EPA/600/R-94/155
September 1994
ENVIRONMENTAL STUDIES
IN THE
NEMUNAS RIVER BASIN, LITHUANIA
Edited by
Robert V. Thurston
•Fisheries Bioassay Laboratory
Montana State University
Bozeman, Montana 59717
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ATHENS, GEORGIA 30605
Printed on Recycled Paper
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DISCLAIMER
The information in this document has been funded in part by the United States
Environmental Protection Agency. Papers describing EPA-sponsored research have
been subject to the Agency's peer and administrative review, and have been approved for
publication. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use by the United States Environmental Protection
Agency.
11
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FOREWORD
Scientists from Lithuania and the United States have been involved in a joint
research program since 1988 on the fate and effects of pollutants in the environment
Cooperators on the Lithuanian side have included scientists from the Lithuania
Environmental Protection Department, the University of Vilnius, and several Institutes
within the Lithuania Academy of Sciences. The U.S. scientists were from the
Environmental Research Laboratory, Athens, Georgia (ERL-Athens), U.S.
Environmental Protection Agency, and the Fisheries Bioassay Laboratory at Montana
State University (MSU), Bozeman, Montana.
Research under this cooperative effort has developed in five technical areas:
(1) Identification of organic and inorganic chemical pollutants and monitoring of
chemical and biological parameters in Lithuanian surface and ground waters;
(2) Ecological and water quality modeling, including evaluation of assumptions for
modeling fate and effects of pollutants; (3) Measurement of transformation and
equilibrium constants for predicting fate of pollutants; (4) Investigation of
microbiological transformation processes; (5) Toxicity testing of identified pollutants in
biological species indigenous to the Baltic Republics.
Since the initial establishment of goals for this joint research effort in 1988, six
scientists from ERL-Athens and MSU have visited Lithuania for 1-4 weeks on ten
occasions, and six Lithuanian scientists have visited the U.S. for 2-18 weeks for training
at MSU and ERL-Athens. During their trips to Lithuania, U.S. scientists have had the
opportunity to work and exchange ideas with several Lithuanian scientists involved in
closely related areas of environmental research. The present report results from this
association. It contains 17 papers authored by 56 scientists who share a joint concern for
the ecological health of this Baltic Republic that has only recently been able to begin to
deal with the consequences of the chemical insults to its environment over the past
several decades.
Rosemarie C. Russo, Ph.D., Director
Environmental Research Laboratory
Athens, Georgia, USA
111
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ABSTRACT
Selected research results from a joint environmental studies project that began in
1988 between Lithuania and the United States of America are presented. Chemical and
hydrological studies address hazardous chemicals, including trace metals, in surface
waters of Lithuania; methods for determining heavy metals (an interlaboratory
comparison); identification of chemicals at pesticide storage sites in Lithuania; and
monitoring pollutant loads in the Nemunas River. Biological studies examine'
biodegraders associated with hydrobionts; toxicity of acetanilide herbicides; water quality
in Kursiy Marios Lagoon; cytogenetic changes in freshwater bivalve molluscs;
morphological changes of bivalve molluscs under heavy metal bioaccumulation;
carcinogenesis research on fishes; toxicity of wastewaters and heavy metals to rainbow
trout; and fish avoidance reactions to pollutants. Radionuclide studies investigate
strontium 90 concentrations in the KurSiu Marios Lagoon ecosystem and thyroid diseases
m Lithuania. Baltic Sea studies examine the mechanism of hydrogen sulfide oxidation
and biological consequences of deep water stagnation.
IV
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CONTENTS
Foreword . . jy
Abstract jv
Acknowledgements YJJ
CHEMICAL AND HYDROLOGICAL STUDIES
Hazardous Chemicals in Surface Waters of Lithuania, 1990-1993.
JJ. Ellington, R.V. Thurston, J. Sukyte, and K. Kvietkus 1
Trace Metal Concentrations in Natural Waters of Lithuania, 1991-1992.
K. Kvietkus, D. Ceburnis, J. Sukyte, JJ. Ellington, and R.V. Thurston ... 19
Intel-laboratory Comparison of Methods for the Determination of Heavy Metals in
Natural Waters.
K. Kvietkus, D. Ceburnis, JJ. Ellington, arid Y. Shane Yu 31
Chemicals Identified at Two Pesticide Storage Sites in Lithuania, 1990-1993.
JJ. Ellington, J. Sukyte, N. Striupkuviene, and J.F. Neuman 39
Problematic Aspects of Nemunas River Load Monitoring.
S. Zareckas, A. Galkus, and K. Joksas 47
BIOLOGICAL STUDIES
Investigation of Microorganisms: Biodegraders Associated with Hydrobionts.
J. Syvokiene and L. Mickeniene 59
Toxicity of Acetanilide Herbicides and their Biodegradation in Pure Pseudomonas
Cultures.
A. Cetkauskaite, V. Jankauskas, J. Berzinskiene, E. Bakiene,
and W.C. Steen 65
Hydrobiological Condition and Water Quality Estimation of Kur§h| Marios
Lagoon, 1991.
A. Antanyniene, A. Baranauskiene, S. Budriene, G. Jankaviciute,
K. Jankevicius, J. Kasperoviciene, A. Kucinskiene, S. Mazeikaite,
G. Slapkauskaite, R. Sulijiene, and I. Trainauskaite 73
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Cytogenetic Changes in Freshwater Bivalve Molluscs from the Nemunas River
and Kursii| Marios Lagoon.
J. Barsiene, T. Virbickas, and D. Barsyte 85
Morphological Changes in Bivalve Molluscs Under Different Levels of Heavy Metal
Bioaccumulation.
L. Lazauskiene and G. Kaspariunaite 95
Carcinogenesis Research on Fishes of Kursiij Marios Lagoon, 1991.
E. Bukelskis and D. Serelyte 103
Toxicity of Factory Wastewaters and Heavy Metal Solutions to Rainbow Trout.
Z. Vosyliene, G. Svecevicius, and S. Syviene 107
Avoidance Reaction to Pollutants by Vimba Under Laboratory and Field Conditions.
G. Svecevicius . 115
RADIONUCLIDE STUDIES
Total jS-Activity and 90Sr Concentration in the Kurshj Marios Lagoon Ecosystem.
R. Dusauskiene-Duz 123
Investigations of Thyroid Diseases in Lithuania in Relation to Radiation Doses
from Chernobyl.
V. Sidlauskas, J. Danys, A. Krasauskiene, A. Aukituolyte,
D. MaSanauskaite, R. Jurkunaite, A. Telksnys, E. Janulionyte,
J. Sviciulyte, T. Nedveckaite, V. Fih'stovicz, and A. Mastauskas 131
BALTIC SEA STUDIES
Mechanism of Oxidation of H2S in the Baltic Sea.
J. Sukyte, V. Zelionkaite, E. Rinkevichiene, and V. Razumovskij 137
Some Biological Consequences of Deep Water Stagnation in the Eastern Gotland
Basin, Baltic Sea in 1980s.
S. Olenin 147
Index by Author 157
VI
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ACKNOWLEDGEMENTS
The Editor and the authors extend their thanks to the many reviewers who have
made comments and suggestions on these papers when they were in manuscript stage.
Reviewers at ERL-Athens were Robert Ambrose, Rochelle Araujo, Leo Azarraga,
George Bailey, Craig Barber, George Baughman, Donald Brockway, Timothy Collette,
Ray Lassiter, MacArthur Long, Nicholas Loux, William Payne, Susan Richardson, John
Rogers, William C. Steen, Luis Suarez, and N. Lee Wolfe. Reviewers at MSU were
Keith Cooksey, Stephan Custer, Dave Dooley, Gil Geesey, Eric Grimsrud, Algirdas
Jesaitis, Andrew Marcus, Gordon McFeters, and Samuel Rogers. Special assistance
during editing stages was provided by Darius Sabaliunas at Vilnius University, Janina
BarSiene at the Institute of Ecology in Vilnius, and Birute Bielinyte and Jonukas
Neuman at MSU. The final document was prepared by Susan Clark.
vn
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HAZARDOUS CHEMICALS IN SURFACE WATERS
OF LITHUANIA, 1990-1993
JJ. Ellington1, R.V. Thurston2,
J. Sukyte3, and K. Kvietkus4
ABSTRACT
Water samples were collected from 45 locations throughout Lithuania between
1990 and 1993 as part of a study to determine the distribution and nature of hazardous
chemicals in Lithuanian surface waters. Organic chemicals were extracted from the
water samples by use of solid phase extraction cartridges or disks in Lithuania, and the
cartridges and disks were taken to the United States for elution and analysis by means of
gas chromatography and infrared/mass spectrometry. The herbicide, alachlor, was
identified in a sample taken from Klaipeda Harbor, and 2-(methylthio)benzothiazole and
a dialkylthiophene were identified in a sample taken from the Nemunas River at
Grigiskes, in addition to the expected hydrocarbons and fatty acids detected at several
locations. Five hydrological water quality variables (pH, temperature, dissolved oxygen,
conductivity, and redox potential) were measured at most of the sampling sites.
Several water bodies were identified as being in a state of decline.
INTRODUCTION
A field study on the quality of surface waters in the Republic of Lithuania was
initiated in 1990 under the sponsorship of the risk assessment program of the United
States Environmental Protection Agency (U.S. EPA), Environmental Research
Laboratory, Athens, Georgia (ERL-Athens). During six separate visits in December
1990, July 1991, June and July 1992, and June and October 1993, water samples were
collected from 45 locations and sediment samples from two locations throughout
Lithuania. These visits were conducted in cooperation with scientists from the Lithuania
Environmental Protection Department, the Lithuania Academy of Sciences, and Vilnius
University. Sampling sites were located on the Nemunas River and five of its tributaries,
Kaunas Reservoir, Kursnj Marios Lagoon, Klaipeda Harbor, the Baltic Sea coast, Lake
Dusia, Lake Druksiai, and the Kulpe River (Figure 1, Appendix A). Organic chemicals
were extracted from the water samples by use of solid phase extraction cartridges or
disks and these cartridges and disks were taken back to ERL-Athens for elution and
analysis by means of gas chromatography and infrared/mass spectrometry.
Environmental Research Laboratory, U.S. Environmental Protection Agency, Athens, Georgia, USA
^Fisheries Bioassay Laboratory, Montana State University, Bozeman, Montana, USA
3
Lithuania Environmental Protection Department, Vilnius, Lithuania
Institute of Physics, Lithuanian Academy of Sciences, Vilnius, Lithuania
1
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During the 1990-1992 expeditions, hydrological water quality variables were also
measured at each site using a remote sampling probe. Water samples were also
collected from monitoring wells at a controlled landfill for pesticide wastes at
Zigmantilkes and from runoff water at an agrochemicals storage facility at Utena; results
of analysis of those samples are reported separately (Ellington et al 1994).
43
Figure 1; Field study water sampling stations.
METHODS
Hydrochemical and Physical Water Quality Variables
On-site water quality measurements in 1990 and 1991 were made with a Hydrolab
Surveyor 2, and in 1992 with a Surveyor 3 (Hydrolab Corporation, Austin, Texas, USA),
equipped with pH, temperature, dissolved oxygen (DO), conductance, redox potential,
and depth sensors. The pH electrode (accuracy +0.2 units, resolution +0.01 unit) was
calibrated with buffer solutions at pH 3, 7, and 10. The temperature sensor
(accuracy ±0.2°C, resolution ±0.01) was verified by simultaneous emersion in a flask of
water with a thermometer certified by the U.S. National Institute of Standards and
Technology.
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The DO sensor (accuracy ±2%, resolution 0.01 mg/L) was calibrated by the saturated
air method as described in the instrument manual. The conductance sensor
(accuracy ±1% of range resolution 4 digits) was calibrated with solutions of potassium
chloride prepared with distilled, deionized water. The redox potential sensor
™T wC7 ? « mV', resoluti™ 1 mV> was 'aerated with quinhydrone dissolved in pH 4
and pH 7 buffer solutions. The stated accuracies are the variations anticipated by L
manufacturer if measurements are made under ideal conditions.
Blanks and Filter Preparation for Collection of Organic Chemicals
nf ffc concentration of organics from the water samples were
of three types: Bakerbond™ SPE cartridges (6 ml) that contained 500 mg of LyfaflSL
500 or ^1000 ±Cn?rar ( r1^ B"^* ***£**" cartridSes (6 **) that contained
50n ™ f ™g nil °f 18,' ?d EmP°re extraction disks (47 mm) consisting of
500 mg of C-8 or C-18 suspended in a Teflon fibril network. The Bakerbond™
cartridges were precleaned at ERL-Athens by removing lettering on the cartridges with a
tissue soaked m hexane and extracting the cartridges in a Soxhlet apparatus overnight
with hexaneracetone (9:1). The Burdick and Jackson cartridges and the Empore^d isles
were precleaned by applying vacuum and pulling through the sorbent bed, in sequence
*vo 5-ml portions of the final extraction solvent, methanol, and organic-free distilled, '
deionized water. The Bakerbond™ and Burdick and Jackson cartridges were capped
with solvent-cleaned aluminum foil and stored in screw-cap glass vials until sample
st±d TH t f mP,°re diSl?. r rC ™PP&d flat in ^It-cleaned aluminum' foil and
stored and transferred in petn dishes. Two blanks of each solid phase sorbent were left
refrigerated at ERL-Athens during each sampling trip and two were taken on the
f 6 thCy Were kept ^ the solid Phase sorbent containers and returned
f extraction and analysis together with the samples. Silicone tubing
) was used for pumping water during some sampling regimes, and thislas
checked for leachable organic contaminants by slicing a 2-cm portion of the tubing into
cross sections approximately 3 mm thick, and extracting these by immersion under ethyl
ether with frequent stirring for 6 hours. Organic chemicals in the ethyl ether extract
were identified before and after treatment with diazomethane. Low levels of silica-
contammg compounds, hydrocarbons, and alkyl fatty acids were detected in the extract
Sampling Techniques in the Field for Organic Chemicals
Water samples were filtered through borosilicate microfiber prefilters (Micro
Filtration Systems), 47 mm diameter, to remove particles > 2.7 Mm at time of collection
or within 24 hours thereafter. The extraction cartridges and disks were activated with '
methanol according to supplier specifications prior to concentration of the organic
chemicals. The C-18 cartridges were used to concentrate nonpolar organic chemicals
from the water samples. Carboxylic acids and phenols were extracted from water
samples by adjusting the pH of the sample < 2 to protonate the anions before passage
of the water through the prefilter and the C-8 cartridge or disk
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A hand pump (Masterflex^) was ujed in th§ field t9 force thf water s
through the prefflter contained in an in-line 47-flim filter hglder, then through the
cartridges at the exit end of th§ sjlicgne tub|ng. When laboratory facilities were
available, water samples were pumped through the prefilters. at the sampling site and
then transported to the labgrafgry where they werg pulled thrgugh the cartridges and
disks by vacuum supplied by water aspiratjgn gr mechanical pump, The amount of
water sampled at each location varied frgrn 0.2. |g 1.5 L? but generally water was pumped
or pulled through the SPE cartridges, gr disks until flgw decreased tg a few ml/minute.
Upon return tg ERL-Athens, the SPg cartridges and disks were stored at 4qC until
ehjtign, and analysis,
Extraction and Deriyatjzation of Organic Chemicals
Prior to eluting the §grbed grganics frgm tfeg £PE cartridges with solvent, excess
water was removed fey applying vacuum, ear^gxyiic acids, and phenols were eJuted from
the C-S cartridges with I m] m*ethangi/acetgne §;1 Neutrals w§r& similarly ejuted frgm
tfte C-18 cartridges with 5 m] he^ane/acetgne §?1. Thf d|§k§ were placed in a test twbe
with | ml of hexane/3cetgn§ g;l and §gnicated fgr 2, minijtes, and, after standing fgr
5 niinutes the solvent wa§ trangferrgd tg §ngther tub§ and the extractign repeated. In
199? the extractign seqiieace fgr the disk§ w§§ ehgnged tp a sequence gf 5 ml of ethyl
acetate, 5 m] of methyiene chjgride, and finally § mj gf ethyl acetate/methylene chloride
(1:1) (U.S. EPA 1991), Extracts were reduced to a final vglume of 1 ml, Methyl esters
gf carbgxylic acids wer§ fgrmed in the extract frgni the C-§ cartridges, which were thgse
lifd fgr extractign of the plf 2 water samples, by feufebjing gaseous diazgmethane
thrgugh the final vgju.me.
The sediment §aniple§ cgjjected for §naJy§J§ of grganic chemicals were extracted
according to U.S. EPA Method §l§l for th§ g^Stlgn gf herbicide§ and U& EPA
Method 3550A fgr the ejctrsstign gf base/neutra}§ (U,S, EPA 1992), The appropriate
surrogate and matrix spil?e§ a,nd Bjethgd blank§ w§r§ P^rfornied m prescribed.
Gas; Chroniatography (GC) Analysis
High resglutlgn QQ §n§lys|s gf sample extr§6t§ was performed with a Hewlett-
Packard model 589Q QC §quipped with both fla.jne ignizatlon (FID) and ejectron
capture (BCD) detectgrg, splifjess inlet, and awt9§anipler (76?3A). Separa,tign gf sample
cginpgnents was achieved using a 30 m x 0.32 miu i.d.". x 0-25 /tm film PEI-5 capillary
column gperatied initially at 4Q"°C fgr 2 m|ni!te§? thefl temperature-prggrammed to 290PC
at 8°/m|nute. Helium carrier gas was used at ft flow rate of 30 pm/second. Detectgr
temperatures were §§t at 300eC^ (FIP) and 3£Q8g (IPD)- Injectjgns gf 1 /*L were made
at an inlet temperature Q
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GC/Mass Spectrometry (GC/MS) and GC/Fourier Transform-Infrared
Spectrometry/Mass Spectrometry (GC/FT-IR/MS) Analysis
Low resolution electron-impact (El) GC/MS was performed on either a Finnigan
4500 (Finnigan MAT, San Jose, California), or a Hewlett-Packard (Palo Alto, California)
mass selective detector (MSD). Chemical ionization (CI) mass Spectrometry with
methane gas was performed on the Finnigan 4500 system. High resolution El GC/MS
analyses of sample extracts were performed on a VG 70-SEQ high resolution hybrid
mass spectrometer (VG Instruments, Manchester, United Kingdom). The GC/FT-IR/MS
analyses were performed on a Hewlett-Packard system equipped with a 5965B infrared
detector and a 5971 series mass selective detector. The gas chromatographs attached to
the mass spectrometers were equipped with capillary columns similar in dimensions,
liquid phases, and flows to the one described above; the temperature program was also
the same.
SAMPLING PROGRAM, RESULTS, AND DISCUSSION
Hydrochemical and Physical Water Quality
In December 1990, the measured pH values ranged from a low of 7.5 in the
Kulpe River at Siauliai (LT-17) to a high of 8.7 at Nida (LT-08) (Appendix B). The
high temperature, low DO, and high conductivity (1.55 mS/cm) at Siauliai were due to
the high volume of industrial effluent released into the Kulpe River as it flows through
an industrial section of Siauliai. The other values reported in Appendix B are within the
ranges expected for the time of year and the high volumes of surface water caused by
heavy rainfall.
In July 1991, the surface DO levels in Kaunas Reservoir were higher at the
sampling sites closer to the dam and above deeper water (LT-19A, -19B, -20, -21,
and -22) (Appendix C). The DO levels in the Nemunas River below Kaunas Reservoir
and continuing into Kurshj Marios Lagoon were also lower than in other surface waters.
The Kulpe River at Siauliai (LT-17) again had a high conductivity value (2.14 mS/cm)
for surface water and extremely low DO (0.53 mg/L).
In June 1992, the measured pH and DO values were higher than those measured
in July 1991 at all but two sampling sites, Melnrage (LT-11) and Rasyte (LT-30)
(Appendix D). The pH value of 9.5 and DO value of 17 mg/L recorded at Lake
Druksiai (LT-43) are the result of extensive photosynthetic activity by the dense plant
growth in the lake, both standing and planktonic. This photosynthetic activity, while
raising the level of molecular oxygen, also increases the hydroxyl ion load with a
corresponding increase in pH.
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Organic Chemicals in Water
In December 1990, water measurements were made and samples collected on
SPE cartridges from 24 sites in Lithuania, including the Vilnia, Neris, Nevezis, Kulpe,
and Nemunas Rivers, Kaunas Reservoir, and Kursnj Marios Lagoon (Appendix B).
Upon return to ERL-Athens, the sorbed organics were eluted and GC-FID and
GC-ECD chromatograms were obtained. The retention times of all the major peaks in
the chromatograms from the surface water samples were identical. GC/MS analysis of
the 1990 surface water extracts identified the major peaks as several dialkyl phthalates,
silica-containing components, saturated hydrocarbons, and some unidentified peaks with
the same mass fragmentation pattern that appeared in every extract. Dialkyl phthalates
are commonly found in environmental samples and stringent precautions are necessary
to assure their presence in the collected sample is not due to accidental contamination.
Hydrocarbons, phthalates, and fatty acids were found in a few of the method and field
blanks but at lower levels than in the samples; the presence of phthalates in the samples
may be real. Absence of man-made chemicals other than possibly phthalates in the
surface water samples was surprising, but could have been caused by the ambient water
temperature (4°C) at this time of year, and the high levels and flows of water from
recent heavy rainfall. Both of these conditions would lower the concentration of organic
chemicals in the surface water, possibly below the level of detection for the volume of
water sampled. Organic chemicals identified in water samples collected during this and
subsequent expeditions are listed in Table 1.
Table 1. Hazardous chemicals identified in surface water samples.
Chemical
Alachlor1
Alkyl hydrocarbons2
Aromatic hydrocarbons2
Dialkylthiophene2
2,4-Dichlorobenzoic acid1
Fatty acids2
2-(MethyIthio) benzothiazole2
Phcnanthrene carboxylic acid2
Phthalates1-3
Silicon organics2
Tctrachlorinated unknowns2
Grigiskes
(LT-31)
X
X
X
X
X
X
X
X
X
X
Kaunas Reservoir
(LT-19-22)
X
X
X
X
X
X
X
Green Harbor
(LT-52)
X
X
X
X
'Identified by GQFT-IR/MS and confirmed with a standard
identified by GQFT-IR/MS but not confirmed with standards
3Diisooctyl-, Butylbenzyl-, Di-n-ocryl-, and Di-n-butylphthalates were observed the most frequently
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In July 1991, 15 of the former sites were sampled, as were eight new locations
including the Salcia River (LT-32) downgradient from the long-term pesticide storage
site at Zigmantiskes (LT-24) (Ellington etal 1994), the Neris River (LT-31) downstream
from Vilnius below a paper manufacturing plant, and the Merkys River (LT-33)
downstream from a combined poultry growing and processing facility (Appendix C).
Low resolution GC/MS indicated the presence of three tetrachlorinated isomers in the
diazomethane-treated sample extracts from the Neris River and Kaunas Reservoir
(LT-20) (Figure 2).
zoooooo -
1000000 -
Figure 2. Total ion mass chromatogram of the solid phase extraction eluant from
Kaunas Reservoir. GC/MS Peak 1 = 2,4-dichlorobenzoic acid methyl
ester. GC/MS Peaks 2, 3, 4 = unknown tetrachlorinated isomers.
High resolution El GC/MS of the three tetrachlorinated peaks in the Kaunas
Reservoir water sample extract yielded best fit empirical formulae of either C7H13C14O5P
or C14H8C14O2. A tetrachlorinated trialkyl phosphate fits the former formula while
tetrachlorinated aromatic esters and other structures containing carbonyl groups can be
drawn for the latter. Both formulae are similar to those of compounds commonly used
as electrical insulators and flame retardants. The compound containing phosphorus was
eliminated as a possibility by further GC analysis of the sample using nitrogen and
nitrogen-phosphorus specific detectors, respectively. The presence of the carbonyl
functional group in the three unknowns was confirmed by GC/FT-IR. The loss of the
m/z 31 ion (loss of —OCH3) during electron impact GC/MS argued for the presence of a
methoxy functional group, possibly a methyl ester. However, the absence of a strong
peak in the 1250-1310 cm"1 region of the IR spectrum and the broad carbonyl peaks
argued against the ester functionality. Detection of the unknowns only after treatment
of the extract with diazomethane is evidence for a reactive hydrogen needed to form a
methoxy group. A tetrachlorinated aromatic ring system containing a carbonyl group,
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methoxy group, and differing chlorine substitution is the most likely structure for the
three unknown isomers. The surface water samples collected at Kaunas Reservoir and
the Neris River at Grigiskes (LT-31) also contained 2,4-dichlorobenzoic acid in addition
to the three tetrachlorinated unknowns. Fatty acids, identified in the Kaunas Reservoir
and GrigiSkes samples as their methyl esters, could be an indication of contamination by
municipal sewage.
In June 1992, samples were collected at some of the sites previously sampled and
new sampling sites were added at Lake Dusia, Lake DrukSiai, and three locations on the
Nemunas River, two of these upstream from Kaunas Reservoir, and one downriver from
Gardinas in Belarus (Appendix D). Lake Druk§ai is both the source of and receiving
body for the cooling water for the Ignah'na nuclear power facility. Chemicals tentatively
identified in surface water samples, but not confirmed with standards, included
2,2-dimethoxy-l,2-diphenyl ethanone, dialkylthiophene, and 2-(methylthio)thiophene.
During June 1993, water samples were collected at Juodkrante, Klaipeda Harbor,
and at three new sampling sites: Amber Cove (LT-51) and Green Harbor (LT-52) in
the Klaipeda Harbor area of Kurshj Marios Lagoon, and the Aitra River (LT-53).
Organjcs were extracted with Empore™ disks from the Klaipeda Harbor water samples,
but only the Green Harbor sample was analyzed by GC/FT-IR/MS. This site is located
at the mouth of a channel that is used for drydock-ship repair, and sewage from the
Klaipeda municipal treatment facility is discharged into this channel. In addition to the
expected fatty acids, hydrocarbons, and phthalates tentatively identified by interpretation
of MS and IR spectra, an herbicide (alachlor) was identified and confirmed with a
standard. The alachlor was present at approximately 3 ppb. The source of the alachlor
remains unknown and it is uncertain if this concentration of alachlor poses a threat to
aquatic organisms. The only compounds detected in the Aitra River sample were sulfur,
trace amounts of phthalates and hydrocarbons, and low molecular weight carboxylic acids
in the 10-40 ppb concentration range.
Organic Chemicals in Sediments
In June 1992, two sediment samples were collected for analysis for organic
chemicals. One of these samples was collected off the mouth of the Gilija River (LT-06)
as it flows into Kursiij Marios Lagoon, and the other from the Neris River at GrigiSkes
(LT-31), downstream from Vilnius. After extraction of the organics, the resulting
acid/herbicide and base/neutral extracts were analyzed by GC/FT-IR/MS. Fatty acid
methyl esters, hydrocarbons and the methyl ester of phenanthrene carboxylic acid were
the only compounds identified with a high degree of confidence using both the spectral
matching library and manual interpretation. The presence of multichlorine substitution
was indicated in the mass spectra of several peaks in each extract but tentative
identifications were not possible. The only compounds other than hydrocarbons
identified in the base/neutral extracts were cholesterol in the Grigiskes sample and the
octyl ester of phosphoric acid in the Gilija sample. Alkyl phosphate esters are
commonly used in lubricating greases, and this is one possible source of the octyl ester.
8
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CONCLUSIONS AND RECOMMENDATIONS
The majority of the population and industry of Lithuania are located in the
Nemunas River basin which includes the Neris River. Anthropogenic chemicals were
detected in only a few of the surface water samples, and we cannot explain the reasons
for this. For the December 1990 samples, this might have been at least in part because
of a combination of the low water temperatures and the dilution of surface waters from
heavy rainfall during that season of the year. Failure to detect a larger number of
chemicals in the samples collected during the summers of 1991-1993 might have been
partially the result of a national economic recession, with commensurate decrease in
industrial activity. The low levels of DO (<5 mg/L) in the Kaunas Reservoir in July
1991 is an indication of a water system in decline, as are the extremely high DO
(17 mg/L) and pH (9.5) measurements in Lake Druksiai.
Other sites of concern are the Kulpe River in Siauliai, outside the Nemunas River
basin, which receives the majority of its total flow within the city limits from industrial
and municipal sources, and the Deime River flowing from Kaliningrad into the southern
end of Kurliij Marios Lagoon. Analysis of sediment samples from rivers and lakes
should be given a high priority, inasmuch as sediments contain the historical burden of
pollution and are one of the eventual sinks for pollutants.
ACKNOWLEDGEMENTS
Ruta SiuSiene, Jonas Masiulionis, Birute Simanskiene, and Albinas Feilius of the
Lithuania Environmental Protection Department, Janina Barsiene of the Institute of
Ecology, and Anolda Cetkauskaite and Valdas Jankauskas of the University of Vilnius
assisted with water sample collections and water quality measurements made in the field.
Nijole Striupkuviene and Svetlana Urboniene of the Lithuania Environmental Protection
Department assisted in preparation of samples prior to transportation to ERL-Athens.
Transportation for our expeditions in Lithuania was provided by the Lithuania
Environmental Protection Department. The LR-EI and CI GC/MS analyses were
performed at ERL-Athens by John Pope and Alfred Thruston, Jr., the high resolution
GC/MS analyses by Susan Richardson, and the GC/FT-IR/MS analyses by Timothy
Collette and George Yager; the solid phase extraction devices and the extracts were
prepared by Terry Floyd. This research was supported in part by U.S. EPA Cooperative
Agreements CR816369 and CR995189 to Montana State University.
REFERENCES
Ellington, J.J., J. Sukyte, N. Striupkuviene, and J.F. Neuman. 1994. Chemicals
identified at two pesticide storage sites in Lithuania, 1990-1993.
In: Environmental Studies in the Nemunas River Basin, Lithuania.
U.S. Environmental Protection Agency, Athens, Georgia, USA.
-------
LEPD (Environmental Protection Department of the Republic of Lithuania). 1993.
Water Quality of Lithuanian Rivers, 1992. Annual Report. Vilnius.
U.S. EPA (U.S. Environmental Protection Agency). 1991. Method 525.1 In: Methods
for the Determination of Organic Compounds in Drinking Water. EPA/600/4-
88/039 Revised July, 1991. U.S. EPA, Washington, D.C., USA.
U.S. EPA (U.S. Environmental Protection Agency). 1992. Methods 8151 and 3550A.
In: Test Methods for Evaluating Solid Waste. Physical/Chemical Methods SW-
846, Revised 1992, Third Edition, Update II. U.S. EPA, Washington, D.C., USA
10
-------
Appendix A. Field study water sampling stations
Number
LT-01
LT-02
LT-03A
LT-03B
LT-04
LT-05
LT-06
LT-07
LT-08
LT-09
LT-10A-B
LT-11A-D
LT-12
LT-13-1,2
LT-14
LT-15A-C
LT-16
LT-17
LT-18
Name
LavoriSkes
Pavilnys
Pioneer Camp
Turniskes
Gediminas Castle
Gariunai
Gilija
Uostadvaris
Nida Station
Juodkrante
Klaipeda Harbor
Melnrage-A-D
Palanga
Sovetskas
Smalininkai
Kulautuva-A-C
Panevezys
Siauliai
Kedainiai
Description
Vilnia River, ca 40 km above Vilnius at hay field
Vilnia River, ca 8 km above Vilnius at dam
Neris River, ca 16 km above Vilnius at Young Pioneer Camp
Neris River, ca 1-2 km below Pioneer Camp, and 102 km above
Turniskes
Vilnia River, ca 200 m above confluence with Neris River
Neris River, ca 2 km below Vilnius wastewater treatment facility,
near field with abandoned Soviet tanks
Kursiij Marios Lagoon off the mouth of the Gilija River
(Matrosovo Kanalas)
Kursiu. Marios Lagoon, ca 10 m offshore, at Uostadvaris
Kursiij Marios Lagoon, ca 200 m offshore at Nida
Kursiij Marios Lagoon, at Juodkrante from the shore at boat
beaching area
Klaipeda Harbor, east side, from the dock, north side of Dane
River mouth
Baltic Sea, at Melnrage, (A) from the end of breakwater, near
grounded freighter, "Rudolph Breitscheid", (B) from boat 100 m
west of "Rudolph Breitscheid", (C) from boat immediately inside
mouth of breakwater, (D) from boat inside mouth of breakwater,
opposite stone tower and ca 100 m downstream from museum
Baltic Sea, at Palanga from the pier 50 m from shore
Nemunas River, At Sovetskas, west of bridge, ca 3 km below one
paper plant and ca 11 km below another, (1) from north bank,
(2) from boat at river center
Nemunas River, at Smalininkai from the north bank near houses
Nemunas River, ca 13 km below Nemunas-Nevezis confluence, (A)
from north bank, (B) south bank at Zapyskis, (C) north bank ca 1
km below Zapyskis
Nevezis River, ca 1 km below discharge from Panevezys wastewater
treatment facility, south bank ca 200 m from abandoned church
Kulpe River, ca 100 m below abattoir and ca 1 km downriver from
Siauliai
Nevezis River, ca 15 km below wastewater treatment facility, from
south bank at collective farm
(Continued)
11
-------
Appendix A. Continued
Number
LT-19A
LT-19B
LT-20
LT-21
LT-22
LT-23A
Name
Nemunas, II Cut
Lapaine
Kaisiadoriu
Dambrava, IV Cut
Pazaislis, VII Cut
Sand Beach
Description
Kaunas Reservoir, upper end inside Nemunas River, before
discharging into the reservoir
Kaunas Reservoir, upper end even farther inside Nemunas River
Kaunas Reservoir at Hydroaccumulation Electric Power (HAEP),
near middle, 200 m offshore
Kaunas Reservoir, near sauna, 200 m offshore
Kaunas Reservoir, near dam, 400 m off Pazaislis Monastery
Nemunas River, below mouth of Neris River and above mouth of
LT-23B
LT-24A-F
LT-26A-B
LT-27
Lampedziai
Pesticide Storage
Site
Raudondvaris
Neris at Kaunas
Nevezis River, on south bank across from heavy industry
Nemunas River, below mouth of Neris River and above mouth of
Nevezis River, on north bank ca 500 m below LT-23A
Ministry of Agriculture Pesticide Storage Site southwest of Vilnius,
ca 3 km north of Salcia River, Monitoring Wells #1-6 inside the
site
NeveSis River, (A) ca 25 km above confluence with Nemunas,
from west bank, (B) ca 1 km above confluence with Nemunas, from
west bank
Neris River at last bridge confluence with Nemunas River, from
north bank
LT-28A Rusne-A
LT-28B Rusne-B
LT-29 Deime
LT-30 Rasyte
LT-31 Grigiskes
LT-32 Zigmantiskes
LT-33 Jasiunai
LT-34 New Vilnia
LT-38 Sredny Gonkolsky
LT-39 LiSkiava
Lt-40 Lake Dusia
LT-41 Atytus
Nemunas River at bridge, ca 10 km before Kurshj Marios Lagoon
Nemunas River, ca 2 km .above bridge
Deime River, immediately inside mouth before flowing into
southernmost section of Kursh} Marios Lagoon
Kurshj Marios Lagoon, approximate center
Neris River, ca 17 km west o£ Vilnius, from south bank
Salcia River, ca 3 km south of Pesticide Disposal Site
Merkys River, ca 25 km south of Vilnius
Vilnia River, ca 20 km above Vilnius and just above New Vilnia
Nemunas River, east bank ca 5 km below Gardinas city limits {in
Belarus)
Nemunas River, west bank, ca 10 km below Druskininkai
Lake Dusia, east side, ca 40 km northwest of Druskininkai
Nemunas River, ca 3 km below Atytus water treatment facility
(Continued)
12
-------
Appendix A. Continued
Number
LT-42
LT-43
LT-44
Name
Visaginas
Lake Drukliai
Crusader Creek
Description
Wastewater treatment facility outflow at Visaginas
Lake DrukSiai, south shore of west arm
Mouth of creek before flowing into Nemunas River,
LT-39
ca 40 m below
LT-45A-E Utena Storage Utena warehouse toxic chemical storage location:
A - access hole to drainage pipe from under warehouse and water
catchment area
B - water catchment under warehouse, east side
C - storage tank inside door on right side
D - storage tank inside door on left side
E - storage tanks LT45C and LT45D combined and treated
Adjacent to the Ministry of Agriculture Pesticide Storage Site
southwest of Vilnius, Monitoring Wells #1-4 outside the fence
Amber Cove Kursiij Marios Lagoon, 1 km north of Juodkrante, ca 400 m
offshore
Green Harbor Klaipeda Harbor, ca 200 m offshore from discharge of Klaipeda
municipal wastewater treatment facility
Girenai Aitra River, east of Girenai, ca 10 km above confluence with Jura
River
LT-46A-D Pesticide Storage
Site
LT-51
LT-52
LT-53
13
-------
Appendix B. Hydrochemical and physical water quality data, November-December 1990
Station
Number
LT-17
LT-01
LT-02
LT-03A
LT-04
LT-05
LT-16
LT-18
LT-19A
LT-20
LT-21
LT-22
LT-23A
LT-I5A
LT-14
LT-13A-1
LT-06
LT-07
LT-OS
LT-09
LT-10A
LT-11A
LT-12
Station
Name
Siauliai
LavoriSkes
Pavilnys
Pioneer Camp-A
Gediminas Castle
Gariunai
PaneviZys
Kidainiai
Nemunas-H
Kaisladoriif
Dambrava-IV
PaSaislis-VII
Sand Beach-A
Kulautuva-A
Smalininkai
Sovctskas-A
Gilija
Uostadvaris
Nida Station
Juodkrantc
Klaipeda Harbor-A
Mclnragc-A
Palanga
Depth1
(m)
Sur
0.6
Sur
Sur
Sur
Sur
0.8
0.6
33
1.0
0.9
0.9
0.7
0.7
0.8
0.7
Sur
Sur
Sur
0.9
0.7
0.7
0.7
Dissolved
Temp.2 Oj^gen3
(C) (mg/L)
Kulpe River
11.17 4.66
Vilnia River
5.22 10.28
4.79 9.70
Neris River
3.27 11.74
4.90 11.78
3.45 11.25
Nevezis River
2.68 11.95
2.62 12.41
Kaunas Reservoir
2.66 11.61
2.53 11.55
2.82 11.30
2.98 11.15
Nemunas River
3.14 10.97
1.60 10.89
2.38 11.01
2.62 7.40
Kursiu Marios Lagoon-
. 3.77 10.43
3.60 11.06
3.95 11.77
3.13 12.00
5.24 1134
Baltic Sea Coast
6.29 11.43
5.59 11.71
PH*
7.50
7.89
7.79
7.98
8.14
8.13
7.86
8.04
8.09
8.06
8.10
8.11
8.12
8.06
8.05
7.62
7.89
8.02
8.27
8.10
7.58
7.79
7.92
Conductivity5
(mS/cm)
1.55
0.444
0.463
0.415
0.472
4.23
0.798
0.876
0.484
0.484
0.486
0.487
0.512
0.784
0.570
0.635
0.546
0.524
0.467
0.480
6.44
11.02
9.29
Salinity
(PPO
0.3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
3.2
5.9
3.8
Redox
Potential*
(mV)
41
91
95
97
111
90
105
127
121
138
148
148
• 145
157
166
120
97
112
106
39
95
101
160
'Depth probe accuracy ±0.45 m, Sur — approximately 0.3-0.5 m below surface temperature
2% *pH accuracy ±0.2 units 5Conductivity accuracy ±1% of range, resolution 4 digits
accuracy ±0.2° 3DO accuracy ±
6Redox accuracy ±20 mV
14
-------
Appendix C. Hydrochemical and physical water quality data, July 1991
Station
Number
LT-17
LT-31
LT-27
LT-32
Station
Name
Siauliai
GrigiSkes
Neris at Kaunas
ZigmantiSkes
Depth1
(m)
Sur
Sur
Sur
Sur
Dissolved
Temp.2 Oxygen3
(C) (mg/L)
21.80
18.53
21.06
15.82
Kulpe River
053
Neris River
830
1451
Salcia River
8.46
PH4
7.69
8.10
858
7.72
Conductivity5
(mS/cni)
2.14
-
0.476
0.453
0.452
Salinity
(PP»)
0.6
0.0
0.0
0.0
Redox
Potential*
-------
Appendix C. Continued
SUUoa
Naarfxr
LT-20
•
•
V
«
V
•
*•
LT-21
«
*•
»
V
»
*
•
LT-22
•
»
•
•
*
•
•
It
II
IT
*
Station
Nam.
