Studies on the toxicity of pulp and paper mill effluents—I. Mutagenicity of the sediment samples derived from Kraft paper mills

Waler Research Vol. 15, pp. 17 to 24 Pergamon Press Ltd 1981. Primed in Great Britain

0043.1354/81/0101-0017$02.00/0

STUDIES ON THE TOXICITY OF PULP A N D PAPER MILL EFFLUENTS= 4. MUTAGENICITY OF THE SEDIMENT SAMPLES DERIVED FROM KRAFT PAPER MILLS NAOHIDE KINAEx, TAKASHIHASH~U~ ~, TOSHIOMAKITAx, ISAOTOMITAx, IKUO KIMURA2 and HISAYUKIKANAMORI3 XLaboratory of Health Science, Shizuoka College of Pharmaceutical Sciences, Shizuoka 422, 2Laboratory of Viral Oncology, Aichi Cancer Center Research Institute, Nagoya 464 and 3Hiroshima Prefectual Institute of Public Health. Hiroshima 7.'~4,Japan

(Received December 1979) Abstract--Sediment samples collected at three different coastal points (Shinguu, Ooigawa and Tagonoura) were extracted with diethylether and then methanol. Each extract was separated into five fractions by high-speed liquid chromatography and they were submitted to mutagenic assay using B. subtilis and S. ryphimurium and to GC-MS analysis. The ether fraction of sediment sample from Shinguu showed DNA damaging potency on B. subtilis and exhibited mutagenic effect on S. typhimurium TA 98, TA 100 and TA 1537. 2A,6-Trichlorophenol, 3A,5,6-tetrachloroguaiacol, dehydroabietic acid. pyrene and fluoranthene were mutagenic substances among 28 substances identified and tested.

INTRODUCTION

ether and then methanol from sediment samples collected from three different points which geographicaUy resemble each other. The extracts were fractionated by the application of high-speed liquid chromatography and submitted to rec and reversion assay with B. subtilis and S. typhimurium respectively and aliquots of them were applied to GC-MS analysis.

Neoplasms and related disorders have been found in marine fish in coastal waters of Japanese islands. The prevalence of skin neoplasms found in yellow fin goby (Acanthooobius flav/manuas) and cresthead flounder (Limanda anoustirostris) and especially in spotted sea trout (Nibea mitsukurii) have received attention since their incidence has been reported high in several areas. Neoplastic lesions may be attributed to a variety of factors; viral or bacterial infection, abrasion, dietary insufficiencies and environmental stress. Genetic factors may also be important. The present study was undertaken to explore the cause of the neoplasms in Nibea mitsukurii with the emphasis on the association of the disease with coastal or estuarine environment. The coastal area where the fish with neoplastic diseases are captured are polluted with many organic substances. They include resinic and phenolic substances from pulp and paper industries and organo chlorine and polycyclic aromatic hydrocarbons which are ubiquitous environmental pollutants. The toxicity of pulp and paper mill effluent on fish and planktons have been reported by Walden (1976) and Stockner (Stockner & Cliff, 1976; Davis, 1973; Davis & Mason, 1973; Seim et al., 1977; Leach & Thakore, 1977; Thakore, 1977). Recently, Ander et al. (1977) have found the mutagenic potency of pulp mill effluents at the chlorination stages using S. typhimurium TA 1535 and TA 1537 though the toxic substances are not known. The present study was undertaken in an attempt to identify mutagenic substances from the sediment samples from the pulp and paper mill effluents. Organic compounds were extracted with diethylw.a 15 I--B

