The use of fish bile metabolite analyses as exposure biomarkers to pulp and paper mill effluents

The use of fish bile metabolite analyses as exposure biomarkers to pulp and paper mill effluents

Chemosphere, Vol. 36, No. 12. pp. 2621-2634, 1998 Q 1998 Elsevier Science Ltd All rights reserved. Pried in Great Britain @M-6535/98 $19.00+0.00 Perg...

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Chemosphere, Vol. 36, No. 12. pp. 2621-2634, 1998 Q 1998 Elsevier Science Ltd All rights reserved. Pried in Great Britain @M-6535/98 $19.00+0.00


PII: SOO45-6535(97)10226-O



Leppanen, H.‘, Marttinen, S. and Oikari, A.


of JyvaskylP, Department

of Biology and Environmental

Sciences, P.O.B. 35, FIN-4035 1 Jyvasky-

Iii, Finland (Received in Germany 25 September 1997; accepted 28 November 1997)

ABSTRACT The exposure of caged one-year-old

whitefish (Coregonus


to wood extractives and chloropheno-

lies from pulp and paper mill effluents in Lake Saimaa (SE Finland) was investigated.

Whitefish were exposed

at 17 locations affected by effluents from pulp and paper miJJs and at 5 reference areas.

Resin and fatty acids

and chlorophenolics

in bile was also mea-

in bile were analysed by GC and CC-MS. Cholesterol concentration

sured. Results show that

despite of


changes to ECF and waste water treatment


employed by the pulp and paper industry in the study area, it is still possible to detect elevated concentrations of these substances in the bile of fish exposed in the vicinity of a pulp and paper mill. Q1998 Elsevier Science Ltd. All rights reserved

INTRODUCTION Exposure of animal organims to industrial xenobiotic chemicals informs about their biological effects. Soutbern Lake Saimaa, SE Finland, has received pulp and paper mill effluents from four mills for over 50 years. These effluents include wood extractives which are nonstructural

wood constituents

solvents or water. They comprise a very large number of individual compounds rophilic types. Important classes ofwood phytosterols,

sohrble in neutral organic

of both lipophilic and hyd-

extractives are terpenoids and steroids, including resin acids and

fats and waxes including fatty acids, and phenolic constituents

including stilbenes, lignans and

flavonoids [I]. Of wood extractives, resin acids are the most acutely toxic components to aquatic life [2]. The LCsO (96h) value for dehydroabietic is approximately

of pulp mill effluents

acid, the most abundant and least toxic resin acid,

1 mg/l for S&no guirdneri [3 - 51. The wood extractives are suspected to cause changes

also in fish reproduction. levels and dminished

These changes include smaller gonad size, alterations in blood plasma sex steroid

secondary sexual characteristics

[6 - 131. Due to biological waste water treatment by


2622 aerobic processes the levels of these toxicants in lake waters receiving pulp and paper mill effluents have decreased so that it may be ditlicult to detect these substances in lake water itself [ 14 - 161. Instead fish bile analyses may be sensitive markers of pulp and paper mill effluents. Resin acids and some chlorophenolics


known to be conjugated in the liver of fish mainly with a glucuronic acid moiety [ 17, 181. Concentration

of a

toxicant and it’s metabolites can be 1000 - 100 000 times higher in the bile than in water were the fish live in [2. I9 - 221. The half life of the body burden of resin acids , both in Bee and conjugated forms, has been observed to be below 4 d [22]. The objective of this study was to assess whether the assay of bile metabohtes of chlorophenolics

and selected wood extractives still may reveal a reliable measure on the exposure of fish un-

der altered technological

solutions, particularly the transition from chlorine-based

bleaching to elemental

chlorine free (ECF) process.



The mills in the area produce hardwood

and softwood pulp. The production

of pulp during the study period

was 1236 t/d for mill A, 66 1 t/d for mill B and 1703 t/d for mill C. The amount of waste water discharged into the lake was 198 276 m3/d for mill A, 57 335 m3/d for mill B and 145 852 m3/d for mill C. All mills have employed elemental chlorine free bleaching. Mills A and C have employed activated sludge waste water treatment plants. During the caging period mill B still used aerated lagoons for waste water treatment.

