of concentrations or exposure times necessary' to achieve a specific survival of test organisms. Where the concentration is fixed, exposure time is expressed as Lethal Time to death of the median fish (LXso*). Where exposure times are fixed, commonly at 24, 48 and 96 h, a range of concentration is examined and the 503;, survival concentration determined by interpolation of three or more survivals in the 0-100q~, range. The resulting value is the lethal concentration permitting 50% survival (LCso+}. In bioassays used for regulation monitoring, exposure time, effluent concentration and fish surxivals are fixed. In the test procedure, if fish survivals exceed the stipulated value, the sample passes: if not. it fails. Acute bioassays are not routinely run for exposure periods exceeding 96 h. That is, the hyperbolic response curve relating concentration to exposure time is virtually flat at exposures greater than 96 h. For further details, see Sprague (1969).

Procedural variables specific to pulp aml paper effluents For bioassays utilizing salmonids the dissolved oxygen should be maintained above 9.0 mg 1- ~: since Hicks and DeWitt (1971) have stated that below this level, the survival time of juvenile coho salmon in bleached kraft effluents, is markedly reduced. This oxygen level is appreciably higher than the 5.0 mg t- ~ recommended by APHA U9711. Where salmonids are used for bioassays, an appropriate temperature for acclimation, maintenance and testing ts 15 0 l ' C . The solubility of oxygen at this temperature is t0.2 mg 1- L Thus, if bioassay data are not to reflecl inadequate dissolved oxygen m test solutions, levels should be controlled between 9.0 and 10.2 mg IOxygen levels in bioassay test solutions can be maintained between 9.0 and 10.2 mg 1-1 provided solutions are replaced every 24 h ~Wafden and McLeay, 1974). Additional requirements are that the solutions, themselves, must have no oxygen demand and the fish loading must be tess than 0.5 g 1- ~ These data are in rough agreement with the figures cited by Sprague (1969), of 0.3-0.5 g 1-~. where 90 ° of the

Toxicity 9,-

I

/

of

pulp and paper mill effluents

UI

/

,,,,"

/

~ 1 1

81/

et

"b

/ ~

,,;u

~.;r

_~/

~<7.,..~- ~

It h ' ft.-"7 V:/I -/ f

-.L J

_, O0

z

II I

*

,~o~"

°:L/,if// I~J"'J" 0

"t""

i 4

I

I 8

I

I I 12 T I M E , hr

I 16

I

I 20

I

I 24

Fig. l. Oxygen demand for periods up to 24 h, of various sewers and outfalls from five bleached kraft mills, at 65°0 concentration (Walden and McLeay, 1974). bioassay solutions are replaced every 8-12 h by flow- able to oxygenate the bioassay solutions directly, through techniques. rather than the larger volumes of replacement soluThe oxygen demand of most bioassay test solutions tions. containing pulp and paper effluents is considerably Walden and McLeay (1974) indicated that an acgreater than the oxygen requirement for fish respir- ceptable procedure, concomitant with minimal loss ation (Fig. 1). Indeed, if oxygen levels are to be mainin toxicity, was to inject minimal amounts of oxygen tained above 9.0 mg l - t by replacement only, solu- directly into the bioassay solution. Injection through tions would need to be exchanged every 4 h (Walden a fine hypodermic needle, mounted at the bottom of and McLeay, 1974). Only where effluents are abhorthe test vesset, produced fine bubbles which were mall}, toxic, i.e. low concentrations in test solutions, almost completely adsorbed into the bioassay solucan oxygen levels be maintained above 9.0 mg l - t , tion. Foaming was avoided, which might have by replacement every 8-12 h. For sulfite effluents, resulted in fractionation of the toxic constituents (Ng, which have substantially higher oxygen demands than Mueller and Walden, 1973). Direct injection of minikraft effluents on which these data were secured, the mal amounts of air did not affect the toxicity of the problem of maintaining oxygen levels by solution more toxic effluents, but did result in some toxicity exchange is proportionately greater. loss in the less toxic ones (Walden and McLeay, The problem of maintaining adequate dissolved 1974). Supersaturation, which occurred with the use oxygen levels in bioassay solutions is illustrated in of pure oxygen, affected bioassay data. Walden and the following example. A typical test solution volume McLeay (1974) concluded that with the above prois 30 1., and requires exchange every 4 h. Over a 96-h cedure, oxygen levels were easier to keep under 10.2 exposure, 690 1. are required for direct exchange and mg 1- t, than with other procedures which were exam5184 1. for a 90'~i; flow-through replacement technique. ined. Automatic control of oxygen levels was not feasFor concentrations applicable to pulp and paper ible with available commercial instrumentation. -o/ effluents, e.g, 6~,o, these numbers are still 449 and In pulp and paper bioassays, solution replacement 3370 1. for the two procedures, respectively. is mandatory to maintain the concentration of the This problem cannot be solved by the on-site bio- toxicants, which otherwise are depleted because of assay of mill effluents, which could supply the their instability and their uptake by test fish. This required volumes with minor pumping facilities. Simi- concentration must be maintained in the range which lar problems are encountered in the preparation of permits partial fish survivals if useful bioassay data the replacement solutions, which must contain 10.2 are to be obtained. For stable toxicants, where deplemg 1-* of dissolved oxygen. Most mill effluents are tion is due solely to uptake by test fish, the concendevoid of oxygen and their concentration in bioassay tration can be stabilized in many instances by mainsolutions is such that the small proportion of dilution taining a low fish weight to solution volume ratio. water cannot furnish the required oxygen to give 10.2 However, where the toxicants are unstable, periodic mg 1-t in the replacement solution. Adequate levels 'exchange of test solutions is necessary to maintain of dissolved oxygen can only be achieved by direct concentration levels. Thus, for bioassay of pulp and oxygenation. Under these circumstances, it is prefer- paper effluents, a suitable combination of fish-to-

6d2

C . C . WALDEN 25

20

0,2

-

A

7-6--

A

5 I0

l

[

12

24

I

I

I

36 T I M E , hr

48

60

A I ,

72

A

1,

I

84

96

Fig. 2. LC~o values of a bleached kraft mill effluent sample, corresponding to three fish loadings and 12-h solution replacement; for different exposure times (Walden et al., 1975). fish was limited when fish loadings were 0.5 g lor less. Toxicant concentrations could be maintained at stable levels for exposure periods of 72-96 h if fish loading was 0.5 g 1-L or less and if bioassay solutions were replaced every 24 h. Replacement of solutions every 12 h permitted use of fish loadings up to 2.0 g 1-t, for any exposure time examined (Fig. 2). Solution replacement may be achieved by two techniques; flow-through or direct exchange. Flowthrough involves continuous addition of a replenishment solution with a corresponding discharge via an overflow. Direct exchange involves rapid transfer of fish from the exhausted bioassay solution to a fresh one, usually utilizing a nylon net lining the bioassay vessel, thus minimizing disturbance of the test fish. Walden et al. (1975) showed that the results achieved by 90~ replacement by flow-through and by solution exchange, produced the same results for individual samples (Fig. 3). Disadvantages of the flow,through

volume ratios and periodicity of solution replacement must be employed so that lower fish loadings or more solution replacement or both, do not affect the measured toxicity. Fish-to-volume ratios have been established for stable toxicants and recently suitable fish loadingsolution replacement combinations have been assessed for pulp and paper effluents. The APHA procedure (1971) stipulates fish-to-volume ratios of at least 1 1. g - t d a y - t , whereas Sprague (1969) recommended 2-3 1. g - t day-1. Davis and Mason (1973) substantiated Sprague's values for bleached kraft effluents for the ETso technique. Their bioassay solution concentrations were approximately 2.5 times those necessary to secure 96-h LCso data, so desirable fish loadings for longer exposures probably should have been lower, Walden et al. (1975) examined various combinations of fish loading and solution replacement, in the bioassay of bleached kraft effluents. For static 96-h bioassays, depletion of toxicants by I00

50--

d

,2hr

-

re) 20--

O Static clmnCeoq,t¢ X 9 0 % replac~mqmt flowtlvo~lh

I0 1.7

I 12

,,I 24

1 48

t 72

I 96

TIME, hi"

Fig. 3. LCso values obtained by flow-through (90~ replacement) and by direct solution exchange at 12 and 24 h, for two bleached kraft mill effluent samples (Walden er al., 1975).

Toxicity of pulp and paper mill effluents Table 2. Effect of pH. alkalinity and hardness of bioassay test solutions on the measured ETso (Rainbow trout, Salmo Gairdneri) of a sample of kraft unbleached whitewater (pulping effluentl Effluen: Concentratlon -: v/v !5

Alkallnity I -m~/l CaCO]

Rardness 1 -mg/l CaCO 3 (EDTA)

ET5( -mir

10

13

126

6.83'4

106

i~3

126

7.93

i35

143

340

pH 1

6.72

6.72

1. 2. 3. 4.

33

29

48

6.73, 4

100

t27

50

7.3 3

125

124

58

pH-6.7; alkal~nl=y-4.0 mg/l; hardness-9.0

occurring. Increases in toxicity during storage were associated with anaerobiosis and the formation of hydrogen sulfide. Samples must be stored in completely filled, t i ~ t l y sealed containers and must be discarded if strong hydrogen sulfide odors are observed.

Miscellaneous procedural rariables for acute bioassavs

Average of [nicial and final values. Diluent water:

643

mg/l.

Dlluen~ water: pH-8.1; alkalinlty-143.0 mg/l; hardness-160,0 mg/l. pH adjusted prioc co commencement of bioassay.

procedure include possible short-circuiting of effluent flow in the bioassay vessel and the large volumes of effluent required as percentage replacement approaches 100°%. For example, 90% replacement every 12 h over a 96-h exposure, requires 18.5 times the original volume. pH Values outside the normal biological range are known to be unacceptable in bioassay procedures (Doudoroff and Katz, 1950; Holland et al., 1960), but recent limited evidence suggests minor variations in pH also affect bioassay data, Ladd (1969) reported that the survival of juvenile coho salmon (Oncorhynchus kisutch) in various concentrations of kraft effluent was longest when the pH was between 8 and 9, and that both survival times and LCso values decreased progressively as pH values deviated from this range. Unpublished results from our laboratory indicate that the measured toxicity is greater at a pH of 6.7, than at 7.3 and 7.9 (Table 2). Leach and Thakore (1974) have shown that the resin acids are more toxic at pH values just below 7.0 than at values just above. Additional evidence is desirable in order to assess the value at which pH should be controlled during bioassay of pulp and paper effluents. An interim value, subject to change, is 7.5. Storage of pulp and paper effluents intended for bioassay, poses problems because of the lability of the toxic constituents. Deterioration of toxic values is indicated even during storage at low temperatures (Howard and Walden, 1965; Servisi, Stone and Gordon, 1966; Webb and Brett, 1972). Davis and Mason (1973) cited instances where toxicity declined with storage, remained the same, and in some instances, increased. Walden and McLeay (1974) demonstrated that the most important safeguard in stabilizing sample toxicity during storage, is the exclusion of air. Under these conditions most samples could be stored for at least four days with virtually no loss in toxicity. Storage at low temperatures achieved little more, except to minimize any possibility of anaerobiosis

The manner in which other procedural variables affect bioassay results is not specific to pulp and paper wastes. It is not within the scope of this presentation to deal with these parameters, except briefly, and the reader is referred elsewhere (Sprague, 1970; APHA, 1971). The selection, care and maintenance of test organisms is critical if the biological variation inherent in bioassay testing is to be minimized. On the basis of availability and ease of maintenance under laboratory conditions, rainbow trout (Salmo 9airdnerii) is the test species of choice. As is documented subsequently, considerable evidence exists to show that as representative of the salmonids, the rainbow trout is at least as sensitive to toxicants as are other readily available species. Limitations on fish-tovolume ratio restrict test fish to small juveniles. No published evidence indicates that sensitivity of rainbow trout to toxicants is dependent on their size. In our laboratories, we showed that young salmonids (coho salmon), which were 50-85 days old, displayed the same sensitivity to bleached kraft effluents as older fish (415-450 days old). Except for the effect of differing pH, the measured toxicity of pulp and paper effluents is not dependent upon the characteristics of the dilution water used in the bioassay. Where seawater is used for dilution, it has exceptional buffering capacity for pH and substantially detoxifies groundwood effluents where zinc dithionate has been used for brightening. Evidence indicates that the detoxification is associated with the toxicity attributable to zinc (Herbert and Wakeford, 1964; Van Horn, 1971), rather than with toxicants of woody origin. Inasmuch as the use of zinc salts is declining, this does not represent a major ongoing problem. If pH adjustment of bioassay test solutions is practised, any high quality water is satisfactory for dilution purposes. Death of test fish during acclimation and maintenance, prior to bioassay, is indicative of stress, and bioassays should not be undertaken while these conditions occur. Adequate precautionary measures for acclimation and maintenance are described by Doudoroff et al. (1951), APHA (1971). and Hunn, Schoettger and Whealdon (1968). It is our experience that limitations on die-offs during acclimation-maintenance, proposed by these authors, is not sufficiently restrictive. Any appreciable die-off is totally unacceptable. Survival of test fish throumh a control bioassay is essential to confirm that stresses, other than those due to exposure to toxicants, are not affecting bioassay data. Although APHA (1971) specifies no more

6-~

C.C.

WALDEN

than t07~, mortality among control fish during any test. continuing losses of this magnitude, particularly with salmonids, indicate serious problems. For example, in our laboratory no control mortalities have occurred in over seven ,,ears of testing. ACUTE TOXICITY OF P U L P ,AND PAPER MILL EFFLUENTS

Van Horn (1961) has reviewed aquatic biology and the pulp and paper industry with a second review covering literature issuing in the interim years (1971). Marier (1973) has published a general review of the environmental impact of pulp and paper wastes. The annual Literature Review issue of the Journal of the Water Pollution Control Federation covers pulp and paper literature in this field. Walden and Howard (1971) and Howard (1973) have specifically reviewed the toxicity of kraft mill effluents.