MATERIALS AND METHODS (1) Sampling Sediment samples were collected from Shinguu, Ooigawa and Tagonoura in March, 1978. Sampling points are as shown in Fig. 1. These samples were placed in glass bottles with tight caps and kept at 4°C in a cold room. (2) Chemical analysis of the samples Chemical analysis of the sediment samples were conducted according to the Standard Method of Analysis for Hygienic Chemists (1973) in Japan. The concentration of lignin and arsenic in samples were determined by Pearl. Bensons method (Felicetta & McCarthy, 1963) and flameless atomic absorption method respectively. (3) Extraction and fractionation of oroanic compounds All sediment samples were dried at room temperature and successively extracted with distilled ether and methanol using Soxhlet extracting apparatus. Both ether and methanol extracts were concentrated to dryness under reduced pressure after drying over anhydrous sodium sulfate (Wako reagent for Fluometric analysis). These extracts were applied on silica gel column in a high-speed liquid chromatography (HLC) with u.v. monitor and eluted with n-hexane containing 1% ethanol and then with methanol. The handling conditions for HLC are shown in Fig. 2. All fractions obtained are designated HLC-I-HLC-5 and were concentrated to dryness by a rotary evaporator and kept at 4°C in brown sampling tubes. These samples were passed through millipore 200 nm membrane filter for the mutagenic assay. 17

18

KINAE e! al.

NAOHIDE

Nogo~

, r ~

~

Togonouro

Shin

shoreline

V

Kumono

seo-shoreline

Pocific Oct,on

Circles

indicate somoling

points

Fig. I. A part of Japanese island showing the sampling points of sediments. n - Hexorm-Emon(~ (99: I )

~honoi

g

| o

2o

,o

3o ~mio.

time (rmn)

HLC Conditionsz Instrument

:

Column size

:

Packing material Sample Flow rate

-Z

:

Hitachi

633 ~8 x 500 mm

Press Temp.

:

8 kg cm

:

Lichlosorb-

Decector

:

25.0 ° 254 Om u.v.

:

0.16

:

20 cm h "I

Sl I00 (30M, m) 1.0 ml 3.0 ml min "I

:

:

Range Chart

speed

Fig. 2. HLC elution pattern of ether extract obtained from Shinguu sediment.

~

l

l

14

18 19

20

O: 0

5

JO

15 Reoctmn

GC-MS Condi tionst Instrument i CGC) Column ~

Nippon

20

Denshi

Column Te~p Injec. temp. C~. 6~s Detector

z : ~

:

TIM sens.

s

3 x 10 "10

:

5 mm rain"I

speed

30

D-300

Glass (2 m x 2 n~n) packed with 2% OV17 on Chromosorb W (80/100 mesh) 220 ° 2500 -Z He, 1.8 kg ~a TIM, ~IC

Chart

25

time (rain}

(MS) Mode Ion source temp. Ioniz. volt Ioniz. curt. accel, volt Multi. volt

Scan. speed Response

Fig. 3. Gas chromatogram of HLC-I.

:

: :

: : :

El, CI 250 ° 70 eV 3OO ~ A 3 kV 1.4 kV 5 s 1

Toxicity of pulp and paper mill effiuents--I (4) Mutaoenic assay (i) Rec-assay. The assay was carried out according to Kada (1975) using B. subtilis H-17 (rec'), M-45 (rec-). Each sample was dissolved in dimethylsulfoxide (DM$O) at an appropriate concentration. Fifty /zl of sample solution was applied to a paper disk on the broth agar plate. The plates were incubated at 37°C for 2 days and the difference of the inhibition length of two strains were measured. Rec effects were represented as follows; - : no difference in inhibition, + : difference in inhibition is less than 1.9 mm, + : difference in inhibition is more than 2.0 ram. 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) was used as the positive reference compounds. Each experiment was done in triplicate. (ii) Reversion assay. The assay was carried out according to Ames et al. (1975) using Salmonella TA 98. TA 100 and TA 1537. The so-called s-9 mixture was prepared from PCB-trcated Wistar male rats and used by supplementing glucose-6-pbospbate and NADP as cofactors. The test samples were dissolved in DMSO at an appropriate con. centration and used after the incubation with or without s-9 mixture. Each tester strain was cultured overnight in Difco broth medium. Each 100/zl of the cell suspension and the sample solution, 500/A of 100mM phosphate buffer (pH 7.4) in the presence or absence of s-9 were mixed and then incubated at 37°C for 15 rain. Then, 2 ml of soft agar containing 0.75° Difco agar, 0.6% NaCI, 0.5 mM histidine and 0.5 mM biotin was added to the above mixture and then poured onto the top of agar plates with Vogel Bonner E minimum culture medium containing 2~o glucose. The plates were incubated at 37°C for 2 days and the numbers of his* revertant colonies were counted. 4-Nitroquiuoline-l-oxide (4NQOk benzo(a)pyrene and 9-aminoacridine were used as the positive reference compounds. (5) GC-MS analysis Aliquots of the sedimcmt samples were used for gas chromatograpb--mau spectrom~er computer system to identify the organic compounds in each fraction. The nwatsuring conditions are shown at the bottom of Fig. 3.

