Analele Științifice ale Universității „Alexandru Ioan Cuza” din Iași, s. Biologie animală, Tom LX, 2014
A SHORT OVERVIEW FOCUSED ON INVERTEBRATE AND
VERTEBRATE BIOINDICATORS FOR FRESHWATER
ENVIRONMENTS CONTAMINATED WITH HEAVY METALS
Ștefan-Adrian STRUNGARU*, Gabriel PLĂVAN, Marius Andrei RĂU and
Mircea NICOARĂ
Faculty of Biology, Alexandru Ioan Cuza University, Bd. Carol I 11, 700506 Iaşi, Romania.
* [email protected]
Abstract. The invertebrate and vertebrate bioindicators are known as biomonitors which are used to indicate the
negative effects caused in time by different pollutants from water, air and soil. These species are living in natural
environments and are not the same model species/organisms which are used in toxicological studies conducted in
laboratory conditions. Can the bioindicator species adapt to the negative effects of the pollutants? Each aquatic
environment is unique and each species on this planet has the capacity to adapt itself to the environmental
conditions. The aim of present study was to investigate the main bioindicators of freshwater environments
contaminated with heavy metals, in order to be used in future studies.
Keywords: analysis methods, bioindicators, heavy metals, sampling.
Rezumat. Un scurt comentariu axat pe nevertebratele si vertebratele bioindicatori ai mediilor acvatice
dulcicole contaminate cu metale grele. Vertebratele şi nevertebratele bioindicatoare mai sunt cunoscute ca
biomonitori care indică efectele negative cauzate în timp de către diferiţi poluanţi din apă, aer şi sol. Aceste specii
care sunt întâlnite în natură nu sunt aceleaşi cu organismele model utilizate în studiile toxicologice în condiții de
laborator. Sunt capabile organismele bioindicatoare să se adapteze la efectele negative ale poluanţilor? Fiecare
mediu acvatic este unic şi fiecare specie de pe această planetă are capacitatea să se adapteze la condiţiile de mediu.
Scopul acestui studiu a fost investigarea principalilor bioindicatori ai mediilor acvatice dulcicole contaminate cu
metale grele, care ar putea fi utilizați în studiile viitoare.
Cuvinte cheie: metode de analiză, bioindicatori, metale grele, prelevare probe.
Introduction
The invertebrate and vertebrate bioindicators are known as biomonitors which are
used to indicate the negative effects caused in time by different pollutants from water, air
and soil. These species are living in natural environments and are not the same model
species/organisms which are used in toxicological studies conducted in laboratory
conditions. A certain species can be classified as a bioindicator of the environmental health
after the analysis of several specific criteria: is it common in many ecosystems? Can it
tolerate a large group of toxicants, those which cannot be tolerated creating disturbances of
the populations by decreasing the number of the individuals and triggering migrations in
order to avoid the pollution sources? One possibility to measure the pollutant effect upon
the bioindicator is the decrease of population density or the absence of the species from an
area where it was previously present in high abundance.
Many studies proposed databases with bioindicator species and many of them
were focused on water quality analysis. For instance, Neumann et al., 2003 presented in
their study the data base of LIMPACT with 39 bioindicators that were observed in the
studied stream (Taxa: Turbellaria, Oligochaeta, Gastropoda, Amphipoda, Isopoda,
Plecoptera, Coleoptera, Diptera, Ephemeroptera, Megaloptera and Trichoptera). The results
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were based on the interpretation of the abundances and rule syntax. These showed the level
of contamination with pesticides correlated with nine water-quality and morphological
parameters. Other authors proposed benthic larvae of insects for pesticides and heavy
metals after complex chemical analyses. The freshwater mollusks are considered to be the
best bioindicators for the contamination with pesticides and heavy metals in freshwater
environments, but the interactions with environmental conditions release questions about
the bioindicators. Can the bioindicator species adapt to the negative effects of the
pollutants? Each aquatic environment is unique and each species on this planet has the
capacity to adapt to environmental conditions. The aim of present study was to investigate
the main bioindicators of freshwater environments contaminated with heavy metals, in
order to be used in future studies.