Kai£iadorii|
»
•
P
*
•
*
*
Dambrava-IV
*
w
II
K
V
•
ff
Pa2abIis-VII
m
tt
n
w
m
m
m
H
it
•
•
Depth1
(m)
05
2.0
3.0
4.0
6.0
8.0
10.0
12.0
05
2.0
4.0
6.0
8.0
10.0
12.0
14.0
OS
1.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
Temp.2
(Q
Kaunas
22.81
22.85
22.85
22.85
22.73
22.71
22.30
22.09
22.48
22.51
2250
22.44
2230
22.17
22.15
22.17
22.22
22.05
21.59
21.45
2137
21.28
21.18
21.12 ,
20.75
20.66
20.67
20.65
Dissolved
Oxygen3
(mg/L)
Reservoir
1.13
1.13
1.05
1.03
0.91
0.65
054
0.55
4.76
4.91
4.99
556
4.49
3.14
2.74
2.71
8.44
5.22
3.45
3.25
2.98
2JS4
2.54
231
1.45
1.21
1.31
1.23
PH*
7.60
7.62
7.64
7.65
7.63
7.63
7.60
7.58
7.72
7.74
7.81
7.83
7.70
7.61
7.59
759
7.93
7.81
7.62
755
7.53
7.52
750
7.49
7.46
7.44
7.45
7.45
Conductivity5
(mS/cm)
0.476
0.477
0.477
0.477
0.478
0.477
0.490
0.496
0.461
0.459
0.458
0.456
0.456
0.456
0.456
0.453
0.443
0.446
0.447
0.488
0.448
0.449
0.447
0.449
0.450
0:450
0.449
0.450
Salinity
(PPO
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0,0
0.0
0.0
0.0
0.0
0.0
0:0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Redox
Potential4
(mV)
114
109
104
101
122
116
115
113
117
115
113
112
115
116
115
99
141
140
146
145
145
144
142
141
142
141
139
138
Nemunas River
LT-23B
LT-15B
LT-14
LT-I3-1
LT-28A
Lamped2iai-B
Zapygkis-B
Smalininkai
Sovctskas
Rusni-A
Sur
Sur
Sur
Sur
Sur
20.97
20.56
20.25
20.55
20.72
12.02
5.14
11.88
10.28
11.70
852
7.47
8.42
7.88
8.21
0.466
0.464
0.462
0.516
0.484
0.0
0.0
0.0
0.0
0.0
73
93
74
75
82
(Continued)
16
-------
Appendix C. Continued
Stalk*
NuMber
SUtion
Nama
Depth1
(a)
Temp.2
(C)
Dissolved
Oxygen9
(mg/L)
PH4
Conductivity5
(mS/cm)
Salinity
(PPI)
Redox
Potential*
(tnV)
Kursiu Marios Lagoon
LT-29
II
•
m
•
«
LT-06
*
LT-30
II
II
LT-07
II
n
it
LT-8
II
LT-10A
LT-11A
LT-12
*
Deime
II
II
ft
•
•
Gilija
•
Rasyte
II
ft
Uostadvaris
m
m
m
Nida Station
II
Klaipeda Harbor-A
Mclnragd-A
Palanga
it
05
2.0
4.0
6.0
8.0
10.0
05
15
05
2.0
4.0
0.5
2.0
4.0
6.0
0.5
2.0
Sur
Sur
Sur
15
21.40
21.21
20.90
20.85
20.84
20.84
20.93
20.29
19.72
19.69
19.17
20.18
20.14
20.12
20.13
19.83
19.70
19.27
Baltic
18.84
19.78
19.73
3.88
3.76
2.89
2.75
2.77
2.66
8.68
935
9.71
9.60
7.28
11.03
10.96
10.75
10.77
7.17
7.20
9.22
Sea Coast
8.45
857
8.40
7.48
7.50
7.47
7.46
7.44
7.43
7.75
8.01
837
833
8.02
8.47
8.45
8.40
836
8.08
8.03
834
8.18
8.48
850
0593
0.593
0.592
0590
0590
0590
0.495
0.481
0.475
0.475
0.480
0.467
0.468
0.467
0.467
0.476
0.477
5.30
11.72
10.25
10.26
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2.4
6.4
5.5
5.5
86,
83 ;••/ ,: ..'
83
81
75
75
105
97
88
87
95
57
53
52
50
106
108
115
78
78
72
'Depth probe not operating - actual depth measurements reported. Sur = approximately 03-05 m below surface
temperature accuracy ±0.2° 3DO accuracy ± 2% 4pH accuracy ±0.2 units
5Conductivity accuracy ± 1% of range, resolution 4 digits 6Redox accuracy ±20 mV
17
-------
Appendk D. Hydrochemical and physical water quality data, June 1992
Station
Number
LT-40
LT-43
LT-34
LT-04
LT-03B
LT-31
LT-26B
LT-38
LT-39
LT-44
LT-41
LT-15C
LT-13-2
LT-28B-2
LT-29
N
H
H
LT-06
H
LT-30
"
LT-07
"
LT-08
LT-11B
H
LT-11C-2
M
LT-11D-2
N
Station
Name
Lake Dusia
Lake DrQkSiai
New Vilnia
Gcdiminas Castle
TurnisTces-B
GrigiJkes
Raudondvaris
Sredny Gonkolsky
Lilkiava
Crusader Creek
AJytus
Kulautuva-C
Sovetskas
Rusne-B
Dcim6
K
ft
ft
Gilija
H
Rasytd
'
Uostadvaris
ft
Nida Station
Mclnrage-B
n
Melnrage-C
n
Melnrage-D
"
Depth1
(m)
Sur
Sur
Sur
Sur
Sur
Sur
Sur
Sur
Sur
Sur
Sur
Sur
Sur
, Sur
Sur
5
10
12
Sur
2.5
0.7
2.2
Sur
4.5
Sur
0.6
8
Sur
10
Sur
10
Dissolved
Temp.2 Oxygen3
(C) (mg/L)
Lakes
19.81 9.32
22.14 17.15 .
Vilnia River
15.80 12.18
17.64 11.44
Neris River
19.23 13.59
19.29 13.20
Ncvezis River
19.41 10.52
Ncmunas River
21.64 9.78
20.16 7.99
11.71 9.73
21.11 9.76
19.53 10.42.
21.45 14.98
21.98 1436
KurSiu Marios Lagoon
22.10 6.07
21.93 5.15
21.65 4.11
26.64 3.95
20.16 11.07
19.98 10.37
20.46 8.79
20.47 8.83
22.08 13.34
21.50 9.89
20.50 7.99
Baltic Sea Coast
7.63 7.81
6.59 7.25
6.83 7.33
6.58 6.95
7.06 6.77
6.89 6.88
pH4
8.46
9.49
8.08
8.17
8.62
8.55
8.52
8.51
8.31
8.10
8.55
8.53
8.97
8.94
8.10
7.99
7.89
7.86
8.95
8.95
8.69.
8.75
8.84
8.57
8.57
7.61
7.57
7.31
7.48
7.58
7.59
Conductivity5
(mS/cm)
0.327
0.320
-
0.509
-
-
0.421
0.377
0.366
0.460
0.399
0.431
0.405
0.400
0.558
0.560
0.558
0.556
0.424
0.428
0.441
0.442
0.411
0.424
0.429
12.56
12.62
12.54
12.57
12.66
12.69
Salinity
(PPfl
0.2
0.2
-
03
-
-
0.2
0.2
0.2
0.2
6.2
0.2
0.2
0.2
0.3
0.3
0.3
0.3
0.2
0.2
0.2
0.2
0.2
0.2
0.2
7.2
7.2
7.2
7.2
7.3
7.3
Redox
Potential*
302
264
297
298
279
276
333
284
305
311
303
322
286
276
330
331
334
334
292
317
319
314
309
322
310
392
389
409
396
420
425
'Depth probe accuracy ±0.45 m, Sur = approximately 0.3-0.5 m below surface temperature
2% *pH accuracy ±0.2 units sConductivity accuracy ±1% of range, resolution 4 digits
accuracy ±0.2° 3DO accuracy ±
6Redox accuracy ±20 mV
18
-------
TRACE METAL CONCENTRATIONS IN NATURAL
WATERS IN LITHUANIA, 1991-1992
K. Kvietkus1, D. Ceburnis1, J. Sukyte2
JJ. Ellington3, and R.V. Thurston4
ABSTRACT
The concentrations of ten metals in natural waters in Lithuania were measured in
1991 and 1992 to understand the influence of industrial wastes on the quality of those
waters. The analyses were performed by atomic absorption spectroscopy. Samples from
the Kulpe River at Siauliai and from the Baltic Sea near Klaipeda Harbor had high trace
metal concentrations. Hydrological parameters such as pH, dissolved oxygen,
conductivity, redox potential, and salinity were also measured; these showed significant
differences among the sampling sites.
INTRODUCTION
Low mineral levels and simple chemical composition are general characteristics of
Lithuanian rivers (Garunkstis 1988). The anthropogenic chemical composition of small
rivers in Lithuania is usually determined by the chemicals used in agriculture, and the
composition of large rivers is influenced mostly by sewage from industrial enterprises
and big cities. Lithuanian rivers are generally alkaline because dissolved calcium,
magnesium, and carbonate ions comprise up to 85% of all dissolved substances. The
abundance of these ions is caused by carbonated sediments and by climatic conditions
that stimulate the leaching of carbonates. The lowest levels of carbonates are found in
the small rivers, flowing from marsh waters (Garunkstis 1988).
Of the area of Lithuania, 70 percent (46,600 km2) falls within the Nemunas River
basin, so the water quality of most rivers in Lithuania influence the quality of the
Nemunas River and Kurshj Marios Lagoon, into which the Nemunas River flows.
Kurshj Marios Lagoon (1,610 km2) is the largest reservoir of fresh water in Lithuania.
In addition to Kursiij Marios Lagoon being the recipient of pollutants discharged from
the Nemunas River, other major sources of pollution to the lagoon are the discharge of
the Deime River from Kaliningrad and several wastewater outfalls from the City of
Klaipeda into Klaipeda Harbor, a major shipping port. Approximately 26 factories
discharge their contaminated waters into the northern part of the lagoon
(Griskevicius 1987).
Institute of Physics, Vilnius, Lithuania
Department of Environmental Protection, Vilnius, Lithuania
3
Environmental Research Laboratory, U.S. Environmental Protection Agency, Athens, Georgia, USA
Fisheries Bioassay Laboratory, Montana State University, Bozeman, Montana, USA
19
-------
The number of analyses performed on river waters to measure trace metals has
greatly increased in recent years due to improvements in analytical techniques used for
geoexploration and environmental management (Salomons and Forstner 1984). There
are very significant regional differences with respect to various processes occurring in
rivers and other natural water basins, to concentrations of different elements, and to
compounds and poisonous effects which they induce (Anikiev et al 1990, Nimmo et aL
1989). Natural water, especially river water, has a direct influence on groundwater,
which is used as drinking water (Pelig-Ba et aL 1991), with resultant effects on humans
(McClain and Becker 1975). Until 1990, there were no detailed investigations of heavy
metals in natural waters in Lithuania, mainly because of the lack of adequate
instrumentation.
The present study was undertaken during 1991-1992 to assess the distribution of
trace metals in Lithuanian surface waters. Water samples were collected and analyzed
for 10 trace metals at 23 sites on five rivers, Kaunas Reservoir, Kursh| Marios Lagoon,
and the Baltic Sea at the mouth of Klaipeda Harbor (Figure 1, Table 1). Twenty of
these sites were sampled during July 1991, a representative climate year, and 15 sites
were sampled during June 1992, following an especially dry spring. Eight of the sites
were sampled both years, enabling comparison between the representative and dry
periods.
Figure 1. Map of surface water sampling sites.
20
-------
Table 1. Description of surface water sampling sites.
Number
Name •
Description
LT-06 Gilija
LT-07 Uostadvaris
LT-08 Nida Station
LT-10A-B Klaipeda Harbor
LT-11A-B Melnrage
LT-13-1-3 Sovetskas
LT-14
LT-15B-C
LT-16
LT-17
LT-18
LT-19A
LT-20
LT-21
LT-22
LT-26B
LT-27
LT-29
Smalininkai
Kulautuva
Panevezys
Siauliai
Kedainiai
Nemunas, II Cut
Kaisiadoriij
Dambrava, IV Cut
Pazaislis, VII Cut
Raudondvaris
Neris at Kaunas
LT-28A-B Rusne
Deime
LT-30
LT-31
LT-42
LT-43
Rasyte
Grigiskes
Visaginas
Druksiai
Kursiij Marios Lagoon at mouth of Gilija River
Kursiij Marios Lagoon from the Neringa Spit 17 km east of Nida
Kursiij Marios Lagoon 1.5 km off the dock at Nida
Klaipeda Harbor (A) north side of mouth of Dane River,
(B) middle of channel off Dane River
Entrance to Klaipeda Harbor at the Baltic Sea, (A) east side of east
breakwater, (B) west side of east breakwater
Nemunas River at Sovetskas, (1) south bank, (2) middle of channel,
(3) north bank
Nemunas River north bank, ca 50 km upstream from Sovetskas
Nemunas River north bank, 20 km downstream from Kaunas and
13 m downstream from Nevezis River-Nemunas River confluence,
(B) south bank at ZapySkis, (C) north bank ca 1 km below Zapyskis
Nevezis River 11 km downstream from Panevezys and 1 km below
the discharge pipe of wastewater treatment facility
Kulpe River at Siauliai
Nevezis River, east bank, 1.5 m below wastewater treatment facility
Nemunas River just before emptying into Kaunas Reservoir
Kaunas Reservoir, 300 m offshore from HydroAccumulation
Electric Power facility (HAEP)
Kaunas Reservoir below HAEP
Kaunas Reservoir at dam, within sight of Pazaislis Monastery
Nevezis River, west bank, 10 km west of Kaunas
Neris River immediately above bridge before confluence with
Nemunas River
Nemunas River, delta region near Rusne, (A) 10 km upstream from
Kursii} Marios Lagoon, (B) ca 12 km upstream from Kursiij Marios
Lagoon
Kursiij Marios Lagoon, southernmost section in the mouth of
Deime River
Kursiij Marios Lagoon, in the "center"
Neris River, 17 km west of Vilnius
Sewer line from Ignalina NPP, near Lake Druksiai
Lake Druksiai, south shore of west arm
21
-------
METHODS
All samples were collected at a depth of approximately 0.5 m. In 1991, samples
were stored in nitric acid-washed 50-ml plastic bottles to which one drop of metal-free
nitric acid had been added, and then stored refrigerated until analyzed within 4 months
of collection. Sampling and analysis in 1992 was identical to 1991, with the exception
that glass bottles were used instead of plastic bottles. One sample from each location
was analyzed by a graphite furnace atomic absorption spectrophotometer Zeeman/3030,
Perkin-Elmer, USA. Ten trace metals—cadmium, chromium, cobalt, copper, iron, lead,
manganese, nickel, vanadium, and zinc—were analyzed, and six hydrological parameters--
pH, dissolved oxygen, conductivity, redox potential, salinity, and temperature—were
measured. Detection limits and calibration concentration ranges of the instrument for
different elements are reported in Table 2. These were determined by methods similar
to those prescribed in the instrument operating manual. No matrix modifiers were used.
Operational conditions are reported by Kyietkus et al (1994).
Table 2. Detection limits and cah'bration concentration ranges of the instrument (AAS).
Metal Calibration concentration
ranges Oig/L)
Detection limit (;ig/L)
Sampling quantity (jiL)
Pb
Cu
Ni
Co
V
Cr
Zn
Fc
Cd
Mn
10-50
10-50
10-50
10-50
10-50
10-50
10-50
20-100
1-5
10-50
05
05
0.8
0.9
1.0
0.5
0.3
1.0
02
03
50
40
80
60
100
20
5
20
40
20
At the same time water samples were collected for trace metal analysis, on-site
water quality measurements were made at most stations using a Hydrolab Surveyor 2
(Hydrolab Corporation, Austin, Texas, USA) equipped with pH, temperature, dissolved
oxygen (DO), conductivity, redox potential, and depth sensors. The pH electrode
(accuracy +0.2 units, resolution +0.1 unit) was calibrated with pH 3, 7, and 10 buffer
22
-------
solutions. The temperature sensor (accuracy +0.2°C, resolution ±0.01) was verified by
simultaneous immersion in a flask of water with a thermometer certified by the U.S.
National Institute of Standards and Technology. The DO sensor (accuracy ±2%,
resolution 0.01 mg/L) was calibrated by the air saturation method. The conductivity
sensor (accuracy ±1% of range, resolution 4 digits) was calibrated with solutions of
potassium chloride prepared with distilled, deionized water. The redox potential sensor
(accuracy +20 mV) was calibrated with aqueous solutions of quinhydrone.
RESULTS AND DISCUSSION
Trace Metals
With the exception of zinc, the measured concentrations of metals (Table 3) at
stations where water samples were taken in both 1991 and 1992 varied by a factor of five
or less. The concentrations of zinc in the 1992 samples were an order of magnitude or
more greater than in samples collected at corresponding locations in 1991. We believe
that in 1992 there was an unidentified error affecting the measurement of zinc, possibly
contamination related to preservation and/or storage of samples prior to analysis.
Accordingly, although we are reporting the zinc values obtained for 1992, they will not
be considered further in the discussion.
Freshwater chronic toxicity criteria (FCT) values recommended by the U.S. EPA
(1992) for six of the metals (cadmium, chromium, copper, lead, nickel, and zinc) are
listed in Table 3 Footnote 1; the maximum contaminant levels (MCL) used in the former
USSR (Bespamiatnov and Krotov 1985) for cobalt, iron, vanadium and manganese are
listed in Footnote 2.
In 1991, out of 200 individual measurements, 18 of these exceeded either the FCT
or MCL criteria, and of these, seven measurements exceeded these criteria by more than
two-fold. In 1992, 150 measurements were made. Excluding zinc, out of the remaining
135 measurements none exceeded MCL criteria, and although 11 exceeded FCT criteria,
only two of these, both for lead, exceeded the criteria more than two-fold. Although the
number of values exceeding FCT and MCL criteria were fewer in 1992 than 1991, part
of the reason for this is that in 1992 we did not resample some of the sites found to be
most polluted based on the 1991 sampling.
Of all metals measured, iron most often exceeded the MCL criterion; this
happened in 13 out of 35 samples. The only location at which iron was markedly higher
than the criterion, however, was the Nemunas River at Rusne (LT-28). We sampled the
Kulpe River at Siauliai (LT-17) in 1991 and obtained measurements for lead, copper,
and chromium that exceeded FCT criteria values by factors of two, three, and 50
respectively. Cadmium, copper, and lead measurements at Melnrage (LT-11), also made
in 1991, exceeded FCT criteria by two-, five-, and 50-fold. Klaipeda Harbor (LT-10) was
sampled in both 1991 and 1992, and although the measurement for lead in the single
sample collected in 1991 was below the detection limit, lead in samples collected in 1992
at two separate locations were five- and ten-fold the FCT criterion.
23
-------
Table 3. Trace metal concentrations in natural waters in Lithuania
(values expressed in /xg/L).
Site
number
LT-06
LT-07
LT-08
LT-10A
LT-10B
LT-11A
LT-13-1
LT-13-2
LT-13-3
LT-14
LT-15B
LT-15C
LT-16
LT-17
LT-I8
LT-19A
LT-20
LT-21
LT-22
LT-26B
LT-27
LT-28A
LT-28B
LT-29
LT-30
LT-31
LT-42
LT-43
Pb1
1991
BD3
BD
0.5
BD
_4
152
BD
_
_
1.8
BD
—
4.7
7.9
BD
BD
BD
BD
0.8
—
BD
0.8
—
BD
0.6
0.6
_
—
1992
BD
BD
0.9
59.0
16.0
_
BD
BD
BD
—
—
BD
_
—
—
—
—
_
_
0.5
—
_
BD
0.6
0.9
_
0.5
BD
Cu1
1991
2.7
1.7
2.9
2.7
—
66.0
63
_
_
10.4
0.8
—
14.3
32.0
3.7
19.6
5.7
1.6
3.1
—
4.6
3.2
—
0.7
1.8
6.0
_
—
1992
5.7
3.6
4.5
2.9
3.0
—
7.4
2.6
2.4
—
—
63
—
—
~
—
—
—
~
32
—
—
4.9
3.8
66
_
0.42
8.4
Ni1
1991
1.8
1.2
0.9
1.2
_
29.5
2.2
—
_
2.8
13
~
18.0
35.0
6.6
2.8
1.0
1.4
13
~
1.6
2.6
—
0.8
1.5
23
—
—
1992
0.8
2.0
1.1
5.5
3.5
—
1.6
0.8
1.0
—
—
2.1
—
—
—
—
—
~
—
BD
—
—
1.4
0.8
2.0
—
1.1
BD
Co2
1991
0.9
BD
BD
3.0
..
29.0
BD
—
..
BD
BD
—
BD
1.0
0.9
BD
BD
BD
BD
—
BD
1.7
—
0.8
BD
BD
—
-
1992
BD
1.7
BD
61.0
160
„
0.9
1.0
1.7
_
—
13
—
—
—
—
—
—
—
BD
—
—
1.7
0.9
BD
—
BD
BD
1991
2.4
13
1.8
BD
..
10.9
1.5
—
—
1.2
BD
—
1.2
1.9
7.7
1.2
BD
1.0
1.1
-
BD
2.2
-
1.1
2.1
BD
—
—
V2
1992
1.1
BD
1.0
6.1
5.1
—
1.5
1.0
1.4
—
—
1.9
—
—
—
—
-
—
—
BD
—
—
1.8
0.8
1.4
—
BD
BD
1UJS. EPA FCT criteria values (see text) in jig/L are Pb 3.2, Cu 12*, Ni 160*, CrVI 11, Cd 1.1*, Zn 110*
(* = hardness dependent criteria, 100 mg/L as CaCO3)
2Former USSR standards (see text) in ng/L are Co 100, V 100, Mn 10,000, Fe 500
24
-------
Table 3.
Continued
Site
number
LT-06
LT-07
LT-08
LT-10A
LT-10B
LT-11A
LT-13-1
LT-13-2
LT-13-3
LT-14
LT-15B
LT-15C
LT-16
LT-17
LT-18
LT-19A
LT-20
LT=21
LT-22
LT-26B
LT-27
LT-28A
LT-28B
LT-29
LT-30
LT-31
LT-42
LT-43
1991
0.6
0.5
0.6
BD
-
BD
1.6
~
~
1.2
BD
-
1.3
520
15
BD
BD
BD
BD
-
0.9
2.7
-
0.7
1.2
3.7
-
— •
Cr1
1992
1.1
1.0
1.5
BD
BD
—
1.4
1.2
0.9
—
—
1.6
~
—
—
—
—
—
—
0.9
—
—
1.7
0.6
2.2
—
BD
BD
1991
120
128
148
120
~
33
202
—
~
158
157
~
66
81
76
175
178
120
60
—
127
207
—
156
102
103
—
—
Mn2
1992
163
206
170
75
68
_
240
350
2120
~
~
270
~
..
..
_
—
—
—
131
_
—
306
163
172
—
34
30
Fe2
1991
730
450
750
120
—
250
780
—
_
450
310
—
270
820
110
350
280
300
30
—
530
2430
_
560
510
290
—
—
1992
276
191
781
133
112
-_
642
426
380
—
508
__
__
—
._
__
170
„
._
707
276
688
—
140
20
1991
BD
BD
BD
BD
....
2.4
BD
«
__
BD
BD
„
0.30
0.45
0.40
BD
BD
BD
BD
„
BD
BD
__
BD
0.27
0.80
-
Cd1
1992
0.23
020
0.20
2.0
1.7
BD
2.0
BD
—
BD
-_
—jj
——
«.
__ -
_
__
0.24
..
«._
0.22
0.21
0.34
BD
0.26
1991
3.5
2.7
4.8
152
4.0
12.9
6.6
2.7
7.2
43.0
3.3
10.4
5.0
1.7
2.9
6.7
7.2
3.3
90
14.5
-
Zn1
1992
320
23.0
740
360
290
570
250
380
17.7
13.5
60.4
13.7
570
63.1
440
BD = Below detection limit
4— = Not sampled
25
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Hydrological Parameters
The hydrological parameters measured in 1991 and 1992 at the stations at which
water samples were taken for trace metal analyses are reported in Tables 4 and 5.
Metal speciation in aquatic solution is dependent upon such factors as temperature, pH,
redox, alkalinity, and concentration of the cations present.
Table 4. Hydrological parameters of natural waters in Lithuania, 1991.
Site
No.
LT-06
LT-07
LT-08
LT-10A
LT-11A
LT-13-1
LT-14
LT-15B
LT-16
LT-17
LT-18
LT-19A
LT-20
LT-21
LT-22
LT-27
LT-28A
LT-29
LT-30
LT-31
GUija6
Uostadvaris6
Nida Station6
Klaipeda Harbor-A
Melnrage-A6
Sovetskas-A6
Smalininkai
Zapyskis6
Panevezys
Siauliai
Kedainiai
Nemunas, II Cut
Kaisiadorii}
Dambrava, IV Cut
Pazaislis, VII Cut
Neris at Kaunas
Rusne-A6
Deime6
Rasyte
Grigiskes
Temp.1
°C
20.9
20.2
19.8
193
18.8
20.6
20.3
20.6
19.1
21.8
21.2
22.7
22.8
22.5
22.2
21.1
20.7
21.4
19.7
18.5
PH2
7.75
8.47
8.08
834
8.18
7.88
8.42
7.47
7.82
7.69
7.84
756
7.60
7.72
7.93
8.58-
8.21
7.48
8.37
8.10
DO3
mg/L
8.7
11.0
7.2
9.2
85
103
11.9
5.1
6.9
0.5
7.2
15
1.1
4.8
8.4
14.5
11.7
3.9
9.7
83
Conductivity4
mS/cm
050
0.47
0.48
530
11.7
052
0.46
0.46
0.73
2.14
0.81
0.48
0.48
0.46
0.44
0.45
0.48
0.59
0.48
0.48
Redox5
mV
105
57
106
115
78
75
74
93
76
-95
61
92
114
117
141
83
82
86
88
61
Salinity
ppt
0.0
0.0
0.0
2.4
6.4
0.0
0.0
0.0
0.0
0.6
0,0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Temperature probe accurate to ±0.2°C
3DO probe accurate to 2%
5Redox probe accurate to ±20 mV
^H probe accurate to ±0.2 units
4Conductivity sensor ±1% of range, resolution 4 digits
""Locations resampled in 1992
26
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Table 5. Hydrological parameters of natural waters in Lithuania, 1992.
Site
number
LT-06
LT-07
LT-08
LT-11B
LT-13-2
LT-15C
LT-26B
LT-28B-2
LT-29
LT-30
LT-43
Name
Gilija6
Uostadvaris6
Nida Station6
Melnrage-B6
Sovetskas-A6
Kulautuva6
Raudondvaris
Rusne-B6
Deime6
Rasyte
Druksiai
Temp.1
°C
20.2
22.1
205
7.63
215
195
19.4
22.0
22.1
205
22.1
PH2
8.95
8.84
857
7.61
8.97
853
852
8.94
8.10
8.69
9.49
DO3
mg/L
11.1
133
8.0
7.8
15.0
10.4
105
14.4
6.1
8.8
17.2
Conductivity4
mS/cm
0.4
0.4
0.4
12.6
0.4
0.4
0.4
0.4
0.6
0.4
0.3
Redox5
mV
292
309
310
392
286
322
333
276
330
319
264
Salinity
ppt
0.2
0.2
Q2
72
02
02
02
02
03
02
02
Temperature probe accurate to ±0.2°C 2pH probe accurate to ±0.2 units 3DO probe accurate to 2%
'Conductivity sensor ±1% of range, resolution 4 digits 5Redox probe accurate to ±20 mV
"•Locations samples in 1991
The pH values of natural waters from which we collected samples for metal
analyses in 1991 were found to be very similar throughout, and were within a range of
7.5-8.6 (Table 4). In 1992, a slightly lower value (7.3) was found in the Baltic Sea at
Melnrage, at the mouth of Klaipeda Harbor (Table 5). According to theoretical
calculations, it has been suggested that the heavy metals in Lithuanian natural waters are
in a carbonated form (Turner et al 1981). This is supported also by the fact that natural
waters of Lithuania are generally alkaline (Garunkltis 1988).
Dissolved oxygen in natural waters is of great importance because it represents
the vital capacity of water. Very low values were found in some parts of the Kaunas
Reservoir (1.1-1.5 mg/L) and Kursiu Marios Lagoon (3.9 mg/L) in 1991, and an
extremely low value of 0.53 mg/L was found in the Kulpe River near Siauliai, where
wastewater discharges from an industrial complex are discharged directly into the river.
In 1992, DO values ranged from 6 to 17 mg/L (Table 5), and at all seven of the
freshwater locations sampled both years the DO values ranged from 1 to 5 mg/L higher
in 1992 than in 1991. Dissolved oxygen values measured at Melnrage, in the Baltic Sea
at the mouth of Klaipeda Harbor, were as much as 1 mg/L lower. It has been suggested
that these higher values in 1992 may be related to a decrease in industrial production
because of the current economic crisis, with corresponding decreases in wastewater
discharges into freshwater bodies.
27
-------
During 1991 and 1992, the conductivity of the water in the Neris and Nemunas
Rivers, Kaunas Reservoir, and the freshwater area of Kursnj Marios Lagoon ranged
from 0.44 to 0.59 mS/cm. Slightly higher values for conductivity, relative to other surface
waters, were observed in the Nevezis River at Panevezys, Kedainiai, and Raudondvaris
(0.73-0.81 mS/cm), and an extremely high value for freshwater of 2.14 mS/cm was
measured in the Kulpe River at Siauliai. Elevated conductivity values were also found in
other more polluted rivers, and inside the mouth of Klaipeda Harbor where high
conductivity values are normal due to infusion of ions and salts from the Baltic Sea.
Salinity at Melnrage on the Baltic Sea was approximately 6 ppt. Speciation of metals in
seawater can be different from that in freshwater, where anions other than carbonates
may prevail (Turner et al 1981): toxicity to aquatic animals of heavy metals can depend
upon that speciation. This has been detailed for copper by Pagenkopf et al 1974 and
Chakoumakos et al 1979.
Temperature, salinity, and conductivity values at all sites were within ranges
considered to be normal, except for the Kulpe River at Siauliai. The negative redox
value measured in 1991 in the Kulpe River at Siauliai (Table 4) is indicative of reducing
conditions and very low DO levels. The products of contaminant degradation under the
reducing conditions at Siauliai would differ from other surface waters where oxidative
conditions exist and positive redox values were obtained.
CONCLUSIONS AND RECOMMENDATIONS
The number of sampling sites visited and samples analyzed for this study were
restricted due to time constraints; but even so, information obtained was sufficient to
confirm two "hot spots," namely Klaipeda Harbor and the Kulpe River at Siauliai.
Except for the chromium concentration in the Kulpe River sample and the high lead and
copper concentrations measured at Melnrage (Klaipeda Harbor), the other metal
concentrations ranged from below detection limits to a factor of 2-3 higher than criteria
values recommended by the U.S. EPA (1992). Because only about one-third of
wastewaters discharged in Lithuania are treated to meet any established standards,
future metal analyses should be focused on the sites downstream from cities and
industries involved in metal plating and other activities that use metals. Detailed
investigations are needed on trace metal concentrations in the rivers flowing into Kursiy
Marios Lagoon. In addition, a study is needed on pollution sources discharging into the
Kulpe River at Siauliai.
ACKNOWLEDGEMENTS
Transportation for our expeditions in Lithuania was provided by the Lithuania
Department of Environmental Protection. This research was supported in part by U.S.
EPA Cooperative Agreements CR816369 and CR995189 to Montana State University.
28
-------
REFERENCES
Anikiev, V.V., E.N. Shumilin, A.A. Lobanov, E.N. Slin'ko, and V.V. Yarosh. 1990.
Geokhimiya 10:1494.
Bespamiatnov, G.P., and N. Krotov. 1985. Maximum Contaminant Levels in the
Environment Leningrad, p. 75 (In Russian)
Chakoumakos, C., R.C. Russo, and R.V. Thurston. 1979. Toxicity of copper to
cutthroat trout (Salmo clarki) under different conditions of alkalinity, pH, and
hardness. Environmental Science & Technology 13:213-219; Correspondence
13:1148.
GarunkStis, A. 1988. Lithuanian Waters. Vilnius, p. 56 (In Lithuanian)
Gri§kevicius, A. 1987. Water Economy of the Cities. Vilnius, p. 30 (In Lithuanian)
Kvietkus,K., D. Ceburnis, J. Ellington, and Y. Shane Yu. 1994. Inter-laboratory
comparison of methods for the determination of heavy metals in natural waters.
In: Environmental Studies in the Nemunas River Basin, Lithuania. U.S.
Environmental Protection Agency, Athens, Georgia, USA.
McClain, R.M., and B.A. Becker. 1975. Toxicology and Applied Pharmacology 31:72.
Nimmo,M., C.M.G. Van der Berg, and J. Brown. 1989. Estuarine^ Coastal Shelf
Science 29:57.
Pagenkopf, G.K., R.C. Russo, and R.V. Thurston. 1974. Effect of complexation on
toxicity of copper to fishes. Journal of the Fisheries Research Board of Canada
31:462-465.
Pelig-Ba, K.B., C.A. Biney, and L.A. Antwi. 1991. Water, Air and Soil Pollution 59:
333.
Salomons, W., and U. Forstner. 1984. In: Metals in the Hydrocycle. Springer-Verlag,
Berlin, Heidelberg, New York.
Turner, D.R., H. Whitfield, and A.G. Dickson. 1981. Geochimica Cosmochimica Acta
45:855.
U.S. EPA (U.S. Environmental Protection Agency). 1992. Water quality standards;
Establishment of numeric criteria for priority toxic pollutants; States' compliance;
Final rule. 40 Code of the Federal Register Part 131. 57(246):60848-60973.
29
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INTER-LABORATORY COMPARISON OF METHODS FOR THE
DETERMINATION OF HEAVY METALS IN NATURAL WATERS
K. Kvietkus1, D. Ceburnis1
J J. Ellington2, and Y. Shane Yu3
ABSTRACT
An inter-laboratory study was conducted by the Ecological Spectroscopy
Laboratory (ESL), Institute of Physics of the Lithuania Academy of Sciences, and the
Environmental Research Laboratory, Athens, Georgia (ERL-A), of the U.S.
Environmental Protection Agency, to compare the analytical methods used by each
laboratory to measure trace metals in natural waters. Duplicate water samples were
collected at 19 locations throughout Lithuania in July 1991, and each laboratory analyzed
these samples for eight metals: cadmium, cobalt, chromium, copper, lead, manganese,
nickel, and zinc. Analyses were performed by graphite furnace atomic absorption
spectroscopy (AAS). Some samples required dilution before analysis in order to reduce
absorbance to levels where accurate background correction and quantitation could be
achieved using the Zeeman effect. Although eight metals were measured, a correlation
equation was determined for only six of these because of considerable differences
reported for zinc, and because of the failure to detect cobalt in any of the samples
analyzed at ERL-A. The regression line plot that was calculated for the six metals had a
slope of 0.957 and a coefficient of determination of 0.829.
INTRODUCTION
Until 1990, no detailed investigations of heavy metals in natural waters had been
performed in Lithuania, mainly because of a lack of adequate instrumentation. Also,
nitric acid with sub parts-per-billion metal content for sample preservation was not
available, and usually only glass containers were available for sample collection and
storage. Bottles made of glass often contain appreciable amounts of metals in then-
walls, and these metals can easily dissolve and contaminate the sample (Ross 1986). To
avoid such contamination, bottles must be washed in strong acid to leach the metals
from the container wall. Polyethylene or teflon bottles are preferred for storing samples
that contain trace amounts of metals because, after acid washing, these containers are
least likely to contaminate the water samples (Moody and Lindstrom 1977).
1 Institute of Physics, Vilnius, 2600, Lithuania
ry
U.S. Environmental Protection Agency, Athens, Georgia, USA
technology Applications, Inc., Athens, Georgia, USA
31
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An additional problem, associated with water analysis for metals, is that metal
concentrations in the bulk aqueous phase can alter during storage due to sorption--
commonly referred to as the "container wall effect." This effect is minimized by
acidifying the samples with nitric acid. Another factor that affects the measured
concentration of metals in water samples is the analytical method, e.g., the conditions of
atomization of the sample and the use of matrix modifiers to minimize interferences and
improve quantitation.
As part of a joint US/Lithuania cooperative research program, water samples
were collected in duplicate for analysis of heavy metals at both ESL and ERL-A. This
inter-laboratory comparison would be useful for two reasons: (1) as a background from
which to discuss and compare the instrument variables and the techniques employed by
each laboratory, (2) as a database through which trends or possible problems associated
with the measurement of some of the metals could be revealed.
MATERIALS AND METHODS
Samples were collected in 50-ml plastic bottles, precleaned at ERL-A by washing
with 3% nitric acid (Fisher Scientific Co., Trace Metal Grade) and rinsing with organic-
free deionized water. Nitric acid (300 j«L) was then added immediately after cleaning to
lower the pH of the water to < 2 to serve as a sample preservative and to minimize
sorption to the wall of the container. The samples were stored under refrigeration and
analyzed (within 4 months of collection) without filtration . All glass and plastic used in
the analyses were acid-washed and rinsed with metals-free water prior to use. The
metals for which analyses were performed were cadmium, cobalt, copper, chromium,
lead, manganese, nickel, and zinc.
Analyses at ESL were performed on a Perkm-EImer Zeeman 3030 atomic
absorption spectrophotometer (AAS) equipped with an AS-60 autosampler, and a
HGA-600 graphite furnace with a L'vov platform. The AAS system was controlled by an
internal microcomputer using a keyboard and video monitor. The internal gas flow rate
was 300 mL/min. ERL-A used a Perkm-EImer Zeeman 5100 PC AAS, equipped with an
AS-60 autosampler, an HGA-600 graphite furnace with a L'vov platform, and an IBM
PS/2 50Z computer with graphics-based application software. The internal gas flow rate
was 300 mL/min, except during atomization when it was 0 mL/min (Tables 1 and 2).