Kraft mill effluents (a) Acute toxicity to fish. Ebeling 11931) in Sweden was the first to report the toxic effects of kraft wastes on fish, namely that these wastes killed fish and imparted a resin-like taste to their flesh. Cole (t935) demonstrated that black liquor was toxic to fish in concentrations of less than 1 part to 200 parts of water. Dilutions as low as 50 times killed all fish exposed to black liquor within 30 min. whereas fish survived 14-24 days at dilutions of 500 times. Bergstr6m and Vallin (1937) studied the toxicity to fish of various components of kraft mill effluents including digester condensates and diffuser liquors (unbleached white water). Extrom and Farner (1943) studied the effects of digester condensates and a waste described as "total mill sewage", which corresponded to unbleached pulping effluent exclusive of recausticizing waste. Using three species of local fish. they demonstrated that, although the process streams and outfalls were toxic, their concentration in receiwng waters did not result tn any mortalities. Normal mill operation was difficult to define in terms of effluent quality: indicative of the varmtton in toxicity values encountered by subsequent investigators. Using various species of local minnows. Daphnia. mayflies and Chironomus larvae. Van Horn, Anderson and Katz (1949, 1950) determined the lethal threshold for a variety of substances known to be present in kraft pulping wastes, i.e. various inorganic sulfides. methyl mercaptan, sulfate soaps, resin and fatty acid fractions of sulfate soaps. Subsequently, lethal concert. trations of these materials were shown to be present in kraft mill outfalls. With adequate dilution, stream toxicity was considered minimal. Similar studies were undertaken by Dimick and Haydu (1952), who examined the behaviour of coho salmon in simulated streams at a 1:20 effluent dilution ratio. Despite early distress in some of the fish. no mortalities were recorded. A pertinent observation was that reduced bivalent sulfur compounds were being oxidized or air

stripped from the effluents under test. Haydu, Amberg and Dimick (1952) confirmed the toxicity of hydrogen sulfide, methyl mercaptan, sodium sulfide and high sodium hydroxide levels--constituents of kraft mill effluents to spring (O. tshawy~scha) and coho salmon and to cutthroat trout (S. clarkii). Although early studies dealt almost exclusively witta the toxicity of unbleached kraft pulping wastes, more recently emphasis has been on bleached kraft pulping wastes. Alderdice and Brett (1957). in a carct\tl piece of work which has received considerable attention. examined the eflbct of bleached kraft pulp mill effluent upon coho salmon under experimental conditions which simulated those which were expected to pertain to the estuary of the Somass River at the head of Alberni Inlet, British Columbia, Canada. Aiderdice and Brett showed that in 2(Y',,,, salinity seawater at 17.8°C, the limiting toxicity of bleached kraft effluent below which survival was complete and independent of the length of exposure, was 4.8°,; vv- t. However. considering oxygen requirements lbr fish respiration and the interaction of effluent with lowered oxygen availability, the limiting concentration recommended for this situation was 2.5}o vv-~ These bioassays simulated specific brackish water conditions, involved no pH adjustment, or control of dissolved oxygen or sulfide content of the discharge. In a major study of the toxicity of industrial effluents, Holland et al. (1960) examined the effects of kraft pulp mill effluents on spring and coho salmon in both fresh and seawater over both short- and tongterm (up to 300 days) exposures, with the long-term exposures involving solution exchange. The lethal thresholds ranged from 3.1 to 677,, vv-~ for coho salmon and from 1.9 to 3.6}~o vv-t for sprmg salmon (see Table 3). Holland et al. examined survivors to assess deleterious effects, short of mortality. Based on distress symptoms or reduction in growth, "'apparent tolerance levels" and "'critical levels" were reported. the critical level being that concentration below which deleterious effects were not observed. During these studies, storage of effluents and air stripping during bioassay eliminated effects due to bivalent sulfur compounds and free chlorine. Howard and Walden (1965) examined the toxicity of individual kraft process streams and combined mill outfalls to common guppies and sockeye salmon unTable 3. Concentrations of neutralized bleach kraft effluents toxic to various salmon species 96-h~ LCSO :[ v/,

3,1 -

671"2

Test S~c~es o~ SaZ~n

Coho ( O ~ c o ~ c h u s

ktsucch)

Iav~s~l&acor~ Holland e~ ~ , .

_I.

:qf,~

1.9 - 3.6 I'z 34 - 6&

Sockeye (0. ~e=ks)

Howard and N41den. :'~b5

All,retie

S p r ; s ~ ~d

22 IZ - 252

I. 2.

Fl~In$

~at*r D~oassay

L e t h & l t h r e s h o l d values

HcLees~. ,96~

Toxicity of pulp and paper mill effluents deryearlings iO. nerkat in fresh water at neutral pH. As shown in Table 3, their data indicated limited toxicity for bleached kraft effluents, 96-h LCso'S ranging from 34 to 64°0 vv -t. An important feature of their study disclosed that as much as 75°0 of the toxicity, ascribed by previous authors to kraft effluents, was due to an imbalance in pH. Howard and Walden also demonstrated that the test fish became acclimated over relatively short periods of time--a matter of days--to increasing concentrations of effluent. For example, test fish which had been exposed to sublethal concentrations of effluent could be exposed to progressively increasing concentrations without mortality, until concentrations were considerably higher than the 96-h LC~o value, as demonstrated by independent bioassay. Betts and Wilson (1966) reported the toxicity of bleached kraft pulp mill effluents and various process streams to Atlantic salmon IS. salar) (Table 3). Their studies dealt primarily with chemical modifications of the effluents in an attempt to reduce toxicity. Servisi et al. (1966) reported 96-h Lcso values for bleached kraft effluent to juvenile sockeye salmon in the range of 12-42",; vv-~ (Table 31. Adult migrant sockeye salmon were exposed to concentrations of neutralized whole mill effluent for periods of up to 49 days. Although the data showed that 2",;, vv -1 effluent was lethal to the adult fish, Servisi et al., because of the difficulties in working with spawning migrants and other experimental problems, indicated that their results did not conclusively demonstrate that the adult fish were more susceptible to effluents than were juveniles. In a comparative study between Atlantic salmon and lobsters (Homarus americanus), Sprague and McLeese (1968) indicated lethal thresholds in the range of 12-25% vv-~ (Table 3). Confirmatory studies by European investigators have produced similar results, although methods for expressing toxicity make intercomparison of data difficult. Podoba (1966) indicated the toxic action of black liquor necessitated a dilution of 1:7500 and concluded that for individual fish species, the fry were more susceptible to kraft pulping wastes than adults or the roe. Donnier (1972) concluded that the threshold for total effluent toxicity in seawater to various orgafiisms, including fish, was as low as 1 part in 20. Reported data in the literature substantiate that bleached kraft effluents do have some slight toxicity to fish. the concentration at which they cause fish mortality being variable, usually in the range of 10-1000o. In more recent years, unpublished mill and regulatory' monitoring of mill discharges have provided substantial confirmation. The toxicity status of bleached kraft discharges presently is sufficiently well documented that additional verification seldom merits publication in the technical literature. Although data of previous investigators indicated variable toxicity of kraft effluents, substantive documentation was provided by Howard and Walden

645

11971) in a study of seven mills throughout a 40-day operating period. This study disclosed that all major process sewers in the kraft mill possessed toxicity and that substantial variation occurred between daily samples. More recently, Bruynesteyn and Walden 11971) demonstrated that samples from the unbleached side of the mill showed considerable variation over time intervals as short as 15 min: whereas with bleachery effluents, sample characteristics seldom remained constant more than 12 h. Loch and McLeod (1973), in an examination of four pulp mills, reported that the fluctuations in toxicity between samples from different mills was not excessive. Howard and Walden t1971) failed to find any correlation between toxicity and BOD5 or TOC of individual mill process sewers and of the combined outfall. Howard and Walden indicated the first caustic effluent to be the most toxic process sewer, followed by the unbleached white water and first chlorination effluent. Betts and Wilson (1966) had reported the chlorination process sewer as the most toxic in the bleached kraft mill. Unpublished information from our laboratory indicates that this apparent difference is an artifact, arising from the bioassay procedures. The doseresponse curves for chlorination and caustic extraction effluents frequently cross, i.e. ETso values are lower for caustic effluents whereas 96-h ucso values are lower for chlorination effluents. Walden, Howard and Sheriff (1971) attempted to correlate the toxicity of mill process sewers and mill outfalls, utilizing the previous data of Howard and Walden (1971) and mill operating data collected concurrently. Although specific correlations were obtained for individual mills, no general conclusions could be made. Substantially similar conclusions were reached by Loch and Bryant (1972), while examining characteristics of a single mill. Bruynesteyn, Walden and Hill (1972) demonstrated that the only two toxic sources in the pulping process were the dilute black liquor, passing through the pulp washing stages without recovery, and the condensates from the multiple effect evaporator. Subsequently, Bruynesteyn and Walden (1973) showed that the use of combined condensates as shower water increased the toxicity of the pulping discharge considerably. However, toxicity of the condensates could be virtually eliminated if foam carryover in the multiple effect evaporator was prevented. Additional studies, specifically on bleachery effluents, have been relatively limited. Seppovaara (1973) indicated that bleachery eftluents were nontoxic to rainbow trout and crucian carp (Carassius carassius) at 1: 10 and 1:25 dilutions, except when free chlorine was present. (b) Acute toxicity to invertebrates. The acute toxicity of kraft mill wastes to aquatic invertebrates was observed first by Van Horn (1947) and coworkers (Van Horn, Anderson and Katz, 1949, 1950), who examined the toxicity of components of kraft mill wastes to various aquatic species, including Daphnia

646

C.C. WIn,bEY

and insect larvae. Generally their data suggested that these invertebrates were slig.htly more resistant to pulping wastes than were fish. and certain species. such as Chironomus. were substantially more resistant. Dimick and Haydu (1952) reported that stonefly and mayfly nymphs were more resistant to most kraft components than were fish. Hood. Duke and Stevenson (1960) documented the toxicity of black liquor to brine shrimp (Artemia salina~ and to unidentified marine zooplankton. Recent studies have been principally by European investigators, who have been concerned about the role of these organisms in the aquatic food chains, as a basis for defining regulatory requirements. Lesnikov (1966)documented the toxicity of black liquor to various species of Daphnia. Litvintsev (1967) examined the toxicity of pulping wastes to various micro-crustaceans, indicating that Gammarus pulex was more sensitive than Oaphnia maqna and Cyclops strennus. Filimonova {1968) ascribed the absence of zooplankters, especially Cladocera and Rotifera, near mill outfalls, to toxic effects, although certain species were highly resistant. Donnier 11972), examined the effects of whole mill outfal[s and black liquor on invertebrates and fish involved in a number of food chains, including phytoplankton, zooplankton (brine shrimp), blue mussels, crustaceans, crabs and sea worms. His general conclusions were that threshold levels for toxicity of whole mill outfalls was approximately 5 °,, v v - , for the various species. Fahmy and Lush (1975) compared the effect of untreated kraft effluent, biotreated effluent, lime-treated effluent, and activated carbon-treated effluent on the mature and young stages, as well as the egg stage of three species of invertebrates, including two microcrustaceans and one mosquito species. The Chironomus species was the most sensitive of the three. with the sensitivity to kraft effluents being approximately the same as that of rainbow trout. An interesting observation is that of Sprague and McLeese (1968), comparing the toxicity of kraft pulp mill effluents to adult and larval lobsters, as well as to juvenile salmon. Their data indicated no marked difference in sensitivity, but a difference in the mechanism of the toxic effects. Although data are substantially less than for fish species, kraft effluents definitely are toxic to various invertebrates. None of the invertebrates are more sensitive than are the juvenile salmonids, and certain species are much more resistant.

Sulfite wastes Early investigators had difficulty demonstrating any acute toxicity for sulfite waste liquors, other than the effects due to the extremely heavy oxygen demand. Williams et aL (1953) first showed that sulfite waste liquors were acutely toxic to fish. Holland et al. (1960) determined the toxicity threshold of sulfite waste liquor over a 30-day exposure in flowing brackish water to be 880 mg I-~ No significant difference in the ammonia-base and calcium-base pulping

liquors could be demonstrated, althoug_h accurate LCso values could not be determined because of mortalities in the controls. These authors could not demonstrate any toxicity to herring roe. Podoba (1966) indicated that juvenile fish were more sensitive to the acute toxicity of sulfite waste liquor than the corresponding adults or roe and indicated mortalit'. m incubating eggs at concentrattons of 200 mg I - " Grande /1964~. invesngating toxic condmons in the River Otra in Norway, attributed some of the toxic effects in the low alkalinity receiving waters to sutfite discharges. In the laboratory fish mcrtalities occurred at concentrations between 1000 and 4000 mg 1 - of sulfite waste liquor. Kondo. Sameshima and Kondo (1973), in determining 24- and 48-hr LC.~o vames tbr neutral sulfite semichemical wastes, indicated that they were approximately one-third as toxic as kraft wastes. Furthermore. Kondo et al. reported that the toxicity did not diminish with storage, as tt did lor kraft wastes. Wilson I.t972/determined the 96-h LCs0 value for sulfite waste liquors to be in the range of 2000-2750 mg 1-~. as measured against Atlantic salmon parr. Wilson and Chappel (19731 examined five representative sulfite mills including a high-yield soda-base mill. a relatively low-yield soda-base mill. a mill making high purity cellulose via the ammoniabase process and corresponding calcium- and magnesium-base low-yield mills, the latter mill with recovery. For the soda-base mills, mill outfatl samples were relatively nontoxic, in the range I I . 6 - 6 0 + " , vv- z : whereas for the ammonia-base mill. LCso values ranged from 8.7 to 25°0, vv-~. For the calcium- and magnesium-base mills, toxicity determinations were limited to red liquor samples, which ranged between 0.18-0.29 and 1.1-3.5°,o v v-~. respectively. Relatively limited information is available concerning the effect of the toxicity of sulfite pulping wastes on species other than fish. DeWitt 11963~ determined that introduction of sulfite waste liquor into artifical streams increased numbers of midge larva and water mites, but concluded that any toxic effect was primarily due to anoxia. Gazdzauskaite ~1971. 1971aj studied the effects of effluents from sulfite mills on various life stages of the freshwater shrimp, Pontogamrm~rua robustoides. Mill outfatls were toxic at 12.5 and 25% v v -1, the shrimp becoming immobilized at lower concentrations, but reviving in fresh water. Despite experimental problems, the toxictty to fish of sulfite pulping wastes is well documented and. although the evidence is somewhat meager, toxmity to other species also occurs. The difficulties of investigators in segregating toxic effects from those due to the oxygen demand emphasize the limited role toxicity plays in natural situations, compared to problems arising from potential oxygen depletion. Many of their data have been derived from the spent digester liquors, rather than outfall samples, again illustrating problems in measuring the low toxicity of the effluents. Results do not suggest any marked species difference in toxic responses.

Toxicity of pulp and paper mill effluents Although information is limited and exceptions undoubtedly do occur, sulfite mill discharges generally are less toxic than those from kraft mills. As subsequent data will show, wood extractives are primarily responsible for the toxicity of pulp and paper discharges, including those from chemical pulping and bleaching. Since spent sulfite cooking liquors are appreciably less toxic than black liquors from the kraft process, the inference is that the destruction of toxic wood extractives during sulfite cooking is substantially greater than during alkaline sulfide pulping.

Other Bark leachates are toxic (Schaumburg and Atkinson, 1970), although Schaumburg and Willard (1973) indicated that leachates from logs in natural waters have little or no toxic impact on the environment. Howard and Leach (1973) and Leach, Gietz and Thakore {1974) in a survey of woodroom effluents, confirmed that these effluents are toxic to fish, their toxicity depending upon the degree of water recycle. Softwood species tended to be more toxic than hardwood species, and no seasonal variation in toxicity was encountered. Literature reports concerning the toxicity of mechanical pulping effluents are limited. Row and Cook (1971) ascribed the major portion of the toxicity of these effluents to resin acid soaps. Howard and Leach (1973al and Leach and Thakore (1974a) surveyed a number of mechanical grot, ndwood mills in Canada and determined that the findings paralleled those encountered for debarking effluents; namely, toxicity depended largely upon water recycle, with softwood species producing more toxic effluents than hardwood species. Wilson (1975) has examined the toxicity of effluents from newsprint operations, utilizing purchased chemical pulp, on representative species of algae, zooplankton and macroinvertebrates. Biotreated effluent had no adverse reaction on any of the organisms examined. Untreated effluent was toxic to some algae, but not others, and was toxic to the zooplankton and invertebrates. The most sensitive species (Daphnia) had a sensitivity of the same order as rainbow trout. One invertebrate species avoided the effluent and its toxicity reaction could not be determined. Martens, Gordon and Servisi (1971) indicated that the toxicity of deinking wastes was a function of detergents used in the process. Confirmatory unpublished information has been obtained in our laboratories. Thomas and Legault (1967) documented the toxicity of wastes from two paper machines, concluding the wastes were relatively nontoxic. The acute toxicity of these other discharges of the pulp and paper industry is of about the same order as the toxicities of effluents from the chemical pulping processes, subject to the greater recycling of process water sometimes practised in the mechanical processes.

