effects of bleached kraft mill effluents on the swimming activity of monoporeia affinis (crustacea,...
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00456535(95)00325-8
Chemosphere, Vol. 31, Nos 11112, pp. 4455-4473, 1995 Copyright 0 1995 Ekvier Science Ltd
Printed in Great Britain. All rights reserved 00456535/9S $9.50+0.00
EFFECTS OF BLEACHED KRAFT MILL EFFLUENTS
ON THE SWIMMING ACTIVITY OF
MONOPOREIA AFFINIS (CRUSTACEA, AMPHH’ODA) LINDSTRbM
Harri Kankaanpaa a*, Marjo Lauren a, Madeleine Mattson b and Magnus Lindstrom b
a Finnish Institute of Marine Research, Helsinki, Finland
b Tvarminne Zoological Station, University of Helsinki, Hat&o, Finland
(Received in Germany 14 March 1995; accepted 3 October 1995)
ABSTRACT
The crustacean Monoporeia affinis Lindstrom was exposed to two different concentrations of bleached krafl mill effluent (BKME) containing, originally, 8.3 mg/g organic chlorine determined as adsorbable organic halogen (AOX). The swimming activity of the animals was recorded by pairs of infia-red sensitive photocells attached to 5.6 1 aquaria having a seawater inflow rate of 12 ml/mm The test sequence included a control phase, an exposure phase and a recovery phase. During the exposure phase the total swimming activity decreased in three of the four test aquaria containing effluent, indicating that the animals had retreated into the sediment. Total activity increased during the recovery phase and, in the case of the lower organic chlorine concentration, activity almost achieved the original control-phase level Statistical differences in swimming activity changes during experiment were evaluated using ANOVA. Experimental methods are explained in detail.
Key terms: Activity recording; amphipod; adsorbable organic halogen; bleached krafl mill effluent; extractable organic halogen; monoporeia affinis; pulp industry
1. INTRODUCTION
The amphipod Monoporeia ufflnis, a benthic deposit feeder, is one of the most abundant macrobenthic species in
the Baltic Sea. The density ofM. uffinis may vary from a few animals to over 20 000 individuals per square meter
(SegerstrHle, 1937). Over 50% of the biomass of the soft bottoms of the Baltic Sea consists of M. uffinis and the
bivalve Mucoma baltica (SegerstriQe, 1933; Ankar & Elmgren, 1976). A4. ufJinis is an important organism in the
Baltic Sea’s food chains.
Light is the main factor governing the diurnal activity of A4. uflnis (Dormer 8c Lindstrom, 1980). During the day
A4. uffinis burrows several centimeters into the sediment (Hill & Elmgren, 1987) and fimctions as a bioturbator
* Correspondence address: Finnish Institute of Marine Research,
PO Box 33, FIN-00931 Helsinki, Finland. + 358 0 613 945 18 (telephone), + 358 0 613 944 94 (fax)
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oxygenating the surface sediments. It has been shown that the daily activity rhythm of this amphipod can be affected
by several organic contaminants (Lindstrom & Lindstrom, 198Ob). Thus the species can be used as an indicator for
sublethal concentrations of marine pollutants.
Bleached kratt mill effluents (BKME) have a noticeable efFect on the biology of the receiving waters, which can be
seen in the disappearance of benthic invertebrates and even in a higher incidence of fish diseases (Sundelin, 1988;
Sodergren et al., 1993). BKME is a complex mixture containing the chlorinated and non-chlorinated products of
the degradation of wood lignin. These components include chlorinated phenols (Paasivirta et al., 1992) guaiacols
and catechols (Paasivirta et al., 1985), which are all well known toxicants. The toxicity and mutagenicity of the
effluents has been shown to be associated with the polar fractions of BKME (Higashi et al., 1992; Rao et al.,
1994).