At each laboratory, calibration standards (1 to 40 /zg/L) were prepared at the time
of analysis by a series of dilutions using organics-free deionized water that was made
0.2% in nitric acid. ESL stock solutions (1,000 mg/L) were obtained from the
Physics/Chemistry Institute of the Ukrainian Academy of Sciences, and ERL-A stock
solutions of each metal (1,000 mg/L) were obtained from Fisher Scientific Co. At ESL,
a programmed non-linear calibration method based on a two-coefficient equation, part
of the 3030 software, was used. At ERL-A, non-linear calibration curves that satisfied
correlation coefficients of at least 0.995 were used.
32
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Table 1. Ramp and hold times for furnace condition (in seconds).
Step
Diy
Char
Cool
Atomize
Clean
Table 2.
Ramp
ESL
Furnace
2
2
0
1
temperature
and
ERL-A
1
1
1
0
1
instrument
Hold
ESL ERL-A
operating
Temperature (°C)
Element
Cadmium
Chromium
Cobalt
Copper
Lead
Manganese
Nickel
Zinc
Cadmium
Chromium
Cobalt
Copper
Lead
Manganese
Nickel
Zinc
Dry
110
110
110
110
110
110
110
110
120
120
120
120
120
120
120
120
Char
250
1200
1000
900
500
1000
1300
400
850
1650
1400
1300
850
1400
1400
700
Cool
-
-
~
-
~
-
—
-
20
20
20
20
20
20
20
20
Atomize
ESL
1600
2300
2200
2000
1800
1900
2300
1600
ERL-A
1650
2500
2500
2500
1800
2200
2500
1800
Clean
2650
2650
2650
2650
2650
2650
2650
2650
2600
2600
2600
2600
2600
2600
2600
2600
10-50
10
3
3
conditions.
Conditions
Wavelength
228.8
357.9
2425
324.8
2833
2795
232.0
213.9
228.8
357.9
242.5
324.8
283.3
279.5
232.0
213.9
50
30
15
5
5
(nm)
Slit
0.7
0.7
0.2
0.7
0.7
0.2
0.2
0.7
0.7
0.7
0.7
0.7
0.7
0.7
0.7
0.7
33
-------
Nitric acid/deionized water blanks were prepared at both ESL and ERL-A by
adding purified water (18 MQ cm"1) to 50-ml plastic bottles that contained nitric acid.
Metal impurities were not detected in ESL and ERL-A blanks. Matrix modifiers were
not available for use in the analysis of samples at ESL; magnesium nitrate was used as a
matrix modifier at ERL-A for each metal except nickel, which was analyzed without the
addition of a modifier.
By agreement, each laboratory analyzed samples using instrument conditions and
sampling techniques that had been used historically at that laboratory. The operating
conditions in Tables 1 and 2 are reflective of the use of matrix modifiers at ERL-A that
allowed higher char temperature, faster ramps, and longer hold times. Higher
temperatures enhance removal of compounds from the matrix that could interfere with
the analysis. The modifiers also shorten the time required for the furnace wall and the
vapor to attain a stable temperature, an essential characteristic of the L'vov platform
technique. A modification of the method described in the instrument operating manual
(Perkin-Elmer 1982) was used to determine the instrument detection limits for each
metal (Table 3).
Table 3. Instrument detection limits.
Element
ESL
Detection
Limit
Sample Amount
Detection
Limit
ERL-A
Sample Amount
Cadmium
Chromium
Cobalt
Copper
Lead
Manganese
Nickel
Zinc
03
0.5
0.9
05
0.5
03
0.8
03
20
20
60
40
50
20
80
5
03
0.5
0.7
0.6
0.9
0.9
1.2
0.6
20
20
20
20
20
20
20
20
34
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RESULTS AND DISCUSSION
Each laboratory analyzed for eight metals in water samples collected
simultaneously from 19 sampling stations; this totalled 152 measurements at each
laboratory, and a combined total of 304 measurements (Table 4). Of these
304 measurements, 119 were reported as below detection limits, 44 by ESL and 75. by
ERL-A. Most of the measurements at ERL-A for cadmium, cobalt, lead, and nickel
were below detection limits; cobalt was not found in any sample, and nickel in only
three. However, at ESL nickel was found in all 19 samples, although cadmium, cobalt,
and lead were detected in half or fewer. Both laboratories reported chromium, copper,
manganese, and zinc in most or all of the samples.
f,
There were two areas where the results at ESL and ERL-A differed significantly.
Measurements at ERL-A for zinc in five of the samples were five to 35 times higher
than at ESL, while measurements for zinc at ESL for two of the samples were five and
10 times higher than at ERL-A. In the case of the seawater samples (LT-ilA), seven of
the metals measured considerably higher at ESL than at ERL-A. Reasons for both of
these large discrepancies will need further study. Elimination of the zinc data, the
seawater sample data, and those data pairs where either of the two laboratories reported
a concentration below detection, left 52 data pairs for six of the eight metals. A log-log
regression plot of these 52 data pairs was calculated using Slide Write Plus™ software
(Figure 1).
1000
100
10.
0.1
A Cadmium
• Chromium
v Copper
a Lead
o Manganese
» Nickel
[ESL] =D.9566[ERL-A]+0.2435
r'=0.829
0.1
.10
[ERL-A]
100
1000
Figure 1. Regression line for metals analyses at Ecological Spectroscopy Laboratory
and Environmental Research LaboratoryrAthens.
35
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Table 4. Results of water analyses by element1 (concentrations reported in,/*g/L).
Sample
LT-06
LT-07
LT-08
LT-10A
LT-11A
LT-13-1
LT-14
LT-15B
LT-16
LT-17
LT-18
LT-19A
LT-20
LT-21
LT-22
LT-27
LT-28A
LT-29
LT-31
Cadmium
ESL ERL-A
BD3
BD
BD
BD
2.4±1.0
BD
BD
BD
03±0.1
0.45±0.05
0.4±0.1
BD
BD
BD
BD
BD
BD
BD
0.8±03
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
1.0±03
BD
BD
BD
BD
BD
BD
BD
1.0±03
Chromium
ESL ERL-A
0.6±0.1
05±0.1
0.6±0.1
BD
BD
1.6±0.1
1.2±0.2
BD
13±03
520±60
15±0.3
BD
BD
BD
BD
0.9±0.1
2.7±0.2
0.7±0.1
3.7±0.2
BD
BD
BD
19.0±2.0
BD
BD
1.0±1.0
BD
1.0±0.5
1300±80
215±35
9.5±0.5
4.0±2.0
3.0±2.0
2.0±1.0
0.5±0.5
2.0±1.0
05 ±05
15±05
Copper
ESL ERL-A
2.7±0.1
1.7±0.1
2.9 ±05
2.7±0.1
66.0±1.0
63 ±0.1
10.4±0.1
0.8±0.1
143±0.1
32.0±1.0
3.7±0.1
19.6±0.1
5.7±0.1
1.6±0.1
3.1 ±0.1
4.6±0.1
3.2±0.2
0.7±0.2
6.0±0.1
25±05
BD
1.0±0.2
1.0±0.2
BD
4.0±1.0
2.5 ±05
BD
35 ±05
27.0±0.2
25±05
25±1.0
6.0±1.0
1.0±02
15±05
15 ±05
1.5±05
BD
65±05
Cobalt omitted from table because all measurements at ERL-A were below
cobalt at ESL were: LT-06, 0.9±2; LT-10A, 3.0±15; LT-11A, 29.0±5.0; LT-
LT-28A, 1.7±03; LT-29, 0.8±0.1
detection limit. Values for
17, 1.0±0.1; LT-18, 0.9±0.1;
36
-------
Table 4.
Continued.
Lead
ESL ERL-A
BD
BD
0^±05
BD
152±5.0
BD
1.8±03
BD
4.7±03
7.9±03
BD
BD
BD
BD
0.8±05
BD
0.8±03
BD
0.6±02
BD
BD
1.0±03
BD
BD
BD
BD
BD
3.5 ±0.5
1.0±03
BD
BD
BD
BD
BD
BD
1.0±03
BD
15±Q5
Manganese
ESL ERL-A
120±1.0
128±2.0
148±2.0
120±1.0
33.0±1.0
202±2.0
158±4.0
157±2.0
66.0±1.0
81.0±1.0
76.0±2.0
175 ±3.0
178±4.0
120±2.0
60.1 ±1.0
127±2.0
207±5.0
156±1.0
103±1.0
120±3.0
100±3.0
120±3.0
80.0±4.5
6.0±0.9
200±3.0
140±3.0
120±3.0
215±05
17.0±15
17.0±1.0
150±10
160±3.0
90.0±10
12.0±0.9
100±9.0
200±9.0
36.0±1.0
185±OJ
Nickel
ESL ERL-A
1.8±0.4
1.2±0.1
0.9 ±0.1
1.2±0.4
29.5±3.0
2.2±0.1
2.8±0.1
13±0.1
8.0±0.1
35.0±0.1
6.6±0.1
2.8±0.1
1.0±0.1
1.4±0.1
13±0.1
1.6±0.1
2.6±0.1
0.8±0.2
23±0.1
BD
BD
BD
BD
BD
BD
BD
BD
45±05
20.0±0.4
4^ ±0.5
BD
BD
BD
BD
BD
BD
BD
BD
Zinc2
ESL ERL-A
35 ±0.1
2.7±0.1
4.8±0.4
152±30.0
4.0±0.2
12.9 ±0.4
6.6±0.1
2.7±0.4
7.2±0.3
43.0±3.0
33 ±03
10.4±13
5.0±0.5
1.7±0.2
2.9 ±0.4
6.7±0.8
7.2±0.1
23 ±03
145 ±0.1
9.0±1.0
2.0±0.2
3.0±0.2
15.0±1.2
25.0±2.0
460±20
150±10.0
3.0±0.2
9.0±0.2
75±25
7.0±0.2
20.0±2.0
25.0±2.0
2.0±0.2
35±0.5
55±05
6.0±0.2
2.0±0.2
200±6.0
inc data were not used in calculating the regression line (Figure 1).
BD Below detection limit of the instrument used.
37
-------
The coefficient of determination (r2) of 0.829 and slope of 0.957 are an indication
that the results of the measurement processes in each laboratory are similar. Of the
52 data pairs, ESL reported higher values for 43, which is reflected in the +0.244 offset
of the plot We cannot determine from the data available whether the frequently higher
values reported by ESL are the result of an unknown background source of metals or
some other cause at either ESL or ERL-A.
CONCLUSION
This inter-laboratory comparison study provided the desired interaction between
chemists at ESL and ERL-A and identified six metals that can be analyzed with
confidence in results. The analysis for zinc presents a problem that must be addressed,
and the analysis for cobalt will require a concentration step to attain easily quantifiable
concentrations. Additional work will be needed to determine reasons for differences in
the results of analyses for metals in seawater. The slope and correlation coefficient for
data on six of the metals measured indicate the data are similar, with a bias toward
frequently higher concentrations at ESL.
ACKNOWLEDGEMENT
Judita Sukyte and Robert V. Thurston assisted in the water sample collections
and preparations in Lithuania. Transportation for our expeditions in Lithuania was
provided by the Lithuania Environmental Protection Department.
REFERENCES
Moody, J., and R. Lindstrom; 1977. Selection and cleaning of plastic containers for
storage of trace element samples. Analytical Chemistry. 49:2264-2267.
Perkin-Elmer. 1982. Analytical Methods for Atomic Absorption Spectrophotometry.
Perkin-Elmer Corp., Norwalk, Connecticut, Sections 8.1-8.2.
Ross, H. B. 1986. The importance of reducing sample contamination in routine
monitoring of trace metals in atmospheric precipitation. Atmospheric
Environment. 20:401-405.
38
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CHEMICALS IDENTIFIED AT TWO PESTICIDE
STORAGE SITES IN LITHUANIA, 1990-1993
JJ. Ellington1, J. Sukyte2
N. Striupkuviene2, and J. F. Neuman3
ABSTRACT
Water samples were collected from two sites in Lithuania being used to store
hazardous chemicals, and these samples were analyzed to determine what chemicals
might migrate from these sites into the surrounding environment. One of these sites, at
Zigmantiskes, is a controlled landfill for banned, unsuitable, and unknown pesticides.'
Water samples were collected from wells inside the boundary of this landfill on five
occasions during the period 1990-1993, and on two occasions during 1993 immediately
outside the landfill boundary. Chemicals considered to be mobile in underground water,
e.g., s-triazine herbicides, were identified in water samples from inside the boundary but'
were below the level of detection in samples from outside the boundary. The second
site is a warehouse near Utena that is being used as a storage facility for toxic chemicals.
A fire occurred at the warehouse in April 1993, and water used to extinguish the fire
seeped into the warehouse basement and its drainage system. Water was subsequently
pumped from the basement into temporary storage tanks to reduce the amount of
contaminated water seeping into the local aquifer. In June and October 1993 water
samples were collected for analysis from the basement, the drainage system, and the
storage tanks. Atrazine and several chlorinated carboxylic acids, including 2,4-D, were
identified in all of these samples.
INTRODUCTION
A field study on the quality of surface waters in the Republic of Lithuania was
initiated in 1990 under the sponsorship of the risk assessment program of the United
States Environmental Protection Agency (U.S. EPA), Environmental Research
Laboratory, Athens, Georgia (ERL-Athens). During the course of this study, two sites
that did not involve surface waters were also investigated, one at Zigmantiskes and the
other at Utena.
The Zigmantiskes site is a controlled landfill established by the Ministry of
Agriculture of Lithuania for the storage of banned, unsuitable, and unknown pesticides
and related wastes; it is located 40 km southwest of Vilnius, between the Merkys and
Salcia Rivers. The perimeter of the landfill is enclosed by a fence and monitoring wells
Environmental Research Laboratory, U.S. Environmental Protection Agency, Athens, Georgia, USA
Lithuania Environmental Protection Department, Vilnius, Lithuania
Fisheries Bioassay Laboratory, Montana State University, Bozeman, Montana, USA
39
-------
have been drilled both inside and outside the perimeter (Figure 1). The second site is a
warehouse near Utena that is now being used as an agrochemical storage facility. It is
located 115 km north of Vilnius, in the Sventoji River watershed. Approximately
32,000 kg of pesticides and unknown powders had been collected and placed in
temporary storage at the warehouse when, in April 1993, a fire started somewhere within
the mass of the stored pesticides. Water, used to extinguish the flames, drained into the
basement of the warehouse, and although much of this runoff water was collected by
pumping into holding tanks, some of it reached an underground drainage system that
empties to a nearby stream.
46D
Figure 1. Zigmantiskes Pesticide Storage Site.
Water samples were collected from the Zigmantiskes site on five occasions
between 1990 and 1993, and from the Utena site on two occasions in 1993. These water
samples were passed through cartridges or disks containing sorbent media to extract the
organic chemicals, and the cartridges and disks were taken to ERL-Athens for elution
and identification of the sorbed organics. This manuscript presents the results of these
analyses.
40
-------
METHODS
Water samples were collected from monitoring wells inside the fenced boundary
at the Zigmantigkes site in December 1990, July 1991, June 1992, June 1993, and
October 1993. In May 1993, monitoring wells were installed outside the fenced
boundary by direction of the Environmental Protection Department (EPD), and in June
and October 1993 samples were also collected from these monitoring wells. Water
samples were collected from three locations at the Utena site in June and October 1993.
The cartridges and disks used for extraction of organic chemicals from water
samples in this study were pre-cleaned at ERL-Athens, and activated in Lithuania. After
water samples were collected, those to be later analyzed for organic acids were adjusted
to pH 2 with sulfuric acid before passage through a cartridge or disk. After return to
ERL-Athens, the cartridges and disks were eluted with organic solvent Those eluants to
be analyzed for organic acids were treated with diazomethane before chromatographic/
spectral analysis. Gas chromatographs (GC) were used to separate the analytes in the
extracts. These were then identified using low and high resolution mass spectrometers
(MS) and Fourier transform-infrared spectrometers (FT-IR) interfaced to the GC. The
methods used for the preparation and activation of the cartridges and disks, collection of
samples, extraction of the organic chemicals, and identification of the extracted organics
has been reported by Ellington et al (1994).
RESULTS
ZigmantiSkes Pesticide Storage Site
Organic chemicals identified from water samples collected at the Zigmantiskes
storage site are listed in Table 1. Dialkyl phthalates are commonly found in
environmental samples, so stringent precautions are necessary to assure their presence in
the collected samples is not due to accidental contamination. Hydrocarbons, phthalates,
and fatty acids were found in a few of the method and field blanks, although at lower
levels than in the groundwater samples. At the present tune, it is not known whether
the phthalates identified in the groundwater samples are representative of the ground
water from which the samples were taken, or whether they resulted from contamination
during collection and/or sample preparation in the field prior to analysis.
Analysis of the extract of a groundwater sample taken from monitoring well #6
(LT-24F) during December 1990 revealed the presence of dimethyl sulfone, caprolactam,
2,4-dichlorobenzoic acid (derivatized to form the methyl ester), simazine, atrazine, and
propazine. The latter three compounds are s-triazine herbicides that have been detected
worldwide in ground and surface waters. Atrazine is one of the most heavily used
herbicides worldwide, with global release in 1980 estimated at 90,000 tons (Rippen
1987). The identities of the s-triazines and the dichlorobenzoic acid were confirmed with
standards. We believe the 2,4-dichlorobenzoic acid is most likely a product of
degradation of a more complex chemical. The 2,4-dichlorobenzene fragment is part of
41
-------
the structure of several agricultural and/or industrial chemicals including
chlorfenvinphos, propiconazole, and polychlorinated biphenyls, whose degradation in the
environment would yield 2,4-dichlorobenzoic acid. Caprolactam is a monomer used in
the synthesis of high molecular weight polymers including Nylon 6, and dimethyl sulfone
is also a solvent for high temperature reactions.
Table 1. Anthropogenic chemicals identified in water samples from monitoring wells at
the Ministry of Agriculture Pesticide Storage Site, Zigmantiskes (LT-24).
Alkyl hydrocarbons1
Atrazine2
Benzophenone1
Caprolactam1
Desmetryn2
Dialtylthiophcne1
2,4-Dichlorobcnzoic acid2
Dichlorprop1
2,2-Dimethoxy-l,2-diphenyl ethanone1
Dimethyl sulfone1
Fatty acids1
Lenacil2
Lindane2
2-(Methylthio) benzothiazole1
Phthalates23
Phytane2
Pristane2
Prometryn2
Propazine2
Pyridine1
Silicon organics1
Simazine2
Tetrachlorinated unknowns1
Identified by GC/FT-IR/MS but not confirmed with standards
identified by GC/FT-IR/MS and confirmed with a standard
3DiisooctyI-, Butylbenzyl-, Di-n-octyl-, and Di-n-butylphthalates were observed the most frequently
In July 1991, new compounds identified in water samples taken from monitoring
well #6 were benzophenone, pyridine, pristane, and phytane. Three compounds that
contained four chlorine atoms each were detected in the acidified sample from well #6
after elution of organics from a solid phase extraction (SPE) cartridge and treatment of
the concentrated eluant with diazomethane. A molecular weight of 348 was determined
by chemical ionization GC/MS for all three compounds, indicating they were isomers.
These isomers were also found in samples from the Neris River and Kaunas Reservoir
(Ellington et al 1994). Best fit empirical formulae of C7H13C14O5P and C14H8C14O2
were determined by high resolution GC/MS. A tetrachlorinated trialkyl phosphate fits
the former formula while tetrachlorinated aromatic esters and other structures
containing carbonyl groups fit the latter. Both formulae are similar to those of
compounds commonly used as electrical insulators and flame retardants. The compound
containing phosphorus was eliminated as a possibility by further GC analysis using
nitrogen and nitrogen-phosphorus specific detectors. The presence of the carbonyl
functional group in the three unknowns was confirmed by GC/FT-IR. The loss of the
m/z 31 ion (loss of —OCH3) during low resolution electron impact GC/MS argued for
the presence of a methoxy functional group, possibly a methyl ester. However, the
absence of a strong peak in the 1250-1310 cm"1 region of the IR spectrum and the broad
carbonyl peaks argued against the ester functionality. Detection of the unknowns only
after treatment of the extract with diazomethane is evidence that they contained a
reactive hydrogen. A tetrachlorinated aromatic ring system containing a carbonyl group,
methoxy group, and differing chlorine substitution is the most likely structure for the
42
-------
three unknown isomers. The presence of pristane and phytane could be an indication of
contamination by crude oil, while benzophenone and pyridine are used in the synthesis
of organics.
In June 1992, samples were also taken from wells #1, 2, 3, and 5 (LT-24A-QE)
which had not been sampled during previous trips. Chemicals in a sample from well #5
(LT-24E), first tentatively identified by GC/MS and/or GC/FT-IR/MS, and subsequently
confirmed with standards, were: atrazine, desmetryn, lenacil, lindane, prometryn,
propachlor, propazine, and simazine. Chemicals tentatively identified in this same
sample, but not confirmed with standards, included 2,2-dimethoxy-l,2-diphenyl ethanone,
dialkylthiophene, and 2-(methylthio)thiophene.
During June and October 1993, water samples were collected from three of the
four monitoring wells (LT-46A-D) installed outside the boundary fence of the landfill in
May 1993. Chemicals tentatively identified by GC/FT-IR/MS in extracts of samples from
wells #01 and 02 (LT-46A and B), downgradient from the underground water flow from
the landfill, were sulfur, dibutyl- and dioctylphthalates, hydrocarbons, and carboxylic
acids. These chemicals were almost identical to those found in the water taken from
well #4 (LT-24D) located 1.5 m inside the boundary and upgradient from well #01
outside the boundary. Except for phthalates and hydrocarbons, none of the chemicals
identified in samples from wells #1, 2, 3, 5, or 6 inside the boundary appeared in
samples from well #4 also inside the boundary, or wells #01 or 02 outside the boundary.
Phthalates and hydrocarbons were detected in trace amounts in the majority of samples,
and we believe their presence was due to contamination from equipment during
sampling procedures. The absence in wells outside the boundary of other chemicals
found in wells inside the boundary, especially the extremely mobile chemical atrazine, is
evidence that chemicals at the Zigmantiskes site have not migrated beyond the landfill
boundary at this time.
Utena Pesticide Storage Warehouse
The chemicals stored in the warehouse at Utena at the time of the fire in April
1993 had been inventoried by the Lithuania EPD and the Ministry of Agriculture. The
inventory showed 31,586 kg of pesticides and unknown powders, with the unknown
powders (23,730 kg) and unknown solids (1,610 kg) comprising the majority of that
inventory. The remaining pesticides consisted of fentiuramas (3,458 kg),
2% ethylmercury chloride (190 kg), ziramas (970 kg), nitrophen (1,070 kg), and DNOC
(2-methyl-4,6-dinitrophenol, 558 kg) (LEPD 1993). After the fire, runoff water was
pumped from the basement of the warehouse into two 50,000-L tanks (LT-45QD) to
prevent environmental contamination from water seeping into the drainage system and
into a nearby creek. The volume of water in both tanks was approximately 26,000 L. In
June 1993. a 100 ml sample of water from LT-45D was extracted by EPA Method 505
(base/neutrals) and EPA method 515.1 (chlorinated acids), and the extracts were
analyzed by GC/MS (U.S. EPA 1991). Compounds identified from this sample are listed
in Table 2.
43
-------
Table 2. Anthropogenic chemicals1 identified in water samples collected at the Toxic
Chemical Storage Warehouse, Utena (LT-45). (Detected concentrations were in
the ppb range.)
Chemicals by class2 LT-45A LT-45D LT-45E
Carboxylic acids
Chlorobutenoic acid
Chloropropanoic acid
Dichloroacetic acid
Dichlorobutenoic acid
Dichloropropanoic acid
Trichloropropanoic acid
Trichloroacetic acid.
Phenols
Chloromethylphenol
Dichlorophenol
Dimethylphenol
Methylphenol
Trichlorophenol
Phenoxycarboxylic acids
Chloromethylphenoxyacetic acid
Chloromethylphenoxybutanoic acid
Dichlorophenoxyacetic acid
Dichloromethylphenoxyacetic acid
Dichlorophenoxybutanoic acid
Dimethylphenoxyacetic acid
Dimethylchlorophenoxyacetic acid
NJNf-Dimethyl-2-(chloromethylphenoxy)-acetamide
Methylphenoxyacetic acid
Tricliloromethylphenoxyacetic acid
s-Triazine/phosphate pesticides
Atrazine
Propazine
Simazine
Vapona
Ureas and thiooxamides
Lenacil
Tetramethylurea
Tetramethylthiourea
N,N,N',N'-Tetramethylthiooxamide.
N,N,N',N'-Tetramethyldithiooxamide
x
x
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
The EPA/NIH Mass Spectral Library was used for tentative identification of the chemicals identified by "x".
Atrazine, simazine, propazine, and lenacil were confirmed with standards.
High resolution GC/MS was used to determine the molecular ions and empirical formulas. Identical empirical
formulas for some peaks indicated isomers but the structures of the individual isomers could not be
determined from the spectral information.
44
-------
Mercury was suspected to be in the water in the holding tanks, so in the summer
of 1993 the Lithuania EPD attempted to precipitate this mercury by the addition of
ferric chloride. In addition, calcium hypochlorite was added to degrade other pesticides.
Water in LT-45C was transferred to LT-45D, and the combined contents were treated
with FeCl3-6H2O and Ca(OCl)2. In October 1993, samples of water from the ferric
chloride-treated water (LT-45E) and from the underground drainage system (LT-45A)
were extracted and the resulting base/neutral and acid fractions were -analyzed by
GC/MS. The compounds identified in these two samples are also listed in Table 2.
Three chemicals, namely, tetramethylurea, and dichloro- and
trichloromethylphenoxyacetic acids, were detected in all three samples. The ferric
chloride/calcium hypochlorite treatment had reduced the concentrations of many
chemicals below their detection level, and the chemicals that were detected in that
sample were factors of 2-8 below the levels previously determined in the untreated run-
off water (LT-45D). The concentration of isomers of hexachlorocyclohexane (HCH),
namely 7-HCH (iindane), and a-HCH, was reduced 50- to 200-fold by the ferric
chloride/calcium hypochlorite treatment from initial concentrations down to
concentrations of 0.06-0.11 ppb. The treatment also reduced the concentrations of
dichlorophenoxyacetic acids from 250 down to 30 ppb, and tetramethylurea was reduced
from 600 to 200 ppb. The corresponding concentrations for these two chemicals in the
drainage system water (LT-45A) were 12 ppb and 30 ppb, respectively. The s-triazine
herbicide concentrations ranged from 250 ppb in the holding tank water to 10 ppb in the
drainage system water, and was not detected in the treated water.
CONCLUSIONS AND RECOMMENDATIONS
The ZigmantiSkes Pesticide Storage Site is only one of several possible
groundwater pollution sources in Lithuania. The ferric chloride/calcium hypochlorite
treatment of the runoff water in the tanks at Utena substantially lowered the
concentrations of many of the chemicals, some to below their detection limits, but the
toxicity of the treated water remains unknown. Chemicals in groundwater have the
potential to migrate and contaminate off-site water supplies, and for this reason
monitoring should be continued in the area of the Zigmantiskes site and should be
expanded to include other such facilities as they are identified. The monitoring at the
ZigmantiSkes site, now that several of the chemicals in the underground water have been
identified, could be based on an immunoassay test for the most mobile chemical.
Atrazine, because of its water solubility and low partitioning to organic carbon, is one of
the more mobile chemicals identified in samples from both Zigmantiskes and Utena.
Commercial immunoassay test kits are available for atrazine and could be used to
monitor its movement in underground water at both sites. These kits are relatively
inexpensive, their use does not require extensive technical training, and the results can
be provided on location within minutes.
45
-------
ACKNOWLEDGEMENTS
Ruta SiuSiene, Jonas Masiulionis, and Robert V. Thurston assisted in the water
sample collections and Svetlana Urboniene, Naste Seskuviene, and Robert V. Thurston
in their preparations in Lithuania. Transportation for our expeditions in Lithuania was
provided by the Lithuania Department of Environmental Protection. The low resolution
and chemical ionization GC/MS analyses were performed at ERL-Athens by John Pope
and Alfred Thruston, Jr., the high resolution GC/MS analyses by Susan Richardson, the
GC/FT-IR/MS analyses by Timothy Collette and George Yeager, and the solid phase
extraction devices and the extracts were prepared by Terry Floyd. This research was
supported in part by U.S. EPA Cooperative Agreements CR816369 and CR995189 to
Montana State University.
REFERENCES
Ellington, J.J., R.V. Thurston, J. Sukyte, and K. Kvietkus. 1994. Hazardous chemicals
in surface waters of Lithuania, 1990-1993. In: Environmental Studies in the
Nemunas River Basin, Lithuania. U.S. Environmental Protection Agency, Athens,
Georgia, USA.
LEPD (Environmental Protection Department of the Republic of Lithuania).
Water Quality of Lithuanian Rivers, 1992. Annual Report. Vilnius.
1993.
Rippen, G. 1987. Handbuch Umweltchemikalien, 2nd ed. Ecomed: Landsberg/Lech,
Federal Republic of Germany, (In German), as cited in: H-R. Buser, Atrazine
and other s-triazine herbicides in lakes and in rain in Switzerland. Environmental
Science & Technology 24:1049-1058.
U.S. EPA (U.S. Environmental Protection Agency). 1991. Methods 505 and 515.1
In: Methods for the Determination of Organic Compounds in Drinking Water.
EPA/600/4-88/039, Revised July 1991. U.S. EPA, Washington, DC, USA.
46
-------
PROBLEMATIC ASPECTS OF NEMUNAS RIVER
LOAD MONITORING
S. Zareckas, A. Galkus, and K. Joksas1
ABSTRACT
Problematic aspects of monitoring are analyzed on the basis of microscopical and
chemical analysis of material composition of water, suspensions, suspended load, bed
load, and bottom core sediments of the Nemunas River during 1990-1991. These
include organic matter, trace metals, and hydrocarbons. Taking into account saturation
of different transport media with anthropogenic ingredients and their spatial/temporal
fluctuations, we have concluded that it is groundless to use only water for monitoring
and assessment purposes. We consider comprehensive knowledge about recent
sedimentation processes to be most important in assessing pollutant trends. We
recommend that some of the transport media, such as bottom sediments and, in certain
cases, water, should be used for identification of local pollution, and others, particularly
sediment load, for assessment of changes of chemical-and physical state. Analysis of
suspended load material, collected by long-term sampling methods which integrate short-
term and accidental fluctuations, is indispensable in the monitoring of dynamic trends of
environmental pollution.
INTRODUCTION
Advances in physical and chemical analytical methods and technical
measurements, as well as an increase of information about contamination of water
bodies, have not only failed to bring the implementation of objective monitoring closer,
but in some cases have confused our ability to assess realistically the state of an aquatic
system at a given time. Objective monitoring is impeded by limited understanding of the
ecological-philosophical approach even among researchers themselves or, at best, failure
to recognize how it may be realized. Furthermore, the concept of the water basin as a
system with an undefined number of controlling variables does not exist. The absence of
a search for causality of an observed state in certain investigations (or self-limitation by
empirical explanations) along with the increase of non-representative measurements not
only fails to help solve the questions of objective monitoring and assessment of
ecological state, but is even harmful as a stereotype.
Institute of Geography, Marine Geochemistry and Sedimentology Division, Vilnius, Lithuania
47
-------
The frequent non-representative character of pollution measurements is
predetermined by an abundance of localized point-sources, the especially complex
structure of pollutant transport, and ignorance about the complexity of natural processes
taking place in aquatic systems. All this forces us to doubt the correctness of available
data and the validity of its interpretation. Technological pollution must be investigated
against the background of natural equilibria and regularities and not vice versa.
The speculations presented here concern all larger water bodies including Kursnj
Marios Lagoon, the Baltic Sea, and the Nemunas River, which, integrating and carrying
the largest part of Lithuanian total sediment load, which should obtain priority in the
national monitoring program. Based on the data collected in 1990-1991 from this main
Lithuanian artery, we shall attempt to define the problematic aspects of the Nemunas
River system monitoring.
Natural and technogenic substances carried by the Nemunas River take part in
the sedimentation cycle (inflow-migration-transformation-precipitation-accumulation on
the bottom), where they change phases and composition, fractionate, and undergo
physical-chemical reactions and related changes of pollutant toxicity. The results of
discrete investigations of only one medium may be biased by random observation of one
link of the sedimentation cycle without knowing whether the point of observation was in
a zone of transition, partial discharge, or rapid sedimentation. Different media and
migration forms of a substance are not equally informative. Some are helpful in
identifying aspects of local pollution, while others are more helpful in characterizing the
state of the aquatic system. Elaboration of the concept of Nemunas River monitoring
demands a preliminary analysis of the character, direction, and intensity of predominant
processes. This prompted us to carry out a qualitative and quantitative analysis of the
interrelation between priority pollutants in different media and migration forms at
different cross-section locations along the river.
MATERIALS AND METHODS
Samples of water, suspensions, suspended load, bed load, and bottom sediments
from cores were taken for chemical and microscopical investigations during April to
October 1990 and 1991 in the lower part of the Nemunas River (Figure 1). Material
composition and particle size of the samples, hydrocarbon content, nature of the
material, and trace metal concentrations (iron, manganese, copper, zinc, cadmium, lead,
and chromium) were determined. Water and samples of suspended matter were taken
from the surface (0-20 cm) and near-bottom (50-100 cm above the bottom) layers in
three to five points of river transverse profile. Suspended load material was collected
with traps (vessels with 0.3 L capacity and 6 radially spread out holes with 0.7 cm
diameter) in the central channel of the river at distances of 0.2, 0.4, 0.6, 1.0, and 1.5 m
above the bottom. Bed load was caught with traps of original construction. Exposition
time of the traps was 1-2 days. Bottom sediments were taken with a vacuum cylinder
(length 1 m, diameter ~5 cm) at five to eight locations along a cross-section of the river
profile.
48
-------
Figure 1. Situation scheme: Station 1-Atmata, Station 2-Pakalne, Station 3-Skirvyte.
Suspensions were collected with the aid of filters containing 0.45 /*m diameter
pores. Suspensions, suspended load material, and bed load samples were prepared for
examination according to uniform methods (Lisitsin 1956, Bogdanov and Lisitsin 1968),
using dyes and clarifying with Canada balsam. Particles under the microscope were
counted separately for the following fractions: >0.05, 0.05-0.01, 0.01-0,005, 0.005-0.0025
and <0.0025 mm. Depending upon concentration, particles were counted in the 2-5th
microscope fields.
For hydrocarbon analysis in different media, we applied our own methods
(Zareckas 1989). In water, suspension, bed load, and bottom sample analyses the same
solvent was used, seeking to avoid methodical complications, and allowing comparison of
results from different media. Hydrocarbon analyses were performed by gas
chromatography (Chrom-5™).
Metals in the water column were investigated in two phases: soluble and
suspended. To distinguish between them, water samples were filtered through Dubna™
lavsan filters with a pore diameter of 0.45 /un. The suspended matter that accumulated
on the filters was dissolved in acid mixture of HNO3 and HCICX in teflon crucibles. The
soluble metals were concentrated and precipitated with Tioxine™ (Vircavs et al 1984),
and the accumulated tioxinates were filtered through Dubna™; these also were
decomposed with an acid mixture of HNO3 and HC1O4. Metals from sediment traps,
bed load, and bottom sediment samples were each also similarly dissolved in acid
mixture. In the resulting five concentrates, the content of metals was determined by
atomic absorption spectroscopy (Perkin-Elmer Model 403).
49
-------
RESULTS AND DISCUSSION
The concentration of soluble trace metals and oil hydrocarbons carried by the
Nemunas River water is not high. Hydrocarbon content is 0.6 to 52 /*g/L, which is close
to or below the maximum permissible limit of 50 /*g/L. (Sachaev and Scherbitsky 1986).
The spatial distribution of oil products in water is especially complex. Lateral and
vertical variations of hydrocarbon within a cross-section (Smalininkai-112 km, Rusne-
15 km) can vary as much as eight times, which is almost the same variation as for the
whole Nemunas River (Figure 2). In addition, occasional variations in the cross-
sectional concentrations of trace metals in the Nemunas River, which can vary as much
as nine-fold, are caused by point-source pollution. There are also marked variations in
metal concentrations in the Nemunas River profile attributable to sorption onto
suspended matter (Figure 3). The concentration of total suspended material may reach
as much as 40 mg/L toward the lower Nemunas River and, as a consequence, the total
load of metals transported on particulates also increases. Because of abrupt and
frequent changes in trace metal concentrations, especially cadmium, lead, zinc, and
copper which can have 20-fold variations, this eliminates either suspended or soluble
trace metal forms as optimum media for continuous monitoring.
max
30
112 108 98 86 56 15 (5) (2) 0 O
DISTANCE FROM MOUTH (Km)
Figure 2. Concentration of hydrocarbons, suspended load (HC ), and
changes of technogenization degree (percent HC«10~4/percent Cor)
in a longitudinal profile of the Nemunas River.
50
-------
"ttJF 86 58 15 _
DISTANCE FROM MOUTH (km)
Figure 3. Substantial and microelemental composition of normally suspended
microparticles (suspensions). Substantial composition percent:
(1) mineralogenic, (2) autochthonous detritus, (3) allochthonous detritus,
(4) phytoplankton skeletons. Chemical composition percent
(ppm = 10~4 .%): Fe x 1; Mn, Pb x ICT2; Cu, Zn, Cr x lO'3; Cd x 10~4.
D5p = medial diameter of transported substance. C = concentration of
suspensions.
Suspended matter collected in the long-term sampling traps may not be
representative of the river runoff suspended matter as a whole because those particles
likely possess higher sinking rates resulting from different granulometric and substance
composition. Nonetheless, the trap material integrates short-term fluctuations of
pollution over a long period of time and permits observation of the dynamics of
transported pollutants and concomitant changes of river "health."
51
-------
With decreasing water current speed and diminishing median diameter (D50) of
transported particles, as well as with mechanical fractioning of suspended material in
which mineralogenic particles predominate, there is an increase in the concentrations of
the trapped trace metals and hydrocarbon material (Figures 2, 4). As the solids
suspended in the Nemunas River begin to deposit in the Nemunas River delta
(Figure 1), the river becomes less polluted (Table 1). The degree of oil pollution
changes correspondingly. Hydrocarbon, expressed as percent of organic carbon (C ),
has been calculated at 4% at Rusne (Figure 2), 15 Km from the river mouth. This
parameter is about 0.05% for natural organic matter in the river. In transported
sedimentary matter of the Nemunas River delta, biogenic hydrocarbons do not exceed
3% of the total of all hydrocarbon.
~9*i
E
•£ H
1-
'Sf
_E
I20
z
g
I
Zn Fe.Mn.Pb.Cr.Cu.Cd
CONCE
9
10-
*.
A
T
•Fe
#Mn
•Zn
TCr
165 112 86 58 15
DISTANCE FROM MOUTH (kml
Figure 4. Substantial and microelemental composition of suspended load
(settleable solids). Substantial composition percent: (1) mineralogenic,
(2) autochthonous detritus, (3) allochthonous detritus, (4) phytoplankton
skeletons. Chemical composition percent (ppm = 10 ~4 %): Fe x 1;
Mn x 10 ~2; Cu, Zn, Pb x 10 ~3; Cd, Cr x 10 ~4. D50 = median diameter
of transported substance. C=concentration of suspensions. V=current
velocity.