647

SUBLETHAL TOXICITY OF PULP AND PAPER )dILL EFFLLENTS Investigations into sublethal toxicity of pulp and paper mill effluents stem from the recognition by biologists that conditions approaching the acutely lethal concentration, as determined in traditional acute bioassays, do not represent a safe concentration [br survival, maintenance and continuity of fish stocks (Walden and Howard. 197l: Fry. 1971~. Acute bioassay results are, in themselves, valueless and must be related to the concentration producing no deleterious effects on the aquatic ecosystems [Warren and Doudoroff, I958). Commonly, acute bioassay data are related to what are considered "'safe" limits, by application factors. However, it is dangerous to do so, unless the effects causing the acute response (deathl and sublethal effects are known to be the same [Warner. 1964). Thus the objective of sublethal investigations is to determine the nature of sublethal stresses or effects due to pollutants and then measure the threshold levels below which no effects occur, Stresses are cumulative and any stress imposed upon an organism reduces its capability to meet other natural stresses, and thus affects survival. At concentrations below this sublethal threshold, in receiving waters, no pollutantoriginating stresses are imposed on fish and other aquatic species. Nonetheless, all sublethal changes induced by pollutants may not be detrimental. For example, Mount and Stephan (1967) cite a 10% reduction in hematocrit, originating from exposure to a pollutant, as not necessarily being an undesirable effect. Sprague (1971) has reviewed general procedures for sublethal measurements and has discussed the problem of ascertaining "safe" levels. For pulp and paper effluents, known sublethal effects are attributable to coniferous fibers, volatile reduced sulfur compounds and nonvolative soluble toxic moieties. The latter are of major environmental concern and are dealt with here. Information concerning fibers and hydrogen sulfide is dealt with subsequently.

Kra~ effluents The progressive visual effects displayed by fish on exposure to lethal concentrations of kraft effluent have been described by Walden and Howard (1968) as follows; loss of schooling, reluctance to eat, respiratory distress, convulsive coughing, abnormal gill movements, excessive mucous production, loss of equilibrium and death. Investigations on sublethal effects necessarily stem from these physical observations. In an early study Van Horn (1952) reported that sublethal concentrations of kraft pulping wastes produced no visible effects on fish organs. This report typifies the frustrations of many subsequent investigators, inasmuch as autopsies on dead fish disclose no obvious reason for mortality. Holland et al. (1960)

('~.<

C . C . WALDEN

reported critical levels, concentrations below which no effects could be observed, and which ranged between 0.29 and 0.56 of the lethal threshold. These observations were based primarily on growth and visually evident distress. Avoidance is an important protection mechanism of fish against toxic materials. Jones et at. 11956) and Dimick er al. (19571 obtained inconsistent results on exposing various salmonid species to increasing concentrations of kraft pulping wastes. Spring salmon avoided modestly higJa levels of waste in some instances but were attracted by the same concentrations of other samples. Coho salmon and steelhead trout IS. 9airdnerii) did not display any avoidance at the concentrations examined. Sprague and Drury /19691 failed to demonstrate avoidance of satmonids to kraft pulp mill effluents at concentrations below 50°,,0 v v- I. The effect of pollutants on fish stamina is an important sublethal parameter and most frequently is measured by swimming performance. Tokar and Owens (1968) failed to demonstrate any effect at effluent concentrations approximating up to 2.5 mg BOD~ l-Z. Specific toxicity data were not furnished, but average data suggest concentrations could represent 0.8 of the 96-h LCso. Howard (1973, 19751 found reduced swimming pertbrmance at concentrations above 0.1--0.2 of the 96-h L¢5o value (Fig. 41. However, test fish recovered completely after 6--12 h recuperation in fresh water. The effect of low concentrations of bleached kraft mill effluent on growth of fish is negligible or even stimu[atory. The minimum threshold level affecting growth, according to Tokar and Owens (1968), is 0.12 of the 96-h LCso value, where growth periods were limited to 2-3 weeks. Warren (19721 related growth of fish and food consumption to effluent eoncen-

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Fig. 4. Mean critical swimming speed values for groups of ten juvenile coho salmon (Oncorhynchus kisutch) exposed for periods up to 7 days in sublethal concentrations of bleached kraft mill effluent, prior to stamina testing in the same effluent concentrations.

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Fig. 5. The relationship of normalized growth rates of spring salmon (Oncorhynchus tshawytscha) to the BOD levels of kraft effluent to which they were exposed; while being fed on high restricted ldaily repletion) rations (Warren. 1972). trations defined in terms of BODs (Fig. 5). In longerterm studies, Webb and Brett (19721 obtained similar data: growth rates were unaffected over 57-day exposures to effluent concentrations of I07,,, v v - t of bleached kraft effluent, whereas at 25'~, v v -I both growth rate and tbod conversion were affected. McLeay and Brown (19741 observed a stimulation in growth of coho salmon exposed for 200 days to bleached kraft mill effluent at concentrations of 0.1 and 0.25 of the 96-h LCs0. Sublethal concentrations of bleached kraft effluents affect fish respiration. Schaumburg, Howard and Walden (1967) measured the pressure change in the buccal (mouth) cavity of salmonids, as affected by increasing concentrations of various pollutants. The change in frequency with which the water flow over the gills was reversed, i.e. a cough, increased directly with increasing sublethal concentration of kraft effluents and exposure time. Walden. Howard and Froud (1970) used this technique to assess quantitatively the threshold levels at which respiration was affected (Fig. 61. The minimum concentration of various bleached kraft effluents causing a response was between 0.08 and 0.18 of the 96-h eCso ',alue. as determined by static bioassay. The respiratory function returned almost to normal on continued exposure (Schaumburg et al.. 1967: Walden er al., 19701. Davis I1973) showed a significant increase n the cough frequency of juvenile sockeye salmon exposed to an effluent concentration of approximately 0.2 of the 96-h LCs0 value. Other measured parameters such as ventilatory water flow. oxygen uptake and buccal pressure were affected at the same effluent concentrations. The arterial oxygen tension was depressed by a 24-h exposure to effluent concentrations between 0.33 and 0.47 of the 96-h LCso value. Present evidence implicates the toxic action as a direct effect on gill tissues, with other effects arisin~ from the stress response occasioned by interference with respiration. In our laboratories we have shown

ToxiciD of pulp and paper mill etItuents

649

longed exposure, while at the same time the number of circulating neutrophils increased. No changes were observed in the red blood cells, although the hematocrit values were decreased on exposure for 25 days. Howard and Walden (1967! previously had reported that hematocrit values of salmonids were affected by exposure to sublethal concentrations of kraft effluents. McLeay {1973) also reported that plasma glucose in.~_ h 5 creased over the first 12 h and decreased over the subsequent 25-day exposure. Various biochemical i oa changes occurred in blood and tissues associated with carbohydrate metabolism at effluent concentrations corresponding to 0.33 and 0.40 of the 96-h kcso value. More recently, McLeay and Brown 11974). in exposure of fish extending up to 200 days. indicated that plasma glucose increased, as did liver glycogen, serum X and muscle lactic acid. At the same time, body protein 0,5 -4% Control i. . . . X. . . . X. . . . X X and serum pyruvic acid levels declined. These changes occurred during exposure to effluent concentrations of 0.1 and 0.25 of the 96-h t,cso value, at the same time that growth was being enhanced. McLeay and I I I I I 0 I 2 3 4 5 Brown (1975) observed similar changes in muscle and EXPOSURE TIME, hours plasma glucose for fish exposed for short periods to Fig. 6. Mean "'cough" frequencies of groups of eight rain- 0.8 of the 96-h t,cso value where additional stresses bow trout juveniles (Salmo gairdnerii) exposed to various were imposed on the test animals by exercise (swimmsublethal concentrations of a neutralized bleached kraft ing). In addition, recovery on resting in water was mill waste IWalden et al.. 1970). enhanced over similar rest periods in effluent. The that impairment of respiration is a major effect of general conclusion of McLeay and Brown (1974) was kraft mill effluent by demonstrating that radiochemi- that the response of the fish was that of a general cally-tagged unsaturated fatty acids were taken into hormetic response stage of resistance to stress, comthe gill tissues, but not otherwise widely disseminated patible with interference with respiration. Only limited studies have been carried out on adult within the fish body and, further, that these acids were salmon. Servisi et ell. (1966) were properly conservadesorbed upon return of exposed fish to fresh water. Since their publication, the data of Fujiya (1961, tive in concluding that lethal levels for adults were 1964) have concerned pollution biologists, because of not statistically lower than for juveniles. No studies the drastic effects measured in a field situation, over have been made concerning the effects of sublethal a short exposure period, He reported substantial his- concentrations of pulp mill effluents upon adult migrants but the importance of olfactory homing and topathological damage to fish (Sparus macrocephalus) caged downstream of a mill outfall where approxi- swimming performance are such that the lethal mate dilution of wastes was between 16 and 30 times. threshold levels developed for juveniles must be These reports, which ha~e received considerable applied with caution where adult fish are concerned. Data concerning roe (Holland et al., 1960: Podoba, attention by biologists, indicated a decrease in RNA, 1966) indicate that threshold levels for this stage of a decrease in glycogen content of the liver, a decrease in pancreatic RNA, degeneration in the kidney, nec- the life cycle of fish are probably higher than correrosis and desquamation of intestinal epithelia, sponding levels for juveniles. For a number of years, Warren and coworkers together with changes in the circulatory system. Although information concerning the acute toxicity have undertaken extremely important investigations of the mill discharge in question was not provided, on the effects of untreated and biotreated kraft mill the low concentrations of 3-6°J(, v v - ' suggest that effluents on productivity in laboratory aquaria, artithe effluent was abnormally toxic. Intensive investiga- ficial laboratory streams and streams artifically tions in our laboratory have failed to corroborate created in a natural environment. Measurements inthese findings on salmonid species {McLeay, 1973). cluded fish biomass and, in some instances, numbers Private discussions with workers at Oregon State and diversity of invertebrates. The 320-ft streams University have disclosed that these investigators also created in a natural location were fed from the nearby Williamette River. After colonization and stocking of have failed to corroborate these findings. McLeay (1973) examined the blood and tissues of the streams, one was reserved as a control and the juvenile coho salmon exposed to bleached kraft mill other two were dosed with effluent from an adjacent effluents over 12+h and 25-day exposures. After 12-h, mill producing unbleached kraft and neutral sulfite the numbers of circulating small lymphocytes de- semichemical pulp. Over the years, the mill effluent creased markedly, returning to normal with pro- initially was treated only to remove primary solids,

050

C, C. WALI)EN

subsequently ~as biotreated and finalt? received have been constructed and experimental studies ha~e more extensive biotreatment and eMuent polishing. commenced, but no results are presently a~ailable. Biological data were related to BOD~ values of In specific instances information concerning the the effluent, although acute toxicity data also ~ere nature of the sublethal kraft mill effluent has been determined. used to measure quantitativel.~ the threshold levels In early laboratory studies by this group. Eliis at which these changes occur. Furthermore. some of 119671 indicated that production of under.yearling these procedures show promise as sublethal bioassa~s spring salmon was reduced in artificial laboratory Ibr the rapid monitoring of these incipient threshold streams dosed with untreated unbleached kraft levels. Walden et al. 119701 developed the increase in effluent in the concentration range 0.14-0.36 of the cough frcquenc~ as a technique ~or measuring the 96-h tc~o value. This effect was the greatest for those threshold concentration at which respiration ~s streams with the highest fish loadings. Effluent con- all~cted. Howard t19731 also reported the threshold centrations of 0.05-0.08 of the 96-h LCso value had level at which the cough frequency is aff~ted to be no effect. The effect of higher concentrations was indi- 0.148 of the 96-h LCs0 value. Although this respiration cated as being due to a toxic effect on the fish. rather bioassay of the sublethal threshold can be completed than on the fish food organisms, which actuall? over the same 96-h interval as an acute toxicity bioincreased in abundance. Subsequently, Seim 119701 assay, necessary sophistication of equipmenl and reported that dosing artificial laboratory streams with technique hardly qualifies this procedure for routine 1.5°o of biotreated effluent during the early and late monitoring. fall and early spring, reduced production of fish bioHoward and Walden 1974j. using quantttauve mass. However, during the summer, fish biomass pro- techniqt,es for measuring swimming performance. duction increased at all dosage levels up to 4°,;, with compared critical swimmmg speed of control- and the greatest increase corresponding to a dosing rate effluent-exposed groups of coho salmon. Further of 1.0'},, v v- ~. Enhanced productivity during the sum- details are presented by Howard tl9731, who indimer was attributed to a diminished effluent toxicity. cated that the threshold effect of bleached kraft All levels of effluent enhanced production of fish food effluents on swimming stamina was at an effluent conorganisms, whereas dosage levels above 1'~,;,were con- centratton between 0.I and 0.2 of the 96-h LCso. This sidered toxic to the fish. Experiments with up to 7.5{!o technique was used to quantitatively evaluate the v v- t of biotreated effluent, comparing untreated with effectiveness of various effluent detoxification probiotreated effluent at approximately the same BOD5 cedures (Howard. Walden and Munro. 1974~. Stamina testing, as a means of mon,tormg sublethal threshand volume levels, resulted in both increased food production and enhanced production of fish biomass olds. although highly reproducible, presently requires (Lichatowich, 1970). During these studies, no toxic relatively large volumes of effluent and sophisticated effects were reported for effluent concentrauons corre- equipment, although equipment redesign might be expected to minimize these disadvantages. sponding to about 0.03 of the 96-h :.C~o value. Howard and Walden (1974l and Howard. Walden Studies with the 320-ft artificial streams involved effluent dosing to maintain BOD5 levels at 0.5 mg and Munro (19741 also have reported a temperature tolerance test, in which the reduction in the critical t -~ (Warren, 1972). With primary treated effluent. effluent concentration was about 0.22% v v- t. corre- thermal maximum is quantitatively related to subsponding to about 0.025 of the average 96-h C%o. lethal concentrations of neutralized bleached kraft Biotreated effluent was fed at about 0.7430 ~ v-~: mill effluent. Using this procedure. Howard 11973) about 0,01 of the 96-h t.cso. As mill effluent was ex- cited the recipient threshold as between 0.062 and tensively biotreated and polished, feeding rates m- 0.230 of the 96-h LCs0. The ecological relevance of this creased to as high as 5','0. Untreated effluent reduced temperature tolerance test has not been established. the abundance of insects in these streams but did not The work of McLeay (1973) in developing a "cheaffect their diversity {Warren, 1972~. Amphipods were mical profile" of the biochemical responses in fish reduced in numbers during injection of untreated exposed to sublethal concentrations of kraft pulpmill effluent, but increased when effluent was biologically effluents, bears promise of furnishing rapid biochemitreated {Warren e t al., 19741. Snail populations m- cal procedures for routine monitoring of threshold creased when streams were fed untreated or biologi- levels. For example, following a few hours of exposure cally treated effluent. The abundance of fish food to sublethal concentrations of kraft mill effluent. organisms was not affected, although some shifts in plasma glucose in fish blood was significantly elepopulations did occur. Utilizing extensively treated vated. McLeay {1973) demonstrated that the threshand polished effluent, no effect on fish productivity old of response was between 0.I and 0.3 of the 96-h was encountered (Caron, 1974). Any effect on the ben- LCs0. Biochemical techniques offer the potential thic community is doubtful, not reaching statistically advantage that sample requirements are small and many procedures have been completely automated. significant levels. The National Council for Air and Stream Improve- in response to requirements of the medical sciences. ment (U.S.A.) is initiating similar studies in Georgia McLeay ~19751 has examined the Carter {1962) bioason warm water species of fish. The artificial streams say as a means of rapidly monitoring the threshold