In addition to the components mentioned above, the effluent contains substantial amounts of high molecular-weight
material (Higashi et af., 1992; Jokela & Salkinoja-Salonen, 1992) which cannot be quantified using traditional
chromatographic methods, consequently the amount of organochlorine compounds present in the BKME, the water
and the sediment samples are determined as a sum. The sum parameters EOX (extractable organic halogen) and
AOX (adsorbable organic halogen) are used to quantify complex organohalogen matrices. It has been estimated that
76% of the total input of EOX to the Baltic Sea is discharged by pulp mills (Wulff et al., 1993) especially those
feeding the large scale pulp and paper industry in the north, in Finland and Sweden. AOX values in BKME-polluted
seawater samples from receiving waters in South-east Finland have been reported at 25-200 ug/l (Kankaanpti &
Tissari, 1994b) and from lake water within 1 km of a Swedish pulp mill at 14-830 ug/l (Grimvall et al., 1992).
This study attempts to demostrate the effects of sublethal concentrations of bleachery effluent on the behaviour of
M. affin under laboratory conditions. The concentrations used correspond to those found in receiving waters near
to pulp mills.
2. MATERIALS AND METHODS
2.1. Sampling and treatment of the animals, sediment and BKME
The sediment and animal samples were taken by the Finnish research vessel Aranda using a van Veen grab sampler
in June 1994. The animals were separated carefully from the sediment with sieves. Animals and sediment were
stored in a refrigerator at +5OC. The bleachery effluent (60 liters) was collected from the outlet of the Sunila Pulp
Mill in Kotka, South-east Finland, in May 1994. This mill uses Chlorine-chlorine dioxide Bleaching. The effluent
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was stored in 30 I plastic canisters at -20°C and brought to room temperature before the experiment. Prior to
applying the BKME to the test aquaria, the liquid was vacuum filtered through a Whatman filter no. 1 in order to
remove the largest particles. The sampling locations are shown in figure 1.
LONGITUDE I...:~..,....:. : ..:. :....; ..:.__. _J 20 2.1. 7.2. Y ;,.-+-
Samphg station for animals
seawater sanlphg
Figure 1. Sampling locations in the Gulf of Finland and the Bothnian Sea.
2.2. Apparatus, activity measurements and maintenance of test animals
The swimming activity of the animals was recorded using the method of Lindstrtlm & Lindstrom (1980a,b) (figure
2). The animals were transferred to 4 test and 2 control glass aquaria (L 33 x W 6 x H 35 cm). Forty animals were
taken randomly from the storage basin and placed in each aquarium which had been filled beforehand with 5.5 litres
of brackish-water with a salinity of 0.6 - 0.7%. All parts in contact with the water were made of glass, silicone or
polypropylene. The bottom was covered with a 2 cm of mud (= 400 ml) from the sampling station. Water entered
each aquarium through a nozzle about 1 cm above the mud bottom and was drained from about 1 cm below the
water surface to keep the water-mass in slow-but-constant movement The seawater flow was adjusted to
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12 mI/min, giving a 90% replacement time of about 16 hours, enough to keep the oxygen level sufficiently high, i.e.
over 8 mg/l (Lindstrom & Lindstrom, 1980a). The mud served as a shelter for the burrowing animals during the
illuminated period and also provided them with food. Water temperature was kept at 6-7 “C.
Three aquaria were placed in each of two light-proof experimental chambers. Fluorescent tubes (24V, SW) above
one opalescent and one light-absorbing acrylic filter served as the light source. The light intensity in the chambers
was about 8 x lOI photons m-2 s-l (about 0.013 FE or 0.0029 W rnm2) with a broad maximum around 600 run.
This intensity is similar to the intensity at a depth of 30 m in the Tvarminn e area (see figure 1). The illuminated
period was 12 hours, from 08:OO to 20:00 hrs.
The swimming activity in each aquarium was recorded with four photocell pairs consisting of infra-red (IR) emitting
diodes and phototransistors. IR light is invisible to the animals (Donner, 1971; Dormer & Lindstrom, 1980). The
photocell pairs were mounted on horizontal frames surrounding the aquaria, at a level about 2.5 cm below the water
surface. The breaking of the IR beam by an animal resulted in one count. The sum of the counts for each unit was
printed once an hour. The photocell recording units were adjusted to respond to a glass capillary with a diameter of
2 mm. The activity recorded in the different aquaria could not, however, be compared directly to each other in
terms of, for instance, number of animals actively swimming, because the size and sensitivity of the detecting fields
of the photocells were slightly different (Lindstrom & Lindstrom, 1980a).