52
-------
Table 1. Suspended matter runoff.
Substance Groups of Detritus (kg/s)
Section
(Distance
from mouth)
Smalininkai
(112 km)
Smalininkai
-bed load material
Rusne (IS km)
Atmata (Delta)
Skirvyte (Delta)
Pakalne (Delta)
Pakalne (Delta)
-bed load material
Q
(m3/s)
515
370
176
161
35
R
(kg/s)
12.23
0.0184
11.19
3.87
5.96
0.98
0.00013
Autoch-
thonous
0.61
1.23
0.13
0.21
0.054
Biogenic
Alloch-
thonous
0.49
0.28
0.17
0.15
0.005
Phyto-
plankton
734
5.09
2.09
1.55
0.627
- Mineral-
ogenic
3.79
4.59
1.48
4.05
0.29
As a result of sorption of oil products, the amount of transported hydrocarbon in
suspended form regularly increases towards the lower Nemunas River when compared
with the soluble form. In the Nemunas River mouth the ratio of suspended to dissolved
hydrocarbons reaches 25. Similar changes in trace metal forms occur (Figure 5).
100-:
80-
|60-
2 4O-
=3
o ?
5
Fe Mn Cu Zn Cd Pb Cr
Fe Mn Cu Zn Cd Pb Cr
112km
Figure 5. Change in metal forms in the lower course of the Nemunas River:
(1) metals in suspension, (2) metals in soluble form.
53
-------
On average, 90% of the zinc, 86% of the copper, 81% of the cadmium, and
67% of the lead are carried in suspended form by the Nemunas River (Table 2). As
background values constantly change, these must be borne in mind when evaluating the
health of an aquatic system.
Table 2. Total chemical runoff (g/s). Percent suspended shown in parentheses.
Section
-bed load material
Rusne (15 km)
Atmata
Skirvyte
Pakalne
Pakalne
-bed load material
(l(T5g/s)
Fe
Mn
Cu
Zn
Cd
Pb
Cr
263
(65)
125
(72)
156
(64)
21
(70)
145
3.20
(35)
130
(45)
3.03
(75)
0.23
(52)
18.10
1.21
(88)
0.32
(97)
1.11
(94)
0.12
(92)
0.36
3.35
(90)
2.60
(88)
2.07
(90)
0.42
(93)
1.66
0.084
(95)
0.025
(20)
0.072
(97)
0.013
(77)
0.030
2.23
(45)
3.29
(76)
1.16
(61)
0.21
(62)
036
1.4
(59)
0.80
(49)
0.75
(61)
0.14
(71)
0.54
HC
Smalininkai
(112 km)
Smalininkai
496
(73)
8830
7.05
(66)
773
131
(79)
13.80
6.43
(86)
20.10
0.050
(60)
1.310
8.20
(71)
20.10
3.12
(51)
0.92
12.01
(7.50)
1.64
33.68
(68)
4.78
(47)
19.45
(66)
1.42
(87)
2.89
The longitudinal distribution of pollutants in the surface layer of bottom
sediments of the Nemunas River is much more complex, generally decreasing towards
the delta, even though there may be occasional maxima (Figures 6 and 7). Fluctuations
of hydrocarbon concentrations can be 1,0.00-fold, thereby altering the typical exponential
dependence of trace metals and hydrocarbon content upon median diameter (Figure 8).
The anomalies are related to intensive accumulation of nonhomogeneous sedimentary
matter below the larger sources of pollution entering the Nemunas River between
Kaunas and the river mouth at 199, 165, 112, and 56-58 km. The concentration of
metals in bottom sediments is directly related to organic substances (Figure 8) which
have good sorptive capacity and transport pollutants to the bottom. An important role
in hydrocarbon accumulation and transportation is played by phytoplankton, the
skeletons of which control the concentration of oil products in the transported
sedimentary matter and bottom sediments (Figure 6).
54
-------
lfe5 158 li2 1O8 98 & 71
DISTANCE FROM MOUTH (km)
Figure 6. Changes of the content of hydrocarbons (HC ) and degree of
technogenization (percent HC»lQ-4/percent C ) in the bottom sediments
of longitudinal profile of the Nemunas River. F = amount of phytoplankton
skeletons (percent) in (1) suspended load, (2) and bottom sediments.
121
11-
9
7
5-
~ 3
^^ I'
Z
o
u
\ -A
A
\
V.
-—TV NiZn
9
g 5 S
-------
E
n.
Q.
%— *•
OT
1U .
1 2
Corg(%)
CO 02 03 ft* Q5 Qg
D50(mm)
op
^04-
£03
(§02
0.1
10
Ig HC(ppm)
woo
Figure 8. Metals concentration dependence in bottom sediments on organic
matter (C ) and median diameter (D50), and hydrocarbons content
dependence on D50 in (1) bottom sediments, and (2) suspended load.
(Circled region contains anthropogenically polluted samples.)
CONCLUSIONS
Distribution of priority pollutants in different media in the Nemunas River, and
the tendencies to change both within and among these media, lead us to conclude that a
better way to monitor trace metals and hydrocarbons in the Nemunas River would be to
analyze sedimentary matter collected from long-term sampling traps. Contamination
intensity must be evaluated in the context of natural variation and trends, and in the
context of types of samples being studied and how the sample types vary in respect to
one another. Furthermore, to be able to assess the condition of those sections of the
river below larger pollution sources, locally accumulating bottom sediments should also
be monitored.
56
-------
REFERENCES
Bogdanov, Y.A. and A. P. Lisitsin. 1968. Distribution and composition of suspended
organic substance in the water of Pacific Ocean. Oceanological Investigations,
Moscow, 18:75-155. (In Russian)
Lisitsin, A.P. 1956. Methods of sampling and investigation of water suspensions for
geological goals. Scientific Works of the Institute of Oceanology Academy of
Sciences of the USSR, Moscow, 19:204-231. (In Russian)
Sachaev, V.G., and B.V. Scherbitsky. 1986. A Reference Book on Environmental
Protection. Kiev, 149 pp. (In Russian)
Vircavs, M.V., O. Veveris, and J.A. Bankovsky. 1984. Concentration of microelements
by precipitation with organic disulphides on the ground of 8,8-dichinolildisulphate.
Geology of Oceans and Seas. Materials of All-Union School of Marine Geology,
Moscow, 1:104-105. (In Russian)
Zareckas, S. 1989. A complex of methodical procedures of extraction and concentration
of hydrocarbons in the investigation of marine ecosystems. Transactions of the
Academy of Sciences of Lithuania, Vilnius, 1(170): 128-134. (In Russian)
57
-------
-------
INVESTIGATION OF MICROORGANISMS:
BIODEGRADERS ASSOCIATED WITH HYDROBIONTS
J. Syvokiene and L. Mickeniene1
ABSTRACT
The ability to decompose oil products is characteristic of the microorganisms in
the digestive tract of molluscs. Molluscs were collected from the Nemunas River and
KurSiu Marios Lagoon in 1991, and their activity is related to using hydrocarbons as a
source of carbon in the processes of water purification. The largest number of oil-
degrading bacteria was obtained from digestive organs of specimen #708
Anodonta piscinalis, collected upstream from Smalininkai. The number of oil-degrading
bacteria exceeded that of saprophytic bacteria in the digestive tract of investigated
molluscs in most cases. En:zymatic systems of microorganisms from the digestive tract of
molluscs take part in an inactivation process of biologically active materials. A. piscinalis
can be used as a biofilter in ecologically polluted waters. Determination of
microorganisms in the digestive tract of molluscs or other hydrobionts which take part in
degradation of oil-products, will strengthen the knowledge base of ecosystem processes
which aid in self-purification.
INTRODUCTION
Ail increase in environmental pollution undoubtedly has a strong influence on
functional activity and properties of microorganisms of hydrobionts. The effects include
the localization and specialization of microflora inhabiting the digestive tract. Because
of changes in the living conditions of hydrobionts, alterations in the community structure
of intestinal microorganisms occur, as do changes in the regularities of functioning of
some populations. Any pollutant entering the environment has a potential negative
effect on water quality, and tends to impact intestinal microorganisms of hydrobiont
populations. Data on microorganisms, located in the digestive tract of molluscs taking
part in decomposition of hydrocarbons, are scarce.
The purpose of this research was to show the direct participation of
microorganisms associated with individual molluscs in the decomposition of pollutants of
organic origin. These investigations are important for two reasons: determination of
common biological regularities, and application of the results in predicting the response
of natural communities of hydrobionts to anthropogenic impact. Participation of
microflora of hydrobionts in water purification processes is not well understood, and has
not been widely studied. Natural water purification through the activities of hydrobionts
may be a very important ecological process.
Institute of Ecology, Vilnius, Lithuania
59
-------
MATERIALS AND METHODS
Two species of molluscs (Unto pictorum L., Anadonta piscinalis Nilsson) were
collected for microbiological investigations in 1991 from the Nemunas River at
Smalininkai and from Kurshj Marios Lagoon at Ventes Cape. Microorganisms isolated
from the intestine, crystalline style, and coelomic fluid of these molluscs were evaluated
according to their ability to grow on agar media containing hydrocarbon. Samples were
taken from the digestive system of every specimen from Smalininkai and of 3-5
specimens of molluscs from the Ventes Cape. The digestive systems were investigated
according to methods described by Mattheis (1964). The ability of microorganisms to
degrade black oil and diesel oil hydrocarbons was investigated by measuring growth
based on colony-forming counts on agar media (Gerhardt 1983), where the hydrocarbon
served as the only source of carbon. Other microbiological indices were evaluated
according to methods of Rodina (1965) and Romanenko (1985).
RESULTS
The data show that microorganisms of molluscs are possibly capable of using
hydrocarbons as the only source of carbon and of growing in such media (Table 1). The
concentration of oil-degrading bacteria was different in separate organs, with the highest
concentration being found in the intestine and crystalline style. The number of
microorganisms in coelomic fluid was less than in the intestine by a factor of 10 or more
(Figure 1), and even fewer were found in water (from 11 x 103 to 19 x 103 cells/ml) and
in soil (25 x 105 cells/g) (Jankevicius et al 1991).
Table 1. Number of microorganisms in the content of intestine (I), crystalline style (CS),
and coelomic fluid (CF) of molluscs from two sampling sites in Kursm Marios
Lagoon (I and CS, millions/ml; CF, millions/g).
Oil degrading bacteria
Saprophytic bacteria
With black oil
With diesel oil
specimen number
and species
I
CS
CF
I
CS
CF
I
CS
CF
Smalininkai
537
538
707
708
Unio pictorum
it
Anadonta piscinalis
n
0.20
5.20
3.40
3.40
1.20
0.30
0.10
3.80
0.02
0.10
0.09
1.20
0.40
1.40
150
5.60
0.00
0.20
0.01
8.20
0.04
0.20
o.oi
0.10
2.10
1.40
2.70
6.80
0.10
0.06
0.30
0.01
0.05
0.03
0.07
0.10
Ventes Cape
U. pictorum
A. piscinalis
4.10
3.50
1.00
3.10
0.08
1.40
1.20
4.80
0.30
6.80
0.18
0.08
2.00
4.70
0.15
0??,
0.02
006
60
-------
10
-D
3
Z
Saprophytic Bacteria
Intestinal Crystalline Coelomic
tract stylo fluid
|S37 Una pfc/erum
Oil-Degrading Bacteria on Block Oil Media
Intestinal
tract
Crystalline Coelomic
style fluid
011-Oea.roding Bacteria on Diesel Oil Media
Intestinal
tract
1538 Unto piclcrum
1707 Anodonte facinola
Crystollline Coelomic
»tyf« fluid
1708 Aaocbnlo fifdnofft
Figure 1. Bacteria counts in intestinal tract, crystalline style,
and coelomic fluid of Unto pictorum and Anadonta piscinalis.
Results indicate that microorganisms from the digestive tract of molluscs might
grow by degrading oil pollutants. In agar containing black oil (Figure 2) and diesel oil
(Figure 3), degradation occurred based on increases in the number of microbial cells. In
contrast, on media containing no black or diesel oil (Figure 4), microbial growth was
negligible.
In all the individuals studied saprophytic bacteria were uniformly distributed,
however, their concentration fluctuated and the highest concentration observed was in
the intestine and the lowest in coelomic fluid (Table 1). Data of Zandmane (1984)
indicate that water pollution may be evaluated by determining the ratio of oil-degrading
to saprophytic bacteria. Our investigations confirmed the findings of Zandmane,
although we also found that in the presence of both black and diesel oil the oil-
degrading bacterial counts were high and rivaled the saprophytic bacterial counts.
61
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Figure 2. Growth of bacteria isolated from the intestine of molluscs
and incubated on agar media with black oil.
Figure 3. Growth of bacteria isolated from the intestine of molluscs
and incubated on agar media with diesel oil.
Figure 4. Growth of bacteria isolated from the intestine of molluscs and
incubated on agar media without either black or diesel oil (control).
62
-------
DISCUSSION
Literature dealing with research on oil degradation by microbial hydrobionts in
molluscs is virtually nonexistent. There is some information about aquatic
microorganisms and intestinal microflora of mussels (Mytilus) taking part in
decomposition of hydrocarbons in fresh water (Mironow 1985), and in brackish water
(Puchenkova 1988a, Gunkel and Dahlman 1986, Shamshoom et al 1990). There are
several opinions concerning the authenticity of oil-degrading bacteria (Prieur 1982,
Minet et al 1987, Ledo et al 1983, Puchenkova 1988b). The large concentration of
microorganisms found reveal that hydrocarbon utilizers are inhabitants of the intestine of
molluscs. It should be noted that enzymatic systems of microorganisms from the
digestive tract of molluscs may play an important role in transformation processes of
biologically active combinations of black oils.
Intestinal microflora react to changes in environmental conditions. When
microbiological systems of organisms are disturbed, biosynthetic activity of
microorganisms appear affected, with a weakening of enzymatic functions.
Microorganisms, however, utilize pollutants and convert them into organic acids, amino
acids, and other products, which serve as a source of energy. Hydrocarbon degradation
was characteristic of microorganisms associated with the content of intestinal tracts of
molluscs collected at Ventes Cape, and in three of the four molluscs collected at
Smalininkai. The functional activity of hydrocarbon-degrading microorganisms is related
to availability of a metabolizable carbon source (oil product), and the concentration of
microorganisms taking part in self-purification processes.
Identification of oil-degrading bacteria in the digestive tract of bivalve molluscs
assists in the evaluation of the health of ecological systems. This study is intended to be
helpful in future monitoring of oil pollution levels. Research should be expanded on oil-
degrading bacteria in the digestive tracts of other species of hydrobionts, Le., in fishes
such as roach (Rutilus rutilus) and stickleback (Gasterosteus aculeatus).
REFERENCES
Gerhardt, F. (Ed). 1983. Methods of General Bacteriology. Mir, Moscow.
(In Russian)
Gunkel, W., and G. Dahlman. 1986. Bacterial degradation of heavy fuel oil in sea
water. Baltic Sea Environmental Proceedings 22:68-80.
63
-------
Jankevicius, K., A. Antanyniene, A. Baranauskiene, S. Budriene, R. Du§auskiene,
J. Ilgevicmte, G. Jankeviciute, J. Kasperaviciene, A. KuSinskiene, S. Mazeikaite,
A. Simanaviciene, G. Slapkauskaite, G. Sulijiene, and I. Trainauskaite. 1991.
Peculiarities of hydrobiological regime and the processes conditioning the water
quality in the Kaunas reservoir and the Kurshj Marios lagoon. In: R. Lekevicius
(Ed.), Pollution of the Nemunas Water Basin and its Biological Effect on the
Ecosystem. Academia, Vilnius, pp. 59-64. (In Lithuanian)
Ledo, A., E. Gouzdess, J. Barja, and A. Tossuso. 1983. Effect of depuration systems on
the reduction of bacteriological indicators in cultured mussels (Mytilus edulis).
Shellfish Research 3:114-121.
Mattheis, T.H. 1964. Ecology of intestinal bacteria of freshwater fish. (Family
Pseudomonadaceae, Genus Pseudomonas). Fishery Journal 12:507-536.
(In German)
Minet, J., T. Barbosa, and D. Prieur. 1987. Formation of the clear model of Mytilus
edulis. Reports of the Academy of Sciences 3:305. (In French)
Mironow, O.G. 1985. Interaction of Marine Organisms with Hydrocarbons of Oil
Products. Nauka, Leningrad. (In Russian)
Prieur, D. 1982. Microflora of the digestive tract of marine bivalves using the mussel
Mytilus edulis as a model. Malacologia 22:653-658. (In French)
Puchenkova, S.G. 1988a. Sanitary-microbiological characteristics of the Black Sea
mussels in the regions of their rearing. Hygiene and Sanitary 7:77-78.
(In Russian)
Puchenkova, S.G. 1988b. Bacteria of genus Acinetobacter in marine environment and
molluscs. Journal Microbial Epidemiological Immunology 3:18-21. (In Russian)
Rodina, A.G. 1965. Methods of Aquatic Microbiology. Nauka, Moscow-Leningrad.
(In Russian)
Romanenko, V.I. 1985. Microbiological Processes of Production and Destruction of
Organic Matter in Inland Water Bodies. Nauka, Leningrad, (In Russian)
Shamshoom, S.M., T.S. Ziara,. A. N. Abdul-Ritha, and A. E. Yacoub. 1990.
Distribution of oil-degrading bacteria in northwest Arabian Gulf. Marine
Pollution Bulletin 21:38-40.
Zandmane, A.K. 1984. Ecology of bacterioplankton of the rivers in Latvian SSR under
the conditions of anthropogenic impact. Synopsis of thesis, Candidate in
Biological Sciences, Minsk, 22 pp. (In Russian)
64
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TOXICITY OF ACETANILIDE HERBICIDES AND THEIR
BIODEGRADATION IN PURE PSEVDOMONAS CULTURES
A. Cetkauskaite1, V. Jankauskas1, J. Berzinskiene1,
E. Bakiene1, and W. C. Steen2
ABSTRACT
The toxicity of acetanilide herbicides was determined using a Photobacterium
bioluminescence quenching method. It has been shown that both propanil and its
primary degradation product, 3,4-dichloraniline, are more toxic, especially at lower
concentrations, than propachlor and its degradation product, aniline. Biodegradation
kinetics for two acetanilide herbicides, propanil and propachlor, were measured using
pure Pseudomonas bacterial cultures grown on either glucose or sodium dodecylsulfate
(SDS) as a sole carbon source. Second-order degradation rate constants were calculated
for primary degradation of both propanil and propachlor. Degradation of propanil was
approximately a factor of 2 higher as compared to propachlor. Pseudomonas strains
grown on SDS as a sole carbon source exhibited a higher primary degradation rate for
propanil than those strains grown on glucose. Differences in propachlor degradation in
cultures grown on SDS or glucose were negligible.
INTRODUCTION
The class of acetanilide herbicides represents a set of compounds possessing an
amide type bond (NH-CO) and the side groups of more complex or simple structure
(Fedtke 1986). Parallel to symtriazine herbicides, acetanilides such as propachlor or
pronamide are still used in Lithuanian agriculture (Sukyte 1990). Residue analyses of
symtriazine and acetanilide herbicides were performed at the Lithuanian Agriculture
Institute, Yoke Branch (Kavoliunaite 1990). Biodegradation of these herbicides was also
investigated in surface water samples (Steen and Collette 1989, Vasiljeva et al 1989).
Some information is available on the biodegradation of acetanilide herbicides using a
pure culture of fungi (Rotmistrov et al 1975). Toxicity studies in conjunction with
biodegradation experiments are anticipated to provide a more complete picture and
elucidation of biodegradation mechanisms. The objective of this research was to
investigate both the toxicity and biodegradation kinetics of acetanilide herbicides using
pure cultures of Pseudomonas.
Department of Biochemistry and Biophysical, Department of Botany and Genetics, Vilnius University,
Vilnius, Lithuania
Environmental Research Laboratory, US Environmental Protection Agency, Athens, Georgia, USA
65
-------
Experimentally a high priority was set for assessment of degradation kinetics and
the reliability of the second-order reaction equation for describing microbial
transformation (Paris et al 1981):
where Kb is a second-order reaction rate constant, B is bacterial biomass, C is
concentration of the test herbicide, t is time, and Kb'B = K, Le., first-order rate constant
K for herbicide disappearance and is linearly dependent upon microbial biomass
concentration.
This equation describes the dependence of xenobiotic biodegradation rate on both
herbicide concentration and bacterial biomass, and likewise provides some insight into
the effects of the chemical structure of xenobiotics when compared across structures
within the same chemical class (Paris et al 1981). The second-order rate constants for
biodegradation and chemical degradation of xenobiotics (alkaline hydrolysis) can be used
to describe the fate of chemical substances in aquatic environments, Le., transformation,
degradation, and sorption using the Exposure Analysis Modeling System (EXAMS II).
MATERIALS AND METHODS
The herbicides propanil and propachlor (99.9% purity, reference standards, U.S.
Environmental Protection Agency, Research Triangle Park, North Carolina, were used in
this study as well as 3,4-dichloraniline (U.S. EPA, technical grade), and acetonitrile
(HPLC grade, Merck, Germany). For microbiological analysis, nutrient agar (USSR)
containing 17 g/L sprat hydrolysate, 11.2 g/L marine agar, 5.9 g/L sodium chloride, pH
7.4 to 7.7, and glucose (sterile ampoules, 40%) or sodium dodecylsulphate (SDS, Merck,
Germany) were used to provide a final concentration of 0.1% as a sole carbon source.
Bacterial strains used in this work were: Pseudomonas sp. 332 (obtained from
Institute of Applied Enzymology, Vilnius, Lithuania), P. aeruginosa 1C (obtained from
I. A. Krivetc, Institute of Colloidal Chemistry and Water Chemistry, Kiev, Ukraine),
Photobacterium phosphoreum (obtained from of A. Ruzgene, Faculty of Nature Sciences,
Vilnius University).
Samples of pure bacterial cultures for biodegradation experiments were prepared
as follows: (1) Pseudomonas cultures were grown on minimal salt medium (KH2PO4,
3 g/L; lyPO^ 7 g/L; NH4NO3, 1 g/L; KC1, 0.5 g/L; MgCl2, 0.1 g/L; pH 7.3) with glucose
or SDS (0.1 percent) as a sole carbon source until stationary growth was observed
(OD590 nm = 0.5-0.9); (2) the bacterial suspension was centrifuged at 3000 rpm for
30 minutes with aseptic precaution, and re-suspended in the same sterile medium to
OD59o nm = 0.1 and volume 50 ml; (3) control samples were autoclaved and after
Goofing the herbicides were added to all samples; (4) sample flasks were incubated in the
heated water bath shaker (Poland) at 28 °C, and shaken at 150 rpm.
66
-------
Bacterial concentration (microbial biomass in number of cells/L), was determined
by direct plate counting, using sprat-hydrolysate/agar medium (35 g/L, pH 7.2).
Disappearance of herbicide in bacterial suspensions was followed by HPLC
analysis following centrifugation at 12,000 rpm for 15 minutes to separate bacteria from
the supernatant. The resulting supernatant was analyzed for both residual herbicide
and/or product
Conditions for HPLC analysis were: (1) reversed phase (C18) Ultrasphere
ODS™ column 4.6 mm x 25 cm (Beckman Instruments); (2) UV detector with
absorption X,,^ = 225 nm for propanil and 3,4-dichloraniline, and X^ = 238 nm for
propachlor; (3) sensitivity of UV detector 0.05 RF for propanil and 0.2 RF for
propachlor ; (4) ratio of solvents acetonitrile:water = 75:25 for propanil and 80:20 for
propachlor; (5) flow rate 1 ml/minute.
Bacterial toxicity of the herbicides was determined using a P. phosphoreum
luminescence quenching method (Microtox™ prototype) (Gil et al 1988).
Photobacterium were grown on sterile sprat hydrolysate/agar medium, pH 7.2, containing
3% NaCl for 12 to 15 hours at 28°C and transferred from the agar medium surface to
3% NaCl solution and mixed carefully. A Luminometer™ Model 1250 (LKB-Wallac,
Sweden) was used for bioluminescence quenching measurements. Luminescence in
samples of Photobacterium suspensions with herbicide and appropriate controls was
measured at 1 minute and at 2 and 20 hours.
Statistical analysis of the data on biodegradation experiments was based on the
linear least square regression. The half-life for herbicide biodegradation reaction and
first-order reaction rate constants were calculated according to the equations of Metzler
(1980). The statistical and regressional parameters, offered by scientists of the U.S. EPA
Environmental Research Laboratory in Athens, Georgia, were used in this work (Paris et
al 1981, Steen and Collette 1989, Vasiljeva et al 1989).
RESULTS AND DISCUSSION
Following 20 hours incubation, cells with propachlor at 10 ppm were observed to
yield diminished bacterial luminescence, an indication of toxicity (Figure 1A). Toxicity
to the enterobacteria species P. phosphoreum was clearly exhibited at the 10 ppm
propachlor concentration. A decrease in luminescence was not observed during the
20 hours incubation at the 5 ppm propachlor concentration, although toxicity might have
been observed had there been a more prolonged incubation period.
67
-------
(A)
(B)
"--,
—
(
...»
._,
1
_„
-..,
>^
—
_,.
< :
' •
~~^
,_.
--
rr=
_
1 !
,_,.
._..
— *L
' ,
^
1
I
-—
"
_, .
1 1
_-.
—
~
1
— -
„_
/
4
?*
— ~.
,,
—
_,.—
SOU
100
so
1
—
--•;•
1 1
i 1
._._...
* 1
S 1
1 I
1
J/
I
3
2
—
— -
-*—
600 - - • - --
2 SOO • --! '.-
£ 400
3 200 --.-— - -
100 —
0 10 20 30 40 50 60 70 80 90 100 110 120 0 10 20 30 40 50 60-70 80 90 100
Time, Sec.
Figure 1. Inhibition of Photobacterium phosphoreum bioluminescence by (A) propachlor,
and (B) aniline after 20 hours of incubation in 3% NaCl. Concentrations of
propachlor: 1 - 0 ppm (Control), 2 - 100 ppm, 3 - 50 ppm, 4 - 10 ppm* 5 - 5 ppm,
6-1 ppm. Concentrations of aniline: 1-0 ppm (Control), 2 - 100 ppm,
3-50 ppm, 4-10 ppm, 5 - 5 ppm, 6 - 1 ppm.
(A)
(B)
|
1400
1200
1000
800
600
400
SOO
C(
P
•" —
•'«-
mtr
/
•op<
; •
— ...
-----
L..J
•A
X
Ml
^
I '
„!„.__
... 4
!,_,[
\: I
•
^~
—
L_,l
1— =•
.__
1 1
- —
__
1 1
]__
V
7
6
5
4
3
?,
_..
—
2500
2000 -
1600
1000
ii n n
0 10 20 30 40 50 60 70 80 90 100
0 10 20 30 40 SO 60 70 80 90 100
Time, Sec.
Figure 2. Inhibition of Photobacterium phosphoreum bioluminescence by (A) propanil,
and (B) 3,4-dichloraniline after 2 hours incubation in 3% NaCl. Concentrations
of propanil: 1-0 ppm (Control), 2 - 100 ppm, 3-50 ppm, 4-20 ppm,
5-10 ppm, 6-5 ppm, 7-1 ppm. Concentrations of 3,4-dichloraniline:
1 - 0 ppm (Control), 2 - 100 ppm, 3 - 25 ppm, 4 - 10 ppm, 5 - 5 ppm, 6 - 1 ppm.
68
-------
Aniline, a degradation product of propachlor, exhibited lower membrane toxicity
for enterobacteria; inhibiting Photobacterium bioluminescence only at the 25 ppm
exposure concentration (Figure IB). On the other hand, the results of our experiment
showed that propanil had a toxic effect on P. phosphoreum. Following 2 hours of
incubation of cells at 1 ppm propanil, a resultant luminescence quenching (Figure 2A)
was observed. The 3,4-dichloroaniline, a primary degradation product of propanil,
exhibited similar membrane toxicity. A slight luminescence quenching Was observed at
the 1 ppm and 5 ppm concentrations following a 2-hour incubation (Figure 2B).
Experimental data from biodegradation experiments with acetanilide herbicides,
using pure Pseudomonas cultures, Pseudomonas sp. 332 and P. aeruginosa 1C, are
presented in Table 1 and in Figures 3 and 4.
Pseudomonas cultures grown on glucose and on SDS as sole carbon sources were
used in biodegradation experiments. The objective of these investigations was to
compare biodegradation rates of cultures grown on different carbon sources and
differences in the degradation of aromatic compounds of the same structural class.
Propachlor degradation investigations were carried out at an initial concentration
of propachlor at 10 ppm. The total number of bacteria, Pseudomonas sp., remained
relatively constant, (2-5 x 1011 cells/L), reflecting a negligible toxicity effect from
propachlor following treatment for 1000 hours.
Cultures of Pseudomonas sp. 332 cells grown on SDS exhibited a greater
biodegradation rate for propachlor than those cells grown on glucose. The mean
degradation half-lives (t1/2) were 900 hours on SDS and 1480 hours on glucose (Table 1).
On the other hand, the mean degradation half-lives for propachlor in cultures of
P. aeruginosa 1C grown on SDS or glucose were 1910 hours and 1340 hours.
In experiments at an initial propanil concentration of 4.2 ppm, both Pseudomonas
332 and P. aeruginosa 1C exhibited higher propanil degradation rates when grown on
SDS than when grown on glucose as a sole carbon source, possibly reflecting the well
known phenomenon of glucose catabolic repression. The arithmetic means of the
degradation half-lives for propanil in glucose-grown and SDS-grown Pseudomonas sp. 332
cultures were 444 hours and 199 hours, respectively. In the culture grown on SDS, the
biodegradation reaction was 2.23 tunes greater. Mean degradation half-lives for propanil
disappearance for P. aeruginosa 1C cultures grown on glucose and SDS were 893 hours
and 318 hours, respectively. Total bacterial concentration in experiments with propanil
ranged from 2-4 x 1011 cells/L during a 600-hour incubation period.
69
-------
0 100 200 300 400 500 600 70
Time (Hours)
0 10020030040050060071
Time (Hours)
0°
Figure 3. Propanil biodegradation of (A) Pseudomonas species 332 and
(B) Pseudomonas aeruginosa 1C cultures, grown on (X) glucose,
or (Y) sodium dodecylsulphate. (Dotted line is least square fit.)
(A)
(B)
1.2
j
W
OB
0.4
0.2
0
Y
X
—
—
« —
^5
— ,
iN
X
^sN
=d
1.2
1;
1
j 0.8
0.6
-~ —
— ^
_.._„...,
.— , -
ss;
"tf~
v^~
^
T^.^.-
T^x,
"^F
»~^,
Y
X"-...
X
,
^
Time, Hours
Figure 4. Propachlor biodegradation in (A) Pseudomonas species 332 and
(B) Pseudomonas aeruginosa 1C cultures, grown on (X) glucose or
(Y) sodium dodecylsulphate. (Dotted line is least square fit.)
70
-------
Table 1. Kinetic parameters of propachlor and propanil biodegradation in pure
Pseudomonas bacterial cultures.
No.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Pstudomonas
bacterial
culture
P.sp. 332,
glc, [SI]
P.sp. 332,
glc, [S2]
P.aer.lC,
glc, [SI]
P.aer.lC,;
glc, [S2]
P.sp. 332,
SDS, [SI]
P.sp. 332,
SDS, [S2]
P.aer.lC,
SDS, [SI]
P.aer.lQ
SDS, [S2]
P.sp. 332,
glc, [SI]
P.sp. 332,
glc, [S2]
Raer.lC,
glc, [SI]
P.aer.lQ
glc, [S2]
P.sp. 332,
SDS, [SI]
P.sp. 332,
SDS, [S2]
Raer.lC,
SDS, [SI]
P.aer.lC,
SDS, [S2]
Initial
concentration
(ppm)
10.05
8.53
8.09
8.96
12.85
12.25
8.76
931
4.17
4.13
432
4.25
4.21
3.85
4.24
3.84
1st order
Degradation degradation
half-life rate constant
(hours) x 10~3
Propachlor
1170
1800
1530
1150
753
1050
1850
1980
Propanil
255
632
710
1080
163
235
165
472
0.593
0.386
0.453
0.605
0.921
0.661
0.376
0350
2.715
1.097
0.976
0.644
4.253
2.948
4.203
1.470
Bacterial
number, N/L
(average)
x!0fl
2.82
2.78
2.98
2.94
2.93
2.93
2.35
238
3.61
4.13
5.62
4.90
3.61
2.88
4.16
330
2nd order
degradation
rate constant
x NT15
2.10
1.39
1.52
2.06
3.14
2.26
1.60
1.54
7.52
2.66
1.74
131
11.8
10.2
10.1
4.45
Abbreviations: P.sp. = Pseudomonas species, P.aer. = P. aeruginosa, [SI] = Sample 1, [S2] = Sample 2
71
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Results of this work indicated a faster primary degradation of propanil as
compared to propachlor in pure bacterial cultures. During other research in our
laboratory, propachlor degradation occurred, slower than that of propanil, in surface
water samples collected from the Neris and Vilnele Rivers (Unpublished data).
Enhanced biodegradation of acetanilide herbicides Pseudomonas by cultures grown in the
presence of SDS was observed. Data also indicate differences in the membrane toxicity
within Photobacterium cultures exposed to acetanilide herbicides and their degradation
products. Propanil was found to be more toxic to the gram-negative enterobacterium
Photobacterium as compared to propachlor. Toxicities to propanil based on
bioluminescence quenching were more pronounced at the 1-5 ppm level than that
observed for propachlor at 10 ppm. Similarly, toxicity to the degradation products
3,4-dichloroaniline and aniline were significantly different.
REFERENCES
Gil, T.A., A.E. Balajan, and D.T. Stom. 1988.. Method of biotesting according to the
luminescence quenching of the luminous bacteria. In: Methods of Water
Biotesting. Chernogolovka, pp. 15-16. (In Russian)
Kavoliunaite, I. 1990. Ecological aspects of the analysis of herbicide detoxication under
conditions of intensive agriculture in Lithuania. Thesis (Summary) on
Dissertation of Doctor's Degree in the Agricultural Sciences. Zodino. 48 pp.
Metzler, D.E. 1980. The Chemical Reactions of Living Cells. Mir, Moscow.
Volume 2:7-8. (In Russian)
Paris, D.F., W.C. Steen, G.L. Baughman, and J.T. Barnett, Jr. 1981. Second order
model to predict microbial degradation of organic compounds in natural waters.
Applied and Environmental Microbiology 41:603-609.
Rotmistrov, M.N., P.I. Gvozdiak, and S.S. Stavskaja. 1975. Microbial degradation of
organic synthetic compounds. Naukova Dumka, Kiev, 223 pp. (In Russian)
Steen, W.C., and T.W. Collette. 1989. Microbial degradation of seven amides by
suspended bacterial populations. Applied and Environmental Microbiology
55:2545-2549.
Sukyte, J. (Ed.). 1990. Report on soil pollution in Lithuania Republic in 1989.
Republican Centre on the Observation of Environmental Pollution.
Hydrometeorologic Administration of Lithuania Republic. Vilnius 10 pp.
(In Lithuanian)
Vasiljeva, G.K., N.D. Ananjeva, and W.C. Steen. 1989. Prediction of microbial
transformation of propanil in the water by the application of the second-order
reaction rate constants. In: Communications of the Academy of Sciences of the
USSR. Series on Biology No. 4., pp. 485-493. (In Russian)
72
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HYDROBIOLOGICAL CONDITION AND WATER QUALITY
ESTIMATION OF KURSiy MARIOS LAGOON, 1991
A. Antanyniene, A. Baranauskiene, S. Budriene, G. Jankaviciute,
K. JankeviSms, J. Kasperoviciene, A. Kucinskiene, S. Mazeikaite,
G. Slapkauskaite, R. Sulijiene, I. Trainauskaite1
ABSTRACT
During 1991, the water pollution level in Kursitj Marios Lagoon, as indicated by
the amount of phytoplankton biomass, reached 6 and 7 based on a scale of 9 (most
polluted). The biomass quantity in the investigated zones amounted to 10.8-48.8 and
58.0-63.5 mg/L. The most eutrophic part of the lagoon was observed in the areas of
Ventes Cape and the mouth of the Nemunas River. The gross primary production and
organic matter destruction ratio in the observed region amounted to 9.2-14.2. According
to the amount of chlorophyll "a", trophic index, and algotest biomass in certain zones of
the lagoon, a hypertrophic level was observed during summer and autumn. Sixty-two
taxons of protozooplankton were registered in the lagoon, 29 of which were saprophytes.
In certain cases the saprobic index was as high as 3. The bacterioplankton and
bacteriobenthos quantity in the lagoon fluctuated from 0.88 x 106 to 8.6 x 106 cells/ml in
the water, and from 45 x 106 to 1600 x 106 cells/g in the bottom sediments. The
mineralization intensity of organic matter in the middle part of the lagoon reached
745 mg dry substance/m3 water/day. An anaerobic organic matter destruction process,
sulfate-reduction, is characteristic of the lagoon. The highest rates of sulfate-reduction
(6.1 mg S /dm3/day) were measured at Klaipeda Harbor during the summer.
Compared with the data obtained in 1990, higher amounts of pyridoxine and vitamin B12
were registered in the lagoon water; nicotinic acid, and thiamine, in addition to
pyridoxine and vitamin B12, were also higher in bottom sediments. The most intensive
organic matter assimilation (Vmax) in the lagoon was observed in July; it amounted to
about 20 jig/L per hour. The turn-over time (T) for natural substrate consumption
ranged from 7 to 37 hours, which is normal for hypereutrophic water bodies. The main
sources of the lagoon contamination are industrial effluents and municipal wastewaters.
Therefore, improvement of Kursnj Marios Lagoon water quality is directly related to
treatment of these industrial and municipal wastewaters.
INTRODUCTION
Most of the Republic of Lithuania is located within the Nemunas River basin.
The Nemunas River flows into Kur§h| Marios Lagoon, which has a major economic and
recreative significance to the Republic including its significance as a major fisheries
resource. Thus, it is very important to know how and at what rate changes occur in
water quality in the Nemunas River basin, and in this connection it is necessary to carry
out direct investigations in Kursnj Marios Lagoon.
lLaboratory of Hydrobotany, Institute of Botany, Vilnius, Lithuania
73
-------
Since 1950 comprehensive investigations have been carried out in the lagoon
(Anonymous 1959). In 1991 specialists from the Laboratory of Hydrobotany of the
Institute of Botany took part in several investigations measuring the following
environment variables: macrophyte condition, composition and quantitative structure of
phytoplankton and bottom sediments diatoms, photosynthetic pigment and biomass, and
B-group vitamins, as well as kinetics of organic matter assimilation, amount of
bacterioplankton and bacteriobenthos, and sulfate-reduction.
MATERIALS AND METHODS
Test samples were collected in northern and central parts of KurSiij Marios
Lagoon in 1991 (Figure 1). Water and bottom sediment samples were taken from seven
stations: Station 2, Kopgalis; Station 3, at the International Ferry Station; Station 3a, by
a cellulose cardboard factory; Station 10, by the town of Nida; Station 11, midway
between Nida and Ventes Cape; Station 12, by Ventes Cape; and Station 12a, the
outflow of the Atmata River. Samples were taken June 7, July 17, and October 1 and 2.
Dan6 River
Klaipeda
Figure 1. Location of water and sediment sampling stations in
the northern and central parts of Kur§h| Marios Lagoon.