Toxicit', of pulp and paper mill ettluents level of sublethal effects of kraft effluents on fish. Under the conditions of the bioassay, the threshold was 0.40.6 of the static 96-h Lcyo. Investigations of sublethal effects, on organisms, other than fish, have centered primarily on molluscs Ioysters), Galtsoff et al. 119381 demonstrated that oysters kept themselves closed in the presence of black liquor, thus reducing feeding periods. Consequently, growth was impaired, glycogen levels declined and condition factor fell off. Subsequently. Galtsoff et al. 11947) indicated that dilutions of pulp mill wastes as low as 1-1000 reduced the time of feeding and dilutions between 1-200 and 1-2000 reduced the rate at which water was pumped over the gills. Fujiya t1962/ also observed that the presence of kraft effluents caused bivalves to close their shells affecting both calcium and nutritive metabolism. Fujiya also reported a number of other effects, including respiration and histopathological and cytochemical effects on the stomach, intestine, digestive diverticula, gills and connective tissues. McLeese (1970, 1973) failed to show any avoidance behaviour of lobsters to kraft mill effluents up to 20'~; concentration, even when superimposed on food odors. In their study with artificial streams, Warren and coworkers (1972) and Warren et al. (1974) failed to note any pronounced effect of kraft effluents in the range of 0.025-0.l of the 96-h LCy0 value, on food organism abundance. Populations shifted, although diversity did not decrease and certain benthic organisms, such as snail populations increased. Woelke (1960) has developed a sensitive oyster larval bioassay based on the abnormalities produced by a 48-h exposure of fertilized oyster embryos to increasing concentrations of effluents. Woelke and coworkers (1965, 1967, 1972), using this bioassay, have demonstrated abnormalities in developing fertilized embryos. Dilution necessary to reduce toxicity from kraft mill discharges below the sublethal threshold level ranged from 170 to, in one instance, as high as 700 times. Individual kraft sewers required dilutions of 525-920 times, to eliminate sublethal effects. Oyster larval abnormalities occurred in one instance at 1.3°o biotreated bleached kraft effluent, from a mill with an excellent record of discharging effluents with no measurable acute toxicity to test salmonids. Although the evidence is considerably less than has been developed for fish, the bulk of the data indicate that the threshold level at which sublethal effects are exerted on fish food organisms and invertebrates corresponds roughly to the same toxic concentration at which the effects appear in fsh. Sulfite effluents

Williams et al. (1953) observed the following sequence of effects, when various species of salmon were exposed to toxic concentrations of sulfite waste liquor: loss of appetite, sluggish movements and reactions, rapid respiration, darkening of the integument, partial obliteration of parr marks, appearance of pale

u_,L

spots on the anterior dorsal area, loss of equilibrium and erratic s~vim/ning, periods of immobility and violent respiration while on the bottom of the aquarium, paralytic spasms, death. Autopsies showed little food in fish stomachs, but other organs were in normal condition. Hoglund ~19611 examined the avoidance of fish in a fluvarium, indicating avoidance to low concentrations of sulfite waste liquor, but not to higher concentrations. Gazdzyauskaite {1971, 1971al examined the sublethal effects of effluents from two sulfite pulp mills on various phases of the life cycle of the freshwater shrimp, Pontoyamarus robustoides. Concentrations of 12.5 and 25°0 immobilized the shrimp. but recuperation occurred on transferral to fresh water. Sublethal levels resulted in reduced food intake. cannibalism, and affected respiration. In subsequent studies, Gazdzyauskaite (1971a) indicated that effluents were nontoxic at 1.56°;, but affected growth, whereas process streams affected fry emergence at 1.56'~oand had an adverse effect on spawning over the 3-12°o concentration range. Evidence suggests that the threshold level at which sulfite waste liquor exerts sublethal effects on bivalves is anomalously low, i.e. effects occur at much lower concentrations than would be anticipated based on effects on other species. Odlaug (19461 indicated that 100 ppm of spent sulfite liquor, reduced the pumping rate of Olympia oysters by 8Y~oon immediate exposure, with complete cessation after 15 days. Stein et al. (1959)indicated that concentrations of ammoniabase spent sulfite liquor, in excess of 55 ppm, affected spawning of Olympia oysters, whereas lower concentrations were stimulatory. Woelke (1960) found that exposure to between 6 and 12 ppm of spent sulfite liquor resulted in the development of over 20% of abnormal larvae and the percentage of abnormalities irfcreased with increasing concentration. Woelke (1965a) bioassayed seawater samples, relating oyster larval abnormalities to the concentration of spent sulrite liquor in the seawater, as indicated by the PearlBenson index {Fig. 7}. The numbers of abnormalities developed in these seawater samples were the same as the abnormalities developed in solutions of sulfite waste liquor of equivalent concentration. The same techniques, applied to seawater samples taken before, during and after mill shutdowns, (Woelke, 1968), showed a decline in larval abnormalities for samples collected during shutdown periods. Subsequently, Woelke. Schink and Sanborn (1970) extended this bioassay to the larvae of various species of clams, obtaining similar effects at l-3 mg 1- t of spent sulfite liquor solids. Throughout these studies, no attempt was made to ascertain the nature of the toxic moieties. Ammonia is well recognized as being toxic to fish in concentrations as low as a fraction of a mg 1(Klein, 1959; McKee and Wolf, 1963). A number of Woelke's publications specifically identify spent sulfite liquor as originating from ammonia-base mills. The

6__

C . C . V¥'.'x.LDEN ~00

resent a special problem, addmonal information is desirable concerning the levels of ammonia in these effluents. Howe~er, the bivalves appear more sensitive to low concentrations of spent sulfite wastes than are other species and this is particularly evident during the embryonic developmental stages.

80

Other

6o

0

During his investigations. Woelke (1967~ examined the effect of groundwood and debarker wastes on the embryonic development of oyster larvae. Effluent dilutions of 59 and 72 times were adequate to eliminate any sublethal effects on larval embryonic development. Literally no other information is reported in the literature on the sublethal effects of other pulp and paper effluents.

b,-

4o

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2G data

0

0

I 6 12 18 24 PEARL-BEN.SON INDICES, ppm

30

36

Fig. 7. The percent abnormalities in Pacific oyster larvae developed 48 h in water samples, with different PearlBenson indices, collected from mill outfall environs (Woelke. 1965). unionized portion of the molecule is the toxic moiety and the fractions thereof of the total concentration have been documented to be highly dependent upon pH values and, to a lesser degree, temperature; within the biological range existing in most receiving waters {Trussell, 1972). At pH values below 7.0, ammonia is virtually nontoxic to fish. However, the 96-h CC5o value for unionized ammonia is 0.4 mg 1 - I At pH values near 8.0, the toxicity of total ammonia is several mg 1-~. Woelke Schink and Sanborn (1972) examined the toxicity of magnesium-, and ammoniabase effluents, using the oyster larval bioassay. Of the two, the magnesium-base effluent was the more toxic. Concurrent measuremnts of ammonia in the ammonia-base effluent suggested that, at the threshold concentration at which larval abnormalities were observed, the unionized ammonia levels did not exceed sublethal threshold levels reported acceptable for long-term exposure of fish (Anon, 1973: Larmoyeux and Piper, 1973). Tabata (1955) ascribed the toxicity of effluents from ammonia-base neutral sulfite semichemical mills to the ammonia content. More recently, Walden and McLeay 11974) document an instance where the toxicity of a discharge from a low-yield sulfite mill was highly dependent upon pH. Toliefson (1974) has indicated that, on the basis of weight of wood pulped in the pulping process utilizing various bases, no differences in toxicity exist in the effluents. At least one field situation is documented (Crown Zellerbach Corporation, 1970: Amberg et aL, t971) where discharge of effluent containing moderately high levels of ammonia has not resulted in ammonia toxicity in receiving waters. Although the bulk of data suggest that ammonia-base effluents probably do not rep-

TOXIC CONSTITUENTS OF PUI.P AND PAPER EFFLLENTS

Pulp and paper effluents are complex mixtures of a relatively larger number of organic and inorganic moieties. Considerable knowledge has now been accumulated concerning the nature of the toxic molecules and their contributions to the impact of pulp and paper discharges on the environment. Suspended solid,~

Anaerobiosis in bottom muds. which is known to result from the settlement of wood fibers in many receiving waters is detrimental to the benthic community, including invertebrates and incubating fish eggs. The effect is due both to the depletion of oxygen and to the toxic action of generated hydrogen sulfide. Most receiving waters contain appreciable quantities of naturally occurring suspended solids, maintained in suspension by water movement or their colloidal size, without apparent effect on the free-swimming nekton. Indeed, Iiterature associated with the discharge of mining wastes indicates that levels of suspended solids can be substantial, without apparent effect on fish. Nonetheless. recent evidence indicates that some suspended fibers of woody origin can be toxic to fish, although this effect does not extend to fish eggs (Smith and Kramer. 1964: Kramer and Smith, 1965, 1966). Smith and Kramer fl9641 indicated coniferous fibers at levels of 2000 mg 1- ~ resulted in substantial mortalities in alevins, whereas aspen fibers did not Groundwood fibers, in turn. were more toxic than chemically pulped fibers (Smith. Kramer and Mac. Leod, 1965). Hematocrits m surviving fish were mcreased (Smith and Kramer. 1964~. At lower fiber concentrations, 100-800 mg t-~. a variety of sublethal effects were reported. Growth was retarded, respiration was affected with a typical "cough response. metabolic rates increased and numbers of mucous cells in the gills were affected fSmith et al.. 1965. Kramer and Smith. 1965; Smith. Kramer and Oseid,

[oxlclty ol pulp and paper mtl! emuents Table 4. Lethal threshold concentrations Img 1-t) of toxicants, knov,n to be ~resent in kraft mill wastes, to salmonid fishes O~cerhv~e~uq

Oncorhv~.chus kisu=¢h (¢oho salmon)

Sa~mo ¢ ! a r k l i

Chemical

:sha~clcscha

Hydrogen su!~ide

0.3

0.7

0.S

Methyl =ercap~an

0.5

0.7

0.9

Sodium sulZide

1.8

1.3

LO

Sodium hydroxide

27

i1.

lO

$odlu~ carbonate

58

4~

33

10,000

2,500

Sodium Sulfate

1966: MacLeod and Smith, 1966). Fish species included walleyes, fathead minnows, brown and rainbow trout, and some exposures extended to 190 days. MacLeod and Smith (1966) also demonstrated reduced swimming performance. Retardation of growth did not recover on replacement in fresh water (Kramer and Smith, 1965). These authors concluded that the presence of relatively small amounts of coniferous wood fibers, in suspension, created stressful conditions in exposed fish.

Kraft pulpin 9 ejfluems Virtually all of the early studies that were undertaken to isolate and identify' toxic materials from pulp and paper effluents were confined to the pulping side of the kraft process. This emphasis reflected the dominant role of unbleached kraft pulp in total chemical pulp production, some 20-30 years ago. (a) Volatiles. Early evidence implicated the volatile reduced bivalent sulfur compounds, which are characteristic of the kraft pulping process, as toxic to fish. Cole (1935a) demonstrated that methyl mercaptan was toxic to fish in concentrations of a fraction of a mg 1-1. Van Horn (1947, 1948) and Van Horn et el. (1949, 1950) demonstrated the toxicity of sodium hydroxide, sodium sulfide, methyl mercaptan and resin salts to various aquatic species and showed that lethal concentrations of these substances existed in the outfalls from five kraft mills. Subsequently, Dimick and Haydu (1952) and Haydu et el. (1952) produced similar data (Table 4) for salmonids. Both methyl mercaptan and hydrogen sulfide are extremely toxic. Recent literature concerning toxicity of hydrogen sulfide cite biological effects at considerably lower values than indicated by earlier authors. For example, Adelman and Smith (1972) cite the toxicity of hydrogen sulfide to goldfish as being in the range 0.036--0.087 mg 1-t. Smith and Oseid (1972) give 96-h L%o values of 0.0744).087 mg 1-' for walleye eggs, with a 96-h LCso value of 0.007 mg 1-1 for fry. Chevalier (1973) emphasized that LCso values of hydrogen sulfide, as measured by flow-through bioassay, are about half that measured by static bioassay. Although these data suggest that the toxicity of hydrogen sulfide is considerably greater than reported by earlier

(cutthroat ~rout)

authors, expressed concentrations apparently relate to the calculated concentrations of the toxic or unionized form of hydrogen sulfide. The ionization of hydrogen sulfide is materially affected by temperature and pH within the normal biological range. The pH values corresponding to the more recent data are not identified, however, the bioassay waters used by Smith and coworkers were definitely alkaline, so that the actual concentration of total sulfide probably was twice as great as cited values. The net role of the volatile reduced bivalent sulfur compounds in the toxicity of kraft pulping etttuents is considered to be low, inasmuch as these compounds are readily air-stripped from or oxidized in receiving waters (Haydu et el., 1952: Walden and Howard, 1971). Substantiating evidence has been secured by Seppovaara and Hynninen (1970) and by Seppovaara (i971), who concluded that the systemic poisoning of fish by kraft condensates was due to the bivalent sulfur compounds; whereas air-stripped condensates displayed no acute toxicity to fish. In an examination of 20 bleached kraft mill outfall samples, from three mills, Ng, Mueller and Walden (1974) showed that the relative contribution to the toxicity of these samples by volatile components averaged 5.4%. Since the bivalent reduced sulfur compounds may not constitute all the volatiles in bleached kraft effluents, their contribution to toxicity in natural situations probably is minor. Moreover. the samples examined by Ng et al. were not biologically treated and such biotreatment would be expected to reduce any residual toxicity still further. (b) Nonvolatiles. As early as 1931, Ebeling implicated resin acids in unbleached kraft effluents by the resin-like taste imparted to the fish flesh and by demonstrating that 5 mg 1-t of resin acids caused fish deaths. Hagman (1936) showed that the safe level of resin acids for fish was 2 mg I-1 or less. Van Horn et al. (1949) cited minimum lethal concentrations of resin acid soaps as 1.0 mg I-L for minnows and 3.0 mg 1-1 for Daphnia. More recently, Mgenp~5., Hynninen and Tikka (1968) have reported a toxicity level of 2 mg 1-~ for resin acid sodium salts to Daphnia pulex. An early indication of the possible role of unsaturated fatty acids as toxic constituents of kraft pulping effluents was furnished by Bergstr/Sm and Vallin (1937).