In aquaria 2-5 the BKME was added to the inflow of seawater by a peristaltic pump during days 20-27. During the
other periods only clean seawater was added. No effluent was added to aquaria 1 and 6, which served as controls.
The flows of seawater and BKME were measured; the latter by means of a micro-flow meter and following this
theoretical AOX concentrations calculated using the BKME intlow and total outflow rates. (Table lb, Figure 3).
2.3. Sampling from the aquaria and p-treatment of the samples
Before the test a 100 ml sample was taken from the seawater stock to establish AOX content. Twice during the test
a 100 ml water sample was taken from the bottom outlet of each of the 4 test aquaria (see section 2.2.), on day 22
and day 27. The sample bottles were stored frozen, at -2O’C, until measured; storage time did not exceed 2 weeks.
Also, at the end of the experiment on day 39, water samples of 100 ml were taken from aquaria 2, 3,4, 5 and 6 for
AOX analysis and, following this, sediment samples of approximately 5 g dry-weight were taken with a small piston
corer from all six aquaria. These sediment samples were then sieved and ah the animals buried in the sample
collected. The sediment samples were frozen, lyophilised (Edwards Freeze dryer, Super Modulyo) and
homogenised in a Fritsch planetary mill (pulverisette 5) and then analysed for EOX microcoulometricahy. Finally,
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the sediment in each aquarium was mixed with water so that the remaining animals could be collected from the
suspension by suction and sieving. The numbers of surviving and dead animals were counted. Animals from each
aquarium, including the control aquaria, were placed in plastic petri dishes, frozen and lyophilised. Homogenisation
of animals was made manually in a mortar.
Figure 2. Schematic diagram of test equipment showing one of six aquarium units. A- brackish-water inlet from the
supply. B- water dispenser to the test aquaria. C- overflow for constant pressure. D- air bubble outlet from water
dispenser. E- syringe nozzle, adjustable in height for fine adjustment of flow. F- light lock for water inlet. G- light-
tight experimental chamber. H- aquarium with bottom sediment. J- water inlet, nozzle diameter 1 mm. K- drainage
from the aquarium. Water samples were drawn through a second outlet parallel to K about 1 cm above the bottom.
L- fluorescent tubes, 24 V, 8 W. M- acrylic filters, light-dispersing white, and light-absorbing dark. N- swimming
activity detector with JR emitters and correspondiig phototransistors. P- event recorder. R- peristaltic pump. S-
test solution (BKME) supply. (The figure is a modification of the original Lindstrom & Lindstrom, 198Oa).
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2.4. AOX method for the water samples
The method is a modification of the DIN standard for water samples (DIN 38409). Briefly, the water samples were
taken to room temperature and approximately 100 mg of activated carbon (Euroglas, maximum chlorine content 15
pg/g) was added to each sample in a 100 ml Sovirel bottle, followed by 5 ml of 0.2 M sodium nitrate solution (pH
2.0 adjusted with nitric acid). The sample bottles were agitated overnight for not less than 16 hours. The activated
carbon was filtered through a 0.4 mm polycarbonate filter (Euroglas, maximum chlorine content 0.5 ug per filter).
The filter and activated carbon were washed with 4 x 10 ml of 0.01 M sodium nitrate solution to displace any
remaining chloride ions. Finally, samples were washed with 4 x 10 ml of ultra-pure water (Millipore Milli-Q UF plus
water). Each sample was then placed in a ceramic crucible, burned at 1 05O’C and analysed microcoulometrically
(Euroglas ECS 2000). The total halogen content of each sample was calculated as chlorine equivalents.
