Effect of pH on toxicity of kraft pulp and paper mill effluent to salmonid fish in fresh and seawater

H'at(,r Re.se(Ir('h Vol. 13. pp. 249 to 254 Pergamon Press Ltd 1979. Printed in Great Britain.

EFFECT OF pH ON TOXICITY OF KRAFT P U L P AND PAPER MILL E F F L U E N T TO S A L M O N I D FISH IN FRESH A N D SEAWATER D. J. MCLEAY, C. C. WALDEN and J. R. MUNRO* Division of Applied Biology, B.C. Research, Vancouver, V6S 2L2, Canada

(Received 10 August 1978) Abstract--In freshwater bioassays with juvenile rainbow trout (Salmo gairdneri), at initial pH values from 4 to 11, kraft mill effluents were considerably less toxic at pH 9-10 than at neutrality. When pH of test solutions was controlled throughout the bioassay period, the least toxic range was 8.5-9.5. Toxicity at typical receiving-water pH values was 50-67~o greater. The acute toxicity of effluent samples to yearling coho salmon (Oncorhynchus kisutch) was identical for these effluents in seawater and freshwater respectively, provided that the pH was adjusted and held at the same value, and that test fish were previously acclimated to the dilution water for several months. Thus seawater constituents other than pH did not affect the acute toxicity of pulp and paper mill effluents appreciably.

INTRODUCTION A number of studies have shown that the pH of pulp mill wastes as discharged to receiving waters can contribute appreciably to their toxicity. Howard & Walden (1965) estimated that up to 75% of the toxicity of kraft mill effluent is attributable to adverse pH. However, in many situations in Canada, effluent is now neutralized, usually to permit subsequent bigtreatment, or because the buffering capacity of receiving waters is limited. The pH variation of these discharges during normal mill operations is minor, but some evidence suggests that even this variation affects bioassay data. Various studies (Leach & Thakore, 1974: Leach & Walden, 1975) have demonstrated that resin acids, which account for a substantial portion of the toxicity of kraft pulp and paper effluents, are considerably more toxic at pH 6.4 than at pH 7.0. Ladd (1969j reported that the survival time of juvenile coho salmon in various concentrations of kraft effluent was longest when test pH was 8-9, and times to death decreased at either higher or lower values. Existing regulatory bioassay procedures for pulp and paper mill effluents in Canada stipulate that bioassays be run in freshwater, without pH adjustment (Anon., 1971, 1972). Since the p H of the natural dilution waters can vary by as much as two pH units, the effect on the measured toxicity may be considerable, even where the mills' discharges are neutralized. No information is available concerning the effect of seawater characteristics other than pH on results of bioassays. The intent of the present investigation was to compare the effect of pH on the acute toxicity to fish of unbleached, semi- and fully-bleached kraft whole

mill effluent and kraft-newsprint effluents, as determined in freshwater and seawater bioassays. A further objective was to determine if equivalent bioassay results could be obtained for pulpmill effluents using either freshwater or seawater as the diluent water and thus provide a meaningful measurement of the inherent toxicity of these discharges as related to the marine environment utilizing freshwater bioassay data. This paper describes the effect of pH values ranging from 4 to 11 on the acute toxicity of six pulpmill effluents in freshwater as measured by mortality-time or lethal concentration, using young fingerling rainbow trout (Salmo gairdneri). Additionally the influence of seawater versus freshwater as dilution water is examined for four pulpmill effluents, with test pH adjusted to the same value. Yearling coho salmon (Oncorhynchus kisutch) acclimated to seawater or freshwater were used for the seawater-freshwater comparisons, since juveniles of this species adapt more readily to full-strength seawater than do rainbow trout or other test fish commonly used for bigassays (Parry, 1960; McLeay & Walden, 1975). MATERIALS AND METHODS

Fish and water supply Rainbow trout, hatched from eyed eggs, were feeding actively for 2 weeks and weighed 0.16 + 0.03 g at the time of the bioassays. Lighting control provided a natural photoperiod with a simulated dawn/dusk. Coho salmon, obtained as swimup fry, were held in freshwater at B.C. Research. A sub-population of 6000 fish was transferred to the Pacific Environment Institute (PEI), West Vancouver three months later. After a two-week recovery period in freshwater (12 + 0.YC), the fish were exposed to an increasing gradient of seawater over a 4-week period, and ultimately held in 27 + 1,',,,, seawater at 11.5 + 1.3cC for 5 months prior to bioassays. An additional sub-group of 1000 coho salmon was transferred from B.C. Research to freshwater at PEI. After a brief