654

C. C, W,'ALDEN

Following the introducton of macroreticular resins for the concentration of the toxic constituents of kraft pulping wastes by Rogers (1973), Leach and Thakore 11973) documented precisely the contribution of all non~olatiles to pulping waste toxicity. The unique feature of the study by Leach and Thakore 119731 was that. during each stage of the chemical separation process, the toxicity of the various isolates was compared by bioassay on a weight equivalent basis to ensure that all of the toxic materials had been retained through the isolation process. Finally'. the workers were able to combine the identified toxlc materials in proportions corresponding to that of the original sample of pulping effluent and demonstrate that this toxicity corresponded exactly to that of the original effluent sample. In an effluent originating from a Douglas fir and western hemlock furnish, the toxic nonvolatiles included three resin acid soaps which were responsible for over 80% of the toxmity and three unsaturated fatty acids. The toxicity of this sample originated solely from the unrecovered dilute black liquor which was washed from the pulp into the final pulping discharge. In corresponding studies. Leach and Thakore 11974) accounted for all toxin constituents similarly, when combined condensates from the multiple effect evaporators were used as wash waters in the final stages of brownstock preparation. These studies involved three additional mills. one from the interior of British Columbia, the other two from Ontario, all in Canada, and operating on a variety of wood furnishes. Information concerning the absolute acute toxicity to salmonids of the more common resin acids found in pulping effluents was obtained by Leach and Thakore 11974, 1975). Using frequent solution replacement during bioassay and analysis of resin acid concentrations at various bioassay stages, Leach and Thakore 11974, 1975) determined the 96-h LCso values to salmonids to be: isopimaric acid. 0,30 mg l-Z: 20

1

w

i~

~lll

~ II

o

pimaric acid. 0.37 mg I-~: sandaracoptmari¢ acid, 0.45 mg t-z: abietic acid. 0.56 mg [~:: deh~droabietic acid. 0.93 mg I-t During their isolation studies. Leach and Tnakore f l973al developed dose-response cur~es tbr various resin and unsaturated fatty acids tFig. 8)_ The slopes of the curves for certain of these toxic molecules, isopimarate, abietate and dehydroabietate in particular. are dependent upon concentration. Moreover, the slopes corresponding to individual fatt? acids and some resin acids also differ. Thus, even it' toxicities are additive, the indicated toxicity of a mixture ~:ould be dependent upon the concentration lexposure timeL The slopes of the curves in Fig. 8 can be segregated loosely into two groups, which bear a striking resemblance to the dose-response curves obtained by Bru.vnesteyn. Walden and Hill 11972) for diluted black liquor and combined multiple e~aporator condensates. Leach and Thakore (19741 also demonstrated that the toxicity of these important resin acids was affected by pH values within the biological range: being much greater at pH 6.4 than at 7.5. The role of fatty acids as toxic moieties in kraft pulping effluents has received minor attention. although their toxicity was identified by Van Horn as early as 1947. Leach and Thakore 11973) demonstrated that the unsaturated. 18-carbon. straight chain fatty acids were jointly responsible for about 18% or the toxicity in a sample in which all of the toxicity was accounted for. Individually, the tmsaturated fatty acids did not result in any acute toxicity at the concentrations at which they were found. This I,'-;",, of the toxicity of the original sample of unbleached whitewater was due to the sodium salts of palmitoleie. oleic, linoleic and linolenic acids. Various other toxic constituents of kraft pulping effluents have been reported at various times, although present data indicate that their contribution to the total toxicity is negligible. Marvell and Wiman I

f

le

i

J i i ii

i

o,.ov-.-...

,.,. I0

. ~.~.,,.£rO,o \¢',. ~x

. y ,, \ \

x&>,,'%.

5 2

~"*.7"'*,_'~',~."" " ' " ; ~ ' * ' .

OC

2

--

I

20

I

I

I 50

1,1

I, tl

,,

I

I

I

I

tOO 200 500 MEDIAN SURVIVAL TIME, mln.

I I I I I I000

L 2000

Fig. 8. Median survival times of rainbow trout juveniles (Salmo .qairdnerii) exposed to different concentrations of the sodium salts of various resin and unsaturated fatty acids (Leach and Thakore, 1973a).

Toxicit~ of pulp and paper mill effluents

(1963i isolated 4-1p-tolyll-t-pentanol as a major toxic constituent of the nondistillable fraction of material extracted from condensate streams. In succeeding studies. Banks (19691 isolated an extremely toxic diol, derived from an aliphatic keto-alcohol whose precise structure was not elucidated. Werner (1963) demonstrated toxicity in sulfur-containing material fractionated from black liquor, but did not complete isolation or identification of molecular entities. Early' evidence did not implicate lignin or any of its degradation products as responsible for the toxicity of unbleached pulping effluents. No relationship could be found between the concentration of phenolies and toxicity (Brebion, Chopin and Humbert, t957), and thiolignin and alkali lignin were nontoxic. Using model compounds, Chopin (1959) examined the toxicity of a number of phenol-like compounds, which were postulated to be lignin derivatives and found only limited toxicity. His conclusions are important: namely that the presence of phenolic-type molecules in kraft pulping discharges did not indicate toxicity despite the known toxicity of the lower aromatic hydroxyls such as phenol and the cresols. Although conventional phenol determinations are not a reflection of toxicity, problems continue to arise involving pulping effluents which concern the interpretation of monitoring data relating to phenol analyses. Pszonka (1973) indicated some of the simple toxic phenols are present in effluents and emphasized the need for further study.

655

The compounds toxic to fish and their relative contribution thereto, which have been isolated from pulping effluents, are listed in Table 5, Similar data are presented for other effluents, which are discussed subsequently'.

Kraft bleacherv e~ue,;ts Studies on the toxic materials in kraft bleachery effluents are all comparatively recent. Das et al. (1969) indicated tetrachloro-o-benzoquinone as a component of bleached kraft chlorination effluents, which was directly toxic or was a precursor of toxic materials to young salmon. The quantitative role of this component in acid bleachery effluents was not indicated. Rogers (1973) has demonstrated the presence of trichloroveratrole as a toxic constituent of bleached kraft effluents, with its origin presumably in the bleaching process. Servisi, Gordon and Martens (1969) examined the toxicity of two chlorinated catechols, using model compounds. Although the authors indicated that these compounds were possible components of kraft pulp mill bleach waste, their actual presence was not demonstrated. Discharge of bleachery effluents containing appreciable amounts of free chlorine may result in toxicity in receiving waters, since the toxicity of chlorine is well documented (McKee and Wolf, 1963). Seppovaara {1973, 1973a) found that the toxicity of chlorination effluents was affected largely by the residual chlorine present. Bteachery effluents normally contain

Table 5. Compounds toxic to fish in pulp mill effluent TOXIC

CONTRIBUTION

KRAFT EFFLUENTS Type of Chemical Compound

Naturally occurring resln acids

Bleachery

Specific Examples Pulping

Able=it, dehydroabiecic, Isoplmarlc, levopimarlc, palustrle, pimaric, sandaracopimaric, neoabletie

Major

Chlorination

Caustic

Minor

Minor

Debarking Effluent

Mechanical Pulping Ef fluent

Sulflte Pulping Effluent

,Major

,Major

Major

Major

Chlorinated llgnins Chlorinated resin acids

Mono- and dichlorodehydroabletie

Unsaturated fatty acids

Olelc, linolelc, llno~ lenio, palmlcoleic

Chlorinated phenolics

Tri- and tetrachlorogualacol

Diterpene alcohols

Pimarol, isoplmarol, dehydroabletal, able~al

Juvablones

Juvabione, Juvabiol,

Intermediate Intermediate

Minor Intermediate Minor

Intermediate Minor

A I -dehydroJuvahione, 41 -dehydroJuvablol, dihydroJuvabione O~her acidics

Epoxystearlc acid Dichlorostearlo acid, pitch dlspersant

Other neutrals

Abienol, 12E-ablenol, 13-eplmanool

Lisnin degradation ~roducts

EuKenol° isoeu~enol 3,31 dimethoxy, 4,41 dihydroxystilbene

Intermediate

minor

Intermediate

656

C.C. WALDEN

limited chlorine residuals, which usually are taken up completely by the organic loadings in the effluentl On the basis of dissimilar bioassay response curves for chlorine and chlorination bleachery effluents which we have examined in our Iaboratories. we have concluded that chlorine is not normally a toxic constituent of bleachery wastes. Leach and Thakore 11974b. 1975a) have recently determined the toxic constituents of the cm~stic bleachery effluents from a western Canadian mill to be: 3, 4, 5-trichloroguaiacol 3.4, 5, 6-tetrachloroguaiacol monochlorodehydroabietic acid (two isomers) dichlorodehydroabietic acid 9. 10-epoxystearic acid

0.75 mg 10.32 mg l - t 0.6 mg l0.6 mg I- t 1.5 mg 1- t

Dichlorostearic acid also has been isolated as a toxic constituent of caustic bleachery waste (Leach and Thakore, 1975b). For this particular sample, the tabulated compounds represented about 80'~; of the total toxicity. These compounds were demonstrated to be present in samples from six other mills and, for some samples, were shown to represent all the toxicity. That is, solutions containing only the pure toxicants in the concentration in which they were determined to be present in the original sample yielded a concentration-toxicity curve identical with that obtained for the original sample. Presumably, the trichloroveratrole, identified by Rogers 11973) as being present in bleached kraft effluents, is formed from the corresponding guaiacol. It is interesting to note that these two chlorinated guaiacols are the only toxic constituents, identified in kraft effluents, which are lignin degadation products. Unchlorinated resin acids usually are minor constituents of caustic bleach effluents, although carryover from the brownstock area can result in these compounds being responsible for as much as 3030 of caustic bleach toxicity (Leach and Thakore, 1974b), Generally, the more stable resin acids, such as the pimaric-type and dehydroabietic acid, are the only ones to survive the first chlorination stage of bleaching (Leach and Thakore, 1975b). Constituents identified in the toxic neutral compounds fraction of caustic bleach effluent include diterpene alcohols and aldehydes related to the resin acids, or other compounds of similarly related structures (Leach and Thakore, 1974b). Rogers and Mahood (1974) have identified these diterpene aldehydes and ketones as being present in the neutral fraction derived from toxic biotreated bteachery effluents, where corresponding resin acids were absent. However, the contribution of these neutral compounds to caustic bleach toxicity is minor (Leach and Thakore, 1975b). No published information is available on the toxic moieties in bleachery chlorination effluent. Preliminary evidence from our laboratories, where this prob-

lem is being examined, indicates that some form of chlorinated lignin is important.

Debarking eJfluents Information concerning the nature of the toxic constituents of debarking effluents has become available only recently. Using a colorimetric assay technique. Zitko and Carson 11971) iadicated high levels of resin acids in debarker effluent from a groundwood mill. Building on information derived for kraft pulping effluents (Leach and Thakore, 1973). Leach. Gietz and Thakore (1974} have obtained substantial information concerning the toxicants contained in debarking effluents. More recently, McKague 11975). working with Leach and Thakore has defined many. if not all. of the toxic constituents in an effluent from softwood debarking. Acidic components accounted for about 90°.; of the recovered toxicity and included C~,s unsaturated fatty acids and various resin acids as follows: pimaric, sandaracopimaric, 7, 15 isopimaric, palustric. abietic, neoabietic, dehydroabietic acid. The C~s unsaturated fatty acids included oleic, tinoleic and linoIonic acid. The toxic neutral compounds inclttded: abienol, 12E-abienol, pimarol, isopimarol and 13-epimanool. McKague 11975) failed to achieve a toxicity balance for his fractionation-isolation study, which was attributed primarily to the lability of palustric acid: a major component of the resin acid fraction. Quantitative evaluation of the toxicity of such minor constituents as 13-epimanool was designated for future study. together with a similar examination of hardwood debarking effluents.

Groundwood effluents An examination of the toxicity of mechanical pulping effluents by Row and Cook (19711 indicated that resin acids were responsible for much of the toxicw. Similar results were secured by Zitko and Carson (1971). Recent studies at B. C. Research IHoward and Leach, 1973a; Leach and Thakore. 1974a~ utilizing techniques developed previously ILeach and Thakore. t973), identified a number of resin and fatty acids. as involved in the toxicity of groundwood effluents. Leach and Thakore 11974a, 19751 undertook a detailed study to isolate and identify all the sigmficant toxic constituents of mechanical pulping effluents. Studies involved effluents from mechanical pulping of softwoods, with particular attention given to etItuent from a mill processing white spruce, lodgepole pine and alpine fir. Acidic constituents represented the major fraction of the toxicity and included the resin acids, abietic, dehydroabietic, and palustric acid. as major constituents. Minor constituents included pimaric, sandaracopimaric, isopimaric and neoabietic acids, and the unsaturated fatty acids: oleic, linoleic and linolenic. Neutral toxicants constituted up to 30". of the effluent toxicity for certain samples ILeach and

Toxicity of pulp and paper mill effluents Thakore, 19751. Toxic materials included the diterpene alcohols, pimarol and isopimarol, and the 'paper factor", juvabione. Derivatives of juvabione, with minor toxicity were identified, ri:, juvabiol and k:-deh.vdrojuvabiol, various diterpene aldehydes. methyl dehydroabietate and manool. Leach and Thakore (1974a. 1975) demonstrated that all the acidic toxicants had been identified by comparing the bioassay response curve of the acidic fraction with that of a synthetic solution (Leach and Thakore, 1973). The concentrations of minor neutral toxicants could not be measured with sufficient accuracy to prepare a synthetic effluent containing all toxicants in the concentration in which they were present in the original effluent. Nonetheless, the data indicated that, if any toxicants remain unidentified, the,,' correspond to an extremely small proportion of the total toxicity of groundwood effluents.

657

effluent toxicity, the observation of Kvasnicka and McLaughlin (19551. that they found no resin acids in liquor from the sulfite pulping of spruce, is pertinent. Nelson and Hemingway (1971) found appreciable quantities of resin acids in the bisulfite waste liquor from the pulping of Pinus radiata. Studies in progress in our laborazories have indicated resin acids as the only toxicants present in spent liquor from a high-yield soda-base mill. Presumably, the survival of resin acids and other wood extractives through the chemical pulping processes is directly dependent upon pulping conditions. The more stringent process conditions associated with the tow-yield sulfite processes probably are responsible for the fact that sulfite waste liquors are only slightly toxic, even where recovery is not practised. Correspondingly, in the kraft process, substantially greater proportions of the toxic wood extractives survive chemical digestion and the practically nontoxic nature of kraft effluent is maintained by the recovery and incineration of the greater proportion of these toxic constituents.

Su(tire e~)h,e,zts Knowledge concerning the toxic constituents of sulrite waste liquors stems primarily from the work of Wilson and Chappel (1973), who identified constituents corresponding to about half of the total toxicity in a sample of the discharge from a high-yield sodabase sulfite mill. Resin acids represented about 26% of the total toxicity, about half of the identified constituents. The two phenolic type compounds, eugenol and trans-isoeugenol represented about 20~o of the total toxicity and were responsible for the darkening of fish integuments, which is a characteristic response to sulfite wastes. Another phenolic-type compound, responsible for about 830 of the total toxicity, was 3, Y dimethoxy, 4, 4' dihydroxystilbene. All of these compounds were known previously to be present in sulfite pulping liquors. In view of the findings of Wilson and Chappel (19731. concerning the role of resin acids in sul~.te

TOXICITY

THRESHOLDS

P,r~c,:

Cauc,nccaciou L

&torch, d~.cce.*

O . t 2 - O.z~ 0.1 - 0.2 0.12 102 v / v • 0.2S

so12~d .& ~ (1960) B ~ a r d (197~) Tokar & Owe~e (1968) Webb &|rec¢ (1972)

O.OS -

W.Ld.n at al. (1970)

~ r 4

:oho

~ho ra~b~

it~ch 2 tms~lc4t~on

vsrt~

O.L8

• o°;I

k~ecochm~:sL

) - 6Z v / v

K~.COC~.,~I

coho ¢oho ccao ~oho

•

b~OC~L~C~I p1,~ |1~co** b~och~c~ (200 d.~.)