2.5. EOX method for the sediment
No commonly accepted standard methods exist for the EOX analyses. The method that we use is a modification of
a cyclohexane-isopropanol method presented by Martinsen et al. (1988) and has been used previously for our
sediment analyses (Kankaanp~ & Tissari, 1994a, b). Each lyophilised and homogenised sediment sample was
weighed and all material (average 5 g dry weight) was placed in a 100 ml Sovirel bottle. To each sample 75 ml
80/20 (v/v) of cyclohexane-isopropanol was added. The samples were sonicated (Branson 3200) for 100 minutes
and then agitated strongly at room temperature for at least 16 hrs. The samples were centrifuged (Heraeus
Megafkge 2.OR) and supematant was transferred to another 100 ml Sovirel bottle. The organic phases were
extracted twice with 25 ml 0.2 M potassium nitrate (pH 2.0 adjusted with nitric acid). The solvent was concentrated
to 0.5 - 1 .O ml using rotary (Heidolph rotavapor) and nitrogen flow evaporation. The concentrates were placed in
Eppendorf tubes and centrifuged to remove the precipitate. This type of precipitate does not contain significant
amounts of organic chlorine (KankaanpU & Tissari, 1994a). From the extract 100 ~1 was burned at 85O’C and the
chloride ions titrated using the Euroglas ECS 2000 microcoulometric system.
2.6. Statistical analysis
For statistical treatment one-way ANOVA (Analysis of variance) was used to compare the total night activities of
the control, exposure and recovery periods of the animals in each aquarium. The significance levels used were:
p > 0.05 not significant (ns.), p < 0.05 almost significant (*), p < 0.01 significant (**) and p C 0.001 very
significant (***). The REGWQ Test (Ryan-Einot-Gabriel-Welsch) @not & Gabriel, 1975) was used to group
periods for each aquarium.
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3. RESULTS
In anaiysing the results, only activity during the dark periods (hours 20-08) has been considered, because the
activity during the illuminated period was very low. The recording of the control period (C) started 13 days after
the animals had been transferred to the aquaria, amount of time which we considered sufficient for steady nocturnal
activity to develope. The effects of sublethal toxicity were recorded during days 20-27 (test period, T) and, finally.
activity during the recovery phase (R), between days 28-39. Malfunctioning photocells caused recording in
aquarium 1 to be interrupted during days 18-20 and 35-39, shortening the C, T and R periods by 2, 1 and 5 days
respectively.
The BKME solutions caused virtually no mortality during the test: the survival rate of the animals in all aquaria was
96- 100% and was not dependent on the AOX concentration.
3.1. AOX in the water stocks and in the aquaria
ARer dilution (1:2) and filtration of the pulp mill effluent, the observed AOX concentration in BRME solutions was
2 590*260 ug/l (average of the two 30 I canisters containing the diluted solution), but the theoretical concentration
should have been half the stock solution’s 8 250 ugil (Table la). This decrease is ascribed to the removal of AOX
containing fibres. Filtration is necessary to ensure an undisturbed flow of the BKME solution. For the AOX
measurements for the water stocks and for the aquarium water concentrations during and after the exposure to
BRME, please refer to Tables la and lb.
Theoretical AOX values for the aquaria were calculated on the basis of the rate of inflow of the BRME solution and
the rate of outflow of BKME in seawater. The water samples anaiysed for AOX taken on the last day of the T
period showed an overall positive bias from the theoretical AOX values. The difference in the observed and
calculated AOX concentrations is seen in Figure 3. In aquaria 2 and 3 the BKME inlet was temporarily blocked,
which explains why the theoreticai AOX values at 96 hours are low.
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SOURCE NO. OF AVERAGE SAMPLES AOX (s)
(i(g/B
PULP MILL EFFLUENT
2 8250 (150)
SEA WATER 2 10.5 (1.5)
DILUTED AND FILTERED PULP MILL EFFLUENT (CANISTER A)
3 2520 (190)
DILIJTBDAND FILTERED PULP h&ILL EFFLUENT (CANISTBR B)
2 2740 (110)
Table 1 a. AOX concentrations in pulp mill effluent, seawater and diluted and filtered pulp mill effluent. s = standard
AQUARIUM DAY N AVERAGE DAY N AVERAGE AOX, AOX, (s) (I@) (s) (k&n)
22 3 122 (9) 27 4 269 (20)
22 2 228 (3) 27 2 656 (19)
22 2 354 (3) 27 3 451 (22)
22 4 143 (9) 27 3 203 (7)
* * * * * *
DAY N AVERAGE AOX, (s) (k4zA)
39 2 41 (7)
39 2 26 (1)
39 2 70 (3)
39 1 58
39 1 62
Table lb. AOX concentrations in the test aquaria and in control aquarium 6. The intlow of diluted BKMB in aquaria 2 and 5 (low AOX) was 33% of the corresponding inflow in aquaria 3 and 4. DAY = day of experiment, N = number of samples, s = standard deviation, * = not applied.