1997 they

also employed activated sludge plant. Mill C uses chemical treatment for cardboard mill waste waters. The volume of cardboard waste water was 49 436 m3/d. Whitefish (Coregorrus lavaretus sensu Sviirdson) of plaukton feeding race were experimentally exposed to water quality by caging at various distances corn the mills (Fig. 1.). The caging took place in depth of 4 - 5 meters and lasted for 30 days in May - June 1996. Each caging site had two cages except site 1 (one cage) and site 2 (three cages). Each cage had 12 juveline fish. The average weight of the fish was 40.4 f 10.7 g and average length 16.5 f 1.3 cm. The exposed fish were sampled on each site on RiV Muikku. Bile samples were placed in liquid nitrogen directly after sampling and later transferred to -80°C in laboratory and kept there until analyses.

Water analyses Lake water samples were collected twice into 2.5 1 glass bottles during the fishcaging period, combined in laboratory and placed in -20°C until analysis. The interval between the sampling was two weeks. Each water sample was a composite

from water column at 0, 1, 2, 3 and 4 meters. Several mill effluent samples were

collected daily with autosampler

and combined to week samples into 2 1 polyethene

bottles in each mill.

Samples were stored in -20°C. The week samples were combined in laboratory to one sample which represented the average quality of mill effluent during the 30-day caging period.

2623 Figure 1. The 1996 study area at southern Lake Saimaa (SE Finland). The small arrows present directions of lake water flow in the area. Effluents from mills B and C are diluted more effkiently than from mill A. Reference sites were 1,2, 10, 21 and 22.

TABLE I. Distances from the caging sites to mills.

site dist. (km) mill

Water sodium (Na+) concentration was used as a tracer of indicating the dilution of effhtent when compared to the sodium concentration of the undiluted effluent. The data was provided by the monitoring program of the Saimaa ‘Water Protection Association Inc., Lappeenranta, Finland.

2624 Fatty and resin acids and phytosterols

from lake water samples and mill effluent samples were analysed ac-

cording to Hemming et al. (1992) I and &&I et al. (1994) [24,27]. Water samples were methylated with diazomethane for fatty and resin acid analyses, acetylated with acetic anhydride for chlorophenolics silylated with N,O-bis(trimcthylsilyl)trifluoro-acetamide Phytosterols

analysed were campesterol,


effluent for one analyses of wood extra&es and bound cblorophenolics

with 1% trimethylchlorosilane

analyses and

for sterol analyses.

and sitostanol. We used 1 1 of lake water and 500 ml of

and 250 ml of effluent for one analyses of chlorophenolics.

were analysed separately according


to Voss et al. (1981) and Paasivirta et al.

( 1992) [25.26].

Bile analyses Resirl mdfatiy


Biles t?om 3 to 7 fish (60 - 120 ul ofbile) were pooled for one analysis. Heptadecanoic

acid was added as an

intemal standard. The samples were extracted with methyl tert-butyl ether at pH 3.5 and methylated with diazomethane.

After the first extraction, which removes t?ee toxicants from bile, samples were hydrolyzed with

KOH in 7O’C in order to release conjugated levopimaric, sandaracopimaric,


abietic, dehydroabietic,

The resin acids analysed were pimaric, isopimaric,

palustric and neoabietic acids. Fatty acids were identi-

fied with GC-MS. Free and conjugated toxicants were analysed separately [ 17,241. All results are averages of 2 - -I IUIlS.

Cholesterol was silylated and analysed semiquantitatively quantitrted

using heptadecanoic

Tom the same extraction as resin and fatty acids and

acid as an internal standard. The cholesterol results are comparable t?om one

site to another. but the absolute values are not accurate, because it was assumed that the detector responses of tire iutemal staudard and cholesterol lcsterol in bile was approximately

are the same. This was not verified though. The average level of cho-

50 &l.

The results are presented

so that the bigbest value of cholesterol in

bile in the whole (site 8) is given value 1 and others are presented in relation to that. Cholesterol was found in bile only in tiee form.

Pooled samples Tom 3 - 7 fish were extracted with hexane at pH 7. After addition of the internal standard (2.6-dibromophenol)

and the tirst extraction, which removes free toxicants from bile? the samples were hydro-

lysed with HCI at 70°C in order to release the conjugated toxicants.

Samples were acetylated with acetic an-

hydride before analyses by GC [ 17.241. Due to limited amount of bile we could not carry out chlorophenolics analyses on every sampling site.