74
-------
Macrophytes were sampled and identified according to the methods of Raspopov
(1985). Phytoplankton and diatoms were investigated according to ecological,
physiological, cytological, and morphological methods of Kiselev (1969), Anonymous
(1975a,b), and Davydova (1985, 1987). Water trophic potential was evaluated by means
of algotests, SCOR-UNESCO (1966), Toom and Leis (1982), and Bednarz (1984).
Primary production and destruction according to Steiman-Nielsen (1952), Vinberg
(I960), and Buljon (1983). Meta- and protozooplankton was investigated according to
Corik (1968), Kiselev (1969), and Mazeikaite (1969). B group vitamins, as the indicators
of the lagoon trophic level, were analyzed according to Odincova (1959), Kuceva (1961),
and £ukova arid Odincova (1967). Organic matter assimilation kinetics were determined
according to Starikova and Korzikova (1972), and Romanenko and Kuznetsov (1974).
Bacterial flora, as a water pollution indicator, was investigated according to Razumov
(1947), Macys and Jankevidius (1971), and Rodina (1965). The intensity of the sulfate-
reduction process was measured according to Kuznetsov and Dubinina (1989), Sorokin
(1982), and Ivanov (1983).
RESULTS
Macrophytes
The principal producers of organic matter in Kursiij Marios Lagoon are
macrophytes and phytoplankton. Over 20 species of macrophytes are usually found in
the northern part of the lagoon and in the Nemunas River delta, dominated by
Phragmites australis, Schoenoplectus lacustris, and Potamogeton perfoliatus.
Microalgae
The greatest variety of microalgae species were determined in June by the
Atmata River outflow. Phytoplankton biomass was determined for the large form
diatom genera Thalassiosira, Stephanodiscus, and Cyclotella. Their total biomass
amounted to 25.9-36.2 mg/L.
In July phytoplankton qualitative and quantitative structure was determined by
the blue-green algae Aphanizomenon flos-aquae which dominated in Kleipeda Harbor
and at Ventes Cape. The biomass of algae in Kleipeda Harbor at Stations 2, 3, and 3a
was 1.6, 29.2, and 29.5 mg/L. The highest phytoplankton biomass registered during July,
near the Atmata River outflow, was 63.5 mg/L. The biomass at the International Ferry
was 59.1 mg/L, and by the cellulose cardboard factory it was 58.0 mg/L.
In October diatoms and blue-green algae dominated in the phytoplankton. The
genera Aphanizomenon, Thalassiosira, Stephanodiscus, and Cyclotella prevailed. The
highest phytoplankton biomass was observed at the Ferry Station, at Nida, and at Ventes
Cape. The biomass in these areas fluctuated within the limits 24.5-34.0 mg/L.
75
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The dominating group of saprobity indicators in KurSiu Marios Lagoon were
j8-mesosaprobes (about 80%) and a-mesosaprobes (about 13%). Research on water and
bottom sediments diatoms was conducted, and 183 species were determined (Table 1).
Seventy-three species of water saprobity indicator diatoms were observed. The saprobic
index ranged between 1.98 and 3.1 (Table 2). These values were consistently high,
indicating a fairly steady degree of pollution at the sites studied in the lagoon.
Table 1. The distribution of diatom algae in plankton in Kursiij Marios Lagoon, 1991.
Sampling
stations
2
3
3a
10
11
12
12a
Water layer
surface
above the sediments
surface
above the sediments
surface
above the sediments
surface
surface
surface
surface
above the sediments
06.17
_
12.45
7.43
11.04
19.15
14.80
Diatoms cells density (106/L)
07.17
• -
338
2.43
3.93
-
29.61
36.47
2430
10.01
3.10
2.14
3.82
5.01
3.42
3.51
-
4.71
4338
12.17
46.04
Table 2. Saprobic indices according to diatom algae data in water samples from
Kurslu. Marios Lagoon, 1991.
Sampling
stations
2
3
3a
10
11
12
12a
Saprobic index (S)
Water layer
surface
above the sediments
surface
above the sediments
surface
above the sediments
surface
surface
surface
surface
above the sediments
06.17
'
-
1.9
3.1
2.57
2.47
2.45
07.17
:
2.02
2.44
2.07
-
2.03
2.12
2.03
10.01
2.24
2.1
2.4
2.05
23
2.06
-
2.1
2.62
2.13
1.98
76
-------
Trophic Index of Water
The amount of chlorophyll "a" in the middle part of the lagoon in June ranged
between 31.2 to 117 jtg/L. Water trophic index according to amount of chlorophyll "a"
was 69.6-88.7. The eutrophication level in the area between Nida and the Atmata River
outflow increased in the direction of the Atmata River. However, on the basis of
pigment index, carotenoid and chlorophyll ratio, and assimilation number, phytoplankton
photosynthetic activity decreased in the direction of the Atmata River outflow.
Pigments
In July the amount of pigments in Kursixj Marios Lagoon increased, pigment
index and day assimilation numbers decreased, while the chlorophyll "a" trophic index
was 75.0-95.8. The eutrophication level at the Ferry Station was high; chlorophyll "a"
amount made up 192.6 ^g/L. In October the amount of pigment in the lagoon water
decreased; however, the potential productivity remained high; algotest biomass amounted
to 53 mg/L.
Phytoplankton photosynthesis rate in the surface water layer in Klaipeda Strait at
Stations 2, 3, and 3a was similar in both June and October, 300-400 mg C/m3 (Figure 2).
In the Nida-Atmata River region this process was the most intensive in June (from 600
to 1200 mg C/m3 per day). This process decreased to 300-600 mg C/m3 per day in July,
while in October it amounted only to 80-150 mg C/m3 per day, with the exception of the
stations at Ventes Cape and the Atmata River outflow. A relatively lower average day
production during the summer season in Klaipeda Strait is related to sea water influence
and pollution effect (Sulijiene and Jankevicius 1978).
X
o
T3
O
en
£
700 r
600
500
400
300
200
100
2 3a 3
Klaipeda Strait Stations
1O 11 12 12a
Nida Section Stations
Figure 2. Mean phytoplankton daily primary production (mg C/m3/day)
in the surface water (0.0-0.5 m) of Kurshj Marios Lagoon, 1991.
77
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Gross primary production (At) and organic matter destruction (R) ratio in KurSiij
Marios Lagoon water indicate that organic matter is synthesized by phytoplankton
(autochthonous) and is transported from the surrounding environment (allochthonous).
The ratio of these two was determined for the surface and trophogenic water layers. On
the surface layer phytoplankton photosynthesis is very intensive and the At/R ratio was
often higher than 1 (in Klaipeda Strait - 0.8-3.0; in the Ventes Cape and the Atmata
River outflow - 9.2-14.2). However, in the whole trophogenic layer the At/R ratio was
different; in Klaipeda Strait it was about 0.2, between Nida and Ventes Cape it was 0.6,
and at the Atmata River outflow it was 1.4 (Figure 3).
15
12
3 -
Surface water fay«r — daily gross production
Surface water layer - daily destruction
Trophogenic lay**" — daily gross production
Trophogenic layer — daily destruction
If
2 3a 3
Klaipeda Strait Stations
1O 11 12 12a
Nida Section Stations
Figure 3. Mean daily gross production and daily destruction of organic
matter in the surface water and trophogenic layers of Kursnj Marios
Lagoon, Summer 1991.
Protozooplankton
There were 62 taxons in protozooplankton observed in Kursiij Marios Lagoon, of
which 29 were saprobic indicators. Holotricha and Spirotricha dominated, and small
amounts of infusoria subclasses Peritricha and Suctoria were observed. The data on the
total protozooplankton quantity are presented in Table 3. The highest amount of these
organisms was observed in the summer in the Atmata River outflow. Thus, water from
the Atmata River is very eutrophic. The saprobic index in June in the middle part of
the lagoon was 1.80-2.75. In July in the middle part of the lagoon it was 2.4-2.7, and in
the northern part it was 2.20-2.67. In October the saprobic index in the middle part of
the lagoon was 1.93-3.00 and in the northern part it was 2.56-2.92. A saprobic index
over 2 indicates pollution (Table 4).
78
-------
Table 3. Common quantity of protozooplankton (cell/L) in KurSiij Marios Lagoon, 1991.
T\a*A
1991
June 7
July 17
October 1
2
-
5396
211418
3a
-
3120
64168
3
-
5510
48800
Stations
10
2640
2546
2448
11
2620
—
3504
12
13918
10880
22260
12a
49600
13981
17520
Table 4. Saprobic index, according to protozooplankton data, in Kursiu Marios Laeoon
1991. 6
Stations
Date
1991
June 7
July 17
October 1
2
-
221
2.56
3a
-
2.67
2.92
3
-
2.62
2.77
10
2.50
2.41
2.80
11
2.75
— •
3.00
12
1.82
256
2.18
12a
250
2.70
1.93
Vitamins
Microalgae form certain structures of vitamins which, in turn, determine structural
succession of phytoplankton. Our investigations on vitamins in Kursiu Marios Lagoon
indicated that diatoms and green algae both synthesize and isolate B-group vitamins into
the environment in larger amounts than do blue algae or pyrophytic algae. Larger
amounts of pyridoxine and vitamin B1? were registered in 1990 in the lagoon water, and
nicotinic acid, thiamine, as well as pyridoxine and vitamin B12 were registered in bottom
sediments. These B group vitamin amounts and dynamics determine quality and
quantitative changes as well as physiological activity of the planktonic organisms
(phytoplankton and bacterioplankton).
Kinetics of Organic Matter Assimilation
The data on organic matter assimilation present information about water self-
purification possibilities; this information also enables evaluation of water quality. The
assimilation intensity of the microorganisms in the surface water layer was investigated
by means of C-labelled protein hydrolyzate. The highest rates of assimilation were
79
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registered in July (Figure 4); average uptake was 20.2 jtg/L per hour. In early summer
and in autumn the process decreased 1.5-fold. The shortest period (7 hours) was
observed in July at the Atmata River outflow, but in autumn this period was lengthened
to 35 hours. Such circulation time is characteristic to hypereutrophic water bodies (Seki
1986). In similar media, like KurSixj Marios Lagoon, microorganisms assimilate 2.7-
23.7 /tg/L per hour of organic matter due to their active transport system (Antanyniene,
Unpublished data).
30
25
1 20
3
15
10
5
0
17 July
&.
^
-------
Sulfate Reduction
Sulfate reduction indicates a high concentration of organic matter and its
degradation under anaerobic conditions. The most intensive sulfate-reduction process
was determined during the summer in the northern part of the lagoon in the region of
the International Ferry, 6.1 mg S2~/dm3 per day. The highest concentrations of
hydrogen sulfide and acid soluble sulfides were measured at the International Ferry and
cardboard factory stations. In the bottom sediments at these stations the concentration
of sulfides amount to 760 and 532 mg/dm3, accordingly. Analysis of the sulfate-reduction
data indicate that water pollution by industrial and municipal organic wastes determine
the intensive sulfate-reduction process and accumulation of hydrogen sulfide and other
sulfides in the bottom sediments of the polluted zone. Because of the resultant high
concentrations of sulfides, considerable damage is caused to the benthic fauna.
RECOMMENDATIONS
KurSiu Marios Lagoon water protection measures have a direct reference to
industrial and municipal wastewater treatment in Klaipeda. Construction of wastewater
treatment facilities, improvement of production technologies, and organization of
circulating water treatment systems must be carried out without delay. In order to
decrease pollution in the Nemunas River system, constant monitoring of surface water
quality is needed.
REFERENCES
Anonymous. 1959. Curonian Bay. Academy of Sciences, Lithuanian SSR, Vilnius,
551 pp. (In Russian, summary in English)
Anonymous. 1975a. Physiology-Biochemistry Research Methods: Investigation of Algae
in Hydrobiological Practice. Kiev, 241 pp. (In Russian)
Anonymous. 1975b. Unified Methods Used for Investigation of Water Quality.
Moscow, 175 pp. (In Russian)
Bednarz, T. 1984. Trophic potential of water by the method of algal test The Botany
News 28, 3:201-210. (In Polish)
Buljon, V. 1983. The Primary Production of Plankton of Continental Basins. Nauka,
Leningrad, 149 pp. (In Russian)
Corik, F.P. 1968. Free Living Infusoria of Moldavia's Water Basins. Academy of
Sciences, Moldavian SSR, Kismiov, 251 pp. (In Russian)
Davydova, N. 1985. Diatoms as Indicators of Holocene Lake Environment Nauka,
Leningrad, 244 pp. (In Russian, summary in English)
81
-------
Davydova, N. 1987. The latest methods of studying diatoms in lake sediments.
Leningrad University Reports 2:29-34. (In Russian)
Ivanov, M.V. 1983. The Sulfur Cycle in Lakes and Reservoirs. The Global Biochemical
Sulfur Cycle and Influence on it of Human Activity. Nauka, Moscow, 433 pp.
(In Russian, summary in English)
Kiselev, J.A. 1969. Plankton of the Seas and Continental Waters. Nauka, Leningrad,
Vol. 1, 656 pp. (In Russian)
Kuceva, L. 1961. Microbiological methods of vitamins determination. In: Vitamin
Resources and their Use. Moscow, pp. 133-139. (In Russian)
Kuznetsov, S., and G. Dubinina. 1989. Methods of Investigations of Aqueous
Microorganisms. Nauka, Moscow, 287 pp. (In Russian)
Macys, J., and K. Jankevicius. 1971. On methods of evaluation of bacterial production.
Transactions of the Academy of Sciences of Lithuania. Ser. B. T. 2(22) :3-9.
(In Russian, summary in Engh'sh)
Mazeikaite, S.J. 1969. About methods of quantitative counting of protozoan plankton
in Onega Lake. Hydrobiological Journal, Bd. 5, 4:132-137. (In Russian, summary
in English)
Odincova, E. 1959. Microbiological Methods of Vitamins Determination. Moscow,
379 pp. (In Russian)
Raspopov, I. 1985. Macrophytes of the Large Lakes in Northwest USSR. Nauka,
Leningrad, 220 pp. (In Russian)
Razumov, A. 1947. Methods of Microbiological Investigations of Water. VODGEO,
Moscow. (In Russian)
Rodina, A. 1965. Methods of Water Microbiology. Nauka, Moscow, Leningrad,
364 pp. (In Russian)
Romanenko, V., and S. Kuznetsov. 1974. Determination of soluble organic matter
using rate in the lake's water. In: Ecology of the Fresh Water Microorganisms.
Moscow, pp. 157-159. (In Russian)
SCOR-UNESCO. 1966. Determination of photosynthetic pigments in sea water. In:
Monographs on Oceanographic Methodology, Working Group 17, Paris, pp. 9-18.
Seki, H. 1986. Organic Materials in Aquatic Ecosystem. Nauka, Leningrad, 187 pp.
82
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Sorokin, J.J. 1982. Bacterial sulfate reduction in bottom sediments of some storage
lakes of Italy. Hydrobiological Journal, Kiev, T. 18, 4:38-44. (In Russian,
summary in English)
Starikova, N., and L.J. Korzikova. 1972. Determination of bound amino acids in the
sea's water and the sediments. Oceanology, 6:1107-1112. (In Russian, summary
in English)
Steiman-Nielsen, E. 1952. The use of radioactive carbon (14C) for measuring organic
production in the sea. Journal of Canadian Exploration 18:117-140.
Sulijiene, R., and K. Jankevicius. 1978. Influence of ecological-physiological factors
upon the intensity of photosynthesis of phytoplankton and upon the processes of
destruction in the northern part of the Lagoon of Kurshj Marios. In:
Physiological-Biochemical Basis of Development of Planktonic Organisms in the
Northern Part of the Lagoon of Kursiu Marios. Institute of Biology, Vilnius,
pp. 74-87. (In Lithuanian)
Toom, M., and M. Leis. 1982. Usage possibilities of algotest method, for determination
water basins condition. In: Biological Aspects of Cryptoindication. Tallin,
pp. 21-22. (In Russian)
Vinberg, G. 1960. The Primary Production in Basins. Academy of Sciences of Belarus,
Minsk, 329 pp. (In Russian)
2ukova, S., and E. Odincova. 1967. Yeast Rhodotorula flava - indicators of PAB acid.
Reports of the Academy of Sciences, USSR., Bd. 172, pp. 706-710. (In Russian,
summary in English)
83
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CYTOGENETIC CHANGES IN FRESHWATER BIVALVE MOLLUSCS
FROM THE NEMUNAS RIVER AND KUR§iy MARIOS LAGOON
J. Barsiene1, T. Virbickas2, and D. Barsyte2
ABSTRACT
A study has been conducted on cytogenetic disturbances of eight species of
bivalve molluscs collected from the estuaries of the Sventoji River, the Kurshj Marios
Lagoon near Dreverna and by Vente Cape, and the Nemunas River. The data show that
the highest level of changes in chromosome sets was present in biotopes from the
Sventoji River estuaries and the Nemunas River. Forty percent of the clams examined
from the estuaries and 50% of those from the Nemunas River had such a high level of
chromosome set disturbances that they were sterile. There was a strong genome
polyploidization trend (42% of bivalves studied) in the Nemunas River population. In
the tissues of polyploid specimens, there was-a dangerously high number of transformed
cells. A significant instability of chromosome sets in snails from Kurshj Marios Lagoon
by Vente Cape was also detected. We presume an ecologo-genetical risk threshold has
been crossed when there are cytogenetical alterations in more than 50% of the cells of
molluscs. Existence of polyploid, mosaic, and hermaphroditic specimens of clams, as
well as presence of high numbers of transformed cells and mitotic suppression in their
tissues, suggest the existence of ecologically dangerous zones in the "Nemunas River -
Kursiu Marios Lagoon - Baltic Sea" hydrosystem.
INTRODUCTION
Anthropogenic pollutants have contaminated many ecosystems in Lithuania, and
for this reason it is important to evaluate the state of the gene pool balance in the
biocenoses, the tendencies for and mechanisms of biological changes, and the
consequences of these for reproduction. It has been shown that effects of pollutants are
usually displayed first at the biochemical, molecular, or genetic level (Veldhuizen-
Tsoerkan et al 1991). Ecologists must find a suitable species for estimating ecological
risk levels in aquatic systems. Molluscs, especially bivalves, may be useful as a
biomarker for regional risk assessment. Different species otAnadonta, Unio, and other
genera of Unionidae are very suitable for studies of bioaccumulation, biodegradation,
and other effects of pollutants because of their distribution, size, sedentary life, and
filtration activity. They are also suitable for studies of relationships between aneuploidy,
growth rate, reproductive peculiarities, and cytogenetic effects of biologically active
pollutants. In the present study, freshwater clams were collected from highly polluted
Institute of Ecology, Akademijos 2, 2021 Vilnius, Lithuania
Vilnius University, Vilnius, Lithuania
85
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areas of the Nemunas River, Kurshj Marios Lagoon, and estuaries of the Sventoji River.
The specimens collected were examined for cytogenetic damage, for bioaccumulation of
pollutants, and, in some cases, for biodegradation of xenobiotics.
MATERIALS AND METHODS
Karyological analysis was performed on specimens of eight species of clams,
Grassland crassa, Unio twnidus, U. pictorum, U. longirostris, Anadonta cygnea,
A. piscinalis, A. subcircularis and Pseudoanadonta anatina, collected during the summer
months of 1989-1991 (Table 1). Tissues were obtained according to the methods of
BarSiene and Grabda-Kazubska (1988). Preparations were produced from cellular
suspensions, stained with 4% Giemsa solution prepared in phosphate buffer (pH 6.8),
and analyzed by means of a Jena Med11^ cytology microscope.
Table 1. Numbers and collection location of mollusc specimens studied.
Sampling sites
Species
Grassland crassa
Unio tumidus
U. pictorum
U. longirostris
Anadonta cygnea
A. piscinalis
A. subcircularis
Pseudoanadonta anatina
Estuaries of
Sventoji River
14
5
17
6
3
2
Kursiu Marios
Lagoon
by Dreverna
3
•
14
Kursiu Marios
Lagoon
by Vente Cape
10
6
28
Nemunas River
above Smalininkai
2
13
4
2
RESULTS
Analysis revealed a diploid number of 38 chromosomes in somatic cells
(Figure 1), although some individuals possessed other modal chromosome sets,
Le., 2n=32, 36, or 37; 3n=57; 4n=76 (Figure 2). A high frequency of chromosome set
abnormalities was detected in tissues of specimens collected from the Sventoji River
estuary. Only three of 23 individuals studied had modal chromosome numbers, which is
well outside the normal range for molluscs from clean water; normally 80-85% have the
modal chromosome number. In half of the clams studied, only 51-84% of the cells were
86
-------
*» «—
+ + •» v»
* > J
^ *,.** *
« ^
IllSXUsSi xsiittaiKH
123 4 5 6 ?s 9 10 11
12 13 1« 15 16 17. IB 19
****
IX XI MM
A& Ml
5 6
•*
8 9
•• ••
1* 15
10
•»
16
11 12
JIA «A ••
17 18 19
Figure I. Metaphase chromosomes and normal diploid karyotypes of:
A-621 Anadonta cygnea, B-444 Unio tumidus (scale lO^m).
-*/
oc *
* <
*«
56
Al A A A«
7 8 9 10 11
12
X • «
13 14 15 16 17 IB
Figure 2. Aneuploid chromosome set of 622 Anadonta cygnea (scale 10
87
-------
normal, while in one-third of the clams as few as 7-50% of the cells were normal
(Table 2). Thirty-two chromosomes predominated in the nuclei of specimen
#673 Anadonta cygnea, 36 chromosomes in #521 C. crassa, 37 chromosomes in
#686 A. cygnea and #683 A. subcircularis, and tetraploid sets occurred in
#685 A. cygnea. Most of polyploid nuclei of #685 A. cygnea have been associated
with the presence of visible tumors; this included fragmented nuclei and nuclei
with highly irregular shapes.
Table 2. Percentage of cells according to number of chromosomes of molluscs collected
from the Sventoji River estuary.
Spec
am
507
509
511
518
521
670
671
675
678
695
672
673
674
676
677
684
785
686
687
689
679
680
683
ilmcn number
32
Crassiana crassa
H
n
it
M
Pseudoanadont
Anadonta piscinalis
H
n
n
A. cygnea
60.0
n
n
tt
25.0
n
tt
tt
tt
A. subcircularis
n
n
Number of chromosomes
33 34 35 36
143
5.0 4.8
4.2 20.8
16.6
75.0
53 6.6
2.2 10.9
16.2
4.4 375 42
14.8
7.7 115
43
11.9 7.1
73 73 73 195
125 125
6.2 13.4
5.8 12.2
2.4 18.2
8.7 43, 8.7 15.0
10.0 10.0
37
143
5.2
4.2
16.6
4.0
10.9
3.8
123
3.8
115
9.8
16.7
4.8
14.6
6.6
67.0
163
8.8
8.7
60.2
38 39 54 74 76 -ISO
71.4
85.0
66.6 4.2
66.8
25.0
84.2
73.8 2.2
80.0
41.6
65.6 7.9 7.9
69.2
30.2
72.7 63
76.2
41.6 2.4
50.0
133 18.9 50.4 10.8
6.7 6.7
65.7
61.8 8.8
523 4.3
66.7 333
19.8
-------
During the summer of 1990, a large A. cygnea population was found in Kursnj
Marios Lagoon by Dreverna. Disturbances in chromosomes were detected in 30-50% of
cells (Table 3). By the summer of 1991 there was only a great number of empty shells at
this same location, and surviving clams had no mitosis.
Table 3. Percentage of cells according to number of chromosomes of molluscs collected
from Kurinj Marios Lagoon near Dreverna.
Specimen number
and species
621
622
623
624
626
628
629
630
631
632
633
704
536
Anadonta
cygnea
H
tl
n
n
n
n
n
n
n
n
«
Unio tumidus
Number of chromosomes
<3S
6.6
45
6.8
95
2.6
2.7
33
45
3.1
6.8
143
35
3.2
33
45
15.9
2.6
35
45
9.4
6.8
45
36
3.2
6.8
23
63
7.9
9.8
9.8
17.6
15.6
13.7
45
28.6
20.0
37
14.0
50.0
23
95
7.9
8.0
5.4
3.2
125
9.1
38
72.0
33.6
72.7
50.8
73.8
72.4
71.6
64.2
563
523
82.0
57.1
80.0
39-48
1.0
1.6
11.4
8.0
2.6
1.8
5.4
3.6
3.1
9.0
9.0
54-57 60-76 90-150
2.6
1.8
33 1.1
1.0 1.4
23
A similar situation was noted in tissues of clams which inhabited the Kursh}
Marios Lagoon biotope by Vente Cape. Only 20% of individuals examined had
38 chromosomes in 80-91% of nuclei. However, 25% of snails had modal amount of
chromosomes in only 25-40% of cells. We noticed chromosome sets changes in
20-40% of cells in the tissues of all remaining molluscs. Some of the bivalves from
Vente Cape were mosaic, for example, specimens #442 and #524 U. tumidus, #690 and
#694 A. piscinalis (Table 4). Also, there were two hermaphroditic clams studied,
#442 U. tumidus and #699 A. piscinalis. The normal diploid number of chromosomes of
#442 U. tumidus was present in'only 35.1% of the cells examined, while only 25% of
the nuclei of #699 ./I. piscinalis were normal.
89
-------
Table 4. Percentage of cells according to numbers of chromosomes of molluscs collected
from Kurslxj Marios Lagoon by Vente Cape.
Specimen number,
sex, and species
441 F
442 FM
443 F
444M
445 F
523 F
524 M
525 F
528 F
620 M
688 F
689 F
690 M
691 M
693 M
694 F
695 M
696 M
698 F
699 FM
Unio tumidus
n
n
n
n
n
ti
n
V. pictorum
Anadonta
piscinalis
n
n
H
n
it
it
H
n
n
H
Number of chromosomes
<35
5.4
23.6
10.1
10.7
7.1
10.0
273
10.7
6.0
3.0
25.0
25.0
35 36
2.9 11.4
5.4 5.4
5.9
8.8 225
7.1 3.6
4.0
333
83
7.1 143
5.0 5.0
143
18.2
9.1
3.6 10.7
47.0
12.1
16.7
42
50.0
37
11.4
17.6
7.4
3.6
16.0
83
5.0
3.6
6.0
83
38
68.6
35.1.
52.9
37.4
67.8
64.0
66.7
833
715
65.0
60.0
85.7
36.4
90.9
67.9
47.0
78.8
50.0
833
25.0
39-48 54-57
5.8
5.4
13
3.6 3.6
16.0
5.0
18.2
3.6
2.1
60-76 90-150
32.4 10.9
125
15.0
30.0
83
2.1
Different tendencies of disturbances of chromosome sets in tissues of bivalves
inhabiting the Nemunas River above Smalininkai were determined. Five out of 12
individuals examined were polyploid. There were also carcinous cells in their tissues.
We counted only 32 chromosomes in 57% of the cells of #544 U. pictorum (Table 5).
Consequently, 50% of the bivalves studied had strong genetic changes. We noticed
significant meiotic anomalies in the formation process of sexual cells. It would appear
that they frequently were unable to take part in reproduction processes and are being
eliminated from biocenoses.
90
-------
Table 5. Percentage of cells according to number of chromosomes of molluscs collected
from the Nemunas River above Smalininkai.
Specimen number
and species
438 Unio tumidus
439 U. longirostris
440
437
533
537
541
544
545
547
548
549
Number of chromosomes
<35
33
6.4
5.7
20.0
57.1
25.0
7.7
10.0
36
125
13.6
10.6
75
28.6
20.0
15.4
125
37 38
6.2 625
45 77.4
133
235
1.9 43.4
80.0
143
25.0 50.0
80.0
15.4
25 175
39-48
45
6.7
17.8
43
5.7
15.4
25
51-57 60-80
18.8
76.7
82.2
6.4 19.0
3.8 10.6
30.8
30.0
90-200
29.8
21.4
15.4
25.0
We found normal karyotypes in only 25% of the individuals among molluscs of
Smalininkai biotopes. Bivalves that inhabited the Nemunas River by Vilkija (below
Kaunas) and the Neris River by Lazdenai (below Vilnius) had no mitosis.
DISCUSSION
Chemical and physical biologically active agents can produce genetic alterations at
subtoxic concentrations (Landolt and Kocan 1984). It is possible for heavy metals to
damage DNA (Choi and Simpkins 1986). Walton et al (1988) were able to determinate
that PAHs caused a considerable number of different chromosomal aberrations in fish
tissues. Exposure of killifish (Fundulus heteroclitus) embryos to methylmercury
decreased mitotic counts 1.5-5-fold, increased chromosomal aberrations 2-2.5-fold, and
retarded development of embryos (Perry et al 1988). Mitotic inhibition was marked in
our karylogical studies of molluscs. This phenomenon was significant in the areas of
highest pollution, namely the Neris River below Vilnius, the Nemunas River below
Kaunas and Tilze (Sovietskas), and Kurshj Marios Lagoon near Klaipeda (Barsiene
1991). We did not observe any mitotic activity in tissues of 75-95% of the specimens we
examined. In 25-40% of specimens examined, potyploid interphase nuclei predominated.
It was presumed that appearance of polyploid cells was a result of pollutant effects on
microtubules and mitotic spindle fibers of nuclei of clams.
91
-------
The greatest abundance of molluscs with damaged chromosome sets was found in
the estuary of the Sventoji River and in the Nemunas River just above Smalininkai. One
of the most significant findings from our study is the correlation between occurrence of
potyploidy in clams and of cancerous cells. There was a notable predominance of
cancerous cells in specimen #685 A. cygnea, and occasionally these cells were found in
potyploid clams from the Nemunas River above Smalininkai. Polyploid hepatopancreas
cells had indications of typical tumor cells. In addition, in tissues of most specimens
studied there were remarkable numbers of aneuploid cells. Earlier it was stated that
chromosomal lesion in mussels might lead to the DNA replication errors and appearance
of potyploid cells (Elston et al 1990). Consequently, chromosome lesions, genome
potyploidization, and carcinogenesis in molluscs might be in close relationships. It would
be interesting to study preneoplastic or neoplastic processes of aneuploid bivalves
specimens.
There is sufficient evidence that chromosomal damages can be the initiating event
in carcinogenesis (Yunis 1981). The progressive nature of hemic neoplasia in Mytilus
edulis was connected with remarkable increases of polyploid cell population and
decreases of diploid cells. In the terminal stage of hemic neoplasia, the neoplastic cells
predominate and fragmentation of giant nuclei (about lOn) is common (Elston et al
1990). In the case of sarcomatoid proliferative disease of M. edulis, aneuploidy as well
as pofyploidy in atypical cell populations has been described (Farley 1969).
The majority of autosomal aneuploids result in a lethal event or in reduction of
viability (Bond and Chandler 1983). Nevertheless, some of the bivalve molluscs that we
studied possessed one to six chromosome lesions and survived in the most polluted
waters. The karyotypes of about 20% of Unionidae clams from the cooling basin of the
Lithuanian Hydroelectric Power Station (HEPS) on the shores of the Kaunas Reservoir
consisted of 32, 36, or 37 chromosomes (Barsiene and Petkeviciute 1988). All these
hypoploid specimens were found in the cold basins zone near the estuaries of the Streva
River which flows into the reservoir. These were the most polluted areas of the basin
because of atmospheric input from the HEPS and input from the Streva River. A
similar pattern of aneuploidy in clams was found in the Sventoji River estuary. These
anomalies have influence to some degree on the growth rate and viability of mussels
(Thiriot-Quievreux et al 1988).
There is considerable evidence that high quantities of polyploid-aneuploid cells in
mollusc tissues, as well as potyploid, aneuploid, mosaic or hermaphroditic specimens of
molluscs exist in ecologically dangerous zones. In our opinion, 12-25% of bivalves
specimens possessing normal diploid non-altered karyotypes in more than 85% of cells
could not maintain a normal population through auto-reproduction. We believe the
clam populations we studied have passed beyond an ecologo-genetic threshold, especially
those from the Sventoji River estuaries and the Smalininkai biotopes.
92
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REFERENCES
Barsiene, J., and B. Grabda-Kazubska. 1988. A comparative study on chromosomes in
plagiorchiid trematodes. I. Karyotopes of Opisthiogfyphe ranae (Frolich, 1791),
Haplometra cylindracea (Zeder, 1800) and Leptophallus nigrovenosus (Bellingham,
1844). Acta Parasitologica Polonica 33:249-257.
Barsiene, J., and R. Petkeviciute. 1988. Cytogenetic studies of Unionidae molluscs from
the cooling water reservoir of the Lithuanian Regional Electric Power Plant
Acta Hydrobiologica Lituanica 7:11-24. (In Russian)
Barsiene, J. 1991. Cytogenetic changes of molluscs. In: R. Lekevicius (Ed.) Nemunas
basin water pollution and biological effects on ecosystem. Academia, Vilnius,
pp. 100-106. (In Lithuanian)
Bond, D. J., and A.C. Chandler. 1983. Aneuploidy. Oxford Monographs on Medical
Genetics. Oxford University Press, Oxford, 198 pp.
Choi, B.H., and H. Simpkins. 1986. Changes in the molecular structure of mouse fetal
astrocyte nucleosomes produced in vitro by methylmercuric chloride.
Environmental Research 39:321-330.
Elston, R. A., A. S. Drum, and S.K. Allen. 1990. Progressive development of circulating
polyploid cells in Mytilus with hemic neoplasia. Diseases of Aquatic Organisms
8:51-59.
Farley, C. A. 1969. Sarcomatoid proliferative disease in a wild population of blue
mussels (Mytilus edulis). Journal of the National Cancer Institute 43(2):509-516.
Landolt, M. L., and R. M. Kocan. 1984. Fish cell cytogenetics: a measure of the
genotoxic effects of environmental pollutants. In: J. O. Nriagu (Ed.) Aquatic
Toxicology. John Wiley and Sons, New York - Singapore, pp. 336-353.
Perry, D. M., J. S. Weis, and P. Weis. 1988. Cytogenetic effects of methylmercury in
embryos of the killifish, Fundulus heteroditus. Archives of Environmental
Contamination and Toxicology 17:569-574.
Thiriot-Quievreux, C., T. Noel, S. Bougrier, and S. Dallot. 1988. Relationships between
aneuploidy and growth rate in pair matings of the oyster Crassostrea gigas.
Aquaculture 75:89-96.
Veldhuizen-Tsoerkan, M.B., D. A. Holwerda, A. M. T. de Bont, A. C. Smaal, and D. I.
Zandee. 1991. A field study on stress indices in the sea mussel, Mytilus edulis:
Application of the "stress approach" in monitoring. Archives of Environmental
Contamination Toxicology 21:297-304.
93
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Walton, D. C, A. B. Acton, and H. F. Stich. 1988. Chromosome aberrations in
cultured central mudminnow heart cells and Chinese hamster ovary cells exposed
to polycyclic aromatic hydrocarbons and sediment extracts. Comparative
Biochemistry and Physiology 89Q395-402.
Yunis, J.J. 1981. Chromosomes and cancer: new nomenclature and future directions.
Human Pathology 12:494-503.
94
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MORPHOLOGICAL CHANGES IN BIVALVE MOLLUSCS UNDER
DIFFERENT LEVELS OF HEAVY METAL BIOACCUMULATION
L. Lazauskiene and G. Kaspariunaite1
INTRODUCTION
Many water bodies in Lithuania are currently in a critical state because then-
ecological balance has been destroyed and intense industrial activity has impaired then-
potential for natural biological self-cleaning. Because they are exclusive filterers,
molluscs play an extremely important role in the process of self-cleaning and self-
regulation of natural communities. They are widely used as biological indicators in
studies of ecological effects of pollutants and for estimation of the environment, in
particular hydroecosystems. The filtering and bioaccumulative capabilities of individual
molluscs depends upon their general morphophysiological state, such as sex, age, size,
and growth rate, as well as on their environment, including hydrochemical and
geochemical conditions (Phillips 1988).
The largest river in Lithuania is the Nemunas, which flows into south-central
Lithuania from Belarus. Its watershed covers 70% of the territory of the republic,
thereby accumulating most of its water outflow with its dissolved components. Except
for some natural water quality recovery taking place along tributaries before they flow
into the Nemunas River, this river reflects the cumulative pollution level of much of
Lithuania as well as some parts of Belarus. The Neris River is the second largest river
in Lithuania; it flows through Vilnius, which is also a significant source of pollution
because it is the country's largest city. Over 100 km downstream from Vilnius the Neris
River joins the Nemunas River at Kaunas, the second largest city in Lithuania, and one
that does not have a water purification system. The Nemunas flows farther westward,
collecting on its way the water from most of central Lithuania brought by a few other
major tributaries, and finally reaches Kursnj Marios Lagoon.
Kursnj Marios Lagoon is triangular-shaped and has only a narrow channel at the
northern end connecting it with the Baltic Sea. The lagoon has water of low salinity
brought by the rivers flowing into it, although some of the higher salinity water at the
Baltic Sea flows into the lagoon through the narrows at Kleipeda Harbor. The ratio of
flow from KurShj Marios Lagoon into the sea is approximately three to one. Because of
the relatively long residence, time of water in the lagoon, it serves as a cumulative
reservoir for waste products, not only from Lithuania, but also from Belarus and,
especially, from the Kaliningrad region of Russia. Thus, a pollution gradient occurs
along the Neris-Nemunas-Kursnji Marios Lagoon aquatic system, and our sampling
stations were distributed along this gradient.
Institute of Ecology, Vilnius, Lithuania
95
-------
MATERIALS AND METHODS
Mollusc samples for evaluating the extent of pollution were taken at the following
stations: the Cape of Ventes and Dreverna (KurSiij Marios Lagoon), Smalininkai and
Vilkija (Nemunas River), and Verkiai (Neris River). The Verkiai station might be taken
for a control; it is located slightly upstream from Vilnius and because it is devoid of
major pollution sources, water from the Verkiai station can be considered comparatively
clean when compared to the other stations.
The following species of molluscs were selected for our investigation, and
collected in the littoral zone at each station: Unto pictorum, U. tumidus, andAnadonta
pisdnalis. The parameters measured were: total weight; the weight, length, width and
height of shell; and the weight of branchiae and liver. Modifications and deformations
of the shell or soft parts of the body, damages to the nacreous layer, its granular
structure, spot patterns and color changes were all evaluated. Attention was also given
to the age and sex structure of the populations and to the state and abundance of
individuals. Spectral analysis was applied to measure the quantities of heavy metals, and
this method has been described in Lazauskiene et al (199 la). Data of the investigation
of benthic communities carried out in 1951-1990 (Ibid) were used for comparison.
RESULTS AND DISCUSSION
Molluscs have played an important role in the benthic communities of Kurjshj
Marios Lagoon. Some 10-15 years ago 22 species of Mollusca were found in the Cape
of Ventes area, and their total biomass reached as much as 5 kg/m2 (Gasiunas 1952,
1972). At present, communities of molluscs and other aquatic organisms have become
impoverished, and their species diversity and biomass has decreased four- or five-fold
since the early 1950s (Table 1). Peleophylic (mud-affiliated) communities with then-
Table 1. Dynamics of the yearly averages of benthic biomass in KurSiu Marios Lagoon.
Year
Average of benthic biomass, (g/m2)
Percent molluscs
1951
1954
1955
1957
1973
1989-90
27.1
16.6
18.1
162
15.1
65
80
80
80
80
55
20
Source: Lazauskiene et id. 1991a
96
-------
characteristic complex of aquatic species have increased their habitat area by 20%.