NcLea7 & l r o ~

(197&)

m u v i , (1973) D4v$1 (199'3)

Fu~lya (1961. 1964)

0.2~

scL~a7 (1973) ItcL~=y (1973)

o.I 0.1~

Mclaay (1973) M¢Le&y & S = ~ Z i l .( s (1967)

•

0.03 0.03 O.05

L L ~ b a c ~ l c h (1970) LLcbat~.eh (1970) w,:=~ (1972)

•

0.0~

Wa~

(ZS72)

•

0.05 0.05

wact~

var~

(1972) (1972)

~e:d

(197.%)

• -

•

(197,)

e~,h

•p c ~

~ts~ b ~ o ~ s

lob.c.¢, &elicit oysters

Znv,lcL&,cor(,)

0.1 - O.3 O.oe

z~*cc.,

0.1S • 20Z v ; v SO'~ v / v 0.6Z */v

.~td~c* sa.~u

a~daac4 e m b U o dsfom i cy

z. w 1 ~ . . x ~ r . . . . d . :~ork4d

vhege

2. S c * ~ 1 ~ c ~ o n

d4ca

:.Iv or .. f r a c r / ~

ENVIRONMENTAL

Substantial data relating to sublethal investigations and estimates of threshold concentration levels imposing stress have been presented and are collected into Tables 6 and 7. Threshold levels have been expressed in fractions of the static 96-h UC~o value; a quantitative measure of the toxicant concentrations. In some instances, where the toxic nature of the effiu~:nts has been described in other terms, data have been reworked to provide values in these same terms, using the interrelationships proposed by Walden et al. (1975). For example, Holland et al. (1960) cited

Table 6. Threshold levels of kraft mill effluent causing incipient changes in various biological organisms

• prtn* ~ 4 cono

FOR

PROTECTION

NcL~.... (1970) Spr*~*

6 C~u~

(196~)

Woelke (1967)

ot 96-hc U:30 .c.=Ic brow...7 v.l... e..~c~..~1 (1974).

p , ~ L ~ . ~ t e d , , s pec W a l d ~

65~

C. C: W.~,LDEY Table 7. Threshold levels ofsulfite mill effluent causing incipient changes in various biological organisms :=v,s=~,:or , 1.6: Y,'.,

gr~tn

2;;:;7........

cszdayf~e

(19~la)

% e~eFomic devllog,en."

~ .

il

~m

wo,~, , e & b (~970)

r

~, . . . o 1 , . .

the lethal threshold in a flowing-water system, i.e. the value below which no kill occurred. These data have been converted to corresponding LOs0 values by dividing by 0.76. Also, the results of Warren (1972) have been adjusted from flow-through bioassay values. The data in Table 6 on kraft effluents follow a consistent pattern, indicating threshold concentrations for various sublethal parameters to be approximately between 0.05 and 0.1 of the 96-h LCso value. At concentrations at or below this value, no sublethal stress has been observed. For invertebrates, although less information is available concermng sublethal effects, acute responses show that these organisms are less sensitive than juvenile fish. Thus. a substantial technical background is available on which to base a rational assessment of the threshold concentration of pulp and paper effluents below which aquatic populations are not stressed. Certainly, this technical information is not all embracing and specific information relating to some areas of environmental concern is unavailable. Despite these deficiencies, no contrary evidence exists to substantiate any sublethal stress in any organism at concentration levels below 0.05 of the 96-h echo value. This concentration value should be expressed in units, which relate solely to the concentration of toxicants in pulp and paper effluents, and which are independent of bioassay procedural variables such as exposure time, fish survival and solution replacement. A system of nomenclature presently is in use amongst biologists involving "'toxic units" and Sprague (1970) indicates that it is finding increasing technical acceptance. Sprague (1970) has indicated that the 96-h LCs0 value is reasonably approximated as unity on the toxic unit scale. Walden et ctl. (1975) have confirmed this observation experimentally for kraft mill effluents and from their calibration data. unity value in toxic units, with adequate solution replacement (24 h) and 50?0 fish survival, is equivalent to 1.96 toxic units with no solution replacement. On this basis, 0.05 of the 96-h static bioassay value is equivalent to 0.1 toxic unit. This 0.1 toxic unit value represents the threshold concentration for pulp and paper effluents below which stressful conditions are not created m receiving waters. Logically, measurement of the concentration of toxic material should be made on samples collected in the field as representative of the receiving waters.

~...

~.ooo

.o~..

Substantial studies have been undertaken on various sublethal bioassay procedures. Although a limited number of these procedures presently can be applied over a short term, they are working at their limit of sensitivity in attempting to detect 0.1 toxic unit. Moreover. in specific instances, the ecological relevance of some of these bioassay procedures is not evident, although the rough equivalence between measurements of various sublethal parameters suggests similar effects are involved. The respiration bioassay undoubtedly is ecologically relevant. Ho~ever. the test is not stmple, and is not adaptable to routine momtormg in most fish laboratories. Thus although the short-term sublethal bioassay of receiving waters may be the method of future choice, as the ecological relevance and the routine nature of the procedures are established: as yet no sublethal bioassay exists which is acceptable as a routine procedure. Direct observations on the environment, over a long-term, may show incipient changes at these threshold levels. Although such studies can be quantitative in nature, they are necessarily retrospective, integrating effects over the period of observation and provide no information concerning the toxicLty of the discharges, particularly the type of information essential for rapid monitoring of effluent toxicity as related to plant operation. Chemical assays cannot be used. Despite recem advances, the nature of the chemical moieties res ponsible for toxicity remains incompletely known The equivalence between biological activity and concentration has not been established for many toxic constituents. Although chemical assays may represent :~ future possibility for routine monitoring, they cannot vet be used for this purpose. The lethal acute bioassay is well accepted in poilu. tion biology as an analytical procedure for measuring the inherent toxicity of industrial discharges. Virtually all the procedural problems specific to the assaylng of pulp and paper effluents have been resolved However. the acute lethal bioassay, as a procedure for pulp and paper samples, has a maximum sensitiwty of 0.8 toxic units, IWalden et al.. 19751 and can only be used where the toxicities are appreciably greater than this value. Consequently, measurements must be made on mill discharges and related to envtronmental levels on the basis of dilution characteristics. For example, if the concentration of mill effluent in receiving waters is no more than 5". then the tox~citv of

Toxicity- of pulp and paper mill effluents the ettq.uent should not exceed 2,0 toxic units. Thus. it is necessary to correlate anticipated maximum toxic levels of the discharge with the corresponding concentrations in the environment. Where multiple discharges occur, total loadings from individual sources can be determined from the product of the concentration of toxicants, in toxic units, and the flow. Toxic loadings, computed in this manner, may be additive ISprague, 1970), thus permitting assessment of multiple discharge situations. Where multiple discharges occur in receiving waters, discharge limits should be established so that a concentration of 0.1 toxic unit will not be exceeded. Allowable toxicity in discharges should be based on existing industrial and domestic outputs, with suitable provision for foreseeable expansion in these activities. Since most pulp and paper discharges are from a single location source, this situation will arise infrequently. A major technical argument against the use of application factors to relate safe and acute levels is based correctly on the possibility that the nature of responses causing death and those causing sublethal stress may be different. Preceding evidence demonstrates that this is not so for pulp and paper discharges, i.e. the effect on respiration is the primary sublethal effect and ultimately is responsible for death. Nevertheless, the use of the acute lethal bioassay to determine safe levels in the environment represents a technical compromise. It may be expected that the use of such a bioassay will, in time, be replaced by an ecologically relevant sublethal bioassay with a sensitivity adequate to make direct field measurements. Available information now permits the design of acute lethal bioassays which are simple, sensitive and accurate. Furthermore, by converting data into toxic units, results are directly comparable even though bioassay procedures may vary. For eminently practical reasons, exposure time should be short, solution replacement should be minimal and the procedures should accommodate the various sizes of test fish available to bioassay facilities at different times of the year. Not all these attributes can be achieved in a single bioassay procedure and some compromise is necessary, which can differ as desirable. Maximum sensitivity is obtained at 100% effluent concentration. Where lesser sensitivity is required, the sample under test can be diluted. Maximum accuracy is achieved with 50'?.0 fish survival. Where enhanced sensitivity is desirable, this can be achieved by increasing measured survivals to 90%, albeit at the expense of accuracy, Exposure time is not critical, but the toxicity of most effluents will necessitate exposures of at least 24 h. At longer exposure times, some solution exchange is essential if bioassay sensitivity is to be maintained. The sensitivity of a number of bioassay procedures for pulp and paper mill effluents, at 100% concentration, has been calculated from the data of Walden et al. {1975) and are listed in Table 8. For exposure

659

Table 8. Sensitivity of ,~arious bioassay procedures at 100% concentration for pulp and paper effluents Sol'ation

~ation

Replacemen=

,r ~_stt Su:'vlvak

~r

Ya~im~ ? i ~ h ~_> 7 o l u ~ ratio/day

S~tasi=ivity --~oxi¢ ual=s

z,'~tter

!2

2:

2~

static

50

2.,3

2.95

9Q

2.3

2.~5

50

2.3

:.68

90

2.0

L.37

50

0.5

~..68

90

0.5

k.37

50

2.3

'-. 3

90

2. ,2

O. 76

50

0.5

L.9,.

90

0.5

1.6~

times not exceeding 96 h, the maximum sensitivity of any acute bioassay procedure, is 0.76 toxic units, corresponding to 90°0 fish survival. When fish survival is 50%, the sensitivity is 1.0 toxic unit. Where lesser sensitivities are'acceptable, shorter-term bioassays can be designed. For example, a 24-h exposure, with a fish loading of 2.0 g 1-1 day- 1 and with solution replacement at the 12-h mark, has a sensitivity of 2.45 toxic units, where fish survivals are at least 90°/0. This procedure could be used to monitor a discharge where the anticipated dilution was at least 245 times (i.e. 2.6% v v -~ of emuent in the receiving waters). In routine monitoring, survivals of 9030 or more would indicate 0.1 or less toxic units in the receiving water. As appropriate, other bioassay procedures in Table 8 could be utilized, adjusting sensitivities to lower values, as desirable, by reducing effluent concentrations in bioassay solutions. Present data are inadequate to stipulate that the above considerations provide adequate protection to oysters and related species in receiving waters. Evidence does not implicate kraft effluents as being stressful at 0.1 toxic unit (Donnier, 1972: Woelke, 1967), but does suggest that sulfite effluents may merit additional attention in specific instances. Available data in the literature relate to actual concentrations of sulfite waste liquor in laboratory situations or indirect measurements on field samples. No absolute basis exists for relating these concentrations to the toxic unit values utilized in this presentation. Nonetheless, the actual concentrations shown to cause sublethal effects are exceptionally low. Evidence indicates that ammonia toxicity is not involved, although ammonia is toxic to fish. Additional research into the nature of the toxic action of ammonia in seawater situations, in conjunction with sulfite effluents, is desirable. Acknowledgements--The author wishes to acknowledge the helpful comments of Dr. T. E. Howard and the financial support of the Canadian Pulp and Paper Association.

660

C.C. WALPE,',REFERENCES

-~delman I. R. & Smith. Jr. L. L. L19721 Toxicity of hydrogen sulfide to goldfish ~Crassius aur,~tus} as influenced by temperature, oxygen and bioassay techniques. J. Fish. Res. Bd Canada 29. 1309-1317. Alderdice D F. & Brett J. R. 11957) Some effects of kraft mill effluent on ~oung Pacific salmon. J. Fish. Res Bd Canada 14. 783-795. Amberg H. R.. Aspltarte T R.. Bymgton K. F. & Ehli J. J. (19711 The design and effectiveness of an aerated lagoon for the treatment of sutfite pulp and paper mill effluents. Presented at the 6th Air and Stream lmprocemen~ Conf.. Tech. Sect._ Can, Pulp Paper As.. Quebec City. Quebec. -~merican Public Health Associatton ~1971) Standard Methods Cbr the Examination of Water and Wastewater. 13th edition. Am. Public Health Assoc.. New York. N.Y. Anonymous 11973) Notes on Water Pollution No. 63. Dept. Environ. (U.K.) ~. pp. HMSO. London. England. Banks R. 11969) I. Isolation of certain toxic components of kraft mill waste and attempts to determine their structure. Ph.D. thesis filed at Oregon State Univ.. Corvallis, OR. Betts J. L.. Beak T. W. & Wilson G. G. (t967) A procedure for small-scale laboratory bioassays. J. Wat. Pollut. Control Fed. 39. 89-96. BergstrOm H. & Vallin S. (1937) The contamination of water by the waste liquors of sulphate pulp mills. Medal Statens UndersOken-FOrsOksants SOtvattenfisket. Kgl. Lantsbruksstyrelson No, 13. Betts J. L. & Wilson G. G. (1966) New methods for reducing the toxicity of kraft mill bleachery wastes to young salmon. J. Fish. Res. Bd Canada 23, 813--824. Blosser R. O. & Owens E. L. (19701 A Guide to the Shortterm Bioassay of Mill Effluents. Tech. Bull. No. 233. Nat, Council Air and Stream Improvement, New York, N.Y, Brebion B.. Chopin J. & Humbert F. 11957) Toxicity to fish (minnows) of some phenolic derivatives formed by the decomposition of lignin m paper pulp factories. Chem. Ind. 77, 1110-I 116. Bruynesteyn A, & Walden C. C. (19711 Origin of Toxicity and Biochemical Oxygen Demand (BOD) in the Bleached Kraft Process. CPAR Report IO--l. Canadian Forestry Service. Ottawa. Ontario. Bruynesteyn A. & Walden C. C. (I973) Effects of condensates on the toxicity of kraft pulp mill effluents. Pulp Paper Mag. Canada 74, T226-T231. Bruynesteyn A.. Walden C. C, & Hill D, A. (1972) Origin of toxic materials in the kraft pulping process. Pulp Paper Mag. Canada 73, T347-T351. California State Water Resources Control Board (19721 Water Quality Control Plan for Ocean Waters of California. State of California, The Resources Agency, Sacramento, CA. Caron A. L. (1974) Unpublished communication. Nat. Council Air and Stream Improvement. Oregon State Univ.. Corvallis, OR. Carter L. 11962) Bioassay of trade wastes. Nature. Lond. I96. 1304. Casey J. P. (Editor)(1961) Pulp and Paper Chemistry and Chemical Technology. 2nd edition. Vol, l-IlL "Interscience, New York, N,Y, Chevalier J. R. (19731 Toxicity of sodium sulfite to common shiners: dynamic bioassay. T A P P I 56(5) 135-136. Chopin J. (1959) Phenolic substances in pulp mill effluents. Ass. Tech. Ind. Papetiere Bull, No. 3. t47-155. Cole A. E. (1935) Effect of industrial (pulp and paper) wastes on fish. Sewage Wks J. 7. 280--302. Cole A. E. (1935a) The toxicity of methyl mereaptan for fresh water fish. J. Pharmac. Expl Ther. 54, 448453. Crown Zellerbach Corporation (1970) Aerated Lagoon