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700
600
500
s 400 3
300
200
100
0 40 70 100 130 160 190 220 250 280
Time (h) from start of T period
theoretical v Aq2 0 Aq3 0 Aq4 0 Aq5 observed v Aq2 . Aq3 8 Aq4 ?? Aq5
Figure 3. Observed average (dark markers) and calculated (black curves) AOX concentrations in aquaria during the
T period.
The reason why the measured AOX increased during test is unclear, but it could have been caused through
concentration of the BKME solution in the temperature controlled room (temperature + 4’C), or by the formation
of a downward concentration gradient in the canister. After closing the BKME it&low AOX levels in the aquaria
decreased markedly to the end of the R period (Table 1 b, day 39) but remained slightly higher than at the start. The
AOX of aquarium 6 (control) was also higher than the AOX for the applied sea water. The reason for this is not
clear, but it is possible that the small amounts of natural EOX compounds present in sea sediments (Kankaanpaa &
Tissari, 1994a) may desorb from the bottom sediment to the aqueous phase.
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3.2. EOX levels in the aquaria after the experiment
To make sure that no BRMB components had been adsorbed irreversibly to the sediment, EOX was measured at
the end of the R period. These results are shown in Table 2.
AQUARIUM NO. OF SAMPLES AVERAGE EOX, (s)
(We dry weight)
1 2 0.76 (0.05)
2 3 0.13 (0.01)
3 2 0.19 (0.001)
4 2 0.20 (0.005)
5 4 0.12 (0.02)
6 3 0.10 (0.004)
Table 2. EOX concentration in sediment from test and control aquaria. s = standard deviation
The EOX levels in all aquaria sediments were low and identical, except for aquarium 1, where a slightly higher
concentration was found. This latter concentration is, however, on a level expected for an uncontaminated
sediment. No substantial changes were seen in the EOX content of M. [email protected] during the experiment (results not
shown). The exposure time was probably too short for bioaccumulation.
3.3. Total dark period activity in the aquaria
During the 12 day adaptation period animals in all aquaria showed increasing activity. From day 13, which was the
beginning of the C period, animals in aquaria 3,4, 5 and 6 continued to become more active before the T period. In
aquaria 1 and 2 the activity decreased slightly. The trends can be seen in Figure 4.
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day number day number
f
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malfimction of the photocells in aquarium 1 is seen as missing values and partly explains the low activity reading for
days 33 and 34.
An example of the hourly and periodical activity changes for aquaria 5 and 6 during days 17-30 is presented in
Figure 5. The animals were usually most active during the first hours of the dark period, after which their activity
levelled off until finally they stopped swimming at the beginning of the illuminated period. The effect of opening and
closing the BKME inflow in aquarium 5 is seen clearly. A more detailed analysis of the changes can be found in
section 3.4.
300
0
-
s-
20 8 20 ? 20 8 20 8 20 8 20 8 20 8 20 8 20 8 20 8 20 8 20 8 20 8 20 8 Hour
17 18 I9 20 21 22 Day of
23 24 25 26 27 28 29 j” expenment
/ m Aquarium 5 (low concentration) 0 Aquarium 6 (control) I
Figure 5. Activity during days 17-30 in aquaria 5 and 6. Illuminated (L) and dark (D) periods are illustrated.
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3.4. Variation of swimming activity within aquaria during experiment according to ANOVA
The variation of swimming activity of A4. afinis in the aquaria during different periods is presented in Figure 6 and
shown as the average activity (X) + % standard deviation (s) of activity. One-way ANOVA was used to check
whether the C, T and R periods were similar or not. The results of ANOVA are shown in Table 4. The activity
differences in aquaria 2, 3 and 5 are significant or very significant, as shown also by the REGWQ grouping: in each
case the C period is in another group than the T and/or R periods.