19 RESULTS

Chemical properties of the sediment samples used in this experiment are shown in Table 1. Chloride ion concentration of Ooigawa sediments was lower than that of others suggesting its low influence by sea-water. The concentration of cadmium, zinc and mercury of Tagonoura sediments were higher than those of others. The color and yield of ether and methanol extracts of sediments are shown in Table 2. The yield of methanol fraction was more than 3 times higher than that of ether fraction in Shinguu sediments. The yields of both ether and methanol fractions from Tagonoura sediments were about 10 times higher compared with those of the sediment fractions obtained from other points. The mutagenic effects of these ether and methanol extracts on S. typhimurium and B. subtilis are shown in Table 3. The ether extracts of sediment samples from Shinguu and Tagonoura showed D N A damaging potency on B. subtilis, though only the ether extract from Shinguu showed mutagenic effects on S. typhimurium TA 1537. Neither ether nor methanol extract from sediment samples of Ooigawa showed mutagenicity. As the ether extract of Shinguu sediments showed both mutageni¢ and D N A damaging potency, it was applied to a high-speed liquid chromatography to separate into five fractions (see Fig. 2). Their weight ratio and their effects on S. "typhimurium and B. subtilis are shown in Table 4 and 5 respectively.

Table 1. Chemical properties of sediment samples ~... Item

Sampling point ~

Shinguu

Ooigawa

Tagonoura

Black Rotten egg 7A0 60.0 5.75 x 103 1.69 x 103 3.24 16.4 0.44 54.0 0.26 3.55

Black Rotten egg 7.42 14.9 4.79 0.76 x 103 15.2 14.3 0.69 143 0.05 1.14

Black Rotten egg 7.83 68.1 5.91 x 103 3.16 x 103 6.35 11.6 2.67 856 0.59 1.42

I

Color Odor pH Loss on drying (%) CI-(ppm) Total-S (ppm) Lignin (ppm) Pb (ppm) Cd (ppm) Zn (pprn) Hg (ppm) As (ppm)

Table 2. Color and yield of Soxhlet extracts from sediments Samples Shinguu: Ooigawa: Tagonoura:

Ether ext. Methanol ext. Ether ext. Methanol ext. Ether ext. Methanol ext.

Color

Yield (nag g- 1 dry weight)

Yellow-brown Green Yellow Yellow-brown Yellow-brown Violet

1.15 3.89 2.01 1.75 17.39 14.22

20

NAOHIDE KINAE et al. Table 3. Mutagenic and DNA damaging potency of sediment extracts S. typhimurium His + revertant colonies plate-1 TA 98 Samples Shinguu Ooigawa:

-$.9

Ether ext. Methanol ext. Ether ext.

40 39 43

Methanol ext. Tagonoura:

Ether ext. Methanol ext. 4NQO (0.3/ag) Benzo(a)pyrene (5.0 #g) 9-Aminoacridine (50/~g) Control (DMSO} AF-2 (0.025 ~g)

TA 100

+$.9 - $ . 9

TA 1537

+$.9 - $ . 9

+$.9

68 63 72

47 102 65

137 144 147

7 8 7

36* !1 18

38

58

60

137

8

13

38 32 189

61 68

82 130 356

137 121

9 9

14 21

430 38

63

411 122

B. subtilis Rec-effect +

+

152

217 118 7

9 +

1000 #g of each sample was used per plate or disk. Numbers in table indicate the mean value in five experiments. * P < 0.001.