All samples were analysed with Perkin-Elmer Autosystem XL gas chromatography equipped with PI and EC detectors. We measured for 17 compounds including chloroguaiacols, -catecols, -vanillins and -phenols. All results are averages of two runs. Compounds were identified by their retention times and with HP 6890 GC equipped with HP 5973 MS. The columus used were NB-54 and HP-5 Statistics Correlations were examined by determing Spearman correlation coefficients. The significance level was set to pCO.05. Statistics were performed by using [email protected]

RESl’LTS AND DISCUSSION The first study on the area was conducted in 1991 when the pulp mill studied (mill A) used chlorine bleaching [ 141. Since then three studies have been performed on the area, one in 1993 [23] and two others on a larger area in 1995 [ I.51 and 1996. The investigations will continue in 1997.

Mill effluents

TABLE II. Concentrations of classes of wood extractives in pulp mill effluent filtrate and in particulate mat-


ter. The filter used was Wbatman GF/C glass microfibre filter @ore size 012 pm). ~MillA filtrate w

MiUB partid. [email protected]






[email protected]




Kesin acids







Fatty acids














TABLE Ill. Total concentrations (DOC-bound + f?ee) oftypical bleaching related chlorophenolics (CC = chloroguaiacols, CC = chlorocatecols, CVa = 6-and 5.6 -chlorovanillins), other chlorophenolics and Na’ in tmfiltered mill effluents. MillA









Other CP’s




Na’ [tug/l]




~-~ CG. CC.CVa

2626 Only dehydroabietic

acid (58% of total amount of resin acids in effluent) and abietic acid (42%) could be

detected in filtered effluent from mill A. In the particulate matter, dehydroabietic (3 1%) and neoabietic were octadecanoic

(17%) acids were present. The most abundant fatty acids in filtered effluent

(57 %) and hexadecanoic

(34 %) acids. In the particulate matter eicosanoic (50 ‘%) and

docosanoic acids (47 9’0)dominated. p-sitosterol(80 and sitostanol(20 dehydroabietic

(25%), abietic (27%), pimaric

% of sterols in effluent and 36 % of sterols in particles)

% in effluent and 30% in particles) were most abundant phytosterols

in mill A. In mill C

(34 ‘%), abietic (30 %), pimaric (21 %) and palustric acids (15 %) were present in filtered ef-

fluent and also in particulate matter. The respective percentages “Gand I7 %. Tbe abundances of fatty acids and phytosterols Hexadecanoic

acid was present in highest concentration


%) of phytosterols

in the particulate matter were 65 %, 8 %, 11

in filtered effluent were very similar with mill A.

of fatty acids (78 %) and p-sitosterol(65

%) and

in particles of mill C.

Compared to other two mills the effluent from mill B had higher concentrations

and different compositions

wood extractives. This was probably due to different waste water treatment. In efiluent the composition resin acids was pimaric 12 %. sandaracopimaric

4 %, isopimaric 11 %, palustiic 4 %, dehydroabietic

abietic 16 % and neoabietic 4 %. Almost the same composition most abundant fatty acids in filtered effluent were octadecanoic the most abundant fytosterol p-sitosterol(71 culate matter (65 %). Docosanoic



49 %,

also prevailed in the particulate matter. The (26 %) and hexadecanoic

acids (25 ?/,), and

%). fSsitostero1 was also the most abundant fytosterol in parti-

(19 %) and tetracosanoic

(22 %) acids were the most common fatty acids

iu particulate matte]‘.

Lake waters The highest concentration

of resin acids were detected on sites 2 (4.5 l&l) and 8 (4.3 &I).

This was unes-

petted since point 2 does not receive BKME and point 8 is theoretically upstream from mill B. A possible explanation for high concentrations

on location 2 is dredging conducted in a nearby harbour in autumn 1995.

The dredged sediments were piled on ice close to sampling point 2. The only resin acid that could be detected in lake waters was dehydroabietic decrease in concentration

acid. In contrast to 1996, in 1991 there was still a clear distance related

of resin acids downstream

from mill A [22]. In investigations

v+hrtn mill A modernized its biological waste water treatment system this concentration [28]. Highest concentrations

conducted after 1992 profile had faded

of fatty acids were on locations 11, 12 and 20, the most abuudant being octade-

canoic acid. Neither resin nor fatty acid concentrations

in water correlated with sodium concentrations

with each other. As some sample bottles were broken during trasport in a deepfieezer, stored inside another container. Surprisingly, the highest concentration


these samples were

of resin acids - up to 170 up/l - were

measured horn those samples. These aberrant values which were caused by melting ice dissolving the glue

2627 that holds the factory label on the bottle are omitted from the data. Fytosterol concentrations in all lake waters were belovv the detection limit (2&l).