Treatment of Su[fite Pulping Effluents. Envm Protection Agency Program No. 12040 ELW, Pro: No WPRO 69-01-68. Washington. D.C. b a s B. S., Reid S. G,. Betts J. L. & Patrick K 19691 Tetrachloro-o-benzoqumone as a component m bleached kraft chlorination effluent toxic to youn~ salmon. J. Fish. Res. Bd Canada 26. 3055-3067, Davis J. C. (19731 Sublethal effects of bleached kraft pulp mill effluent on respirauon and circulation in socke?e salmon ,Oncorhynchus ,erka~. J. Fish. Res Bd Ctma&l 30. 369-377. Davis J. C. & Mason B. J. • 1973) Bioassa', procedures to evaluate acute toxicity of neutralized bleached kraft pulp mill effluent to Pacific salmon J. Fish. Res. Bd Canada 30, 1565-1573. DeWitt J. W. D. 119631 Effects of potlutional conditions on stream organisms with special emphasis on stone fix naiads. Ph.D. thesis filed at Oregon State Univ.. Corvallis. OR. Dimick R. E. & Haydu E. P. (19521 The effect of kraft mill waste liquors and certain of their components en certain salmonid fishes of the Pacific Northwest. Tech. Bull. No. 5l. Nat. Council Stream Improvement. New York. N.Y. Dimick R. E.. Warren C. E.. Jones B. F.. Doudoroff P. & Amberg H. R. (1957) Some preliminary observations on the avoidance reactions of salmonid fishes to pulp mill effluents. Tech. Bull. No. 93. Nat. Council Azr and Stream Improvement. New York, N.Y. Donnier E. [1972)Toxicity of pulp and paper mill effluents on sea environments. Rer. Int. Oceangr. Medicate 28. 53-93. [A.B.I.P.C. 44, 6249], Doudoroff P.. Anderson B. G., Burdeck G. E, Galtsoff P S.. Hart W. B.. Patrick R.. Strong E, R.. Surber E. W. & Van Horn W. M. IL951) Bio-assay methods for the evaluation of acute toxicity of industrial wastes to fish. Sewage Ind. Wastes 23, 1380--1397 Doudoroff P. & Katz M. {1950) Critical review of literature on the toxicity of industrial wastes and their components to fish. I. Alkalies. acids and inorganic gases. Sewa~je Ind. Wastes 22, 1432-1458. Ellis R. H. (19671 Effects of kraft pulp mill effluent on the production and food relationships of juvenile chinook salmon in laboratory streams, Tech. Bull. No. 210. Nat. Council Air and Stream Improvement. New York. N.Y. Ebeling G. 119311 Recent results of the chemical mvesngation of the effect of waste waters from cellulose plants on fish. Vom Wasser 5, 192-200, Extrom J. A, & Farner D. S. [1943) Effect of sulphate mill wastes on fish life. TAPPI. Tech. Ass. Papers Ser, 26, 193-198. Fahmy F. K. & Lush O. L. 11975) Sensitivity of major aquatic food chain organisms to treated kraft milI efl]aents. CPAR Rep. No. 356-1. Canadian Forestr.', Service. Ottawa. Ontarzo. Farvolden S, & Thakore. A. N. (1974) Effects of resin and fatty acids on young salmon. B.C. Research, Vancouver. B.C. (Private communication J. Filimonova Z. I. (t968) Effects of effluents from pulp and paper mills on zooplankton in natural waters. Santr. Gidrobiol. Vodn. Toksikol. 2, 197-200. Finney D. J. (1941~ Probit Analysts. (..ambrldge Umvers]ty Press, Cambridge, England. Fry F. E. J. (1971) The effect of environmental factors on the physiology of fish, Fish Physiol, Vol. 6, pp, 1-98. Academic Press, New York. N.Y, Fujiya M (1961) Effect of krali pulp mill wastes on fish. J. War. Pollut, Control Fed. 33, 968--977. Fujiya M. (1962) Physiological studies on the effects of pulp mill wastes on aquatic organisms. Chapter IV Evaluation of effects on bivalves. Nakai Reyional Fish. Lab. Bull. 17, 1-100.

Toxicity of pulp and paper mill effluents Fuji?a M. (1964) Physiological estimation of the effects of pollutants upon aquatic organisms. Adcances Water Polh~ri,,n Research Vol. 3, pp. 315-33l. Pergamon Press. Oxford. Galtsoff P. S., Chipman W. A., Hasler A. D. & Engle J. B. 11938) Preliminary report on the cause of the decline of the o?ster industr? of the York River, Virginia. and the effects of pulp-mill pollution on oysters. Incestiqationul Rep. No. 37. Bur. of Fish., U.S. Dep. of Commerce, Washington, D.C. Galtsoff P. S.. Chipman W. A., Engle J. B. & Calderwood H. N. (1947)Ecological and physiological studies of the effect of sulfate pulp mill ~astes on oysters in York Ri~er, Virginia. Fish. Bull. No. 43. U.S. Fish and Wildlife Service. Washington, D.C. Gazdzyauskaite I. B. (197l) Effect of effluents from the So~ctsk and Neman mills on the biology of Pontoga,narus robustoides. (1) Vitality. (2) Respiration intensity. Liet. TSR Moksl,t. .4kud. Darbai Ser. C No. 2, 93-I07, 109-I 16. [A.B.I.P.C. 43, 10694). Gazdzyauskaite 1. B. 11971a) Effect of effluents from the So~etsk and Neman sulfite pulp and paper mills on the fertility of Pontogamarus robustoides (Grimm) sars. R yhokhoz. [ zuch. Vnutr. ~bdoemoc No. 6, 29-31. [A.B.I.P.C. 43, 3026]. Grande M. (19641 Water pollution studies in the river Otra. Norway--effects of pulp and paper mill wastes on fish. Int. J. Air Wat. Pollut. 8, 77-88. Hagman N. (1936). Resin acids in fish mortality. Paperi ja Puu 18(1) 32-41. Haydu E. P., Ambcrg H. R. & Dimick R. E. (1952} The ett)ct of kraft mill waste components on certain salmould fishes of the Pacific Northwest. TAPPI 48, 136-141. Hemingway R. W. & Greaves H. (1973) Biodegradation of resin acid sodium salts. TAPPI 56(12), 189-192. [-terbcrt D. M. & Wakcford C. M. (1964) The susceptibility of salmonid fish to poisons under estuarine conditions. 1. Zinc sulfate. Int. J. Air War. Pollut. 8, 25t-256. Flicks D. B. & DeWitt J. W. (1971) Effects of dissolved oxygen on kraft pulp mill effluent toxicity. Water Res. 5, 693-701. Histed J. A. & Nicolle F. M. A. (1973) Water reuse and recycle in kraft bleacheries. Pulp Paper May. Canada 74, T386-T397. Hoglund L. B. (1961)The reactions of fish to concentration gradients. A comparative study based on fluvarium experiments with special reference to oxygen, acidity, carbon dioxide and st, lphite water water. Repc Inst. Fresh~ater Research. Drottiningholm. Sweden. [Water Pollut. .4hstr. 35, 2220]. Holland G. A., Lasater.J.E., Neumann E. D. & Eldridge W. E. (1960} Toxic effects of organic and inorganic pollutants on young salmon and trout. Res. Bull. No. 5. State of Washington Dep. of Fish., Olympia, WA. Hood D. W.. Duke T. W. & Stevenson B. (1960) Measurement of toxicity of organic wastes to marine organisms. J. ~,l,at. Pollut. Control Fed. 32, 982-983. Ho~ard T. E. {1973) Effects of kraft pulp mill effluents on the swimming stamina, temperature tolerance and respiration of some salmonid fish. Ph.D, thesis filed at the Univ. of Strathclyde, Glasgow, Scotland. Ho~ard T. E. (1975) Stamina of juvenile coho salmon in pulp mill effluent and in water after effluent exposure. J. Fish. Res. Bd Canada 32, 789-793. Howard T. E. & Leach J. M. (1973) Identification and treatment of the toxic materials in woodroom effluents. CPAR Rep. No. 148-I. Canadian Forestry Service, Ottawa, Ontario. Howard T. E. & Leach J. M. (1973a) Identification and. treatment of the toxic materials in mechanical pulping effluents. CPAR Rep. No. 149-1. Canadian Forestry Service, Ottawa. Ontario.

66I

Howard T. E. & Walden C. C. 119651 Pollution and toxicit,, characteristics of kraft pulp mill effluents. T.-IPPI 48, 135-[4l. Howard T. E. & Walden C. C. 41967} Hematocrit Variation at Sublethal Concentrations of Kraft Mill Wastes. Presented at the 97th Ann. Conf. Am. Fish. Soc.. Toronto, Ontario. Howard T. E. & Walden C. C. (1971) Effluent characteristics of bleached kraft pulp mills. Pulp Paper May. Canada 72, T3-T9. Howard T. E. & Walden C. C. 11973) Basic bioassay techniques. Pulp. Paper 3,lu9. Canada 74, T285 T289. Howard T. E. & Walden C. C. 119741 Measuring stress in fish exposed to pulp mill effluents. TAPPI 57(2}, 133-135. Howard T. E., Walden C. C. & Munro J. R. 119741 Rapid Monitoring of Mill Effluent Quality. Presented at the 9th Air and Stream Improcement Conj'. Tech. Sect., Can. Pulp Paper Ass.. Toronto, Ontario. Hunn J. B., Schoettger R. A. & Whealdon E. W. (1968) Observations on the handling and maintenance of bioassay fish. Progr. Fish-Culturist 30, 164-167. Jones B. J.. Warren C. E., Bond C. E. & Doudoroff P. (1956) Avoidance reactions of salmonid fishes to pulp mill effluents. Sewage Ind. Wastes 28, 1403-1413. Klein L. (19591 River pollution. 1. Chemical Analysis. Butterworth's. London, England. Kondo R., Samcshima K. & Kondo T. (1973) Spent semichemical pulping liquor. 131. Toxicity characteristics of SCP spent liquor and reduction of its toxicity. Japan "E4PPI IlL 476-485. [.4,B.I.P.C. 44, 12026.] Kramcr R. H. & Smith L. L. (1965) Effects of suspended wood fiber on brown and rainbow trout eggs and ale~ins. Trans. Am. Fish. Soc. 94, 252-258. Kramer R. H. & Smith L. L. (19661 Survi,,al of walleye eggs in suspended wood fibers. Print. Fish-Culturist 28, 79-82. Kvasnicka E. A. & McLaughlin R. R. {19551 Identification of spruce sulphite liquor components. Can. J. Chem. 33, 637-645. Ladd J. M. 11969) Effects of pH on the acute toxicity of kraft pulp mill effluent to juvenile coho salmon, Oncorhynchus kisutch. M.S. thesis filed at Humboldt State Coll., Arcata, CA. Larmoyeux J. D. & Piper R. G. (19731 Effects of water reuse on rainbow trout in hatcheries. Prog. Fish-Culturist 35, 2-8. Leach J. M., Gietz W. C. & Thakore A. N. (1974) Identification and treatment of the toxic materials in pulp and paper woodroom effluents. CPAR Rep. No. 148-2, Canadian Forestry Service, Ottawa. Ontario. Leach J. rvl. & Thakore A. N. (1973) Identification of the constituents of kraft pulping effluents that are toxic to juvenile coho salmon (Oncorhynchus kisutch). J. Fish. Res. Bd Canada 30, 479-484. Leach J. M. & Thakore A. N. (1973a) Isolation of the toxic constituents of kraft pulp mill effluents. CPAR Rep. No. 11-3. Canadian Forestry Service, Ottawa, Ontario. Leach J. M. & Thakore A. N. (1974) Isolation of the toxic constituents of kraft pulp mill effluents. CPAR Rep. No. l 1-4. Canadian Forestry Service, Ottawa, Ontario. Leach J. M. & Thakore A. N. (I974a) Identification and treatment of the toxic materials in mechanical pulping effluents. CPAR Rep. No. 149-2. Canadian Forestry Service, Ottawa, Ontario Leach J. M. & Thakore A. N. (1974b) Identification of the toxic constituents in kraft mill bleach plant effluents. CPAR Report No. 245-1. Canadian Forestry Service, Ottawa, Ontario. Leach J. M. & Thakore A. N. (1975) Toxic constituents in mechanical pulping effluents. Presented at the 8th Int. Mechanical Pulpin9 Corgi, TAPPI., San Francisco, CA.

602

C . C . WALDEN

Leach J. M, & Thakore A. N. 11975a/Isolation and identification of constituents toxic to juvenile rainbow trout tSalmo gairdneril in caasnc extraction effluents from kraft pulp mill bleach plants. J. Fish. Res. Bd Canada 32. 1249-1257. Leach J. M. & Thakore A N. tl975b, Identification of the toxic constituents in kraft mill bleach plant effluents. CP.4R Rep. No. 245-2. Canadian Forestry Service. Ottawa. Ontario. Lesnikov L. A. (19661 Effect of unpurified and purified sulfate waste waters of the pulp and paper industry on aqueous invertebrates. Biol. Prod. Vodoemov Sib.. Dokl. Sot'eshch 273-276. [.4.B.I.P.C. 42, 8t82]. Lichatowich J. A. [19701 Influences of kraft mill effluents on the production of salmon m laboratory stream communities. M Sc. thesis filed at Oregon State Univ.. Corvallis. OR. Litchfield J. T. & Wilcoxon F. 11953~ The reliability of graphic estimates of relative potency from dose-percent effect curves. J. Pharmac. Expl Ther. 108, 1,8--25. Litvintsev A. N. {19671 Effect of waste waters from a woodhydrolyzing factory on aquatic organisms. Biol. Nauk. 100). 20-22 IWater Pollut. Abstr. 42, 17101. Loch J. S. & Bryant W. J. 119721 Toxicity and other pollution characteristics of unbleached kraft mill effluents. Report No. TR72-3. Resource Development Branch. Fish. Service, Environment Canada. Winnipeg, Manitoba. Loch J. S. & McLeod J. C. 11973} Fish toxicity survey of four prairie province pulp mill effluents. Tech Report No. CEN T-73-4. Fish. and Marine Service. Environment Canada. Ottawa. Ontario. Macdonald R. G & Franklin J. N. IEditors) 119651 Pulp and Paper Man@tcture. 2nd edition. Vol. I-Ill. McGraw-HilL New York. N.Y. McKague A. B. 119751 Identification and treatment of the toxic materials in pulp and paper woodroom effluents. CPAR Report No. 148-3 Canadian Forestry Service. Ottawa. Ontario. McKee J. E & Wolf H. W. [t9631 Water Quality Criteria. Publication No. 3-A. California State Water Quality Control Board. Sacramento. CA. McLeay D. J. (1973) Effects of 12-h and 25-day exposure to kraft pulp mill effluent on the blood and tissues of juvenile coho salmon [Oncorhynchus kisutchl. J. Fish. Res. Bd Canada 30, 395--400. McLeay D. J. (1976) A rapid method for measuring the acute toxicity of pulp mill effluents and other toxicants to salmonid fish at ambient room temperature. J. Fish. Res, Bd Canada (in pressl. McLeay D. J. & Brown D. A. 119741 Growth stimulation and biochemical changes m juvenile coho salmon tOncorh)~whus kisutch~ exposed to bleached kraft pulp mill effluent for 200 days. J. Fish. Res. Bd Canada 31, 1043-1049. McLeay D J. & B r o w n D A. 119751 Effects of acute exposure to bleached kraft pulp mill effluent on carbohydrate metabolism of juvenile coho salmon IOncorhynchus kisutch|. J. Fish. Res. Bd Cana~kt 32. 753-760. McLeese D. W. 11970) Behaviour of lobsters exposed to bleached kraft mill effluent. J. Fish. Res. Bd Canada 27, 731-736. McLeese D. W. (t973) Response of lobsters Homarus anuericanus to odor solution in the presence of bleached kraft mill effluent. J. Fish. Res. Bd Canada 30, 279-282. MacLeod J. C. & Smith L. L. t19661 Effect of pulpwood fibre on oxygen consumption and swimming endurances of the fathead minnow IPimephales promelasL Trans, Am. Fish. Soc. 95, 71-84. M ~ i e n l ~ R.. Hynninen P. &. Tikka J. 11968~ On the occurrence of abietic and pimaric acid type resin acids in the effluents of sulphite and sulphate mills. Paperi ja Puu 41"a1. 143-150. Marier J. 11973} The effects of pulp and paper wastes, with