1 (control)’ 6 (control) 2 (low) 5 (low) 3 Aquarium
(high) 4 (high)
T period n R period 1
Figure 6. Variation of activity in aquaria. The variation is presented as X+O.5s
In aquarium 4 differences in the activity levels of the three periods do not represent significant statistical differences.
But, in the overall activity picture (Figure 4) an activity decline from day 22 to day 28 is clear, as is a sharp activity
increase just after the beginning of the R period. In aquarium 6 there was a slight difference in the activities of the
three periods (p=O.O392), but this is not seen in the REGWQ where the C, T and R periods are grouped together.
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AQUARIUM PERIOD REGWQ AVERAGE PERIOD N P SIGNI- GROUPING ACTIVITY FICANCE
NORMALSED TO NUMBER OF ANIMALS
1 (control)
6 (control)
2 (low)
5 (low)
3 (high)
4 (high)
C A T A R A
C A T A R A
C A T R A
C A T R A
C A T R
C A T A R A
42.8 45.0 40.8
47.1 7 60.8 8 0.0392 *
50.2 12
41.7 B 21.7 B 28.1
31.6 B 9.4
34.6
37.0 B 10.2 B 19.7
26.8 18.2 15.3
5 7 0.7704 n.s.
8 0.0063 **
12
8 0.0001 ***
12
8 0.0006 ***
12
8 0.1006 P.S. 12
Table 4. Results of one-way ANOVA of swimming activity changes in the aquaria. C period: days 13-19, T period:
days 20-27, R period: days 28-39. p-values and significances refer to differences in swimming activities within each
aquarium, not between aquaria. N means the number of observations. Limits: p > 0.05 not significant (n.s.),
p < 0.05 almost significant (*), p < 0.01 significant (**), p < 0.001 very significant (***).
4. DISCUSSION
The changes observed indicate that the circadian activity ofA4. u~nis is affected by the BKME levels used in the
experiments. Changes were obvious in three cases. In Aquarium 4, with high BKME concentration, swimming
activity during the T period tirst did not change, then decreased to almost zero at the end of the period. The
measured concentration of AOX in aquarium 4 in the end of the T period was intermediate (45 1 ti2) between the
highest concentration in aquarium 3 and the low concentrations in aquaria 2 and 5 (Table lb). The aberrant
behaviour of the animals in aquarium 4 is difficult to interpret. It is known that A4. afJinis may react to foreign
substances also by increased swimming activity (Lindstriim & Lindstrem 1980b). It is clear that the BKME affected
behaviour negatively. The statistical result from aquarium 4 (showing a 10.06% probability of periods being the
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same) can be questioned due to the disability of ANOVA to detect other than average value differences, e.g.
differences in slopes or trends in activity. Although the average activities during periods T and R in aquarium 2
were quite different (Table 4), ANOVA did not find any difference between the two periods. The ANOVA result
for aquarium 6 (control), showing some difference between the periods, can be explained by the high T-period
activity.
In all experiments, the activity of the animals increased when the flow of BKME was closed. In the experiments
with the lower concentration, the activity increased almost to the level of the control period within two days In the
experiments with higher concentration the activity was not equally high during the R period. This activity decrease
may indicate some level of poisoning, despite the low mortality in all experiments. This behaviour is much the same
as described during phenol experiments (Lindstrbm & Lindstrdm, 1980b).
A possible tendency to migrate away from areas polluted by BKME cannot be deduced from these results.
However, M. uffinis is a regressive species of the first order, which means that it is lacking in polluted areas
(Lepplkoski 1975) and may disappear with even low concentrations of pollutants (e.g. Luotamo & Luotamo 1979,
Rosenberg ef al.. 1975). The reason for this is not obvious, and the decrease in population size may be the sum
effect of several causes. In the present experiments swimming activity of the M. c&finis population decreased under
exposure to BKME in much the same way as Lindstram & Lindstrijm (1980b) demonstrated with exposure to
phenol and p-chlorophenol. This may be due to some kind of toxic influence on the central nervous system, but also
to an escape reaction into the sediment, where the BKME concentration is lower.