Table 4. HLC fractions of ether extracts obtained from Shinguu sediments Fractions

Eluted with

Weight ratio (%)

HLC-I HLC-2 HLC-3 HLC-4 HLC-5

n-Hexane-ethanol (99:1) n-Hexane--¢thanol (99:1) n-Hexano--ethanol (99:1) n-Hexane-ethanol (99:1) Methanol

53.4 1.5 8.6 6.6 29.9

Recovery: 108.30/o. The fraction of HLC-4 showed mutagenicity on TA 98 with a n d without s-9 mixture. H L C - I and HLC-5 showed mutagenicity with s-9 mixture on TA 100 a n d TA 98 respectively. Furthermore, all fractions induced the high frequency of his* revertant colonies on TA

1537 in the presence of s-9.The fractions,HLC-I and HLC-3 showed significantlyhigh mutagenic potency (P < 0.001). But, only HLC-I showed a weak D N A damaging potency on B. subtilis.The identificationof organic compounds included in the five fractions was carried out by G C - M S computer system. The gas chromatogram of HLC-I is shown in Fig. 3. The El and CI spectra of several peaks in the gas chromatogram are given in Figs. 4, 5 and 6. 2,4,6-TrichlorophenoL 3,4,5,6-tetrachloroguaiacol, juvabione (methyl-4-41,5-dimethyl-3.oxohexyl)-I-cycIohexane carboxylate), fluoranthene, pyrene, 2,6-ditert-butyl-4-ethylphenol and retene were identified. The gas chromatograms of HLC-2 and mass spectra of Iongifolene, 2,6-di-tert-butyl-4-methylphenol and dihydroactinicfiolide (5,6,7,Ta-tetrahydro-4,4,7atrimcthyl-2MHbbenzofuranone) are shown in Fig. 7.

Table 5. Mutagenic and DNA damaging potency of HLC fractions from ether extract of Shinguu sediment $. typhinmriura His + revertant colonies plate-1 TA 98

TA 100

TA 1537

Samples

- S-9

+ $.9

- $.9

+ $.9

- $.9

+ $.9

B. subtilis Rec-effect

HLC-1 HLC-2 HLC-3 HLC-4 HLC-5 4NQO (0.3 #g)

35 49 43 54* 31 171

48 68 56 72* 97*

132 110 156 113 134 362

128" 104 125 100 103

16 11 14 15 14

47+* 29* 55~ 46? 32?

_+ -

Benzo(a)pyrene (5.0~g) 9-Aminoacridine (50/~g) Control (DMSO) AF-2 (0.025#g)

252 34

63

298 137

IIl

1000 ~g of each sample was used per plate or disk. Numbers in table indicate the mean value in five experiments. * P <0.05: ~" P <0.01; ~ P <0.001.

177 156 12

19 +

Toxicity of pulp and paper mill effluents--I

21

Peok 3 2,4,6-Trichlorophenol

,ooo

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./.

olt

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CI

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o.... ......

......................

I00

150

200

ooo

,. . . . . . .

250

m/e

Fig. 4. El spectra of peaks 3 and 7 from HLC-I. Other compounds were identified by mass spectral analysis. As se¢n in Table 6, 28 organic compounds were identified from the ether extract of Shinguu sedin~nt. The effects of some of the compounds identified above on B. subtilis and S. typhimurium are shown in Table 7. 2,4,6-Trichlorophenol, 3,4,5,6-tetrachloroguaiacol exhibited high DNA damaging potency at 0.5 mg and 0.1 mg per disk re, ix~tively. Fluoranthen, and pyreoe showed weak mutagenicity on TA 100 and TA 1537 rmpectively at 100/~g per plate and th¢ir mutag~nic effects were dose related. DISCUSSION The present investigation was undertaken in an attempt to explore the mutagmaicity of sediment samples derived mainly from the pulp and paper mill