* Present address: Water Resources Branch, Limnology and Toxicity Section, Ontario Ministry of the Environment, P.O. Box 213, Rexdale, Ontario M9W 5L1. 249

250

I). J. M( L~!AY. C. C. WALI)FN and J. R. M t N r o

(24 h) period in freshwater these fish were exposed to a rapidly increasing seawater gradient within the next 2 days and held for the subsequent three weeks in full-strength seawater (27 + 1",,, salinity, 10.5 4- I'C). At the time of the bioassays the latter group of coho salmon was 11-12 months old. Both fish species were fed Oregon Moist Pellets and maintained according to standard hatchery practice (Leilrilz, 1969). The supply of freshwater for rearing fish at B.C. Research and for bioassays was dechlorinated Vancouver City tap water. Water quality characteristics as determined b? weekly analyses included pH 6.5 + 0.2, alkalinity 3.7 _+ 0.9 mg CaCO 3 1 ~, EDTA hardness 5.0 + 0.004 mg CaCO 3 I 3, conductance 14.1 + 3.7j~fl ~ cm ', and dissolved oxygen 10.9 + 0.4 mg 1 ~. Water temperature was held at 12.0 + 1.5'C using a stainless steel heat exchanger. The seawater used as diluent water for bioassays and for transporting fish was withdrawn from Georgia Strait. Its characteristics included pH 7.6 + 0.2 and salinity 27 + I",,,,.

Fish ('onditinn The condition of both sub-populations of coho salmon held in freshwater or seawater was monitored at weekly intervals during the 4-week acclimation of one group to an increasing seawater gradient: and subsequently at monthly intervals. Testing for fish condition included measurements of length, weight and condition factor (cW L 3), tolerance to hypoxia, and tolerance to a reference toxicant, Lindane (gamma isomer of the insecticide benzene hexachloride). Lindane was dissolved in methanol, followed by appropriate dilution in freshwater or seawater. The tolerance of coho salmon to hypoxia was determined in sealed jars at ambient room temperature (McLeay, 1976). Jars were filled with either fresh or seawater, saturated with oxygen and at 20 + 0.5'C. Fish, acclimated to either fresh or seawater, were added to corresponding jars at Ioadings of 4-5 g fish 1-z. Jars were completely filled and sealed. Time to death of the fish was

recorded, together oxygen value.

with

the

corresponding

dissolved

Effluent Effluents used for these studies were collected as grab samples from British Columbia coastal or interior (sample No. 6, Table 1| mills during normal operation. Subsamples were mixed in a large fibreglass tank at B.C. Research. During transportation and subsequent storage at 4 C , samples were held in completely filled, sealed polyethylene barrels. Characteristics of the nine samples of kraft or kraft-newsprint effluents are given in Table 1. Bioassays were completed within 7 days after effluent sampling.

Lethal bioassay procedures All bioassays were conducted at 12 _+ 1 C. Lighting for the LT50 (time to death of 5050 of the test fish) bioassays was by constant overhead illumination, whereas the LC50 (concentration causing 50% mortality) bioassays were conducted using a natural photoperiod. Ten fish were used per test solution and all fish loadings were held at 0.5 g l :. Tests were performed in 4-I. glass jars, 50-1. polyethylene tanks or 200-1. polyethylene barrels. Adjustment of pH was with 1 N NaOH or H2SO4. In some instances (freshwater LC50 bioassays), pH of test solutions was controlled to within 0.2 pH units by manual addition of NaOH or H2SO,,. In other instances (seawater/freshwater comparisons), pH control utilized Mcllvaine's citrat~phosphate buffer (Sober, 1968), with corresponding addition of buffer solution to the control bioassays. Dissolved oxygen content was maintained at >_9 mg 1 ~ by continuous minimal aeration. Where exposures extended past 24 h, test solutions were replaced at 24-h intervals. Test temperatures, pH, dissolved oxygen, and (in some instances) conductance were monitored a minimum of once every 24 h. The LT50 values were determined as per Litchfield (1949), utilizing a computer program. The LC50 values and corresponding 95°,, confidence limits were computed