particular attention to fish and bloassay procedures for assessment of harmful effects. ,VRCC .Vo. 1350t Nat Res. Council of Canada. Ottawa. Ontario. Martens D. W.. Gordon R. W. & Ser,,isi J. A. ~[971t Foxicity and treatment of de-inking wastes containin~ detergents. Proq. Report No. 25. Int. Pacific Salmor Fish. Comm.. New Westminster, B.C. Marvell E. N. & W i m a n R. t19631 -t-tp-tofylI-i-pentanol in Douglas fir pulping products. ! Ora. Chem. 28. 1542-1545. M o u n t D. I. & Stephan C. E ~1967t A method for establishing acceptable toxicant limits for fish-Malathion and the butoxyethanol ester of 2, 4D. Trans..4m. Fish. Soc. 96, 185-193. Nelson P. J. & Hemingway R. W. ~19711 Resin m blsulfite pulp from Pinus radiata wood and its relationship to pitch troubles. TAPPI 54, 968-97l. Ng K S,. Mueller J. C. & Walden C. C. tl9731 Detoxification of kraft mill effluents by foam fractionation. Pldp Paper Mag. Canada 74. T187-T191. Ng K. S Mueller J. C. & Walden C. C. 11974~ Stud.~ of foam s e p a r a n o n as a means of detoxifying bleached kraft mill effluents, removing suspended solids and enhancing biotreatability. CPAR Rep. No. 233-1 ,Part II. Canadian Forestry Service. Ottawa. Ontario. Odlaug T. O. 11946) Effects of stabilized and unstabihze~i waste sulfite liquor on the Olympia oyster. Ostrea lurida. Trans. Am. Microscopic Soc. 68. 163-I82. Podoba Z. P. 119661 Effect of biologically purified waste waters of the pulp and paper industry on fish. Biol. Prod. Vodoemor Sib. Dokl. Sot'esch. 253-258 ~.4.B.I.P.C 42. 81951. Pszonka B. 19731 Phenolic c o m p o u n d s in spent hquors and pulp mill effluents. Pr:eglad Papier 29. 254-256. Rapson W. H. 119671 The feasibility of recovery of bleach plant effluent to eliminate water pollution by kraft pulp mills. Pulp Paper May. Canada 68, T635-T640. Rogers I. H. (1973~ Isolation and chemical identification of toxic components of kraft mill wastes. Pulp Paper Mag. Canada 74. T303-T308. Rogers 1 H. & M a b o o d H W. 119741 Removal of fishtoxic organic solutes from whole kraft effluent by biological oxidation and the role of wood cxtractives. Tech. Rep. No. 434. Fish. Res. Bd Canada. West Vancouver. B.C. Row R & Cook R. H. [19711 Resin acid soaps toxiclt> and treatability. Presented at the 6th Air and Stream hn provement Conf., Tech. Sect.. Can. Pulp Paper Assoc.. Quebec City, Quebec. Schaumburg F. D. & Atkinson S. II9701 B O D 5 and toxicity associated with log leachates. Presented ar Conj,, Western Div.. Am. Fish. Soc.. Victoria. B.C. Schaumburg F. D.. Howard T. E. & Walden C. C. ~1967 A method to evaluate the effects of water pollutants on fish respiration. Water Res. 1, 731-737. S c h a u m b u r g F. D. & Willard H. K. 119731 Influence of log handling on water quality. U.S. Natl. Tech. Inform. Serv.. PB Report No. 219824/0. Sup. of Documents. Washington. D.C. Seim W. K. 119701 Influences of biologically stabilized kraft mill effluent on the food relations and production of juvenile chinook salmon in laborator~ streams M.Sc. thesis filed at Oregon State Univ. Corvallis. OR. Seppovaara O. t19711 Biological testing of the toxicity oi waste water of a sulfate pulp plant. Suont. Kemistitehti A. 44, 5-6, 103-107 [Chem. Abstr. 75, 672869]. Seppovaara O. {19731. The toxicit~ of the sulfate pulp bleaching effluents. Paperi :a Puu 55. 71"~-715. 717720. Seppovaara O. (1973aL Sulfaatisellutehtaiden .l~.itevedet ja nuden toksissus. Oso II Va[kaisuj~itevedet. Seloste 1130. Oy Keskuslaboratorio. Helsinki. Finland.

Toxicity of pulp and paper mill efiluents Seppovaara O. & Hynninen P. 119701 On the toxicity of sulphate mill condensates. Paperi ju Puu-Papper och Tra 52, 11-23. Ser',isi J. A.. Gordon R. ~,V. & Martens D, W. 11969! Toxicity of two chlorinated catechots, possible components of kraft pulp mill bleach waste. Prog. Rep. No. 18. Int. Pacific Salmon Fish. Comm.. New Westminster, B.C. Servisi J. A,, Stone E. T. & Gordon R. W. 11966) Toxicity and treatment of kraft pulp bleach plant waste. Prog, Rep. 13. Int. Pacific Salmon Fish. Comm.. New Westminster, B.C, Smith L. L. & Kramer R. H. (1964~ Some effects of paper fibers on fish eggs and small fish. Proc. lgth Ind. Waste Co,~tl. Purdue Univ., Lafayette. IN. Smith L. L., Kramer R. H. & MacLeod J, C. (19651 Effects of pulpwood fibers on fathead minnows and walleye fingerlings, a. Wat. Polha. Control Fed. 37, 130--140. Smith L. L,, Kramer R. H. & Oseid D. M. (19661 Longterm effects of conifer groundwood paper fiber on walle?es. Trans. Am. Fish. Soc. 93. 60--70. Smith L. L. & Oseid D. M. (1972) Effect of hydrogen sulfide on fish eggs and fry. Water Res. 6, 711-720. Sprague J. B. 11969) Measurement of pollutant toxicity to fish. I. Bioassay methods for acute toxicity, Water Res. 3, 793-821. Sprague J. B. {19701 Measurement of pollutant toxicity to fish. II. Utilizing and appl',ing bioassay results. Water Res. 4. 3-32. Sprague J. B. (1971) Measurement of pollutant toxicity to fish. 11I. Sublethal effects and "safe" concentrations. Water Res. 5, 245-266. Sprague J. B. & Drury D. W. (1969) Avoidance reactions of a salmonid fish to representative pollutants. Adrances Water Pollution Re.search, Vol. 4. pp. 169-179. Pergamon Press. Oxford. Sprague J. B. and McLeese D, W. (1968) Toxicity of kraft pulp mill effluent for adult and larval lobsters, and juvenile salmon, ll"ater Res, 2, 753-760. Stein J. E., Petersen R. E., Denison J. G., Clark G. M. & Ellis I. E. {1959) The spawning of Olympia oysters {Ostrea lurida) kept in spent sulfite liquor ISSL). Olympia Research Dic. Report Rayonnier, Shelton, WN [Ioc. cir., Van Horn W. M. (1961)]. Tabata K. i19551 Systematic studies on industrial w a s t e s with reference to the tolerance of aquatic lives. II. On acute toxic components digester waste from ammoniabase semichemical pulp mills. Nakai Regional Fish. Lab. 42, II. Thomas P. M. & Legault R. O. t 19671 The effects of industrial wastes from Charmin Paper Products Company on fish of the Cheboygan Ri~er system. Water Res. 1, 217-229. Tokar E. M. & Owens E. L. /1968) The effects of unbleached kraft pulp mill effluents on salmon. I. Growth, food consumption and swimming ability of juvenile chinook salmon. Tech. Bull. No. 217. Nat. Council Air and Stream Improvement, New York, N.Y. Tollegon R. D. (1974) ITT Rayonnier Res. Lab.. Shelton, WN {Personal communication). Trt, ssell R. P. (19721 The percent un-ionized ammonia in aqueous ammonia solutions at different pH levels and temperatures. J. Fish. Res. Bd Canada 29, 1505--1507. Van Horn W. M. 11947) The toxicity of kraft pulping wastes to typical fish food organisms. Tech. Bull. No. 10. Nat. Council Stream Improvement, New York, N.Y. Van Horn W. M. 11948) A study of the toxic components of the waste waters of five typical kraft mills. Tech. Bull. No. 16. Nat. Council Stream Improvement. New York. Van Horn W. M. (1952) The effect of sublethal concentrations of kraft pulping wastes and their components on fish organs. Tech. Bull. No. 49. Nat. Council Stream Improvement. New York. N.Y. Van Horn W. M. (19611 Aquatic biology and the pulp

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and paper industr,, IReport No. II. Tech, Bull. No. I48. Nat. Council Stream Improvement. New York. N.Y. Van Horn W. M. (19711 Aquatic biology and the pulp and paper industry (Report No. 2). Tech. Bull. 5,'o. 251. Nat. Council Air and Stream Improvement. New York. N.Y, Van Horn W. M.. Anderson J. B. & Katz M. 1l'949) The effect of kraft pulp mill wastes on some aquatic organisms. Trans. Am. Fish. Soc. 79, 55-63. Van Horn W. M., Anderson J. B. & Katz M. t19501 The effect of kraft pulp mill wastes on fish life. T.4PPI 33, 209-2l 2. Walden C. C. & Howard T. E. 11968! A cooperative research program on kraft pulp mill effluent quality. Pulp Paper .~,la~b Canada 69, T107-TI 1 I. Walden C. C. & Howard T. E. (197l) The nature and magnitude of the effect of kraft mill effluents on salmon. Proc, Int. Syrup. htent, 3,1easurement q( Enrironmental Pollutants, 363-369, Nat. Res. Council of Canada, Ottawa, Ontario. Walden C. C.. Howard T. E. & Froud G. C. {I9701 A quantitative assay of the minimum concentration of kraft pulp mill effluents which affect fish respiration. Water Res. 4. 61-68. Walden C. C., Howard T. E. & Sheriff W. J. (1971/ The relationship of kraft pulp mill operating and process parameters to pollution characteristics of the mill et'fluents. Pulp Paper Mag. Canada 72, TS2-T87. Walden C. C. & McLeay D. J. il974} Interrelationships of various bioassay procedures for pulp and paper mill effluents. CPAR Report No. 165-l. Canadian Forestry Service. Ottawa, Ontario. Walden C. C.. McLeay D. J. & Monteith D. D. 119751 Comparing bioassay procedures for pulp and paper effluents. Pulp Paper Mag. Canada 76, T130-TI34. Warner R. E. (1964) Quantitative studies of toxicantinduced behavioural pathology in fish. Ph.D. thesis filed at Univ. of Calif., Berkeley. CA. Warren C. E. 11972) Laboratory and controlled experimental stream studies of the effects of kraft effluents on growth and reproduction of fish. Tech. Bull. No. 259. Nat. Council Air and Stream Improvement, New York, N.Y. Warren C. E. & Doudoroff P. (1958) The development of methods for using bioassays in the control of pulp mill waste disposal. TAPPI 41, 211-216. Warren C. E., Seim W, K., Blosser R. O., Caron A. L. & Owens E. L. {1974) Effect of kraft effluent on the growth and production of salmonid fish. TAPPI S712j. I27-132. Webb P. W. & Brett J. R. (1972) The effects of sublethal concentrations of whole mill bleached kraft pulp mill effluent on the growth and food conversion elt]ciency ofunderyearling sockeye salmon. J. Fish. Res. Bd Camnhl 29, 1555-1563. Werner A. E. (t963) Sulfur compounds in kraft pulp mill effluents. Can. Pulp Paper Ind. 16 (3). 35-42. Williams R. W.. Mains E. W.. Eldridge W. E, & Lasater J. E. (1953) Toxic effects of sulfite waste liquor on young salmon. Res. Bull, No. 1. State of Washington Department of Fisheries. Olympia, WA. Wilson M. A. (19751 Assessment of the sensitivity of major aquatic food chain organisms to newsprint mill efiluents which are not acutely toxic to fish. CPAR Report No. 328-1. Canadian Forestry Service, Ottawa, Ontario. Wilson M. A. & Chappel C. I. (1973) Reduction of toxicity of sulphite effluents. CPAR Report No. 49-2. Canadian Forestry Service, Ottawa. Ontario. Wilson R. C. H. (19721 Acute toxicity of spent sulphite liquor to Atlantic salmon (Salmo salarl. J. Fish, Res. Bd Canada 29, 1225-1228. Woelke C. E. {1960) Effects of sulfite waste liquor on the normal development of Pacific Oyster {Crassostrea

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stiya.,) larvae. Rc~. Bull. No. O. State of Washington Department of Fisheries. Olympia, WN. Woelke C. E. (1965) Preliminary anabsis of the relati,,e toxicity of samples from 27 different pulp mill waste streams. U.S. Public Health Service, Washington. D.C. Woelke C. E. i1965al Bioassays of pulp mill wastes with oysters. Biologicai Problems in [4"ater Pollution. Third Seminar. Tech. Rep..Vo. 999-WP-25. Robert A. Taft Sanitary Engineering Center. U.S. Public Health Ser',ice. Cincinnati. OH. WoeIke C. E. 119671 Measurement of water quatlty v, ith the Pacific oyster embryo bioassa',. Water Quality Crit e r i a . . 4 S T M S T P 416. Am. Soc. Testing Materials. Philadelphia. PA. Woelke C. E. (1968) Application of shelltish bioassay

results to the Puget Sound pulp mm polluuon problem. .\'l)rth~vest 3"~i. ..I.21-.I-). 125 -[ 33. Woelke C. E.. Schmk Y. D. & Sanborn E. V,, i~07~'~ L3e,,elopment of an In Sire Marine Btoa,~av ~)th C[ar's. 4nn. Rep. to tire {..S. Fed. W~tter QuL£it~ Admin.. D'apt. of the Interior Grant No. Ig050 DO'. Washington D.C. Woelke C. E.. Schmk T. & Sanborn E. (lg'-2, El! :t of biological treatment on the to,udtv of three Dpes of pulping wastes to Pacific oyster embryos. Report prepared under EPA Contract No. 6,";-0l 37". Washington D.C. Zitko V & Carson M. V ti971} Resin acrd.; and other organic compounds in groundwood and sulfate m,l eflluentsand lbams. 3,/s.~. Rcp. Scr \ , [134. Fish Res Bd Canada. Biologlcal Station, St. Andrev, s. N.B