effluents. Sediment samples were col}¢cted from thr¢¢ points where large pulp and paper mills exist in the neighborhood and large amounts of effluent are discharged every day. After the samples were extracted With ether and methanol, the extracts were submitted to mutagenic assay. As the ether extract from Shinguu sediments showed high DNA damaging potency, it was applied to a high-speed liquid chromatography to separate into five fractions. As s~n in Table 5, fraction HLC-1 showed a w¢ak DNA damaging potency in rec =may and gave a ggni~cant positive mutagenic effect on S. typhimurium TA 1537 strain in the presence of s-9 mixture. Neoplastic disease was found frequently in Nibea mitsukurii caught at Kumano bay. The incidence was 50.4% of 246 fish examined in 1978. Our previous studies have indicated that the level of bcnzo(a)pyrene of fish at Kumano bay was abnormally higher

Peo~ Io

o duvabione

'°°°E -~

I

1:::::

[

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C H~ CH -Cl-~- C " - C H~CH,cH o

1:

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.,c,

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150

Peak 13

200

250

m/e

Fluoranthene lO00r

1 1 ~

2 0 2 ( M +)

%

FLI 50

I00

150

200

250

m/e Fig. 5. El spectra of peaks 10 and 13 from HLCd.

22

NAOHIDE KINAE et al. Peal( 15

Pyrene

°°°I

~

%

202 ( M+)

l

•

30,00

i,..J ,.

,

IoO

)50

o,.. .........,........................ ........ .............. ..................... .=..,...., ............ ..m.6o

50

250

200 m/e

Peak 16

2 , 6 - D i - )err- butyl - 4 - ethyl~enol

.~ E

~'~";~ II

oL .........,..,L..~..,..-.,..:~.. ~,.............................. 5O

Ptlok 18

I 234(M~120'00

II 1

--,

_z

%

.C(CH3)3

IOOOl~

1(30 m/e

150

Re,the

-I

........ !o.oo

~..~..,~..,.~..!

2OO

--

250

e,~,H3

%

LTL

'

O ..,..~.-.!...,. .................. -~"............. ;.........=............... c.:................. 50 K~O 150 200 250 m/e Fig. 6. El spectra of peaks 15, 16 a n d 18 from H L C - I .

OO

Gas chromalo~am of H L C - 2 I

6

0

5

I0 Rq~mfion time tmin)

15

20

El ~ectrum of peak I 2 , 6 - Oi- tert - butyl - 4 - mefhytl2he¢lol

%

'O00rf

H3C ~ o H - - C ( C ~ 3

I:

-'~ C (CH3)3

/ZZO(M*) ]

.,, ....

~ !

-~ of

,

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I

t-{ ZS.OO

,Loo

............ ~ ................ ~ 8 .............. ~ 8 °.............. " ~ ' ........... ;'~8" m/()

EI specs'urn of I~H3k 2 Dit~,droO¢tin idiol id•

50

H3C" CH3

I(30

El spGctrum L

., onglro~ne

150

%

200

250 m / e

of peok 3 ~

CH3

%

CH 3

~= E [JJJi,~lg...,~ ,) L~..................,..'.1O.O0 .. ,....'..,,...,-L'-..T...........','--'=,.:-=--.:,.-'--;.,.'-r."

--

O 'L..,,-:.,

50 ~oo 15o zoo zso m / e Fig. 7. G a s c h r o m a t o g t a m of H L C - 2 a n d EI spectra of peaks 1, 2 and 3.