Table 1. Characteristics of kraft mill effluent samples

Sample No. 1

2

3 4 5 6 7 8 9

Description Semi-bleached kraft whole mill effluent plus newsprint effluent Unbleached kraft whole mill effluent plus newsprint effluent Bleached kraft whole mill effluent Bleached kraft whole mill effluent plus newsprint effluent Bleached kraft whole mill effluent Bleached kraft whole mill effluent Bleached kraft whole mill effluent plus newsprint effluent Bleached kraft whole mill effluent Semi-bleached kraft whole mill effluent plus newsprint effluent

Total organic carbon (mg 1 ~)

BODs* (mg 1 - 1)

Sodium (mg 1 - h

Mill

pH

Conductance (I~fU ~ c m - q

Color (APHA unitsj

A

4.7

1300

2050

260

234

210

A

10.5

785

1250

259

217

150

B

5.3

1420

2600

258

207

21(7

A

2.5

1250

1500

255

203

213

B

3.5

1600

1350

158

130

245

C

6.8

1320

1300

150

127

158

A

5.7

1700

2060

216

190

185

B

10.9

1580

3900

331

220

300

A

2.5

1670

1860

302

308

350

* 5-Day biochemical oxygen demand.

251

Effect of pH on pulpmill effluent toxicity

[

according to the probit technique of Finney (1971). As appropriate, differences between bioassay values were confirmed with Student's t-test (Litchfield & Wilcoxon, 1949). Other facets of the bioassay procedure conformed to Standard Methods (APHA, 1975).

't

pH vs LT50 values in freshwater The effect of test pH on the acute toxicity of three effluent samples (Nos. 1-3, Table 1) in freshwater was determined by LT50 (time to 50% mortality) bioassays. For each sample, replicate bioassays were undertaken at concentrations ranging from 30 to 100% effluent by volume for each of eight pH values (initial values 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and I1.0). The survival of control fish (freshwater only) was determined at each pH. Tests were terminated at 1440 rain (24 h) for surviving fish. The pH of test solutions was not controlled during the bioassays: however the pH value at the time of death of the last fish in each jar was recorded. pH vs LC50 values in freshwater The effect of test pH on the acute toxicity of three additional kraft mill effluents (Nos. 4-6, Table 1) in freshwater was determined by 24-h LC50 bioassays. For each sample eight separate concentrations were examined at each of eight or nine pH values ranging from 5.0 to 10.0. Seawater vs freshwater bioassays The effect of pH and dilution water (i.e. seawater or freshwater) on the acute toxicity of pulp and paper mill effluents (Nos. 1, 7, 8 and 9, Table 1) was determined as LC50 values with bioassays using seawater-acclimated and freshwater-acclimated coho salmon respectively. After adjustment to pH values of either 6.4-6.5 (freshwater) or 7.5 (seawater), pH was controlled by addition of buffers. Test conditions are outlined in Table 2. Controls for each bioassay included seawater-acclimated fish held in freshwater and freshwater-acclimated fish held in full-strength seawater. RESULTS The effect of pH in freshwater bioassays LT50 values. In the control bioassays, fish exposed to pH 11 died within 1 h. Those exposed to pH 4 had an LT50 value of 1215 min, with 4 out of 10 fish surviving for 1440 min (24 h). All other control fish exposed to initial pH values from 5 to 10 survived for 24 h. The hydrogen-ion concentration of all solutions changed during the bioassays. Mean pH changes were as follows: Initial pH 4.0 5.0 6.0 Final pH

4.5

5.7

6.4

(S.D.)

(0.2)

(0.5)

(0.3)

Bioassays with samples of unbleached, semibleached or fully bleached kraft mill effluent gave similar results. At each concentration, the least toxicity (longer LT50 values) was displayed at initial pH values of 9-10. Figure 1 illustrates data obtained for fully bleached kraft effluent. This sample was least toxic at pH 9-10 irrespective of effluent concentration. The unbleached kraft sample was similar; whereas for the semi-bleached sample, low concentrations were least toxic at pH 9 and higher concentrations least toxic at pH 10. At pH 4, all LT50 values were less than 500 min.

,,

12OOL

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,o.,./I;

.

_/ I/

k, ',i lI

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!