Some of the physiological and toxicological effects of BKME on marine organisms have been shown by, for
instance, Higashi et a/. 1992, Munkittrick et al. 1991, Pesonen & Andersson 1991 and Sodergren et al. 1993.
Negative changes in benthic animal communities caused by pulp mill discharges have been observed in the Baltic
Sea (SBdergren et a%, 1993). Reproduction and mortality rates in A4. affiis are affected by BKME contaminated
bottom sediment (Sundelin, 1988). On the other hand some organisms show no clear response to pulp mill effluents
(e.g. Miikelii et al., 1992). The acute toxicity of BKME and the relation of toxicity to AOX and EOX content varies
from species to species; in fact a direct correlation does not seem to exist (e.g. Craig et al., 1990). Furthermore, it is
also possible that the density of M. affinis may increase in relation to a fall off in the interest of predators during
BKME exposure (Leonardsson, 1993).
An alternative to toxicity tests is the monitoring of sublethal changes, which can be performed with species like
U @nis under laboratory conditions. The obvious problem in the mathematical analysis of results is that animals
do not always show stable activity before the actual test. In such cases a compromise must be made between a
prolonged control period starting time and the stability of the animals.
4470
Pesonen and Andersson (Pesonen & Andersson. 1992) showed that both bleached and unbleached krafl mill
effluents increased cytochrome P450-dependent 7-ethoxyresorufin-o-deethylase (EROD) activities in rainbow trout
(Oncor&rc~s @c&) hepatocytes. Furthermore, both p-chlorophenol and phenol have been found to inhibit
A4. a$% activity (Lindstom & Lindstrom, 1980b). According to these results we can expect analogically that the
changes in the swimming activity of A4. afJlis found cannot be attributed solely to the chlorinated compounds in
BKME. Most likely the effect is caused by the whole pool of organic compounds present in the effluent. That the
sum parameters AOX and EOX are used to express the BKME levels applied is a question of convenience.
Due to the long residence-time of EOX and AOX substances in sediments (Wulff et al., 1993) benthic animals can
be exposed to sublethal concentrations of organohalogens for substantial periods of time. Even if the BKME
concentration is not affecting the physiology of animals in a toxic way, on a longer time-scale decreased activity
might have a negative effect on the M. uflnis population simply by decreasing the probability of animals meeting to
copulate during the maturation time in autumn (Lindstrom and Lindstrom, 1980b).
5. CONCLUSION
On the basis of the activity changes observed our conclusion is that bleached krafi mill effluents diminish the
nocturnal swimming activity of A4. A&is but do not change the rhythmicity (for rhythmicity of A4. u&fmis, see
Dormer & Lindstom, 1980). Recent control and reduction of the chlorine bleaching process in the pulp and paper
industry works to prevent chlorinated compounds accumulating in the bottom sediments, but these measures may
not be sufficient if the lethal and sublethal effects of pulp mill effluents are caused in part by the non-chlorinated
compounds in those effluents.
ACKNOWLEDGEMENTS
For the animal and sediment samples we thank Ms. Ann-B&t Andersin from the Finnish Institute of Marine
Research (FIMR). For assistance in obtaining the effluent we thank Ms. Riitta Saares (National Board of Waters and Environment) and Mr. Ilppo Kettunen (Kyrni Water and Environment District). Ms. Riitta Olsonen (FIMR) gave us invaluable help with statistical calculations. We also thank Mr. Kari Lehtonen (FIMR) for valuable comments on the manuscript and Mr. Richard Thompson Coon for correcting the English language.
4471
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Craig, G., P. Orr, J. Robertson & W. Vrooman, 1990. Toxicity and bioaccumulation of AOX and EOX. Pulp Paper Can., Vol. 91 (9) pp. 39-45.
Dormer, K. O., 1971. On vision in Pontoporeia affinis and P. femorata (Crustacea, Amphipoda). Comment. Biol.,
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