Toxicity of pulp and paper mill e~uents--I Table 6. Compounds identified in ether extract of Shinguu sediment

Compounds identified Hydrocarbons: Tetradecane Pentadecane Heptadecane Octadecane Docosane Fatty acids: Pentadecanoic acid Palmitic acid Margaric acid Stearic acid Nonadecanoic acid Fatty acid esters: Methyl myristate Methyl pentadecanoate Methyl palmitate Phthalic acid esters: Di-n-butyl phthalate (DNBP) Di-(2-ethylhexyl)phthalate (DEHP) Phenols: 2, 4, 6-Trichlorophenol 3, 4, 5, 6-Tetrachloroguaiacol 2, 6-di-tert-butyl.~-methyiphenol

M*

Identified from

198 212 240 254 310

HLC-1,-3 HLC-I, -3 HLC-1,-3 HLC-3,-5 HLC-3

242 256 270 284 298

HLC-5 HLC-5 HLC-5 HLC-5 HLC-5

242 HLC-5 256 HLC-5 270 HLC-1, -5 278 HLC-1, -3 390 HLC-I, -2 198 HLC-1 262 HLC-1 220 HLC-2, -4, -5 2M HLC-1

2, 6-di-tert-butyl-4-ethylphenol Others: Anthracene Fluoranthene Pyrene Trifluorotrichioroet hane Dihydroactinidiolide Juvabione Dehydroabietic acid Retene Longifolene

178 202 202 187 180 266 300 234 204

HLC-5 HLC-I HLC-1 HLC-5 HLC-2,-3 HLC-1,-3 HLC-5 HLC-I HLC-2

23

than that of the fish captured at two other areas with low frequences of abnormalities (Tomita et al., 1977). As shown in the text, 28 organic compounds were identified from Shinguu sediment samples. They include 5 aliphatic hydrocarbons, 8 fatty acids and their esters, 2 phthalic acid esters, 4 phenolic compounds, anthracene, fluoranthene, pyrene as polycyclic compounds and dehydroabietic acid, retene and longifolene as resinous compounds. Juvabione as juvenile hormone of insects (Negishi & Sabanski, 1976) and dihydroactinidiolide from the volatile flavor compound of tobacco (Bailey et al., 1968) were also identified, but the origins of them are obscure as yet. Rogers (1975), Keith (1976) and Yamaoka (19791 have studied on the effluents and sediments from paper mills and identified terpenoides, phenols, fatty acids, fatty hydrocarbons and resin acids. Thakore (1977) disclosed the toxic components to fish in pulp mill eiltuents using nuclear magnetic resonance. Among 28 compounds listed in Table 6, fluoranthene, pyrene, 2,6-di-tert-butyl-4-methyiphenol, dihydroaetinidiolide and juvabione are newly identified compounds of the sediments from paper mill. The mutagenicity of each compound will be reported in the next paper. Ackdlow|edOements--Thanks are due to Mr Takeo Kawa. kami for his help in sampling sediments and fishes. This work was supported by a Grant from the Science and Technology Agency of the Government of Japan. REFERENCES

Ames B. N., MeCann J. & Yamasaki E. (1975) Methods for detecting carcinogens and mutagens with Salmonella/ mierosome mammalian mutagenicity test. Mutation Res. 31,347-364.

Table 7. Mutagenic and DNA damaging potency of chemicals identified in sediment samples S. typhimurtum His + revertant colonies plate

Samples Palmitic acid Methyl palmitate 2,4,6-Trichlorophenol 2,6-di-tert-butyl-4-methylphenol 3,4,5,6-Tetrachloroguaiacol Fluoranthene Pyrene Dihydroactinidiolide Dehydroabietic acid Juvabione 4NQO Benzo(a)pyrene 9-Aminoaeridine Control (DMSO) AF-2

Amount used (g8 plate- 1) 100 100 1 10 1 100 100 100 10 100 0.3 5.0 50

TA 98

TA 100

TA 1537

Amount used - S-9 + S-9 - S-9 + S-9 - S-9 + S-9 (ms disk- ~) 31 36 29

50 55 60

147 131 120

118 133 126

9 12 8

8 16 19

10 10 0.5

31 36 36 36 30 34 32 172

60 62 70 52 59 63 56

114 151 130 132 126 137 1(30 362

142 144 215" 143 133 133 131

10 11 9 13 11 10 7

14 10 23 40* 10 14 22

10 0.1 10 1 10 10 0.5

248 33

60

302 133

Numbers in table indicate the mean value in five experiments. * P < 0.01.