/ II

-'7"°'"e~s%

soo

as

0

,

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73~ I

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zoo

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\~-, Q~"11

Sjo

~

'tl

ioo%

"~1

ol I'" 4

l I I I I I S 6 7 S 9 I0 II INITIAL pH VALUE OF TEST SOLUTION

Fig. 1. Effect of pH on times to death of juvenile rainbow trout exposed to a range of concentrations of bleached kraft pulpmill effluent in freshwater.

LC50 values. In 24-h LC50 bioassays where the pH of test solutions was maintained within +0.2 units by external pH control, the pH range giving the highest LC50 values for the three bleached kraft whole mill effluents tested was consistently between 8.5 and 9.5. Throughout the pH range of 5-10, sample No. 4 was least toxic (highest LC50) at pH 9.0-9.5; effluent No. 5 was least toxic at pH 9.0 and No. 6 at pH 8.5-9.0 (Fig. 2). All three samples were considerably (P < 0.05) more toxic at neutral pH than at these alkaline pH values. With an increase of test pH from 6.0 to 8.5 (typical of the pH range for Canadian receiving waters [Anon., 1975, 1976]), LC50 values for samples 4, 5 and 6 increased by 62, 67 and 50% respectively (Fig. 2). The highest conductance in any test solutions, at termination of the bioassays, was 1800 #fl-~ cm -1, representing an increase of only 200/~Q- l c m - ~ due to external pH control.

7.0

8.0

9-.0

10.0

I 1.0

6.8 (0.3)

7.0 (0.2)

7.4 (0.5)

8.4 10.7 (0.8) , (0.5)

Seawater vs freshwater bioassays Condition factor for the sub-population of coho reared in seawater was <1.0 immediately after transfer to PEI and during exposure to salinities of up to 10%,,. Otherwise condition factor for both freshwater and seawater populations at various times was >_ 1.0, and similar for the two groups. The fish in seawater grew more rapidly than the sub-population in freshwater. At the time of the bioassays with the mill effluents the coho in seawater measured

252

D.J. Mt'LL~AY, C. C. WALDENand J. R. MINRD 7(3

-J

with rapidly 'acclimated' fish, gave significantly lower LC50 values in seawater bioassays at pH 7.5 or 6.5 than those obtained in freshwater bioassays with freshwater-acclimated fish at the same pH values lTable 2). In both freshwater and seawater, measured effluent toxicity was greater at pH 6.4-6.5 than at pH 7.5 (samples 1 and 9. Table 2) under otherwise identical conditions. No mortalities of control fish occurred over the 96-h exposures, including freshwater-acclimated fish in seawater and either group of seawater-acclimated fish in freshwater.

. i/

(~ 30 Ill1 2o

DISCUSSION

I'

IO

--o

Sample No. 6

pH VALUEOF TEST SOLUTIONS (~0.2 pH)

Fig. 2. Effect of pH on LC50 values for three bleached kraft mill effluents in freshwater bioassays with rainbow trout. Bars represent 95°,i confidence intervals. 8.3 ___ 2.3 g and 8.6 + 1.5 cm, whereas those in freshwater were 5.1 + 1.1 g and 7.5 + 0.6 cm. Mortalities of fish in fresh or seawater were negligible throughout the study period. Both groups were disease-free when examined for possible bacterial infections. The residual oxygen values obtained for fish held in freshwater or seawater were consistent (1.8 + 0.2 to 2.1 + 0.2 mg 02 1-1 ) and typical of values reported for this species in freshwater at ambient room temperature (McLeay, 1976). No difference between freshwater and seawater sub-populations could be demonstrated (P > 0.05) except for the group of fish rapidly 'acclimated' to seawater, where the value was elevated (3.0 __+0.6 mg 1-1). The LT50 data indicated that the fish group gradually acclimated to seawater was less tolerant to Lindane than the freshwater group (P < 0.05) during the 6 weeks after introduction into a seawater gradient. However, 3 and 5 months after transfer to seawater, LT50 values for Lindane bioassays with both groups were nearly identical (P > 0.05). Unlike these results the group of fish 'acclimated' rapidly to seawater died considerably faster in 0.1 mg Lindane 1 1 than did the freshwater-acclimated fish (P < 0.001t: using either seawater or freshwater as the diluent. The LT50 values (mint were as follows:

Test fish

seawater-acclimated (95°i~ confidence limits) freshwater-acclimated (95% confidence limits)

D i l u t i o n water Seawater F r e s h w a t e r

258 (248,269) 366 (322,417)