‘TABLE IV. Sodium. resin acid (RA) and fatty acid (FA) concentrations on the study area during the whitefish caging period. Sodium was used as an efauent tracer. Reference sites underlined. nd = not determined.



7 =_











































































Fish bile analyses Whitefish are exposed to toxicants via sediment particles and water. The highest amount of resin acids in bile was in fish caged at site 2 (89 t.q/ml). This indicates that the explanation for exceptionahy high concentration

ofresin acid in water at this site can really be the dredging.

Also on other sites the concentration of resin

acids in bile correlated with the concentration ofresin acids in water (p = 0.64, p
The lugbest concentration of total fatty acids in bile was at site 8 (800 pgM) and lowest at site 21 (120 r&l). resin

The concentration of total fatty acids in the bile of whitefish correlated with the concentration of bile acids (p =: 0.56. p
most abundant free fatty acids in bile were 8-octadecenoic acid and hcxadecanoic acid. Hydrolyzable fatty :tcids consist


and bexadecanoic acids. The average amount of free fatty acids

ill tile u as 47 % of total amouut. There was no correlation between bydrolyzable fatty acids of bile and water Na* concentration (representing BKME) as has been reported earlier [2].

2628 Figure 2. The concentration of resin acids in the bile of caged white&b. Columns present the mean values and points individual measurements of pooled samples. The highest value in point 2 (166 @ml) is not presen ted in the figure.


Site at the map (Fig. 1.)

In vertebrate animals cholesterol is excreted by the liver into the bile. In bile it is present in i%eeform and as bile salts [29]. The major bile salt glycocholate is derived from cholesterol via trihydroxycoprostanoate and cholyl CoA [30]. It has been reported earlier [2,3 1,321 that the concentration of “bound” cholesterol (esterifted or conjugated) in ti

bile can increase in dose-dependent way by exposing rainbow trout to pulp

mill effluent. The increase was considered to reflect an altered metabolic status of fish, possibly due to increased cholesterol synthesis in the liver [2]. However, by using the same hydrolysis procedure, we could not find any bound cholesterol, nor bound or free fytosterols in bile. The treatment of whitefish bile by alkali did not release any cholesterol, which means that the cholesterol found in bile was in free form Plankton feeding whitefish seems thus differ physiologically from rainbow trout in this respect. Cholesterol level in bile was highest at site 8 where it was ten times higher than at site 21 where it was the lowest. The cholesterol level correlated with both the amount of resin acids and the amount of fatty acids in bile (cholesterol vs. resin acids p = 0.52, pcO.02, n = 21; cholesterolvs. fatty acids p = 0.81, p
2629 Figure

3. The: concentration of conjugated fatty acids in the bile of whitefish. Cobs

present the mean va-

lues and points individual measurements.

800~ 700














Site at the map (Fig.1)

The concentration of chlorophenolics (CPs) in bile showed a distance related correlation downstream from mill A. The highest amount of CPs was measured at site 4 (2.1 pg/ml). The BCFbacfor CPs varied from 500 to 2000. The iassayed concentration of CPs correlated with sodium concentrations (p = 0.68, p ~0.05, n = 9), however, the ~amountof typical BKME (bleach km8 mill effluent) related CPs (chlorocatecols, -guaiacols and 6-monochloravanillin) correlated with sodium concentration even better (p = 0.82, p
2630 Figure 4.

Cholesterol level in bile of white&h. Cholesterol is presented in relation to highest mean cholesterol

level at the study area (site 8).








Site at the map (Fig. 1)

CONCLUSIONS In the light of the results of this study it is evident that in bile analyses chlorophenolics and especially chloroguaiacols, chlorocatecols and 6-monochlorovanillin, are the best exposure biomarkers of pulp and paper mill effluents even though the pulp industry has employed ECF bleaching. Wood -active concentrations (fatty and resin acids) in bile correlated with each other but it is impossible to directly associate those concentrations to pulp and paper mill ef&nts BKME tracer.

if sodium concentration iu water is considered as the

2631 Figure 5. The sum concentration of chloroguaiacols, chlorocatecols and chlorovan.iJhns in the bile of whitefish. Concentrations correlate with sodium concentrations in the area (p = 0.82, p













Site at the map (Fig. 1) -

ACKNOWLEDGEMENTS The authors would like to that& M.Sc. Marita Knuutila and Nina K#mWinen for analytical help and the cooperation of the crew of R/V Muikhu is greatly appreciated.

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