The occurrence of so-called "dead zones", where no aquatic life is possible, has also been
noticed because of increases in water and bottom sediment pollution in Kursii} Marios
Lagoon. Some 150 various chemical compounds have already been identified in Kursnj
Marios Lagoon (Anonymous 1992). Lazauskiene et al (199 la) reported that sediment
concentrations of heavy metals such as manganese, titanium,, vanadium, chromium, and
cobalt exceeded those found in the macroscopic zoobenthos, but the concentrations of
cadmium, nickel, copper, lead, and mercury had an opposite distribution pattern, being
in the higher aquatic organisms, including Mollusca.
Mercury concentrations in biotic and abiotic components of the northern Kursii}
Marios Lagoon ecosystem have also been reported (Table 2).
Table 2. Mercury content in various objects from the northern Kursii; Marios Lagoon
ecosystem at Dreverna.
Mercury content (mg/kg)
Objects
Water
Bottom sediments
Oligochaeta
Polychaeta
Chironomidae
Mollusca
Gamaridae
Mysidae
Mean
0.25
0.10
0.13
0.08
0.08
0.11
0.90
0.70
Range
0.01-0.68
0.01-0.27
0.04-0.41
0.08-0.19
0.02-020
0.01-0.27
0.02-1.80
0.09-0.91
Source: Lazauskiene et al. 199la
Mercury has a high toxicity level, easily accumulates, and is highly persistent in
organisms. Direct interdependence between the mercury levels in the ground, water,
and macroscopic zoobenthos was established. The highest mercury levels were found in
Gamaridae, Mysidae, and Mollusca. Accumulation of chemical compounds containing
heavy metals in molluscs, regardless of species, is much more intense in the soft parts of
the body than in the shell (Table 3).
97
-------
Table 3. Accumulation of chemical elements in the soft tissues and shell of molluscs
collected at Dreverna. Averages in mg/kg of ash for each fraction are presented.
Chemical elements
Analyzed
portion
Soft tissues
Shell
Mn
2400
600
Ti
1000
100
V
36
3
Cr
12
5
Co
65
2
Ni
8.4
20
Cu
80
20
Pb
18
5
Ag
1
0.1
Sr
380
330
Ba
120
30
Source: Lazauskiene et d. 199 Ib
Chronic exposure to pollutants kills aquatic organisms gradually, impairing their
normal biological functions. Heavy metals enter the tissues of organisms as macroscopic,
microscopic, and ultramicroscopic compounds or in simple elemental form, and thereby
accumulate in the environment. Organisms can acquire resistance to a certain extent
and adjust themselves to the modified environment, due to the processes of detoxication
and adaptation. Yet the adaptation mechanisms have their limits, and when these limits
have been reached various changes occur. Ecological crisis of an organism starts when
morphophysiological and biochemical declinations from the normal state can be
detected. The weight and size parameters of molluscs of the Unionidae family in the
Nemunas River and Kursiij Marios Lagoon indicate that growing conditions are
unfavorable (Figures 1-3).
Nemunas River
Neris River
10 -
£
ra
§
in
1
%
' %•'*
t
.*
•••
«*«.
I *k
....
»iv
...
"
!
I ,
1
: -....•..:.:.-. •.::' <: . : .-.
.' :
• J I
'. • »
:
t : : . : :
-• ;
:
* • ^
: • •
•*":
:-
r :i
I'
...
•**
e
*.
%
i*-.
-
i. . .
t
10 100 1 10 100
Weight (g)
Figure 1. Comparison of weight and length of Unto pictorum sampled in the
Nemunas River (at Smalininkai) and Neris River (Control, at Verkiai).
98
-------
Kursiti Marios Lagoon
10
o
10
10
1
100 1
Weight (g)
Neris River
,«*:'
10
100
Figure 2. Comparison of weight and length of Unio timidus
sampled in Kursn; Marios Lagoon (at Cape of Ventes)
and in the Neris River (Control, at Verkiai).
Kursiq Marios Lagoon
10
E
o
c
o
10
100
Weight (g)
Figure 3. Comparison of weight and length oiAnadonta piscinalis
sampled in KurSiij Marios Lagoon (at Cape of Ventes).
99
-------
Three species of molluscs were distinguished according to their age structure, with
individuals of the age of 3 and 4 years being the most numerous. There was a distinct
difference in numbers between males and females; the proportion of females at the Cape
of Ventes area was almost twice as great as that of males. Damage of various kinds was
found in 94% of molluscs sampled at the Cape of Ventes (Table 4). The damage
consisted of obvious shell deformations, many swellings, and spots on the nacreous layer
of shell, and the liver of some individuals was considerably enlarged.
Table 4. Morphophysiological changes in shell and soft tissues of two Unio species.
Sampling location
Species
Percent damage
Smalininkai (Ncmunas River)
Verldai (Neris River)
Verkiai (Neris River)
Cape of Ventes (Kurshj Marios Lagoon)
U. pictorum
U. tumidus
40
54
41
94
Mollusc communities in the Nemunas and Neris Rivers in 1951-1980 were
previousty more dense and diverse; as many as 32 species of molluscs were once
reported, with total biomass up to 8 kg/m2 (Gasiunas 1972). At present, the
communities are affected by increased water and sediment pollution, and total biomass
of molluscs has decreased to 0.2 kg/m2 (Lazauskiene et al 1991a). Many empty shells of
dead molluscs have been found in the littoral zone. Currently the communities are
predominated by U. pictorum. The male/female ratio was 1:1.4. Even more sharp
distinction of senior age groups (5-6 years old) has been be detected in length class
analysis of the molluscs from the Nemunas River, compared to populations in Kursnj
Marios Lagoon. Defects of shells and the nacreous layer have been found in all the age
groups of molluscs from the Nemunas River, yet they were less severe than those found
in molluscs from Kurshj Marios Lagoon. Defective individuals comprised 40% of the
total in the Nemunas River mollusca communities at Vilkija and Smalininkai, which were
located .closer to the sewer water discharge from Kaunas. The Neris River, generally
less polluted than the Nemunas River (Lazauskiene and Kirlys 1991), had higher species
diversity and productivity of bivalves. Both young and mature individuals were found at
the stations sampled, and their sex ratio was closer to 1:1 (Table 5). Defects of the
molluscs of this area consisted mostly of shell deformations and there was less swelling
in their nacreous layers.
100
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Table 5. Sex composition of the bivalve populations of two Unio species.
Sampling location
Species
Percent Males Percent Females
Cape of Ventes (Kursiij Marios Lagoon) U. tumidus
Verkiai (Neris River) "
Verkiai (Neris River) U. pictorum
Smalininkai (Nemunas River) "
Vilkija (Nemunas River) "
35
465
50
40.6
44.4
65
535
50
59.4
55.6
SUMMARY AND RECOMMENDATION
The results of the studies of bivalve communities in the Nemunas and the Neris
Rivers indicate that productivity and species diversity of representatives of the family
Unionidae have decreased significantly in the last decade, indicating severe
environmental degradation. Increased pollution of these hydroecosystems, especially the
accumulation of heavy metals, is having a negative impact on the population structure of
the molluscs. Accumulation of heavy metals damages the vital functions of molluscs,
including morphometric changes and shifts in sex and age structure of their populations.
Based on these data, and the fact that young individuals are almost absent, we predict
that the Kursuj Marios Lagoon population of Unionidae is doomed to extinction. This is
confirmed by cytogenetic changes in these molluscs (Barsiene 1991). Analysis of the
changes in sex structure of the populations suggests that females are more resistant to
pollution than males, and may possess some additional mechanisms for ecological
adaptation and compensation. To generalize, it can be predicted that the ecological
situation in the Nemunas River-Kursiu. Marios Lagoon-Baltic Sea aquatic ecosystem will
get worse. To control this situation, we suggest introduction of conservation measures
such as artificial reefs and biofilters (Lazauskiene and Kirlys 1991). Introduction of the
artificial biotopes and new habitat substrata will enable increased filtrating capacity of
bivalves, and thereby soften the negative ecological effects of pollutants.
REFERENCES
Anonymous. 1992. Ecological assessment of the Klaipeda seaport. Report on an
Integrated Study for the Institutes of Ecology, Geography, Geology, and Balticeco
Company. Vilnius. 223 pp.
Barsiene, J. 1991. Cytogenetic changes of molluscs, pp. 100-107. In: Prediction of
Ecological Impacts of Environmental Pollution in the Nemunas River, Vilnius
Academy. (In Lithuanian)
101
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Gasiunas, I. 1952. Kursiij Bay Zoobenthos. Kurshj Marios, pp. 191-291.
(In Lithuanian)
Gasiunas, I. 1972. Bottom fauna.
(In Lithuanian)
In: The Nemunas. Mokslas, Vilnius, pp. 44-82.
Lazauskiene, L., I. Jagminiene, and V. Klimasauskiene. 1991a. Ecological effects of
hydroecosystem pollution on the structure of hydrobiont populations and
communities, pp. 65-73. In: Prediction of Ecological Impacts of Environmental
Pollution in the Nemunas Basin, Vilnius Academy. (In Lithuanian)
Lazauskiene, L., I. Jagminiene, V. Klimasauskiene, and K. Joksas. 199 Ib. Peculiarities
of heavy metals' migration and accumulation in the King Wilhelm Canal in the
northern part of Kursnj Bay and in the Baltic near-shore in 1989. pp. 12-19.
In: Prediction of Ecological Impacts of Environmental Pollution in the Nemunas
Basin, Vilnius Academy. (In Lithuanian)
Lazauskiene, L., and V. Kirlys. 1991. Ecological effects of human activity on the Couric
Lagoon and the coastal zone of the Baltic Sea. pp. 317-319. In: Ecological
Engineering for Waste Water Treatment. Swedish Press.
Phillips, J. H. 1988. Use of macroalgae and invertebrates as monitors of metal levels in
estuaries and coastal waters, pp. 20-30. In: Heavy Metals in the Marine
Environment. CRC Press, Inc.
102
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CARCINOGENESIS RESEARCH ON FISHES
OF KURSiy MARIOS LAGOON, 1991
E. Bukelskis and D. Serelyte1
ABSTRACT
Between May and October 1991 fishes were collected for tissue analysis from the
Nemunas River near Vilkija, the Nemunas River delta, and Kursiu Marios Lagoon. We
found changes in the microstructure of the liver of roach and bream, and we also found
tumors in a crucian carp. We attribute these tissue alterations to environmental
pollution by industrial effluents.
INTRODUCTION
Contamination of water that disturbs the stable environment of hydrobionts can
cause preneoplastic and neoplastic changes. Such changes occur especially in those
water basins that receive industrial effluents (Sorensen 1980, Bogovskij and Kudolej
1987). In Lithuania such water basins are the Nemunas River and Kurshj Marios
Lagoon. In this paper we will report additional research on a study started in 1990 on
preneoplastic and neoplastic changes in fishes.
MATERIALS AND METHODS
Fishes for this study were caught from May to October 1991 in the Nemunas
River near Vilkija, the Nemunas River delta, and Kursu| Marios Lagoon near
Vente Cape. Histologic preparations of the livers of 60 breams (Abramis brama)
and 50 roaches (Rutilus rutilus) were prepared for the study. Two breams, in which
visual preneoplastic changes of the bowels could be observed, were used for the
scientific research. The breams and one crucian carp (Carassius auratus) were caught in
KurSh} Marios Lagoon. Over 3,000 breams were caught, of which 111 were used for this
study.
Liver tissues for histologic study were prepared as follows: pieces approximately
2-3 cm3 were excised and fixed in 15-20 times that volume of 10% neutral formalin
solution. These pieces were then washed in running water for 24 hours, and then
dehydrated by placing them progressively in solutions of 70%, 80%, 96%, and twice in
100% ethanol, each for 24 hours. Samples were then fixed in paraffin by immersing in
50/50 ethanol/o-xylene solution for 3 hours, o-xylene for 3 hours, 50/50 o-xylene/paraffin
at 37°C for 3 hours, and finally, paraffin at 57°C for 2-3 hours. Samples were cooled
and then cut to a thickness of 5-1 ^ with a rotary microtome. The cuts were glued on
Department of Zoology, University of Vilnius, Vilnius, Lithuania
103
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clean objective glass-bits with the help of an egg-white and glycerine mixture (1:1).
Before staining, the paraffin was removed from the cuts by dipping the samples into
o-xylene for 3-5 minutes, into ethanol at 96°C for 2-5 minutes, into ethanol at 75°C for
2-5 minutes, and finally into distilled water for 30 minutes. Samples were stained with
Erlich hematoxylene and cosine, and then clarified with a xylene-carbolic acid mixture.
Cover glass-bits were glued with Canada balsam. Preparations were marked with India
ink, examined under a microscope, and photomicrographs were taken.
RESULTS
The liver of roaches partially encases the duodenum. Although generally the
livers were dark red in color, they sometimes were light brown. We did not find any
macromorphological or micromorphological changes in the roach livers we studied.
There were no distinct lobules. The secreting lamellae were made from cellular tissues.
Hepatocytes were small, oblong, and had anomalous forms when compared with those of
a variety of other fishes. The ratio of sizes of the nucleus and cytoplasm was 1 to 4.
Cytoplasm was granular, filled up the volume of the cell, and contained very little fat.
Liver parenchyma was furrowed with blood capillaries but these were not abundant.
Ducts of the bile branched out between the lamellae. Bile secretion was of medium
activity. We found a triad made from artery, vein, and bile. The roaches which were
caught at various locations did not exhibit any differences in micromorphological
structure (Figure 1).
»&«&&*
*?%fc
tis&gb
Figure 1. Photomicrograph of a liver of a roach.
The livers of the bream were of a dark red color, with little variation among
specimens. The liver was compact. Hepatocytes of the bream liver were bigger when
compared to the roach liver hepatocytes. Between the lamellae (which consisted of
some cells) there were several capillaries that were filled with formal blood elements and
bile ducts, but bile secretion was almost invisible. The micromorphology of the bream
104
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livers differed in cases where these fish were caught at different locations. The liver of
the breams from the Nemunas River delta had evenly distributed hepatocyte cytoplasm
and vacuolization was rare.
Vacuoh'zation of hepatocytes was very distinct in the livers of breams caught in
Kurshj Marios Lagoon near Vente Cape (Figure 2). All the hepatocytes had vacuples,
occupying 50% or more of the cell, and as a consequence the cells contained a
considerable amount of fat. Cytoplasm of hepatocytes was around the nucleus. The
nuclei were distinct, and not contracted. Such fatty tissue was perhaps caused by the
intoxication influence of the fermentative system of the liver cells. Decomposed
lipoprotein was apparent in the wounded hepatocytes. The microstructure of the breams
from the Nemunas River near Vilkija appeared unstable because we saw the
vacuolization of most hepatocytes. The nuclei were distinct with micronuclei in them.
There were no vacuoles in some hepatocyte cytoplasm, or they occupied only a very
small part of the cell.
Figure 2. Photomicrograph of a liver of a bream.
In October 1991 in the Kurshj Marios Lagoon near Vente Cape we caught two
breams, in the liver of which distinct morphological changes were seen. After making
micropreparation of the liver we established that general view was typical. The
functional triads looked like the cavities of the large blood vessels that were located side
by side. In most hepatocytes a distinct vacuolization was seen. Cytoplasm could be seen
only around the nucleus.
In many places it was not possible to establish boundaries between the
hepatocytes. Parenchyma cells seemed disordered, and the nuclei were not seen.
Caryolysis and cytolysis had already begun. In the large bile duct we could see bile
secretion. Necrotic areas were dispersed, and not focused in any single location. In the
liver of one of these breams, hepatocytoma had begun to form.
105
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The crucian carp caught in Kurshj Marios Lagoon near Vente Cape had a large
tumor on its gills. Microscopic analysis showed that this tumor formed from reticulary
cells which produce reticulary fibres that were weaved among the tumor ceDs. The
tumor was many-lobuled, with necrosis in the center of these lobules. Between the
tumor cells there were masses of amorphycal connective tissue which were formed after
cariorexis, cariolysis, and cytofysis (Figure 3). These symptoms allowed us to
characterize the changes which formed on the gills of the crucian carp as a diffusive
malignant histiocytic type of reticulosarcoma.
Figure 3. Photomicrograph of a reticulosarcoma in the gills of a crucian carp.
In conclusion, our histological sampling of fishes of the Nemunas River, its delta,
and Kursh} Marios Lagoon suggests that fishes in these waters may have received
environmental insults. Additional histological examinations could form the basis for a
baseline that could be used to monitor progress toward bioremediation of these waters.
REFERENCES
Bogovskij, S.P., and V. V. Kudolej. 1987. Tumors of fish, the meaning of their increase
on the economy; a research perspective. Questions of Parasitology and Pathology
of the Fish. Works of the Institute of Zoology Science Academy of S.S.R.
171:126-134. (In Russian)
Sorensen, E.M.B. 1980. Cytological changes in the fish liver following chronic,
environmental arsenic exposure. Bulletin of Environmental Contamination and
Toxicology 25:93-99.
106
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TOXICITY OF FACTORY WASTEWATERS
AND HEAVY METAL SOLUTIONS TO RAINBOW TROUT
Z. Vosyliene1, G. Svecevicius1, and S. Syviene2
ABSTRACT
A study has been undertaken on the lethal effects to rainbow trout (Salmo
gairdneri) of galvanic wastewaters of some Vilnius factories, and of two solutions of
heavy metals representative of the heavy metals in these wastewaters. Composition of
these model solutions was based on those of wastewater discharges from Vilnius and
Kaunas. The effects of the wastewaters and the model solutions on feeding, behavior,
and respiratory indices have been estimated. The relationship between the toxicity of
the wastewaters and of the model solutions has been compared. Suggested safe
concentrations for fish of the model mixtures have been calculated.
INTRODUCTION
Studies of toxic effects of industrial wastewaters in Lithuania have mostly been
limited to research into hydrobiont communities and their quantitative and qualitative
changes in a given water body. The most complete among such was conducted in Kurshj
Marios Lagoon and in the Nemunas River (Gerulaitis and Valu§iene 1991, Joksas 1991,
Lazauskiene et al 1991). It was found that due to industrial and agricultural activities
there has been an increase of chemical pollution in Kursnj Marios Lagoon. Oil
products, heavy metals, and fertilizers have all contributed to an increase in the
proportion of fishes of low commercial value, and a decrease in the harvest of more
commercially desirable species. Because the pollution is multicomponent containing
heavy metals, oil products, and other organic substances, it is very difficult to estimate
the toxic effect of specific pollutant groups on the ichthyofauna. Special studies on the
toxicity to fishes of single pollutants and their mixtures are needed to do this, as well as
to set effluent discharge limits.
The first studies to estimate toxic effects of heavy metal mixtures on fishes were
started at the Lithuania Academy of Sciences, Institute of Ecology in 1991. Heavy
metals (zinc, copper, nickel, chromium, iron) are lost in electrodeposition processes, and
if discharge purification systems at electrodeposition facilities do not work effectively,
these metals can escape into wastewaters. These metals are often found in rivers
downstream from many of the larger cities of Lithuania.
Institute of Ecology, Lithuanian Academy of Sciences, Vilnius, Lithuania
Environmental Protection Department, Vilnius, Lithuania
107
-------
The purpose of the present study was to examine the toxicity to rainbow trout of
wastewaters from some Vilnius factories where galvanic processes are common, and to
compare the data obtained to those on the toxicity of two different model solutions of
five heavy metals, representative of those metals in industrial discharges.
MATERIALS AND METHODS
The tests were conducted in July, September, and October, 1991, and performed
in duplicate. The fish under study were juvenile rainbow trout (Salmo gairdneri), total
length 2.5-3.5 cm in July and 7-8 cm in October; the weight ranges were 0.5-2.5 g and
5-9 g, on those same dates. Trout were obtained from the £eimena Hatchery, near
Vilnius. They were kept in holding tanks of about 1000-L capacity supplied with aerated
flow-through artesian water of average total hardness 250 mg/L CaCO3, alkalinity
approximately 200 mg/L HCOJ, and pH 7.2. The fish were transferred from holding
tanks to 20-L aquaria for the experiments. They were kept until acclimated to the new
medium, and until they started swimming freely and were feeding well. The water
temperature during the experiments was 11-12° C, and dissolved oxygen was 8-10 mg/L.
The effect of wastewater was observed over a 2-hour period after diluting it in
proportion 1:1. After diluting in different proportions the effect was also observed at
24, 48, 72, and 96 hours. Every day the water was changed, the number of dead fish was
recorded, both the feeding and behavior of live fish were estimated (in grades), and, if
possible, respiratory indices such as gill ventilation frequency and "coughing" rate were
recorded. CtiemicaLanalysis of wastewaters was performed by the Environment
Protection Department.
The formulation of model mixtures was based on the available analytical data of
the amounts and relative proportions of five common heavy metals (copper, nickel,
chromium, zinc, and iron) in the discharges from the cities of Vilnius and Kaunas into
the Neris and Nemunas Rivers (Table 1). Copper was in lowest concentration;
proportions of all other metals were set in relation to copper. The following chemically
pure substances were used in making the model solutions: CuSO4, NiSO4, K2CrO4,
ZnSO^ and FeSO4. The data from the acute tests were processed by regression analysis
of log-probit transformation using the methods of Miller and Tainter referenced in
Belenkij (1963).
RESULTS AND DISCUSSION
A Study of Wastewaters of Vilnius Factories
Electrodeposition wastewater discharge of Vilnius Amalgamated Enterprise of
Construction-Finishing Machines: Wastewater for testing was diluted to 50, 12.5, 10, 7.5,
5, and 0.5 %. The concentration of wastewater that could cause a lethal effect was very
low (7.5-12.5%). All test mortalities occurred within 48 hours, so the 48-hour and
96-hour LCjQ values were both 9.2% (Table 2).
108
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Table 1. Amount, mean concentration, and proportion relative to copper of selected
heavy metals discharged in wastewaters from Vilnius and Kaunas.
Discharge
source
Vilnius (1989-90)
Kaunas (1989)
Kaunas
Vilnius (1989-90)
Kaunas(1989)
Kaunas
Vilnius
Kaunas1
Receiving
river
Neris
Neris
Nemunas
Neris
Neris
Nemunas
Neris
Neris and
Nemunas
Flow
(m3/sec) Cu
100 93
169 3.04
420 7.08
0.11
0.15
Gill
1
1
Heavy metal
Ni Cr Zn
Amount (tons/year)
10.1 12.6 21.0
2.43 7.05 7.70
837 6.44 23.8
Mean concentration (mg/L)
0.12 0.15 0.25
0.12 035 0.38
0.13 0.10 037
Ratio relative to copper
1.1 1.4 23
1.0 1.7 2.9
Fe
243
48.7
155
2.9
2.4
2.4
26
18
Ratios reported for Kaunas are based on mean of concentrations reported for discharges into both the Neris
and Nemunas Rivers.
Table 2. Acute toxicity characteristics to rainbow trout of wastewaters from Vilnius
factories.
Factory wastewater source and type
(percent of discharge)
48-hours
96-hours
Amalgamated Enterprise of Construction-Finishing
Machines, Electrodeposition discharge
Amalgamated Enterprise of Construction-Finishing
Machines, Surface run-off
Polishing Machine-Tool Plant, Surface run-off
Fuel Equipment
9.2
38.8
625
57.2
9.2
38.8
46.9
54.8
109
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Changes in feeding pattern andbehavior were observed at concentrations of
5-10%; the fish showed unrest, often rising to the surface. These respiratory
disturbances were noticed as soon as the test water was introduced. Gill ventilation
frequency increased from 92.5 ±8.5 to 115 ±2.7 minute. After 24 hours, however, fish
ventilation frequencies did not differ considerably from controls.
Surface run-off water from the grounds of Vilnius Amalgamated Enterprise of
Construction-Finishing Machines: Wastewater for testing was diluted to 45, 40, 32.5,
and 10%. As in the case of the electrodeposition wastewater, the range of lethal
concentrations was very small, and the toxic effect was very acute. All test mortalities
occurred during the first 24 hours. At wastewater concentrations of 45-32.5% the
feeding and behavior of the fish changed significantly. Within 1-2 hours after exposure
to wastewater of acutely toxic concentrations, gill ventilation frequency increased to
120-150/minute and the "coughing" rate to 20-30/minute. After 24 hours, ventilation
frequency of the two surviving fish decreased to 77 ±2.5/minute and 83 ±2.6/minute.
Surface run-off water from Vilnius Polishing-Machine Tool Plant: As the amount
of wastewater available for study was small, we analyzed only three concentrations:
75, 50, and 25%. During 96 hours at 75% wastewater concentration, eight out of
10 fish died and at 50% concentration all the fish survived. However, the effect of
50% concentration was rather toxic because the fish were not feeding, arid gill
ventilation frequency after 24 hours exposure decreased significantly to 81.0 ±3.2/minute
(as compared to the control of 101 ±3.0/minute).
Wastewater of Vilnius Fuel Equipment Plant: Because most of the fish died within
48 hours, the calculated 48- and 96-hour LC50 values were only slightly different, 57.2%
and 54.8%. Fish at the next lower concentration, 40.45%, stopped feeding and gill
ventilation frequency increased.
Wastewater of "Sigma" Plant: Wastewater was diluted as follows: 60, 50, 25, and
15%. No deaths of fishes were recorded. At concentrations of 50% and 60%, however,
the fish stopped feeding, gill ventilation frequency was 135-142/minute, and at 24 and
48 hours ventilation frequency decreased to 110-115/minute. The "coughing" rate
remained unchanged.
A Study of the Effect of Model Solutions of Common Heavy Metals
When studying the effect of both model solution No. 1 (Vilnius variant) and
model solution No. 2 (Kaunas variant) on the survival of rainbow trout, a number of
common relationships were found. After introducing the model solutions into the
aquaria, the majority of fish died within 48 hours; the fish that survived recovered very
soon and within 24 hours after exposure appeared to be in satisfactory condition. An
obvious qualitative transformation of the solutions was discovered: after introducing the
solutions to the test aquaria, the color of the water changed to brown, and in time a
brown precipitate settled out, indicating the ratio of metal concentration in solution was
110
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no longer that of the original stock solution. There was a sharp boundary between the
lethal and sublethal concentration zones. No significant differences in rainbow trout
survival were established between the effects of the two model solutions (Table 3).
Table 3, Acute toxicity characteristics of heavy metal model solutions to rainbow trout.
Exposure time
(hours)
48
96
48
96
Effective concentration (percent solution)
LC0
Model solution No. 1
57.8
572
Model solution No. 2
55.2
55.7
LCjo
- Vilnius variant
70.0
69.2
- Kaunas variant
68.1
68.0
LC100
84.7
83.7
83.8
83.0
R2
0.95
0.93
0.86
0.89
With regard to behavior, the locomotor activity of fish significantly increased after
introducing the model solutions at lethal concentrations; random swimming, body
tremors, and respiratory disorders were observed. When the solution concentration
exceeded 70% the fish did not respond to food, but at concentrations of 55-65% the fish
were feeding like those in the control group.
The effect of the Kaunas variant solution on respiratory indices of rainbow trout
after exposure for 1 hour was studied. Lethal concentrations of the solution had an
effect upon respiratory indices of the trout (Table 4). Gill ventilation frequency
decreased as much as 26% while the "coughing" rate increased 10- to 15-fold.
Table 4. Respiratory indices of rainbow trout after 1 hour exposure to model solution
No. 2 (Mean ± SE, n = 10).
Concentration of model solution
(percent)
Control
55
60
65
80
85
Gill ventilation frequency
(number/minute)
102.9 ±1.4
79.0±1.4*
86.0±1.6*
1053 ±2.8
96.0±0.6*
765±0.8*
"Coughing" rate
(number/minute)
1.5±0.2
21.4±0.6*
143±0.2*
14.0±0.8*
13.0±0.6*
15.5±03*
Significant differences from control (P<0.05).
Ill
-------
Data on the chemical composition of the wastewaters prior to dilution, and the
estimated concentrations of the wastewater components at the 96-hour LC50 values are
presented in Table 5. The most toxic wastewater was from Vilnius Amalgamated
Enterprise of Construction-Finishing Machines: the 96-hour LC50 was 9.2%. During the
tests of the wastewater of the "Sigma" plant, although all fish survived the 96-hour test
period, they were obviously intoxicated. The 96-hour LC50 of both model solutions were
much alike: 69.2% (Vilnius variant) and 68.0% (Kaunas variant). The difference in
chemical composition of wastewaters was distinct, but it is clear that the concentrations
of the five heavy metals in the model solutions that caused 50% mortality of fish was
considerably higher than those found in the industrial wastewaters. Based on the data
obtained, one may suppose that the reason for deaths of fish in different wastewater
concentrations is not the heavy metals alone. It is quite possible that the toxicity of
wastewaters was due to high pH, or a quantity of unknown suspended substances that
were not analyzed. It is also possible that the toxicity of wastewaters was caused by
factors not established analytically.
Table 5. Chemical composition of wastewaters of Vilnius factories and lexicological
characteristics of wastewaters and model solutions (concentrations in mg/L).
Percent wastewater
or model solution
Suspended
substances
_pH.
Cu
Ni
Cr
Zn
Fe
Electrodeposition wastewater of Vilnius Amalgamated Enterprise of Construction-Finishing Machines
100 4.0 10.7 0.005 0.04 0.125 0.16 0.13
9-2; 037 - 0.0005 0.004 0.012 0.015 0.012
Ram sewerage wastewater of Vilnius Amalgamated Enterprise of Construction-Finishing Machines
100
38.8"
100
46.9*
100
54.8*
692*
68.0*
72 9.7 0.011 0.06 0.055 0.08 0.44
2.8 - 0.004 0.023 0.02 0.03 0.17
Rain sewerage wastewater of Vilnius Polishing Machine-Tool Plant
114.7 8.41 0.14 0.00 0.00 0.15 1.7
53.6 - 0.065 0.00 0.00 0.07 0.8
Wastewater of Vilnius Fuel Equipment Plant
773 853 0.45 0;49 031 ,1-3 7.0
42.4 - 0.25 0.27 0.17 0.7 3.8
Model solution No. 1 (Vilnius variant)
7.2 - 0,69 0.76 0.97 1.59 18.3
Model solution No. 2 (Kaunas variant)
7.2 - 0.68 0.68 1.2 1.98 12.6
112
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Based on data from the Lithuania Environmental Protection Department on the
annual amounts of discharges of common heavy metals from the cities of Vilnius and
Kaunas, and information on the average annual flow rates of the Nemunas and Neris
Rivers (Table 6), theoretical concentrations of heavy metals in the rivers were calculated.
Using the 96-hour LC50 values of the model solutions, the suggested safe concentrations
were estimated at 0.01 times these values. Such concentrations would be between 0.1%
and 0.6%, depending upon the kind of wastewater.
Table 6. Estimates of representative heavy metal concentrations in the Nemunas and
Neris Rivers and suggested safe concentrations for fishes.
Location or solution
Cu
Ni
Cr
Zn
Fe
Nemunas River
below Kaunas
Neris River immediately
above confluence with Nemunas River
Neris River below Vilnius
Model solution No. 1
(Vilnius variant)
Model solution No. 2
(Kaunas variant)
Estimated concentrations (mg/L)
0.0005 0.0006 0.0005 0.002 0.012
0.0006 0.0005
0.001
0.001
0.007
0.009
0.003 0.003 0.004 0.007 0.077
Suggested safe concentrations* (mg/L)
0.01 0.016 0.18
0.008
0.007 0.007
0.012
0102
0.13
0.01 times the 96-hour LC50
The data above would imply that the concentrations of heavy metals that are
suggested as safe are higher than the actual concentrations of these metals in the Neris
and Nemunas Rivers. Such conclusions may be too hasty. It should be borne in mind
that natural waters contain background levels of heavy metals and that river flow rates
are not constant. Pollution is usually local and zones of more polluted water can be
formed. To suggest more exact safe concentrations of wastewaters and their
components, physiological and behavioral tests are needed. In addition to the data from
such biological tests, one should have more exact information on the physical and
chemical equilibria composition of the receiving water, as well as data oh the
degradation of the pollutants and their accumulation in hydrobionts (Anonymous 1990).
To study the toxic effect of model mixtures on fishes further, it would be necessary to
determine the toxicity of each single component, to establish the relative toxicity of the
components, and to find interdependence of the components in the given mixture.
113
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REFERENCES
Anonymous. 1990. Biological-chemical characterization of industrial wastewater.
Swedish Environmental Protection Agency, Norstedts Tryckeri, Sweden.
Belenkij, M.L. 1963. Elements of quantitative estimation of pharmacological effect.
State Publishing House of Medical Literature, Leningrad. (In Russian)
Gerulaitis, A., and V. Valusiene. 1991. Heavy metals in fishes and their effect on
ichthyofauna of the lower Nemunas and the Kur§h| Bay. In: Nemunas Basin
Water Pollution and its Biological Effect to the Ecosystem. Academia, Vilnius,
pp. 73-81. (In Lithuanian)
Joklas, K. 1991. Peculiarities of heavy metals migration and accumulation in the King
Wilhelm Canal in the northern part of the Kursnj Bay and in the Baltic near
shore in 1989. In: Nemunas Basin Water Pollution and its Biological Effect to
the Ecosystem. Academia, Vilnius, pp. 14-27. (In Lithuanian)
Lazauskiene, L., I. Jagminiene, and V. Klimasauskiene. 1991. Ecological effects of
hydroecosystem pollution on the structure of hydrobiont populations and
biocenoses. In: Nemunas Basin Water Pollution and its Biological Effect to the
Ecosystem. Academia, Vilnius, pp. 71-80. (In Lithuanian)
114
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AVOIDANCE REACTION TO POLLUTANTS BY VIMBA
UNDER LABORATORY AND FIELD CONDITIONS
G. Svecevicius1
ABSTRACT
Avoidance reactions of adult and juvenile vimba, Vimba vimba (L.), to two heavy
metals and three organic pollutants were studied under laboratory and field conditions.
The metals were zinc and copper, and the organic pollutants were a commercial
sulfpnate detergent, phenol, and oil products. Although fish demonstrated significant
avoidance responses to the sulphonate detergent and heavy metals, avoidance to phenol
was less pronounced and there was no avoidance reaction to the oil products. The
avoidance of the sulphonate detergent by vimba was very consistent and independent of
testing conditions. Individual adult and juvenile fish avoided zinc and copper in the
laboratory equally, although these thresholds were considerably lower when the fish were
tested in groups. Avoidance thresholds were not related to pollutant toxicity and were
generally at sublethal levels, except that the concentration range of effective avoidance
of phenol was close to its acutely toxic concentration. From the point of view of
ecological significance to habitat occupied by vimba, the pollution with sulfonate
detergents would impose the greatest danger, and it may be that less danger is caused
by zinc and copper pollution.
INTRODUCTION
Avoidance reaction by fishes is one form of phenotypic adaptation to water
pollution (Flerov 1989). An active retreat of fishes out of polluted areas, however,
results in disturbance to their migration patterns and distribution. This is especially true
in the case of anadromous fishes. Therefore, a reduction of their normal area of habitat,
as well as their resources, can occur.
Great attention has been given to the study of avoidance reaction to pollutants by
fishes during recent decades, and a considerable amount of experimental data based on
laboratory experiments are available (Giattina and Garton 1983, Beitinger and Freeman
1983). Literature data, however, are often fragmented, contradictory, and difficult to
compare. Although avoidance of pollutants by fishes has been observed in nature, it is
supposed that threshold avoidance concentrations in nature are much higher and more
variable than under laboratory conditions where direct or masking factors capable of
effecting fish behavior are absent (Sprague 1971, Beitinger and Magnuson 1976,
Weber ei al 1981, Giattina et al 1981). That is why it is necessary to conduct
field studies to compare with laboratory tests.
Institute of Ecology, Lithuanian Academy of Sciences, Vilnius, Lithuania
115
-------
Our study deals with vimba, an anadromous fish, and although it is one of the
most valuable commercial species in Lithuania, its population is being reduced each
year. Avoidance reactions to pollutants by vimba have not been studied. The aim of the
present work is to study regularities of occurrence of avoidance reaction by this fish to
two heavy metals, phenol, a commercial detergent, and a diesel fuel. These are
representative of the most common pollutants in the rivers of Lithuania.
MATERIAL AND METHODS
The tests were conducted at the Experimental Aquarium, Institute of Ecology,
Lithuanian Academy of Sciences. Test fish were vimba of both sexes, 23-30 cm total
length, collected in 1983-1988 by means of electrofishing during spawning season (May-
June) in the Sirvinta River, and juveniles 7.5-10 cm total length, collected during autumn
by sweep net in the Neris River 13 km above Vilnius. The fish were acclimated and
kept in the laboratory in tanks supplied with aerated artesian water of good quality with
total hardness approximately 250 mg/L as CaCO3, alkalinity approximately 200 mg/L
HCOJ, and pH 7.2.
Acute toxicity tests were conducted on juvenile vimbas. The following effective
concentrations were set for 48 hours exposure: maximum acceptable concentration
(LC0), median lethal concentration (LC50), and absolutely lethal concentration (LC^),
found by regression analysis of log-probit transformation applying the method of Miller
and Tainter, referenced in Belenkij (1963).
The test chamber was similar to those described by Fava and Tsai (1976) and
DeGraeve (1982). The gradient chamber was 1500 x 600 x 300 mm in size, and the total
flowing capacity was 6 L/minute. Fifteen minutes after the introduction of test solution
into one of the canals of the chamber, three zones formed: Zone A, control water;
Zone B, solution; and Zone C, partial mixing zone.
The field experiments for determining avoidance reactions to pollutants were
performed on groups of adult fishes consisting of ten individuals, half male and half
female, in a stream where a working area with a gravel bottom about 12 m2 was
arranged (SveceviSius 1989); the depth was 15-20 cm with a flow rate of 16-17 L/second.
The water quality was similar to the characteristics of non-polluted rivers of Lithuania
with total hardness of approximately 150 mg/L as CaCO3. Test solution was added so
that in the stream two zones were formed, equal by area and volume: one polluted and
one control. A filming camera was used to take a picture every 30 seconds to record the
fish distribution in the stream. The ecological characteristics of the test area, such as
temperature, substratum, and flowing rate, was close to natural for vimba at
migratory/spawning season. The effects of the following substances were studied:
CuSC>4, ZnSO^ phenol, Lotos™ (a sulfonate detergent 22% alkylsulfonate and
alkylbenzosulfonate), and oil products (diesel fuel in emulsion and solution).
116
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Estimation of avoidance reaction under laboratory conditions was conducted as
follows: a single fish, or a group of 10 fish, was placed in a gradient chamber and
acclimated for 0.5-1.5 hours, followed by a 10-minute control period during which the
location of the test fish in one or another zone of the test chamber was recorded visually
at 15-second intervals. The testing was performed during 10-minute periods at 30, 60,
90, and 120 minutes from the start of addition of the test solution. The nature and
intensity of reaction were estimated by the index of reaction through the formula-
Reaction index = 50(2-NT/NC),
where Nc is the number of recordings during the control period of a fish in Zone B
divided by the total number of recordings in both Zone A and Zone B; NT is the
number of recordings during the test period of a fish in Zone B divided by the total
number of recordings in both Zone A and Zone B. When testing was conducted on a
group of fish, the numbers recorded were changed correspondingly.