246 (224,269) 357 (319,4001

The acute toxicity of each of the first three kraft mill effluents tested in seawater/freshwater bioassays was similar when the pH of each test solution was adjusted to 7.5 (Table 2). The fourth sample, as tested

Studies with control fish indicated that the lower lethal limit for pH was approximately 4.0, whereas the upper limit was between 10 and 11. These pH limits have been reported previously (Creaser, 1930: Jordan & Lloyd, 1964: Lloyd & Jordan, 1964; Robinson et al., 1976). At these pH values, any effects due to effluent are obscured. Consequently any impact of pH on effluent toxicity is only measureable inside this range. Therefore subsequent studies were conducted at pH values of 5 to 10. The freshwater bioassays with six samples of unbleached, semi- or fully-bleached kraft whole mill effluents show clearly that these effluents are considerably less toxic at alkaline rather than neutral pH values. In the initial studies, where the pH was adjusted initially to the desired value, the least toxic pH range was 9.5-10.0. However initial pH values of the alkaline solutions declined substantially during the exposure period. Where pH values of test solutions were held at +0.2 pH units throughout the bioassays, the least toxic range was determined to be somewhat lower: i.e. pH 8.59.5. Thus variation within the pH range of 6--8.5, which occurs naturally between different receiving waters, can alter the toxicity of neutralized kraft whole mill effluent appreciably. The maximum elevation of conductance in test solutions attributable to external pH control was only 200 ~ - ~ cm ~. Thus pH control during these bioassays did not create an osmotic stress which altered the test results appreciably. Moreover preliminary studies (McLeay & Walden, 1976) showed that juvenile rainbow trout all survived 96-h exposure to seawater concentrations with conductances of 12,300 ll~ 1 cm-1 or less. Buffers of sufficient strength to hold the pH of test solutions constant at some of the more alkaline values under examination, i.e. pH 9-10, enhanced the toxicity of pulpmitl effluent (McLeay & Walden, 1976) and could not be used. In flowthrough bioassays, involving exposure times of 96 h and single effluent concentrations for each of 4 bleached kraft mill samples from one mill, Ladd (1969) found that the survival of coho salmon was optimum at pH values of 8-9. Since the pH of his test solutions varied by up to 2 pH units the least toxic pH value could not be determined precisely: nonetheless his results support the present findings.

Effect of pH on pulpmill effluent toxicity

253

Table 2. Acute toxicity of kraft whole mill effluent to juvenile coho salmon in seawater vs freshwater bioassays Effluent sample No.*

Test fish seawater-acclimated cohos freshwater-acclimated coho freshwater-acclimated coho seawater-acclimated coho:[: freshwater-acclimated coho seawater-acclimated coho* freshwater-acclimated coho seawater-acclimated coho~ freshwater-acclimated coho seawater-acclimated coho~ freshwater-acclimated coho

Dilution water

pH of dilution water

pH of test solutions

seawater

7.5

7.5

freshwater

6.4

7.5

freshwater

6.4

6.4

seawater

7.5

freshwater

LC50"t (% by volume) 96 h

24 h

48 h

49 (48,50) 46 (43,49) 35 (33,36)

--II

7.5

> 50

6.4

7.5

> 50

43 (42.8,43.2) 43 (42,45)

39 (37,40) 42 (40,43)

seawater

7.5

7.5

> 60

freshwater

6.4

7.5

> 60

48 (47,50) 48 (47.8,48.2)

48 (41,55) 46 (44,47)

seawater

7.5

7.5

freshwater

6.5

7.5

seawater

7.5

6.5

freshwater

6.5

6.5

24 (23,25} 39 (37,41) 21 (20,22) 33 (31,34)

---

m

n

m

* As outlined in Table 1. t Concentration causing 50% fish mortality in time specified (95% confidence limits in parentheses). :~Fish exposed to increasing seawater gradient over 4 weeks and subsequently held in 27 _+ 11!~, seawater for 5 months prior to bioassays. Fish exposed to increasing seawater gradient over 3 days and subsequently held in 27 + 1'~,,, seawater for 3 weeks prior to bioassays. II Not determined.