135

188 212 10

14 2.5 x 10-s

B. subtilis Rec-effect

w

+ +

24

NAOHIDE KINAEet al.

Ander P., Eriksson K. E.. Kolar M. C. & Kringstad K. (1977) Studies on the mutagenic properties of bleaching effluents. Svensk PaperstidnM~. 14, 454--458. Bailey W. C., Bose A. K. Jr., Ikeda R. M., Newman R. H., Secor H. V. & Gorden R. W. (1975) Fish toxicants in kraft effluents. Tappi 5& 136-140. Davis J. C. (1973) Sublethal effects of bleached kraft pulp mill effluent on respiration and circulation in Sockeye salmon (Oncorhynchus nerka) J. Fish. Res. Bd. Can. 30, 369-377. Davis J. C. & Mason B. J. (1973) Bioassay procedures to evaluate acute toxicity effluent on neutralized bleached kraft pulp mill effluent to Pacific salmon. J. Fish. Res. Bd Can. 30, 1565-1572. Felicetta V. F. & McCarthy J. L. (1963) Spent liquor X. The Pearl-Benson, or nitroso method for the estimation of spent sulfite--liquor concentration in waters. Tappi 46, 337-347. Kada T. (1975) Mutagenicity and carcinogenicity screening of food additives by the Rec assay and reversion procedures. International Agency for Research on Cancer Scientific Publication No. 12:105-115 Proceeding on the WHO-IARC meeting. Keith L. H. (1976) Identification of organic compounds in unbleached treated kraft paper mill waste water. Environ. $ci. Technol. 10, 555-564. Kimura I. (1976) Studies on tumor formation: tumors in lower vertebrate animals. 19aku no Ayurm (in Japanese) 96, 216--225. Leach J. M. & Thakore A. N. (1977) Compounds toxic to fish in pulp mill waste streams. Proo. Wat. Technol. 9, 787-798.

Negishi N. & Sabanski M. (1976) An efficient synthesis of Juvabione and todomatuic acid via hydroboration-carbonylation. Tetrahedron 32, 925-926. Rogers I. H., Davis J. C., Kruzynski G. M., Mahood H. W., Servizil J. A. & Gordon R. W. (1975) Fish toxicants in kraft effluents. Tappi 58, 136-140. Seim W. K., Lichatowich J. A., Ellis R. H. & Davis G. E. (1977) Effects of kraft mill effluents on juvenile salmon production in laboratory streams Water Res. 11, 189-196. Standard Method of Analysis for Hygienic Chemist. The Pharmaceutical Society of Japan (1973). Stockner J. G. & Cliff D. D. (1976) Effects of pulp and paper mill effluent on phytoplankton production in coastal marine waters of British Columbia. J. Fish. Res. Bd Can. 33, 2433-2422. Thakore A. N. (1977) Structures of toxic constituents in kraft mill caustic extraction effluents from t3C and ~H nuclear magnetic resonance. Can. J. Chem. 55, 3298-3303. Tomita I., Kina¢ N. & Saitou S. (1977) Studies on aquatic pollutants in relation to neoplastic disease of marine animals. Aquatic Pollutants: Transformation and Biolooical Effects (Edited by Hutzinger O. H.), pp. 481--482. Pergamon Press. Oxford. Walden C. C. {1976) The toxicity of pulp and paper mill effluent and corresponding measurement procedures. Water Res. 10, 639-664. Yamaoka Y. (1979) Identification of terpenoid compounds in the sediment in Hiro Bay by gas chromatographymass spectrometry. Agric. Biol. Chent 43, 1143-1144.