The value of the index 100 denoted maximal avoidance, while 0 denoted maximal
preference and 50 denoted indifference. The significance of reaction was estimated by
Student's t test with P ^ 0.05.
RESULTS AND DISCUSSION
An avoidance threshold concentration of phenol by adult vimba was estimated to
be 2.8 mg/L, and by juveniles it was estimated to be 4.6-4.8 mg/L (Table 1). The
response difference between adults and juveniles was not significant (P>0.1). The
reaction index reached a maximum value at 50 mg/L (Figure 1), and variations in
reaction intensity with time were not significant. The contact of vimba with phenol at
5 mg/L or more caused external symptoms of poisoning, namely sudden spurts of speed,
body tremor, and increase in the rate of respiratory disturbances which manifests itself
though a gill-cleaning reflex referred to as "coughing." Under field conditions, there was
no apparent avoidance reaction to phenol by groups of adult vimba over the range
0.1-1 mg/L, although an increase in locomotor activity was noted at 1 mg/L. tests at
higher concentrations were not conducted.
The most intensive avoidance occurred for the detergent Lotos™. A significant
reaction was found in single adult fish under laboratory conditions within the range
0.1-1 mg/L, and in single juvenile fish within the range 0.5-10 mg/L. At 5 mg/L the
detergent caused the strongest avoidance reaction in juvenile vimba, but the intensity of
reaction decreased considerably at 10 mg/L (P<0.001). Variations of reaction intensity
to the detergent over time were not significant. Groups of juveniles under laboratory
conditions, and adult vimba in field conditions, also intensively avoided Lotos™.
A significant reaction was noted within the concentration range of 0.1-1 mg/L.
117
-------
Table 1. Avoidance threshold concentrations of pollutants by vimba under different
testing conditions (values reported in mg/L with 95% C.I.).
Pollutant
"Phenol
Lotos™
Zinc
Copper
Single juveniles
4.6
(1.8-8.4)
0.061
(0.024-0.13)
0.69
(034-2.0)
0.12
(0.07-0.25)
Laboratory tests
• Group of juveniles
4.8
(2.2-9.7)
0.037
(0.012-0.13)
0.16
(0.084-034)
0.044
(0.014-0.13)
Single adults
2.8
(1.8-317)
0.05
(0.02-0.09)
0.6
(0.1-1.8)
0.1
(0.05-03)
Field tests
Groups of adults
>1
0.047
(0.016-0.11)
0.026
(0.0057-0.077)
0.005
(0.0024-0.0087)
100
90
80
30
Y=20.63+19.30 logX
R2=0.99
P<0.001
10
15
25
50
100
Phenol Concentration (mg/L)
90
80
70
60
50
40
30
1=79.81+10.66 logX
R^O.99
P<0.001
0.1 0.5 1 5 10
Lotos™ Concentration (mg/L)
Figure 1. Avoidance reaction in juvenile vimba to phenol, and
to the detergent Lotos™. Circles (o) indicate the mean
values of the reaction of single fish. Vertical lines
denote +1 SE. Threshold values are marked by arrows.
118
-------
The intensity of avoidance reaction to zinc and copper by single adult and juvenile
vimba in the laboratory was found to be significantly different over time. Long latent
periods of reaction were noticed. Thus, adult vimba significantly avoided zinc at 2 and
4 mg/L and copper at 1 mg/L after 90 minutes into test, and copper at 0.5 mg/L after
120 minutes into test. The juvenile vimbas significantly avoided zinc at 2-4 mg/L after
90 minutes into test, and at 10 mg/L after 30 minutes into test Copper at
concentrations of 0.5-1 mg/L was also significantly avoided after 90 minutes into test,
while at 1.5 mg/L the reaction occurred after 60 minutes. Groups of juveniles avoided
zinc more strongly than did individuals. Zinc was significantly avoided even at
concentration of 0.4 mg/L, and the reaction intensity was stable over time. Copper was
avoided at 0.1 mg/L, although the latent period of avoidance was long. Under field
conditions, adult vimba intensively responded to heavy metals: significant avoidance
reaction to zinc was found at concentrations of 0.22, 0.4, and 2 mg/L, and to copper at
0.05, 0.1, and 0.5 mg/L.
The avoidance reaction to zinc and copper by vimba was strongly dependent on
testing condition. Single adults and juveniles avoided zinc and copper equally. Under
laboratory conditions, however, juveniles in groups avoided zinc and copper more
intensely than did single fish, either adult or juvenile. Among the latter, the reaction to
zinc and copper was notable for a long latent period. The results of field tests on groups
of adult vimba were found to be greatly different from those in laboratory; avoidance
thresholds were 0.026 mg/L for zinc and 0.005 mg/L for copper, an order of magnitude
lower than in the laboratory.
It is known that vimba often form schools. The testing on single fish in the
laboratory has shown, by external symptoms, that some are more sensitive than others to
the same pollutant concentrations. It is very likely that in the avoidance reaction to
metals by groups of vimba, some display of group behavior was taking place, le., the
capability of less sensitive individuals to follow and imitate the behavior of more
sensitive ones.
Our data obtained on the avoidance reaction of vimba to zinc and copper do not
agree with data reported by other authors for Atlantic salmon (Salmo salaf) (Sprague
et at 1965, Saunders and Sprague 1967). These authors report that salmon parr in the
laboratory had a median avoidance concentration of 0.0024 mg/L for copper and
0.054 mg/L for zinc. Simultaneously abnormal slopes of adult salmon migrating for
spawning were recorded as 0.017-0.021 mg/L for copper and 0.21-0.258 mg/L for zinc.
These authors also reported that spawning migration was completely stopped at
concentrations of copper over 0.038 mg/L and zinc at 0.48 mg/L. Consequently, under
natural conditions the Atlantic salmon is less restrained by these metals, and this
phenomenon was explained by the authors as a lack of strong motivation in the
laboratory. The motivation to migrate was probably absent in our test fish, although the
stretch of the stream used for field experiments corresponded in all ecological
parameters (substratum, depth, and flowing rate) to the biotope, attractive for adult
vimba during migratory/spawning season.
119
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None of the fish tested, under either laboratory or field conditions, evidenced
avoidance to oil products when these were added to water to make nominal
concentrations within the range 0.1 to 100 mg/L. A longer contact of fish to oil products
at 100 mg/L caused a number of external symptoms of poisoning. These were decrease
of locomotor activity, respiratory disturbances evidenced by an increase in "coughing"
rate, and, in some cases, loss of equilibrium.
The avoidance response of male and female adult vimbas under laboratory test
conditions was equal; no differences between sexes in reaction insensitivity were noted
(P>0.1). Avoidance reaction intensity to phenol and to Lotos™ by single juvenile
vimba tested in the laboratory is illustrated in Figure 1. Regression analysis confirmed
that a linearly proportional interdependence exists between concentration and avoidance
reaction (P<0.001). The threshold concentration of avoidance is defined as that
concentration value of the regression line at which the reaction index is 50 (Hoglund
1961, Fava and Tsai 1976, Anestis and Neufeld 1986).
Data on acute toxicity of the pollutants studied in juvenile vimba were compared
to threshold avoidance concentrations (Table 2). The data show that the avoidance
thresholds of phenol are the closest of these to maximum acceptable concentration
(68.2-71.6% of LC0). Thus the range of effective avoidance concentrations are close to
acutely toxic levels of phenol. This agrees with the conclusions that some cyprinid fishes
are capable of avoiding phenol only at lethal levels (Ishio 1965, 1969). Avoidance by
vimba of the detergent Lotos™ occurs at low sublethal concentrations (0.3-0.5% LC0) as
does avoidance of heavy metals. The data obtained indicate absence of correlation
between "toxicity of pollutant and intensity of its avoidance.
Table 2. Comparison of 48-hour acutely lethal concentrations to threshold avoidance
concentrations in juvenile vimba (values reported in mg/L).
Pollutant
Lethal concentration
Threshold avoidance
concentration
LCn
LC,
'50
LC
100
Single
Group
Phenol
Lotos™
Zinc
Copper
Oil products
6.7
122
16.8
0.45
9.6
10.9
19.2
20.1
0.69
14.9
17.7
30.1
24.0
1.08
23.0
4.6 4.8
0.061 0.037
0.69 0.16
0.12 0.044
absence of avoidance
120
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SUMMARY
The present study enables us to estimate the ecological danger of pollution to
vimba; one should be guided by the data of field tests as these best reflect possible
reactions of fishes in natural media. Vimba avoided the detergent Lotos™ more than
they avoided any of the other pollutants tested. The avoidance threshold to Lotos™
amounted to 0.037-0.061 mg/L and to 0.0081-0.013 mg/L as sulfonate. These are very
low concentrations, and such concentrations may very well be in Lithuanian rivers along
the migration routes of vimba. Even so, maximum permissible concentrations for
alkylsulfonate and alkylbenzosulfonate detergents are as high as 0.5 mg/L (Anonymous
1975). Heavy metal concentrations in Lithuanian rivers vary within the range
0.001-0.01 mg/L, and may be much higher near pollution sources; zinc and copper can be
considered ecologically potentially dangerous for vimba. Phenol was avoided by vimba
at concentrations over 2.8 mg/L, a level seldom found in Lithuania under natural
conditions, so from this point of view phenol is less harmful. Oil products, generally not
avoided by vimba, may be harmful in those cases when a fish swims into oil solutions at
concentrations high enough to cause unfavorable lethal or sublethal reactions.
REFERENCES
Anestis, I., and J. Neufeld. 1986. Avoidance - preference reactions of rainbow trout
(Salmo gairdneri) after prolonged exposure to chromium (VI). Water Research
20:1233-1241.
Anonymous. 1975. Criteria for protection of inland surface waters from wastewater
pollution. USSR Ministry of Land Reclamation and Water Management,
Ministry of Health, and Ministry of Fish Farming. Moscow, 82 pp. (In Russian)
Beitinger, T.L., and L. Freeman. 1983. Behavioural avoidance and selection of fishes to
chemicals. Residue Reviews 90:35-55.
Beitinger, T.L., and J.J. Magnuson. 1976. Influence of social size rank and size
thermoselection behavior of bluegill (Lepomis macrochirus). Journal of the
Fisheries Research Board of Canada 32:2133-2136.
Belenkij, M.L. 1963. Elements of quantitative estimation of pharmacological effect.
State Publishing House of Medical Literature, Leningrad. (In Russian)
DeGraeve, G.M. 1982. Avoidance response of rainbow trout to phenol. Progressive
Fish-Culturist 44:82-87.
Fava, J., and C. Tsai. 1976. Immediate behavioral reactions of blacknose dace,
Rhinichthys atratulus, to domestic sewage and its toxic constituents. Transactions
of the American Fisheries Society 105:430-441.
121
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Flerov, B.A. 1989. Ecological and physiological aspects of toxicology in fresh-water
animals. Nauka, Leningrad. (In Russian)
Giattina, J.D., D. A. Cherry, J. Cairns, and S. R. Larrick. 1981. Comparison of
laboratory and field avoidance behavior of fish in heated chlorinated water.
Transactions of the American Fisheries Society 110:526-535.
Giattina, J.D., and R. A. Garton. 1983. A review of the preference-avoidance responses
of fish to aquatic contaminants. Residue Reviews 87:43-90.
Hoglund, L.B. 1961. The reactions of fishes in concentration gradients. Fisheries
Board, Swedish Institute of Freshwater Research, Drottningholm, Report
43:1-147.
Ishio, S. 1965. Behavior of fish exposed to toxic substances. Advances in Water
Pollution Research 1:19-33.
Ishio, S. 1969. Discussion. Advances in Water Pollution Research 1:181-184.
Saunders, R.L., and J. B. Sprague. 1967. Effects of copper-zinc mining pollution on
spawning migration of Atlantic salmon. Water Research 1:419-432.
Sprague, J.B., P. F. Elson, and R. L. Saunders. 1965. Sublethal copper-zinc pollution in
a salmon river - A field and laboratory study. International Air and Water
Pollution Research 9:531-543.
Sprague, J.B. 1971. Measurement of pollutant toxicity to fish - III. Sublethal effects
and "safe" concentrations. Water Research 5:245-266.
Svecevicius, G. 1989. Avoidance response to pollutants in the vimba under field
conditions. Lithuanian TSR Academy of Sciences, Publication Series C(4):69-74.
(In Russian)
Weber, D.D., D. J. Maynard, W. D. Gronlund, and V. Konchin. 1981. Avoidance
reactions of migrating adult salmon to petroleum hydrocarbons. Canadian
Journal of Fisheries and Aquatic Sciences 38:779-781.
122
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TOTAL ^-ACTIVITY AND 90Sr CONCENTRATION
IN THE KURSiy MARIOS LAGOON ECOSYSTEM
R. Dusauskiene-Duz1
ABSTRACT
A great amount of radioactive matter was dispersed in the atmosphere after the
accident in 1986 at the Chernobyl Atomic Power Station in Belarus. The area impacted
included the Nemunas River basin and Kursiu Marios Lagoon ecosystem. There is a
continued need to monitor the radioactive matter from Chernobyl to document its
distribution in water basins, migration, bioaccumulation, transformation of chemical
forms, and bottom deposition. The objective of this monitoring is to fix the beginning of
global and technogenic influence and changes of natural systems caused by the accident.
Our studies show a continued threat to benthic organisms by long-term pollution sources
m the bottom sediments of Kursiu Marios Lagoon.
INTRODUCTION
Fission products, together with wet and dry atmospheric deposition, accumulates
on the earth's surface and in its aquatic systems. An interaction with the environment
then begins, and radioactive matter is diluted and distributed among the main
components of the ecosystem. Although Kursiij Marios Lagoon is a specific water basin,
it is greatly influenced by water regime changes in the Baltic Sea, thereby influencing the
aquatic life of the lagoon. At the same time, there is a great amount of suspended
organic matter and nutrient material discharged into Kursnj Marios Lagoon from the
Nemunas River, contributing to eutrophication of the lagoon.
Radioecological investigations in the Kursi\| Marios Lagoon ecosystem began in
1967; prior to that no such work had been done on this water basin. The first step was
to measure strontium-90 (90Sr) concentrations in water, bottom sediments, plants, and
fish (Dusauskiene-Duz 1978). Later on, the influence of chemical composition factors,
such as domestic and paper manufacturing wastewater, on the 90Sr concentrations in
hydrobionts was estimated (DuSauskiene-Duz 1978). Still later, as eutrophication in the
Kur&u Marios Lagoon became intense, measurement of lead-210 (210Pb) concentrations
in water, biota and bottom sediments were, included into the investigation program
(Dusauskiene-Duz 1978, 1983). This enabled us to determine migration peculiarities,
accumulation mechanisms, and the levels of two naturally different radionuclides. The
marked °Pb enabled us tp date the bottom sediments of the lagoon and to establish
sedimentation rates (Dusauskiene-Duz 1978). Total ^-activity (5^) allowed us to locate
the most radionuclide-contaminated regions and their components, as well as to estimate
the role of atmospheric fallout in forming the levels of radioactivity in the basin.
Sector of Radioecology, Laboratory of Hydrobotany, Institute of Botany, Vilnius, Lithuania
123
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MATERIAL AND METHODS
The concentrations of 90Sr in water, biota, and bottom sediments was estimated
according to the carbon method of Sokolova (1971), in which 90Sr is measured according
to its daughter-product yttrium-90 which strikes a balance after 14 days. Chemical
measurements of ^Sr made up 85-89% of total Sr. Radiometry was carried out by low
intensity equipment (UMF-1500™), with intensity of 3.5-4.0 cpm; measurement
efficiency was 17-20%.
RESULTS AND DISCUSSION
Eg-actrvity and ^Sr concentration was measured in the surface-water microlayer
(SWML), 300-400 /on, and surface-water layer, 0.5 m (Table 1). From this, one can
draw a conclusion that the 90Sr concentration level in the Kursixj Marios Lagoon
ecosystem is caused by atmospheric deposition.
Table 1. Total E^-activity and ^Sr concentration in rain, surface-water micro layer
(SWML), and water samples (Mean values in Bq/m3).
Rain
SWML
(% rain)
Water
(%SWML)
90Sr
1100
243
847
(77)
173
(71)
551
(65)
28
(16)
It was established that E^-activity in the SWML depends upon the time and place
of sample collection. The E^-actrvity in SWML samples from the northern part of
Kurs*h} Marios Lagoon (Figure 1) was lower than that in samples from the central part.
There was a slight difference between filtered and unfiltered water. The E^-activity was
slightly higher in unfiltered water samples collected from the central part of Kursiij
Marios Lagoon during spring, while there was a significant difference between filtered
and unfiltered samples collected in autumn because of a greater amount of suspended
matter (Table 2).
Table 2. Mean (and range) E^-activity in the surface water micro layer (Bq/m3).
Station
number
233*
10,11,
12,12«
Unfiltered
-
743
(647-925)
June 1991
Filtered
-
666
(206-1200)
October
Unfiltered
521
(471-555)
1010
(906-1430)
1991
Filtered
638
(617-700)
580
(370-755)
124
-------
28.0
Nemunas R(ver~
Figure 1. Mean concentration of 90Sr in Kurshj Marios Lagoon (Bq/m3), 1978-1990.
Surface-water layer E^-activity was higher in filtered water from the northern part
of Kurshj Marios Lagoon as well as in unfiltered water from the central part (Table 3)
This can be explained by the fact that the northern part of the lagoon is under strong
anthropogenic influence, which includes wastes from the Cellulose and Pasteboard Plant
in Klaipeda (CPPK), the city's sewerage, and the Ship Repairs Plant These activities
cause changes in radionuclide chemical forms. This is evidenced by the'prevalence of
soluble-ionic radionuclide forms over nonsoluble-suspended forms, which poses great
danger for living organisms because it increases migration properties of radionuclides
This conclusion is supported by the data of Joksas (1991), indicating that concentrations
of heavy metals in the waters of the northern part of Kursiu Marios Lagoon are in
soluble form, not suspended particulate form.
125
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Table 3. Mean (and range) E^-activity in surface-water layer (Bq/m3).
Station
Number
June 1991
October 1991
Unfiltered
Filtered
Unfiltered
Filtered
233-
10,11,
12,12"
—
477
(199-781)
—
304
(853-616)
647
(616-679)
786
(308-1110)
987
(802-1170)
610
(370-780)
Ep-activiry in the surface water of the northern part of Kursig Marios Lagoon is
higher than that in SWML because of the influence of surrounding factors. This
indicates that the surface pellicle is destroyed under the influence of water streams in
wastewater channels. In other water bodies, we have seen the same situation when Eg
of non-filtered water is higher than that of filtered water.
Considerable differences between the So-activiry in the bottom sediments of the
northern and central parts of Kursnj Marios Lagoon was not established (Table 4).
Knowing that conditions of hydrodynamic sedimentation are more favorable in the
central part of Kurshj Marios Lagoon, one can assert that E^-activity is higher in the
northern part. Thus, taking into account that the greatest concentration of suspended
terrigenic matter lies in the northern part of the lagoon in the region of Klaipeda
Harbor (JokSas 1991), water mixing results in active coagulation, adsorption/absorption,
and desorption processes. Very intensive degradation processes take place in this zone
under the influence of anthropogenic factors. Small-dispersion muds of small particle
size produce high rates of contamination accumulation. They can be found as soluble-
migratory forms in waters of this part of the lagoon.
Table 4. Mean (and range) E^-activiry in bottom sediment (Bq/kg).
Station Number
Concentration
23,3'
10,11,12,12*
581
{411-732)
533
(300-685)
126
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Aquatic plants are of great importance when forming bottom sediment
radioactivity. It has been established that phytoplankton, which frequently have great
biomass, enrich themselves with heavy metals and their radionuclides (Joksas 1991
^JfU S^? » 1978)' ^ytoP1™**™ ^activity in the central part of the lagoon was
in the 10-136 Bq/kg range, and varied depending upon its gathering place as well as on
intercourse of its physiological groups.
™-7/o Concentration of ^ m SWML reactivity was 28.2% of the total
(247/874 Bq/m ). Water surface pellicle is a short-term concentrated product, dispersing
and diluting in the slightest wind or water movement, and only 11.3% of its primary
activity was in the surface-water layer. Concentration of 90Sr in the near-shore water of
Kursiu Marios Lagoon varies slightly because it is under the constant action of near-
shore water currents; these flow from the Baltic Sea south into the west side of the
lagoon and then go back to the Baltic Sea by flowing north along the east side.
concentration of ^ was measured in the middle part of the lagoon
(Y90o • V1 * ™here influence of near-shore water currents is the lowest. Concentration
ol bt in KurSm Marios Lagoon undoubtedly depends on meteorological conditions such
as amount of precipitation, prevailing wind direction, wind speed, and waving; this
argument is supported by the seemingly active random measurements of 90Sr (Figure 1).
10-70 i™n obtained 9°Sr concentrations in water from long-term measurements (data of
1978-1990) depending upon sampling place and time (Figure 1). This is illustrated by
the results of one year of measurements of 90Sr in Kursiij Marios Lagoon (Table 5) It
was established that the ^Sr concentrations measured in Kursiu Marios Lagoon are
characteristic of other flowing type water basins, and were 2.5 times lower than those in
secluded basins such as Druksmi Lake in northeast Lithuania (Dusauskiene-Duz 1992b).
Table 5. Concentration of ^Sr in KurSiij Marios Lagoon (Bq/m3) in 1990.
Sampling Station Month
Smiltyne
Juodkrante
Pervalka
========
02
-
40.7
03
18.2
31.0
31.0
04
28.4
30.4
29.0
05
293
34.9
37.0
=
06
-
275
18.8
—
07
22.9
37.0
31.8
—
08
25.9
.
26.6
==^=
09
24.4
19.9
10
285
25.8
-
The vitality of plankton and ability to accumulate 90Sr decreases in the northern
part of the lagoon under the influence of salt water from the Baltic Sea. The 90Sr
accumulation coefficient (AC), defined as the concentration of 90Sr in phytoplankton
over the concentration in water, was 26 in the northern part of Kur§iu Marios Lagoon
127
-------
while in the central part it was 54. According to our data, in the northern part of Kursiij
Marios Lagoon single-valent metal radionuclides are absorbed less than those of bi- or
tri-valent metal radionuclides with characteristic high adsorption levels on suspended
matters. The phytoplankton/water AC of 90Sr and^10Pb was 40 and 300, respectively.
Certain aquatic plants are active water quality stabilizers because they purify
mechanically, biologicalfy, and physically-chemically. Nitellopsis obtusa, Potamogeton
perfoliatus, and P. lucens are significant in this purification process. Concentrations of
^°Sr exceeded that in water 190 times for Nitellopsis and 240 times for Potamogeton.
Alga are indicators of ^Sr contamination because they accumulate large amounts of
easily exchangeable Ca, and duckweeds are able to form Ca inlays on leaves with 90Sr
in their structure.
Concentrations of ^Sr in bottom sediment averaged 17 Bq/kg, and varied from
7 to 30 Bq/kg, mainly depending on bottom structure and morphology. Maximum rates
were 15-30 Bq/kg in small-dispersed type bottom (mud), and 7-13 Bq/kg in sand type
bottom. The most variable conditions for the accumulation of 90Sr in bottom sediment
are found in zones with prevailing constant water currents, large vegetation association,
and good sedimentation conditions.
Molluscs, which constitute an especially great biomass in Kur§nj Marios Lagoon,
are in close contact with bottom sediments. Because 90Sr is a calcium analogue, it can
take place in Ca exchange and thus form shells. Concentrations of 90Sr in molluscs from
KurShj Marios Lagoon (Table 6) exceeded the concentrations of 90Sr in phytoplankton
and in rooted aquatic plants.
Table 6. Concentration of 90Sr in molluscs from Kursh| Marios Lagoon (Bq/kg).
Species
Month
05
06
09
Anadonta piscinalis
Dreissena polymorpha
31.1
39.2
11.1
11.5
12.0
22.4
Concentrations of ^Sr in fish organs and tissues, independent of species and
feeding type, indicate the analogous distribution character of this radionuclide (Table 7).
Aquatic life accumulate 90Sr through water or with food. The data indicate that 90Sr
accumulates in fish organisms, as well as in molluscs, mainly from the water.
Experimental investigations indicated that only 30% of 90Sr in shells of molluscs was
assimilated with radioactive food (MarSiulioniene et al 1979). Investigations indicated
that the ratio of ^Sr concentrations in fish and water was 10.6, while in fish and food it
was 0.46. Thus, one can draw a conclusion that assurance of stability of water quality is
the essence of normal ecosystem function.
128
-------
Table 7. Distribution of 90Sr in fish organs and tissues (percent of total).
Organs, tissues
Backbones
Muscles
Head bones
Skin with scale
Fins
Internal organs
Jaws
Pike-perch
19.9
05
15.9
283
29.8
0.4
5.4
Burbot
35.1
0.7
31.8
30.4
Not detected
2.0
Not detected
It has been established that 90Sr, once in a water basin, distributes unequally
among ecosystem components (Table 8). Its maximum level has been fixed in water
where it is constantly replenished through atmospheric fallout, and in molluscs which use
Sr as a calcium analogue in shell forming. Phytoplankton, as well as macrophytes
which accumulate 90Sr, fall to the bottom when they finish their existence, and thereby
take part in bottom sediment activity. Phytoplankton has limited capability to
accumulate Sr, but because of its large biomass and short existence (5-6 days) it can
contribute a significant portion of the radioactivity that is found in bottom sediments.
Table 8. Distribution of 90Sr in ecosystem components of Kursnj Marios Lagoon.
Component
Percent ""Sr of total
Water
Molluscs
Bottom sediments
Water plants
Plankton
Fishes
33.0
33.0
19.0
10.6
4.4
1.0
Source: Dusauskiene-Duz 1978
Because of anthropogenic factors that cause degradation in water quality the
physiological state of aquatic life deteriorates, and as a consequence the ratio between
production and destruction processes is reduced. Thus, destruction processes begin to
dominate and lead to new ecosystem equilibria and total disappearance of some of the
original components. Mechanical sedimentation activity processes, rather than biological
processes, are characteristic of the Kursitj Marios Lagoon ecosystem. This increases the
accumulation of pollution in the bottom sediments, thereby causing formation of a
second-rate long-term pollution source which is of great danger to benthic organisms.
129
-------
REFERENCES
Dus"auskiene-Duz, R. 1978. Accumulation of 90Sr and 210Pb in water plants and bottom
sediments of the northern part of the Kursixj Bay. Physiological-Biochemical
Basis of Plankton Organisms Development in the Northern Part of the KurShj
'Bay. Institute of Botany, Vilnius, pp. 244-255. (In Russian)
Dusauskiene-Duz, R. 1983. Radionuclides (strontium-90 and lead-210). In:
Biogeochemistry of the Kursixj Marios Lagoon. Academia, Vilnius, pp. 124-130.
(In Russian)
Dusauskiene-Duz, R. 1992a. Behaviour of lead-210 in Lake DrukSiai. In:
Radiochemoecological Situation in Lake Druksrai-copling water reservoir of the
Ignalina NPP. Academia, Vilnius, pp. 62-74. (In Russian)
Du§auskiene-Duz, R. 1992b. Investigation of 90Sr migration in Druksiai Lake and the
Baltic Sea. In: Contamination by Radionuclides in Lithuania. Academia,
Vilnius, pp. 71-78. (In Lithuanian)
Joks"as, K. 1991. Migration and accumulation of heavy metals in the King Wilhelm
Channel, northern part of Kurshj Bay and Baltic Sea near shore region in 1989.
In: Contamination of Nemunas Basin Waters and its Biological Influence to the
Ecosystem. Academia, Vilnius, pp. 14-21. (In Lithuanian)
Marciulioniene, D., R. Dusauskiene-Duz, and G. Polikarpov. 1979. Uptake of ^Sr,
137Cs, 144Ce and 106Ru by animal organisms from water and food. Works of the
Lithuanian Academy of Sciences. Ser. B 2(86):145-151. (In Russian)
Sokolova, J.A. 1971. Calcium, strontium-90, and strontium in marine organisms. Kiev,
pp. 237. (In Russian)
130
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INVESTIGATIONS OF THYROID DISEASES IN LITHUANIA
IN RELATION TO RADIATION DOSES FROM CHERNOBYL
V. Sidlauskas1, J. Danys1, A. Krasauskiene1, A, AukStuolyte1,
D. MaSanauskaite1, R. Jurkunaite1, A. Telksnys1
E. Janulionyte2, J. Sviciulyte2
T. Nedveckaite3, V. Filistovicz3
A. Mastauskas4
ABSTRACT
Iodine deficiency in the soil in some parts of Lithuania, especially the southern
regions, jjontributes to thyroid abnormalities in these regions. The prevalence of thyroid
problems in southern Lithuania was tremendously increased by the atmospheric
distribution of iodine-131 as a consequence of the Chernobyl accident in 1986. Studies
are now underway in Lithuania to expand knowledge of baseline formation and the
extent of damage as a consequence of the Chernobyl accident.
INTRODUCTION AND BACKGROUND
The epidemiology of thyroid diseases varies in different countries, and there are
no precise data about this problem in Lithuania. According to Kiauleikiene and
Kiauleikis (1989), 35-50% of children and teenagers examined have thyroid
abnormalities. Investigations of the Institute of Endocrinology of Kaunas Medical
Academy showed that 15% of school children in Kaunas have thyroid disease. The most
frequent abnormalities encountered were hyperplasia of thyroid gland, autoimmune
thyroiditis, and nodular goiter. It has been noted that thyroid disease in Lithuania is on
the increase (Janulionyte et al 1990, Nedveckaite et al in press). There are many
factors that influence thyroid tissue and its functions. These factors include the
radioactive iodine isotopes that comprised a large part of the atmospheric pollution that
fell on Belarus, some regions of Ukraine and Russia, Poland, Sweden, and Lithuania
after the accident at Chernobyl (Figure 1).
Institute of Endocrinology, Kaunas Medical Academy
2Medical Faculty of Vilnius University, Vilnius, Lithuania
Institute of Physics, Laboratory of Radiation Safety
Lithuanian Republic Ministry of Health, National Centre of Hygiene
131
-------
0 200 400
> 10 mSv
1-10 mSv
Figure 1. Predicted doses of 131I to adult thyroids accumulated from
26 April to 1 May 1986, by Lawrence Livermore National Laboratory.
Radionuclide uptake in thyroid is usually measured by special detectors placed
directly over this tissue. Such measurements were not routinely performed in Lithuania
after the Chernobyl accident for reasons beyond control of the experimenters. Instead,
reconstruction of radioactive dose-equivalent in thyroid was performed by means of a
three-compartmental mathematical model for the study of iodine transport kinetics in
the human body. This involves a deterministic ICRP model (Figure 2) and dose factor
values (Anonymous 1978) which convert measurable quantities of radioiodine activity in
air and food (Styro et al 1992) into an estimate of thyroid dose-equivalent.
The initial thyroid dose-equivalent assessment by this means for inhabitants of
Lithuania was performed in May 1986. There are uncertainties associated with model
predictions, such as errors in model structure and environmental variability, as well as
uncertainties in parameter values (Werner and Ingbar 1978). A preliminary examination
of radioiodine thyroid dose-equivalent to healthy adults was determined by a stochastic
Monte Carlo approach having regard for the uncertainties mentioned above, and was
9.8 ±1.0 mSv. This preliminary analysis showed that estimates of dose may be made
from various starting points. A purely deterministic approach does not make allowance
for the magnitude of real doses. Results of a stochastic Monte Carlo-generated
distribution predicted that 30% of the adult population received less than this value, and
5% of the adult inhabitants would have received thyroid doses ranging from 37 to
100 mSv (Figure 3). Infants and children were exposed to radiation doses up to
10 times higher.
132
-------
GUT OR
LUNG
BLOOD
STOOL
THYROID
BODY
URINE
Figure 2. Model for the study of iodine transport kinetics in human body.
0.08 -i
: MODE (4,5 mSv)
0.07 :
DETERM.SOLUTION (9,8 mSv)
MEAN (13 mSv)
95 th PERCENT. (37 mSv)
50 75
Dose, mSv
100
Figure 3. Monte Carlo generated frequency distribution of dose (mSv)
to the thyroid gland of healthy adults from ingestion of milk products
in southern part of Lithuania, May 1986.
According to Galle (1987) Lithuanian inhabitants received thyroid radiation doses
which may have a negative influence on the development of thyroid cells, but not their
function; under this influence, nodules and autoimmune alterations can appear. The
ecological and economical situation in Lithuania, separate from the influence of the
Chernobyl Accident, contribute to the frequency of thyroid pathology.
133
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RESEARCH PROGRAM AND OBJECTIVES
As a consequence of the thyroid diseases problem, especially among children, the
Open Society Fund of Lithuania is supporting a Research Project "Relation between
Radioactive Iodine Isotope Doses and Thyroid Diseases after the Chernobyl Accident
(Epidemiological and Clinical Study in Lithuania)." In addition, the Lithuania
Department of Health has initiated a program entitled "Investigation on Epidemiology of
Thyroid Diseases in Lithuania," under the direction of the Kaunas Medical Academy
Thyroid Laboratory of the .Institute of Endocrinology, together with the Medical Faculty
of Vilnius University and the Laboratory of Radiation Safety of the Institute of Physics.
The program was initiated in collaboration with the Institute of Biomedical
Investigations of Kaunas Medical Academy, location of the International Prophylactic
Program of the World Health Organization (WHO).
The main purposes of these research projects and the overall program is to
determine the incidences of thyroid diseases in the population of Lithuania, to
investigate the relations between thyroid pathology and radioactive thyroid dose
assessment, and the radioecological situation of the region. The main tasks are:
1. To investigate the child and adult populations from different regions
and cities, evaluating their thyroid condition, diagnose disorders,
and develop treatment strategies.
2. To evaluate the radioecological status throughout Lithuania.
3. To establish the relationship between incidence of thyroid diseases
and exposure to radioiodine and other radionuclides.
4. To investigate iodine-deficient areas and evaluate the influence of
this deficiency to thyroid pathology.
5. To map the incidence of thyroid pathology in Lithuania.
6. To confirm the findings and make recommendations to the Department of
Health regarding preventive measures and treatment of thyroid disorders.
These research projects and program will permit partial estimation of the impact
of the Chernobyl accident on Lithuania. Current understanding of the period of latency
of thyroid diseases as a consequence of the influence of ionizing radiation varies between
5 and 30 years. It must be kept in mind, that at the present time in Belarus malignant
thyroid diseases have undergone a rise, especially among infants and children
(Polianskaja et al 1991). Within the framework of these research projects and programs,
investigation of thyroid burden in children, especially malignant diseases, will be carried
out as a separate program.
The Thyroid Laboratory of Institute of Endocrinology has prepared a standard
questionnaire; this information will be computerized according to the guidelines of
WHO, "Investigations on Chernobyl Accident Medical Consequences." International
collaboration will then be possible to compare the result of our research work with the
results of others.
134
-------
PROGRESS TO DATE AND FUTURE PLANS
Some regions of Lithuania were visited in 1993, and result of examinations
conducted are presented in Table 1. Ultrasound examinations were conducted using
"real time" method which makes it possible to measure the size of thyroid by volumetry
These volumetric measurements were made using methods of the Belarus Radiation
Medicine Institute; these are simple, although the equipment operator must be
experienced. Instrumentation was made available through the Open Society Fund of
Lithuania. Results are presented in Table 2. Control groups of healthy adults in each
region have been selected and are now being studied to establish the median size of
adult thyroid in Lithuania for comparison.
Table 1. Prevalence of thyroid gland pathology in some regions of Lithuania, 1993.
Region
Kai&adoriu.
Varcnos
KupiSkio
KupiSkis
Salfininkai
Zarasai
Persons
examined
adult
adult
adult
students
students
students
=====
'Number
300
400
100
372
111
220
Abnormal
thyroid, %
22
23
24
475
70
57
^^s^^gj^gg^g^™'^"''".—
Nodules in
thyroid, %
25
4
6
12
10
125
.
Increased
ATMA,%
15.1
14.7
not tested
not tested
not tested
11.6
Table 2. Thyroid volumetric data from some regions of Lithuania, 1993.
Region
Kaisiadorii}
Varcnos
KupiSkio
Men
163 ±1.0
20.8 ±3.6
22.7 ±2.6
Thyroid volume, ml
Women
153 ±0.8
17.0 ±3.4
14.8 ±23
Collaboration with the Belarus Institute of Radiation Medicine helped us to carry
out investigations on iodine concentration in urine. Twenty people from the Varenos
region were randomly selected for determination of the iodine excretion using the
Cerium-Arsenite method. It was determined that the average iodine concentration in
the urine was 65.5 ±8.2 /tg/L, as compared with a norm of more than 100 jtg/L
(Sidlauskas, unpublished data). Concentrations from 50 to 100 ng/L indicate mild iodine
135
-------
deficiency. The result of these investigations show that in Lithuania there is a problem
of iodine deficiency, separate from the problem of radioactive pollution. The turnover
of the stable iodine will be investigated in other regions as well.
Future plans include of investigation of nitrite-nitrate concentration in ground
water in some regions of Lithuania. There is some information in the literature
suggesting that agricultural chemicals and other types of ecological contamination are
instrumental in provocation of thyroid disorders.
ACKNOWLEDGEMENT
This work was made possible by a grant from the Soros Foundation, Open Society
Fund, Lithuania.
REFERENCES
Anonymous. 1978. Limits for Intakes of Radionuclides by Workers. In: ICRP
Publication 30, Part 1. Pergamon Press, Oxford, New York, Toronto, Sydney,
Paris, Frankfurt, 135 pp.
Galle, R.P. 1987. Immediate medical consequences of nuclear accident. Journal of the
American Medical Association 258:625-628.
Janulionyte, E., J. Monkeviciute, V. Maslauskaite, G. Katiliene, A. Masiulionis,
J. Kiauleikis, and G. Grybauskas. 1990. The increasing prevalence of thyroid
pathology in Lithuania. Sveikatos Apsauga 12:34-35. (In Lithuanian)
Kiauleikiene, M., and J. Kiauleikis.
(In Lithuanian)
1989. The Thyroid. Vilnius University, 30 pp.
Nedveckaite, T., V. Filistovicz, V. Sidlauskas, A. Krasauskiene, E. Janulionyte,
G. Rimdeika, L. Zimaniene, and A. Kesminiene. In press. The impact of the
disaster in Chernobyl on the public health in Lithuania. International Symposium
"Thyroid - 92" Reports.
Polianskaja, O.N., L. N. Astachova, V.M. Drozd, S.V. Markova, A.M. Dubovcov, and
T.A. Mitiukova. 1991. Thyroid system of adult and children after the Chernobyl
accident in Belorussia. In: Chernobyl Accident and the Health of Belorussian
Republic Inhabitants, Minsk, pp. 62-69. (In Russian)
Styro, B., T. Nedveckaite, and V. Filistovicz. 1992. Iodine isotopes and radiation safety.
Hidrometeoizdat, Sankt-Peterburgas, 251 pp. (In Russian)
Werner, S.C., and Ingbar, S.H., (Eds.), 1978. The Thyroid. Harper and Row,
Hagerstown, Maryland, 1047 pp.
136
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MECHANISM OF OXIDATION OF H2S IN THE BALTIC SEA
J. Sukyte1, V. Zelionkaite1, E. Rinkevichiene2, and V. Razumovskij3
ABSTRACT
The mechanism of oxidation of H2S in Baltic Sea water has been modelled in the
laboratory based on our hydrochemical investigations data from the Baltic Sea. Our
preliminary potentiometric and analytical investigations enable us to evaluate existing
mechanisms of sulfide oxidation in seawater and to propose a new one through a
complicated chain reaction caused by active forms of oxygen. The reaction enables
formation of intermediate unstable sulfur compounds including HSO~ and HSO2.