pH is known to have a pronounced effect on the toxicity of many materials to fish. The toxicity of ammonia (Hemens, 1966) and zinc (Mount, 1966) is increased at alkaline pH values, whereas hydrogen sulfide (Bonn & Follis, 1967; Sano, 1976), cyanide (Doudoroff, 1956) and antimycin (Marking, 1975) are more toxic at acid values. Kraft pulp and paper mill effluents can now be included in the latter group. The comparative bioassays in seawater and freshwater demonstrate that the toxicity of kraft mill effluent is equivalent in either diluent, provided the pH of the test solutions is the same. Water quality factors other than pH do not differentially affect results obtained from bioassays utilizing either freshwater or seawater as diluent. With both diluent waters, the effect of pH is the same; i.e. a change in one pH unit within the range 6-8.5 can affect results drastically. The buffer salt solutions used to maintain pH values during the seawater/freshwater comparative bioassays did not affect the test results. Preliminary studies (McLeay & Walden, 1976) showed that both freshwater-acclimated and seawater-acclimated coho salmon survived 96-h exposures to Mcllvaine's citratephosphate buffer at ten times the concentration used here and that, at pH 7.5, LT50 values were nearly

identical with or without the use of buffer in the bioassay procedure. Present studies indicate that where seawater bioassays are to be undertaken, test fish require considerably longer periods of acclimation than is currently practised in bioassay laboratories. Certainly where test fish were acclimated to seawater over the short period of 3 days and then used in bioassays within three weeks, the apparently greater toxicity measured in seawater as compared to freshwater (Table 2, sample 9) was due to stresses associated with the rapid 'acclimation'. Hypoxia bioassays and tolerance to Lindane provided substantiative evidence. Even where acclimation of test fish to seawater was considerably more gradual (over a 4-week period), two additional months in seawater were necessary before fish stresses disappeared. Biological results in this regard were not consistent. Fish condition factor and tolerance to hypoxia indicated more rapid acclimation. Nonetheless, under the controlled conditions practised in these studies, reduced tolerance to Lindane of seawater-acclimated fish can only be attributed to stress associated with the transfer from fresh to seawater. Moreover, bioassay data with Lindane did indicate that this stress disappeared with longer acclimation periods.

254

D.J. MCLEAY, C. C. WALDEN and J. R. MUNRO

Lindane was chosen as a reference toxicant since its chemical structure indicates that it should not be affected by water characteristics. The similar LT50 values found in this study for a given fish group tested in Lindane diluted with either freshwater or seawater support this supposition. O t h e r investigators have indicated that pulp mill effluents and wood extractives are more toxic to fish in seawater t h a n in corresponding freshwater situations. In bioassays with kraft-newsprint and kraft effluents, E n v i r o n m e n t C a n a d a researchers indicated 12 of 13 neutralized effluent samples were more toxic to juvenile coho in seawater t h a n in freshwater (Anon., 1975}. Rogers et al. (1975) indicated that seawater-acclimated underyearling chum salmon (O. keta) were less tolerant to a neutral extract of lodgepole pine in seawater, over a 3-week exposure, than their freshwater counterparts to a similar exposure in freshwater. In both studies, pH of bioassay solutions was not closely controlled, although current findings suggest that pH control would only have accentuated the reported differences. These authors did not compare the condition and tolerance of their fish groups acclimated to seawater or freshwater at the time of the bioassays, using reference toxicants or other appropriate methods such as those employed here. We suggest that their reported findings could be associated with a stressed condition of the seawater-acclimated fish as a consequence of insufficient acclimation, disease or stressful holding conditions. Acknowledgements---This research was funded by the Canadian Forestry Service, Ottawa, through its Co-operative Pollution Abatement Research Program (CPAR Project No. 402). The assistance of the Pacific Environment Institute in acclimating and maintaining coho salmon in seawater for these studies is gratefully acknowledged. REFERENCES

American Public Health Association (1975) Standard Methods for the Examination of Water amt Wastewater. 14th Edn. New York. Anon. (1971) Fisheries Act. Pulp and Paper Regulations, Codes and Protocols, Report 1. Water Pollution Control Directorate, Environment Canada, Ottawa, Ont. Anon. (1972) Guidelines for the Pulp and Paper Effluent Regulations Promulgated Under the Fisheries Act. Regulations, Codes and Protocols, Report 2. Water Pollution Control Directorate, Environment Canada, Ottawa, Ont. Anon. (1975) Brief presented to the Pollution Control Board Inquiry into the Pollution Control Objectives for the Forest Products Industry of British Columbia. Septemher 1975. Environment Canada. Anon. (1975, 1976) Water Quality Data. Inland Waters Directorate, Water Quality Branch, Environment Canada, Ottawa, Ont, Bonn E. W. & Follis B. J. (1967) Effects of hydrogen sulphide on channel catfish, lctalurus punctatus. Trans. Am. Fish. Soc. 96, 31-36. Creaser C. W. (1930) Relative importance of hydrogen ion concentration, temperature, dissolved oxygen, and car-

bon dioxide tension on habitat selection by brook lroul. J. gen. Physiol. 4, 305 317. Doudoroff P. (1956) Some experiments on the toxicity of complex cyanides to fish. Sewa~w ind. Wa.~te~ 28~ 102(~ 1040. Finney D. J. (1971) Prohit Anah'sis. Cambridge Univ. Press, Cambridge. Hemens J. J. (1966) Toxicity of ammonia solution to the mosquito fish (Gambusia affinis). Proc. Inst. Sew. Puri[i 265- 271. Howard T. E. & Walden C. C. (1965) Pollution and toxicity requirements of kraft pulp mill effluents. TAPPI 48, 136--141. Jordan D. H. M. & Lloyd R. (1964) Resistance of rainbow trout (Salmo gairdneri Richardson) to alkaline sotutions. Int. J. Air Wat. Pollut. 8, 405-409. Ladd J. M. (1969) Effects of pH on the acute toxicity of kraft pulp mill effluent to juvenile coho salmon, Oneorhynchus kisutch. M.Sc. Thesis. Humboldt State Coll., California. Leach J. M. & Thakore A. N. (1974) Isolation of the toxic constituents of kraft pulp mill effluent. CPAR Rep. 11-4. Can. For. Serv., Ottawa, Ont. Leach J. M. & Walden C. C. (1975) Identification and treatment of the toxic materials in mechanical pulping effluents. CPAR Rep. 149-3. Can. For. Serv., Ottawa, Ont. Leitritz E. (1969) Trout and salmon culture. Cal{[i Oep. Fish. Game Fish Bull. 107, 169 p. Litchfield J. T. (1949) A method for rapid graphic solution of time-percent curves. J. Pharmac. exp. Ttwr. 97, 399-408. Lit,chfield J, T. & Wilcoxon F. (1949) A simplified method of evaluating dose-effect experiments. J. Pharmac. exp. Ther. 96, 99-113. Lloyd R. & Jordan D. H. M. (1964) Some factors affecting the resistance of rainbow trout (Salmo gairdneri Richardson) to acid waters. Int. J. Air. War. Pollut. 8, 393-403. McLeay D. J. (1976) A rapid method for measuring the acute toxicity of pulpmill effluents and other toxicants to salmonid fish at ambient room temperature. J. Fish. Res. Bd Can. 33, 1303-1311. McLeay D. J. & Walden C. C. (1975) Marine toxicity studies on drilling fluid wastes. B. C. Research Report No. 6114. Prepared for Working Group A, APOA/ GOVT Research Program on Drilling Fluid Wastes, EPS, Environment Canada. McLeay D. J. & Walden C. C. (1976) Effect of pH on bioassays in fresh and seawater. CPAR Rep. 402. 75 p. Can. For. Serv., Ottawa, Ont. Marking L. L. (1975) Effects of pH on toxicity of antimycin to fish. J. Fish. Res. Bd Can. 32, 769-773. Mount D. I. (1966) The effect of total hardness and pH on acute toxicity of zinc to fish. Int. J. Air War. Pollut. 10, 49 56. Parry G. (1960) The development of salinity tolerance in the salmon, Salmo salar and some related species. J. exp. Biol, 37, 425-434. Robinson G. D., Dunson W. A., Wright J. E. & Mamolito G. E. (1976) Differences in low pH tolerance among strains of brook trout (Salvelinus fontinalis). J..fish Biol. g, 5-17. Rogers I. H., Davis J. C., Kruzynski G. M., Mahood H. W., Servizi J. A. & Gordon R. W. (1975) Fish toxicants in kraft effluents. T A P P ! 48, 136-140. Sano H (1976) The role of pH on the acute toxicity of sulfite in water. Water Res. 10, 139-142. Sober H. A. (1968) (Ed.) CRC Handbook of Biochemistry. Selected Data for Molecular Biology. p. J-195. The Chemical Rubber Co., Cleveland, Ohio.