These compounds in reaction with other products of a chain process, form the final
products that have been analytically observed: HS~, S°, S2O|~ and SO2.".
INTRODUCTION
At very irregular intervals, some parts of the bottom of the Baltic Sea may be
entirely devoid of fauna for months or years as a result of oxygen deficiency and the
subsequent presence of hydrogen sulfide. At its worst, these deserted areas may
comprise up to 100,000 km2, equivalent to approximately 25% of the total surface area
of the Baltic Sea. These major stress factors fluctuate intermittently and irregularly
(Andersin et al 1977, Voipio 1981). Hydrogen sulfide is oxidized in the sea water redox
zone by microbiological as well as by chemical processes (Leonov and Aizatullin 1987).
Using antiseptics suppressing microbiological activity, it has been determined that HoS is
oxidized by a chemical pathway (Sorokin 1970). The products of hydrogen sulfide
oxidation by oxygen in sea water has been determined by analytical and indirect
methods. The basic products are as follows: elemental sulfur (S°), polysulfides (S2~)
sulfites (SO2-), thiosulfates (S2O|-), sulfates (SO2.-). Dithionite (S2O|-), dithionate'
(S2°6 )» and tetrathionate (S4O| ), this last in acidic media at pH 7, have also been
reported (Leonov and Aizatullin 1987).
From the variety of oxidation products, an assumption could be made that the
mechanism of oxidation is very complicated and still not fully understood. It is
considered that hydrogen sulfide oxidation is going on in accordance with a chain
reaction mechanism (autocatalytic reaction) and polysulfide ions S2," serve as catalysts
(Bowers et al 1966, Chen and Morris 1971). However, other oxidation reaction
products, such sulfate and thiosulfate ions, act as inhibitors and slow the reaction.
Central Environmental Research Laboratory, Environmental Protection Department, Vilnius, Lithuania
Kaunas Technological University, Kaunas, Lithuania
Klaipeda Hydrometeorological Observatory, Klaipeda, Lithuania
137
-------
Oxidation reactions of hydrogen sulfide are slowed down or speeded up by
catalysts, such as calcium or magnesium ions, heavy metals, phosphates, pH, contact
surfaces, and organic substances. Such a great sensitivity of reaction kinetics to various
admixtures and mediums makes it difficult to assess the theoretical hydrogen sulfide
oxidation rate in seawater based on studies under natural conditions. The most reliable
factor here would be analytical data on seawater, as well as on laboratory experiments
modelling natural conditions. For this purpose, H2S oxidation has been modelled in
water of the Baltic Sea under laboratory conditions.
MATERIALS AND METHODS
Hydrochemical Investigations
Anaerobic processes in the Baltic Sea bottom have been investigated by seasonal
expeditions carried out in 1981-1991 by the Klaipeda Hydrometeorological Observatory,
Lithuania Environmental Protection Department (Figure 1).
Szczecin
Figure 1. Location of sampling stations in the Baltic Sea.
138
-------
Determinations of H2S by a colorimetric method with p-phenylenediamine-
dihydrochloride (Fonselius.1976) were carried out ahnost 20 years ago (Anonymous
1976-1991). Since 1981 H2S has been analyzed continuously at a depth of 80 m and
deeper under-the National Baltic Sea Monitoring Programme.
Modelling of Hydrogen Sulfide Oxidation Reactions
Modelling of RjS oxidation was carried out in samples of water from the Baltic
Sea under laboratory conditions. Recrystallized Na^ was used to prepare a 0.2 M
solution in freshly boiled distilled water and kept isolated from air. In the absence of
dissolved oxygen, the mean H2S concentration in the Baltic Sea is about 0.1 mM. The
solubility of oxygen at a temperature of 5°C is 55 ml/L or 1.5 mM. Thus, in order to
have sufficient amount of dissolved oxygen for oxidation, 0.1-1 mM concentrations of
H2S solution should be used for laboratory testing.
Stock solutions of sodium sulfide were dissolved in distilled water or seawater and
stored in flasks with corks slightly loose so that oxygen might penetrate. After dissolving
Na^ in distilled water, the pH of the resultant solution was 8.5; it was reduced to the
seawater ptt level of 7.5 by adding a drop of 0.1 M HC1. After dissolving Na^ in the
seawater, the pH level was unchanged, apparently due to the buffering capability of the
seawater. The tests have been carried out at a temperature of 20 °C. Periodically the
samples were taken and titrated by iodine solution in alkaline medium in order to
determine the amount of H2S which had not been oxidized by oxygen. A platinum
electrode together with a saturated calomel electrode as a reference electrode was used
to indicate the end-point. •
RESULTS
Formation and Change of Hydrogen Sulfide Zones
The redox conditions in the bottom water and sediment surface have fluctuated
widety during the last 400 years (Hallberg 1974). During this century the oxygen
deficiency as well as the formation of hydrogen sulfide in water layers below the salinity
halocline at a depth of 70-90 m and in isolated basins was observed over 60 years ago
(Granqvist 1931). The interchange of irregular aerobic and anaerobic periods are
presumably determined by the intensity of the inflow of the North Sea waters together
with an anthropogenic impact (Fonselius 1962, 1969).
The present regime of oxygen in the deep bottoms could be dated to 1980.
According to Nehring and Matthaus (1990), there has been no dissolved oxygen detected
in the Gotland Deep at a depth of 248 m since 1980, and at a depth of 200 m since
1983. According to our data of 1984-1991, dissolved oxygen has never been observed in
the Gotland Deep below 200 m. During the same period in the Gdansk Deep at a
depth of 100 m and below, hydrogen sulfide was observed to be occasionally formed and
139
-------
this hydrogen sulfide zone persisted for 4-5 months. In the Gotland Deep, however, a
hydrogen sulfide zone was constantly being formed, with the upper boundary at a depth
of 130-140 m.
During the period 1981-1991, slight decreases in hydrogen sulfide concentration
have been observed in the bottom layer of the Gotland Deep, and for the most recent 3
years there is an apparent positive trend (Figure 2). An intermediate redox zone, where
both dissolved oxygen and hydrogen sulfide are observed, exists in the deep layers of the
Baltic Sea above the hydrogen sulfide zone (Table 1). Hydrogen sulfide oxidation in this
redox zone is one of the main chemical processes upon which changes of other
hydrochemical variables depend, as well as does the general condition in the bottom
layers of the Baltic Sea.
40 -i
4.0-
1.0 -
0.0
. i Mi i . ]i I I |ii • I M i |-i i 1tiitii i!i..|i . i|i••| in,r»T|T
12 K *3 -
0.0
Figure 2. Long-term trends of annual means of H2S in the bottom water
(below 200 m) of the Gotland Deep (source Anonymous 1976-1991).
In the central part of the Baltic Sea, the redox zone is between 80-150 m below
the surface. In the Landsort Deep (station Tin), the redox zone is between 140 and
250 m below the surface. Dissolved oxygen concentration in this zone was 0.0-1.0 ml/L,
although occasionally as high as 5.5-6.0 ml/L, and.the pH was 6.9-7.8. Relative alkalinity
affects the oxidation process of hydrogen sulfide and the resulting sulfur forms in
solution. The predominate form of H2S at pH 7 is HS~.
140
-------
Table 1. Background ranges of pH, (X and H2S in the redox layer of the Baltic Sea in
1981-1991.
Station
24
25
27
30
31
36
37
38
42
43
50
55
62n
78
69n
71
71n
Depth
(m)
100-105
80-83
100-150
80-118
150
80-108
80-100
150
80-100
150-160
80-100
150-158
80-100
150
83
95-104
80-88
78
100-108
100
150-195
150
200-249
pH
732-736
756-7.88
7.18-735
7.46-7.47
6.88-756
.7.16-751
7.00-7.68
7.19-751
7.17-7.86
731-7.62
730-7.47
7.18-7.67
7.27-750
732-7.75
7.28
7.28-7.44
736-7.72
7.21
7.20-7.49
733
6.96-736
7.00-7.85
7.21-7.43
°2
(ml/L)
i.04-1.71
3.60-3.73
0.25-0.99
1.94-230
0.24-0.68
0.29-232
139-3.63
0.09-0.91
054-3.49
039-0.91
2.62-4.17
023-051
0.81-555
0.49-2.72
0.76
0.14-2.11
0.13-258
1.08
0.23-6.02
0.27
0.12-1.08
0.13-1.11
0.15-0.73
H2S
(ml/L)
0.06-0.18
0.06-0.08
0.11-0.17
0.08-0.15
0.08-0.10
0.06-0.17
0.06-0.10
0.07-0.70
0.08-0.14
0.07-0.16
0.07-0.08
0.16-0.17
0.06-0.19
0.15-0.18
0.15
0.06-0.14
0.09-0.16
0.06
0.07-0.19
0.08
0.06-0.18
0.06-0.17
0.05-0.15
Peculiarities of the oxygen and hydrogen sulfide concentrations in the bottom
layers of the Black Sea (reported by Skopintsev 1975) and of the Baltic Sea are shown in
Figure 3. The concentration of oxygen is higher at the Baltic Sea bottom: it depends,
presumably, not only on water dynamics but also on physical processes such as
temperature. At the temperatures in the Baltic Sea bottom layers (4.0-6.3 °C) H^S
oxidation processes occur more slowly (Figure 4). The rates of redox reactions are
partially dependent upon concentrations, and the assumption is made that there are
minimal biochemical processes occurring.
141
-------
zoo
of 0,3 qj,H2S,ral/L
Figure 3. Changes in oxygen and hydrogen sulfide concentrations (ml/L)
in the bottom layers of (1) the Black Sea (from Skopintsev 1975),
and (2) the Baltic Sea (source Anonymous 1976-1991).
t;c
s,s
s
• - 19S6
D - 19*7
o -138«
X - «9
-------
The second leap (8 eqv. tymol H2S) should correspond to full H2S oxidation, according
to the equation:
HS- + 4I2 + 4H2O - HSO} + SHI.
During the period of H2S oxidation, the negative initial potential decreases, and after all
HS has been transferred to potysulfides, it remains at -100 ±60 mV (Figure 5).
200
100
O
-4OO
-200
-300
-•too
• —olist.water
X— seawatki-
•123456749 10 12*456
O.CHI N Iz, ml
Figure 5. Potentiometric titration curves of 0.85 mM/L H2S by iodine in
alkaline solution: 1 - initial solution; 2 - after 20 hours oxidation;
3 - after 70 hours oxidation at 20 °C.
Kinetic H2S oxidation curves (Figure 6) show that H2S oxidation in the sea water
is going on a bit slower than in distilled water: it is conditioned by total impact of other
ions existing in the sea water. According to our calculations, intermediate oxidation
products account for 10% of the initial amount of hydrogen sulfide. The remainder is in
the form of elemental crystallin sulfur (S8) which is deposited, and of sulfates, the origin
of which is difficult to determine because of their great amount in seawater.
1.1>t—Jisi uaier
2.2 — sea aakr ,
1.2 —a.9S? mmol HjS/C
f.2.'— 0.6Z5maol H2S
—-' • ' ' ' • ' ' « _ * __-
20 40 60 SO 400 120 14O 160 1%0 t vol.
Figure 6. Kinetics curves for oxidation of H2S (pH 7.5) at 20°C.
143
-------
DISCUSSION
Mechanism of Oxidation of H2S
As it has been mentioned, the mechanism of oxidation of H2S by soluble oxygen
is still not fully understood. Reactions presented in the literature, such as Leonov
(1987), are as follows:
2HS~ + O2 ->• 2S + 2OH-
(n-l)S + HS- -* Sj- + H
(1)
(2)
These reactions aye not correct because the product is elemental sulfur which later
reacts with hydrogen sulfide, to form polysulfides. Besides, this reaction is tri-molecular,
and the occurrence of such reactions is rather doubtful.
Through investigation of thiosulfate acidic decomposition, H2S and SO2
interaction in Wackenroder's liquid and other polythionate reactions, it has been
determined that elemental sulfur appears in the form of S&. This molecule is formed
through a number of intermediate products. For example, thiosulfate acid
decomposition, expressed by the simple equation
s2of-
2H
H2S03
(3)
is being carried out through the intermediate product sulfan-monosulfonates HSnSO3H
where n = 1 to 8 (Yanitskij et al 1971). Only after the product HS8SO3H with the
greatest amount of sulfur has been formed does the molecule of S8 appear, and the rest
is sulfite acid. Intermediate products of this reaction, sulfan-monosulfonates, were
isolated as complex nitron (Nt) salts (Yanitskij et al 1971).
Our investigations conclude that the main product of hydrogen sulfide oxidation is
molecular sulfur. It should be considered that molecular sulfur results from a number of
already identified intermediate products, namely polysulfides. Isolation of molecular
sulfur by reaction (2) is doubtful. There are data in the literature indicating that the
initial hydrogen sulfide oxidation stage is a chain reaction, although radicals or ion
radicals participating in the reaction are unknown. The most believable initiator of the
chain reaction is active oxygen, which can exist in several forms.
Thus, the first stage of the reaction would be the reaction with oxygen:
HS- + ox -<» HSO- (4)
This hypothetic unstable compound could pose the further chain reaction:
HSO
O
HSO
Ox
(5)
144
-------
The sulfoxilic acid anion HSO 2 is isolated as a salt and is a very active
compound. In the further interaction of HSO^ with hydrosulfide, polysulfides can be
formed:
HSOJ + HS- -» HS2O~ + OH-
(6)
HS2O
+ HS- -*
+ OH-
(7)
When the stage of HS8S formation is reached, molecular sulfur (S8) is formed.
However, sulfoxsilate, because of its reactivity, can react with other substances
participating in the reaction, resulting in hydrosulfite, thiosulfate, sulfate:
HSO>2 + O* -> HSO:
(8)
HSO
0
HS04
HSO2 + HSO2
+ OH-
(10)
These reactions are much more probable since they are homogenic and
bimolecular: similar mechanisms have been proven for many other reactions. These
reactions also correspond to the contemporary attitude towards chemistry, and are
confirmed by the variety of products found in hydrosulfide oxidation by oxygen;
ACKNOWLEDGEMENTS
The authors thank Professor David Dyrssen, Department of Analytical and
Marine Chemistry, University of Goteborg, Sweden/ and Dr. Stig H. Fonselius,
Oceanography Laboratory, Swedish Meteorological and Hydrological Institute, Goteborg,
for their valuable review of this manuscript.
REFERENCES
Andersin, A.B., J. Lassig, and H. Sandier. 1977. Community structures of softbottom
macrofauna in different parts of the Baltic. In: B.F. Keegan, P. O'Ceidish, P.Y.S.
Doaden (Eds.), Biology of benthic organisms. Pergamon, Oxford, New York, pn
7-20. ™'
Anonymous. 1976-1991. Scientific-Technical Reports of Cruises of R/V Oceanograph,
R/V Lev Titov, and R/V Rudolph Samoilovich, Hydrometeorological Observatory,
Klaipeda. (In Russian)
Bowers, J.W., M. J. A. Fuller, and J. F. Packer. 1966. Autooxidation of aqueous sulfide
solution. Chemistry and Industry 2:65-66.
145
-------
Chen, K.Y., and J. C. Morris. 1971. Oxidation of aqueous sulfide by O2: 1. General
characteristics and catalytic influences. Advances in Water Pollution Research 2.
p. III-32/I-III-32/17.
Fonselius, S.H. 1962. Hydrography of the Baltic Deep Basins. Fishery Board of
Sweden, Series Hydrography, Report No. 13, Lund, p. 9.
Fonselius, S.H. 1969. Hydrography of the Baltic Deep Basins III. Fishery Board of
Sweden, Series Hydrography, Report No. 23, Lund, p. 36.
Fonselius, S.H. 1976. Determination of hydrogen sulfide. In: K. Grasshoff (Ed.)
Methods of seawater analysis. Weinheim, pp. 71-78.
Granqvist, G. 1931. Annual Report: Croisiere thalassologique et observations en
lateaux routiers en 1931. Havsforskningsinstitutets skrift Nr. 81, p. 24. (In
French)
Hallberg, R.O. 1974. Paleoredox conditions in the Eastern Gotland basin during the
recent centuries. Merentutkiniuslait. Julk./Havsforkningsinst. Skr. 238:3-16.
Leonov, A.V., and T. A. Aizatullin. 1987. Kinetics and mechanism of oxidation of
hydrogen sulfide in the seawater. Water Resources 1:89-103. (In Russian)
Nehring, D., and W. Matthaus. 1990. Current trends in hydrographic and chemical
parameters and eutrophication in the Baltic Sea. International Reviews
Gesamten Hydrobiology, Berlin.
Skopintsev, B.A. 1975. Formation of the present state of chemical composition of water
of the Black Sea. Leningrad, Hydrometeoizdat, 336 pp. (In Russian)
Sorokin, J.I. 1970. Experimental research of the rate and mechanism of the oxidation
of hydrogen sulfide in the Black Sea by 35S. Oceanology 10:51-62. (In Russian)
Stanev, E. 1986. On determination of the deep of the redox zone in the Black Sea.
Oceanology 26:439-445. (In Russian)
Voipio, A. 1981. The Baltic Sea. Elsevier Oceanographic Series No. 30. Elsevier,
Amsterdam, 418 pp.
Yanitskij, J.V., V. J. Zelionkaite, and V. J. Janitskis. 1971. Isolation of intermediates
products of acid decomposition of thiosulfate. Journal of Inorganic Chemistry
16/3:617-621. (In Russian)
146
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SOME BIOLOGICAL CONSEQUENCES OF DEEP WATER
STAGNATION IN THE EASTERN GOTLAND BASIN,
BALTIC SEA IN 1980s
S. Olenin1
ABSTRACT
The current stagnation period in the Eastern Gotland Basin of the Baltic Sea has
resulted either in total destruction of the bottom communities and formation of so called
benthic deserts, or in shifting of the dominant life forms of macrozoobenthos in the deep
water areas. Benthic deserts now occupy the slope of the Eastern Gotland Basin below
the primary halocline (80-85 m) in the northern part (near Saaremaa Island), below
100-110 m in the central part (Ventspils and Liepaja areas), and below 120 m in the
southern part. In the southern areas the slowly moving infaunal deposit feeder Scoloplos
armiger (Orbiniidae, Potychaeta) has been displaced by the active hemipelagic predator
Antinoella sarsi (Harmothoe, Polynoidae, Polychaeta).
INTRODUCTION
Strong deficiency of oxygen and almost constant presence of hydrogen sulfide in
the deep-water layers have become one of the main features of the Baltic Sea ecosystem.
The negative consequences of this phenomenon include destruction of bottom
macrofauna and formation of large benthic deserts in all the Baltic deeps. The largest of
benthic deserts in the Baltic Sea exists in the Eastern Gotland Basin, where the present
stagnation period has lasted more than 15 years; this is the most serious stagnation event
ever recorded in the Baltic Sea. The drastic changes in environmental conditions include
mean decrease in salinity, temperature, and oxygen content, as well as an increase in
hydrogen sulfide concentration (Kalejs and Ojaveer 1989, Nehring et al 1989, RELCOM
1990, Matthaus 1991). This paper describes changes in macrozoobenthos as a
consequence of the present stagnation process in the Eastern Gotland Basin.
MATERIALS AND METHODS
Samples were collected in 1985-1989 in the northern, central, and southern parts
of the basin along latitudinal (Sections 1-3, 5) and longitudinal (Section 4) directions
starting from 70-90 m below the surface and at approximately every 10 m down to
110-140 m (Figure 1). At each station one to six bottom samples were taken with a
0.1 m2 van Veen grab. Most samples were sieved through 0.5 mm meshes (a 1.0 mm
mesh sieve was used in 1987 only) and preserved with 4% formalin neutralized with
NaHCO3. A total of 338 samples were collected as follows: Section 1 - 107; Section 2 -
54; Section 3 - 111; Section 4 - 35; Section 5-31.
Centre for System Analyses, Klaipeda University, Klaipeda, Lithuania
147
-------
20
21
57
56
Figure 1. The zoobenthos sampling stations in the northern (Section 1),
central (Section 2,3), and southern (Section 4,5) parts of
the Eastern Gotland Basin in 1985-1989.
RESULTS AND DISCUSSION
Ecological Zones on the Slopes of the Eastern Gotland Basin
Due to strong vertical stratification of the water body in the area of the Eastern
Gotland Basin, a clear ecological zonation of the sea bottom exists. Three different
zones have been distinguished on the slopes of the Eastern Gotland Basin, based on
literature data (Elmgren 1975, 1978, Andersin et al 1978, Zmudzinski 1978, Jarvekulg
1979, Seire 1988) and our studies (Jarvekulg and Olenin 1989, Olenin 1989). Every zone
has clear biological features, which in many respects are determined by properties of the
different water layers (Figure 2). This scheme of vertical hydrological structure is
compiled from papers by Kalejs (1984) and Kalejs and Tamsalu (1984).
148
-------
Depth, a
0
10
20
30
4O
SO
60
70
80
90
1OO
110
120
130
140
ISO
UPPER
MIXED
WELL OXYGEHATED
LAYER
XX
. . XX
. .XX
. . XX
. .XX
. . XX
. . XX
. . XX
. . XX
. . XX
,..xx UPPER
. .XX
.. xx 8 E H T H I C
PRIMARY
. . .HALOCLIHE. . .
. ACTIVE DEEP LAYER xx
XX
.,.xx
. .XX
. . XX
. XX
XX T R A H ;
BEHTHIC
I T I O K A L
Z O S E
..SECONDARY HALOCLINE.
. -. BEAR-BOTTOM-. -.-!-!-
- xx—
. . XX
.-.-. LAYER. -.-.-.-.-.-. -xx
160 -.-.-.-.-.-.-.-.-.-.-.-xx
170
180
BEBTHIC DESERT
<"DEAD BOTTOM")
.—.-xx
-. xx
Figure 2. Ecological zonation of a sea bottom
on the slopes of the Eastern Gotland Basin.
Upper Benthic Zone. This zone is situated in the well-oxygenated and mixed
upper water layer. The lower border of this zone generally coincides with the lower
border of the primary halocline (about 80-85 m). The macrofauna are comparatively
diverse and abundant: the number of species is about eight to 12 per sample, total
abundance fluctuates within the limits 1,000-10,000 individuals/m2, and biomass is
between 10-100 g/m2. Commonly the benthic biomass is dominated by infaunal deposit
and suspension feeding species with low mobility. The main biological feature to
determine the border of the upper zone is the presence of a comparatively well-
developed and mature bottom community, dominated by the bivalve mollusc Macoma
baltica.
Transition Benthic Zone. Location of this zone generally depends on oxygen
content in the active deep water layer which is situated below the primary halocline.
Along this layer, which occupies the depths 100-130 m, influxes of saltwater from
Skagerrack and Kattegat usually enter the eastern Baltic Sea (Kalejs and Tamsalu 1984).
The transitional benthic zone is inhabited with a poor and sparse bottom community; the
number of species is one to five per sample, total abundance usually is less than
10 individuals/m2, and total biomass usually varies within 0.001-1.00 g/m2. This
community never reaches maturity because development is often interrupted by
fluctuations of the water layer containing hydrogen sulfide. Only a few species resistant
to low oxygen conditions are able to survive in this zone; these are hemipelagic
149
-------
polychaete Antinoella sarsi, crustaceans Saduria entomon, Diastylis rathkei and
Pontoporeia femorata. Usually only single specimens of the A, sarsi occur at the lower
border. All species listed are able to settle in this zone at larval and/or adult stages, in
the latter case migrating actively from the upper (cleaner) sections of the sea bottom.
Benthic Desert. This zone is located mainly in the near-bottom layer of the
Gotland Basin which is oxygen-poor and continuous!/ stagnant, and often contains
hydrogen sulfide. The main biological feature of the benthic desert is absence of
macrofauna, and synonyms for this zone are often seen in the literature as "lifeless zone,"
"azoic zone," and "dead bottom."
Changes in Location of Ecological Zones in the Eastern Gotland Basin
Northern Part of Eastern Gotland Basin (Section 1). In the upper part of the slope
(70-80 m), sampling was carried out at the beginning of the investigation period (May
and August, 1985) and at the end (July, 1988; July, 1989). Only single specimens of
M. baltica were found at 80 m in May, 1985 (24 individuals/m2, 10.56 g/m2) and a very
poor community was detected at the same depth in August 1985, dominated by P.
femorata (75 individuals/m2, 0.01 g/m2) (Figure 3). A mature "Macoma baltica
community" (eight-ten species per sample; 1310-5880 individuals/m2, 21.6-118.4 g/m2) was
noted at 75-79 m in 1988-1989. The critical depth for single adult specimens of M.
baltica was 83 m. The border of the upper ecological zone was located approximately in
the depth range of 75-80 m.
Depth,
7O
IX -. VII : VII
1987 : 1986 : 1989
Figure 3. Vertical distribution of bottom macrofauna in the northern part
of the Eastern Gotland Basin. One circle indicates one sample: open
circle = "Macoma baltica" community; circle with a cross = community
of the transitional zone consisting of few species; circle with a
star = Antinoella sarsi only; filled circle = no macrofauna.
150
-------
_ In the upper part of the transition zone (81-90 m) seven species were found
However, six of 16 samples taken at these depths during the study period were "empty"
and in the other samples macrofauna were not abundant (10-140 individuals/m2; 0 05-
1.83 g/m ). At 91-110 m, macrozoobenthos were represented by occasional specimens of
A. sarsi until May 1986; only one crustacean, D. rathkei, was found (at 100 m) In
September 1987 an expansion of the benthic desert was detected; only one specimen of
A. sarsi was found in 25 samples taken at 100-140 m. In 1988-1989 no macrofauna were
found in any sample below 85-89 m. Thus, in 1985-1986 the transitional zone covered
the slope of the northern part of the basin approximately from the depth of 80 m down
to at least 100 m. After 1987, this zone was completely displaced by the benthic desert
Central Part of the Eastern Gotland Basin in the Ventspils Area (Section 2) In
1986, sampling was carried out between depths of 90-120 m, which was characterized as
the transition zone. The bottom communities included two or three species per sample
(A. sarsi, S, entomon, P. femorata, juveniles of M baltica) at 90-110 m, and only one
species (A. sarsi) at 120 m (Figure 4). The biomass reached 4.9-6.1 g/m2 due to
presence of big specimens of S. entomon; in the absence of that species the biomass
Depth,
70
Section 2
80
90
100
110
120
130
-ceo-
MOM
•ooe
Month VIII VII
Year 1986 1987
VII
1988
60
70
Section 3
1987 : 1988 : 1989
Figure 4. Vertical distribution of bottom macrofauna in the central part
of the Eastern Gotland Basin in the vicinity of Ventspils (above)
and in the vicinity of Liepaja (below). (Conventions see Figure 3)
151
-------
did not exceed 0.01-0.06 g/m2. In 1988-1989 sampling carried out in the uppermost part
of a slope showed that the mature "Macoma baltica community" occupied the bottom
down to 80-81 m (four to nine species per sample; 50-1990 individuals/m2, 12.0-
57.8 g/m2). The critical depth for a single adult specimen of M baltica was 90 m.
Environmental conditions below 90 m became critical between 1986 and 1987: in
1986 macrofauna were found in all samples, however, in 1987-1988 27 of 34 samples
taken at 90-120 m were "empty." The maximum depth at which macrofauna were noted
was 109-110 m. Although expansion of the benthic desert took place in the central part
of the Eastern Gotland Basin in 1987-1988, this expansion was not as abrupt as that in
the northern area.
Central Part of the Eastern Gotland Basin in the Liepaja Area (Section 3), In May
and Juty 1985, a bottom community consisting of six to nine species with low biomass
(0.63-1.33 g/m2) was found in the upper part of the slope, at 80 m. There were few
species characteristic of the upper benthic zone; these included the priapulid Halicryptus
spinulosus, and the potychaetes Pygospio elegans, and Terebellides stroemi, but did not
include adult M baltica (Figure 4). In 1988-1989, the comparatively mature "Macoma
baltica community," including six to nine species per sample, was found at 68-70 m
(up to 2260 individuals/m2, and up to 20.97 g/m2). Furthermore, adult specimens of
M baltica were detected at 85 and 92 mf Concurrent with this, "empty" samples or
samples without M baltica were obtained at 70-92 m. Thus, the picture of ecological
zonation in the upper part of the slope in the Liepaja area was not as clear as in
Sections 1 or 2, apparently due to gentle sloping of the sea bottom in the upper part.
Similar phenomena can be observed in littoral zones; the zonation is clearer on a steep
rock shore than on a gentle beach.
The transition zone extended down at least 100-110 m in 1985 and 110-120 m in
1986. Unfortunately, due to lack of data at the lowermost depths, the border between
this zone and the benthic desert could not be delineated more precisely. In 1987-1989,
there were no benthic macroinvertebrates in 19 of 24 samples taken at 100-110 m; in one
of the remaining five samples only A. sarsi and Mysis oculata were present, and in the
other four samples orityA. sarsi. At that time no macrofauna were found at 120 m and
deeper. So, the border of benthic desert rose from approximately 120 m in 1986 to 100-
110 m in 1987. On the whole, changes in 1986-1988 in the boundary of the benthic
desert in the central part of the basin in the vicinity of Liepaja were similar to that in
the Ventspils area.
Southern Part of the Eastern Gotland Basin (Section 4). In 1988 the macrofauna at
94-122 m was represented by A. sarsi with the exceptions of a single adult specimen of
M baltica at 97 m and of P. elegans at 108 m (Figure 5). There were no benthic
macroinvertebrates at 130-140 m. In 1989 only A. sarsi and Ostracoda were detected
between 99 and 118 m; four of seven samples taken at 110-120 m, and all the samples
obtained at 125-138 m, were "empty." The border of the benthic desert was located
approximately at 120-125 m.
152
-------
Section 4
130
140
Month VII : y—
Tear 1988 : 1989
•Depth, a
7O
Section
60
90
100
10
120
130
-oo-
Xonth
Year
V VIII
1985
V
1986
V
1987
V
1988
VIII
1989
Figure 5. Vertical distribution of bottom macrofauna in the southern part
of the Eastern Gotland Basin at meridianal (above) and longitudinal
(below) sections. (Conventions see Figure 3)
Southern Part of the Eastern Gotland Basin (Section 5). In 1985 the fairly diverse
"Macoma baltica community" was found at 80 m (10-11 species per sample;
1100-1400 individuals/m2, 12.6-49.5 g/m2) (Figure 5). The macrofauna at the lower
depths in 1985-1986 were also comparatively diverse; the bottom communities included
immobile infaunal and epifaunal invertebrates (H. spinulosus, P. elegans, Ampharete sp.,
Scoloplos armiger) and "active swimmers" (A. sarsi, Mysis sp., P. femorata).
Deterioration was pronounced at the farthest west station of Section 5 at
approximately 120 m. In 1985-1987, the bottom community consisted of two to five
species with total biomass 2.48-6.63 g/m2, dominated by S. armiger. In 1988 only A sarsi
was noted, with biomass 0.15 g/m2, and in 1989 no macrofauna were found.
153
-------
During the 1960s and 1970s the "Scoloplos armiger community" occupied the
deepest sections, from 90 to about 140 m, of the slopes of the Northern and the Eastern
Gotland Basins (Andersin et al 1978, Jarvekulg 1979). During the 1980s complete
deterioration occurred in the Northern and Central parts of the Baltic Sea, and at the
present time the deep areas, represented earlier by the polychaete S. armiger, are void of
infaunal deposit feeding animals. Because of this, there is no bioturbation of the
sediments below the primary halocline, and organic matter, sinking from the upper layers
of the water column, is no longer utilized as food for resident macrofauna. Several
remarkable changes in the functioning of the Baltic Sea ecosystem have occurred as a
consequence.
CONCLUSIONS AND RECOMMENDATIONS
The benthic desert now occupies ah" the slopes below the primary halocline
(80-85 m) in the northern part of the Eastern Gotland Basin, has reached depths of
100-110 m in the central part, and has reached 120 m in the southern part. Data on the
collection of macrozoobenthos in the Eastern Gotland Basin clearly demonstrate that the
benthic deserts has expanded in recent years.
Because of practical and scientific importance, regular information on changes in
the bottom communities of the deep areas of the Baltic Sea are urgently needed.
Benthic studies in the open Baltic Sea are the only investigations that clearly show
biological long-term changes of the kind that may be expected due to eutrophication
(Larsson et al 1985). At the present time, almost all zoobenthos investigations in deep
water areas are being conducted by different researchers operating independently within
a framework of national programs; there are no regular sampling programs in the deep
waters such as there are in inland waters. In addition, the grid of the RELCOM Baltic
Monitoring Programme stations is too sparse for evaluation of ecological changes in the
deep water areas of the Baltic Sea. Accordingly, in order to document the dynamics of
the ecological zones in the Eastern Gotland Basin, annual observations should be
continued.
ACKNOWLEDGMENTS
I am deeply grateful to Dr. R. Rosenberg for critical reading of this paper, to my
colleagues from the former Hydrometeorological Observatory of Klaipeda (now the
Lithuanian Marine Research Laboratory) for assistance during sampling time, and to
Ms. O. Jaganova for her help in sorting of macrofauna samples.
154
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REFERENCES
Andersin, A.B., J. Lassig, L. Parkkonen, and H. Sandier. 1978. The decline of
macrofauna in the deeper parts of the Baltic Proper and Gulf of Finland. Kieler
Meeresforschungen 4:23-52.
Ehngren, R. 1975. Benthic meiofauna as indicator of oxygen conditions in the Northern
Baltic Proper. Merentutkimuslaitos Havsforskningsinstitutet. 239:263-271.
Ehngren, R. 1978. Structure and dynamics of Baltic benthos communities, with
particular reference to the relationship between macro- and meiofauna. Kieler
Meeresforschungen 4:1-22.
RELCOM (Helsinki Commission). 1990. Second periodic assessment of the state of the
marine environment of the Baltic Sea, 1984-1988; background document. Baltic
Sea Environment Proceedings, Helsinki, No. 35B.
Jarvekulg,x A. 1979. Bottom fauna of the eastern part of the Baltic Sea. Tallin. Valeus
382 pp. (In Russian)
Jarvekulg. A., and S. Olenin. 1989. Zoobenthos. Main Trends in Evolution of the
Ecosystem. Project "Baltika", Leningrad. Hydrometeoizdat 4:102-105.
(In Russian)
Kalejs, M. 1984. The oxygen regime of the Baltic Sea. Essays on Biological
Productivity in the Baltic Sea. Moscow 1:68-82. (In Russian)
Kalejs, M., and R. Tamsalu. 1984. Water exchange through the Danish Straits. The
salinity regime of the Baltic Sea. Essays on Biological Productivity in the Baltic
Sea. Moscow 1:27-50. (In Russian)
Kalejs, M., and E. Ojaveer. 1989. Long-term fluctuations in environmental conditions
and fish stocks in the Baltic. Rapport Proces-Verbal de la Reunion Conseil
International pour 1'Exploration de la Mer. 190:153-158.
Larsson, U., R. Ehngren, and F. Wulff. 1985. Eutrophication and the Baltic Sea:
causes and consequences. Ambio 14:9-14.
Matthaus, W. 1991. Long-term trends and variations in hydrographic parameters during
the present stagnation period in the central Baltic deep water. International
Council for Exploration of the Sea 1991/Variability Symposium, No. 38, Session 4.
155
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Nehring, D., S. Schulz, and D. Rechlin. 1989. Eutrophication and fishery resources in
the Baltic. Rapport Proces-Verbal de la Reunion Conseil International pour
1'Exploration de la Mer. 190:198-205.
Olenin, S. 1989. Benthic desert and transitional zone in the eastern part of the Baltic
Sea. Oceanology 29:1006-1009. (In Russian with English summary)
Rosenberg, R., R. Elmgren, J.P. Flescher, G. Persson, and H. Dahlin. 1990. Marine
eutrophication case studies in Sweden. Ambio 19:102-108.
Seire, A. 1988. Benthic fauna in the deep areas of the Gulf of Finland and Eastern
Gotland Basin in 1984 and 1985. Proceedings of the Academy of Sciences of the
Estonian SSR. Biology, Tallin. 37:67-73.
Zmudzinski, L. 1978. The evolution of macrobenthic deserts in the Baltic Sea.
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156
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INDEX BY AUTHOR
Antanyniene, Aldona ............... .......... . . ..... . ...... . . ...... 73
Auk§tuolyte, Asta ......... . ........ ................. ............. 131
Bakiene, Elena ..... . . . . . ........ ................................. 65
Baranauskiene, Aldona ................. . . ; .................. ...... 73
Barsiene, Janina ....... ; ..... . ......................... .... ..... ..85
Barsyte, Dab'a ........... .................................... .... 85
Berzinskiene, Janina . . ....... .... ....... .............. ....... ..... 65
Budriene, Stase .............. ....................... ..... ........ 73
Bukelskis, Egidijus .................... . . . ................ . . . ..... 103
Ceburnis, Darius ............................ ........ ...... ..... 19, 31
Cetkauskaite, Anolda ........ . .................... ..... . . . ....... . . 65
Danys, Jurgis .................................... ". . • ....... ^ ..... 131
Dusauskiene-Duz, Rima . . ........................................ . 123
Ellington, J. Jackson ......... ........................... ... 1, 19, 31 39
Filistovicz, Vitoldas ............... ............ , ........... . ...... 131
Galkus, Arunas . ................... . . . . ...... ..................... 47
Jankauskas, Valdas ......................... . . ....... ............. 65
Jankaviciute, Genovaite ..... ............... ...... ......... . . . . . ..... 73
Jankevicius, Karolis ................ . ....................... ........ 73
Janulionyte, Edita ...... ............. ........ . .............. ...... 131
Joksas, K^stutis ................. ................ ..... ...... . ...... 47
Jurkunaite, Rasa ......... . ...... . ........... ........... . . . ...... 131
Kaspariunaite, Giedre ......................................... ..... 95
Kasperoviciene, Jurate . . . . ............... ................ . ..... .... 73
Krasauskiene, Aurelija ........................... ........... . ..... 131
Kucinskiene, Ale ............................................. .... 73
Kvietkus, K^stutis ................... ......................... \t igt 31
157
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Lazauskiene, Lilija .." 95
Masanauskaite, Daiva ,. . 131
Mazeikaite, Stase 73
Mastauskas, Albinas 131
Mickeniene, Liongina 59
Nedveckaite, Tatjana 131
Neuman, John F. ... . . 39
Olenin, Sergej 147
Razumovskij, Vladimir 137
Rinkevichiene, Elvyra 137
Serelyte, Dalia . . . . v 103
Shane-Yur Y. , .. . . 31
Sidlauskas, Vygandas ..'. 131
Slapkauskaite, Giedre 73
Steen, William C . .. 65
Striupkuviene, Nijole 39
Sukyte, Vide-Judita ... 1, 19, 39,137
Sulijiene,. Rita 73
Svecevidius, Gintaras _ 107, 115
Svi5iulyt6, Jiirate 131
Syviene, Stase 107
Syvokiene, Janina 59
Telksnys, Arturas ...... . . 131
Tmirston, Robert V, 1, 19
Trainauskaite, Izabela ............; 73
Virbickas, Tomas 85
Vosyliene, Zita 107
Zareckas, Saulius 47
Zelionkaite, Vaclova 137
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