Materials and methods required for heavy metals analysis in freshwater
ecosystems
The first step in this investigation is the choice of the analyzed environment that is
important for humans and is rich in biodiversity. All the freshwater environments are
important to sustainability of the life forms on this planet. There is a hierarchy of the
importance of freshwater ecosystems based on: biodiversity richness, endangered species,
economical activities, freshwater usage for drinking. The second step is based on the
identification of the possible heavy metals pollution sources based on anthropogenic
activities e.g.: heavy industry, mining, agriculture, car traffic, waste waters and wastes
disposal. Next step is the choice of the bioindicators. The scientific literature provides
valuable information about bioindicators; if not, we must find the species which respond to
the specific questions. The indicators for heavy metals in fresh water ecosystems are: water,
sediments and biota.
In case of biota there are several ways of finding the indicators of heavy metals
pollution: analysis of the food web (it is very complex, there are necessary many resources;
the analysis should begin with producers-planktonic algae and end with highest
consumer/predator-fish, birds, mammals and humans), organisms with high capacity of
metal absorption from environment (benthic organisms like gastropods, bivalves,
macrophytes) or species with a short life cycle (their population can be damaged very fast
by the activity of the pollutant).
Very important are the sampling period (season of the year) and the number of
samples (for sediments, water and biota) to prove the heavy metals pollution in the studied
environment. The main problem that may appear is the accidental contamination of the
samples. That is why the person that runs the sampling must use uncontaminated bottles
and bags metal free made from PET. The measurement of water parameters (pH,
conductivity, ORP, TDS, water temperature, salinity, dissolved oxygen, oxygen saturation
and others) is useful because the correlation between them and metal content (water,
sediment, biota) may offer a complex image about metal absorption and toxicity.
Once the samples are collected they must be preserved for metals analysis in
laboratory. The biota and sediment samples can be preserved by freezing at -20⁰C in PE
bags. Water samples are preserved by acidification with nitric acid (Marcovecchio et al.,
2007; Biziuk et al., 2010). The equipment and technique used may be different. One of the
most frequent technique used for these types of analyzes is AAS (Atomic Absorption
Spectrometry). The AAS heavy metals analysis is a modern technique for measuring the
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Analele Științifice ale Universității „Alexandru Ioan Cuza” din Iași, s. Biologie animală, Tom LX, 2014
metals concentration in life forms and environment, that was used since 1955 (L’vov,
2005). It is a technique that is still developing nowadays. It is split in two main methods of
analyses: GF-AAS (on graphite furnace with inert gas as transporter) for low concentrations
of metal in samples (µg g-1) and Fl-AAS (flame-AAS with air-acetylene mixture) for high
concentrations of metal in samples (mg g-1). Before the analysis, the samples must be
weighted and digested with acid mixtures; this is different for each sample type. Microwave
digestion system with pressure teflon vessels is often used. The biometric measurements
and biological age of the organisms are important variables for correlations. The AAS must
be calibrated with certificated standard solutions that are not expired and are kept in
conditions recommended by the producer. The blank sample and water for the dilution must
be ultrapure with no content of the elements that will be analyzed, in order to avoid possible
errors. All the bottles and pipettes must be uncontaminated; this can be proved by
absorbance measurements. The metal element with its specific wave length does not have
any absorbance if not present in the sample. The reference materials for sediments and
biota are required to test the accuracy of the device, and QC samples are also required. It is
recommended to firstly test the presence of the metal in samples, to do the calibration
curves and to dilute the sample if the concentration exceeded (Fig. 1).
Figure 1. The absorbance and spectrum number that prove the existence of
cadmium in sample.
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The calibration (Fig. 2) of the device is most important for the measurements and
data validation. If the metal concentrations in samples exceed the modern devices capacity,
the samples must be diluted. All the results and data must be statistical validated and
interpreted.
Figure 2. Examples of calibration for nickel and chromium, using GF-HR-CS-AAS ContrAA 600
AnalitikJena.
Groups of invertebrates and vertebrates with potential in the monitoring of
heavy metal pollution
Macroinvertebrates are the most important bioindicators in heavy metal pollution
because they can provide valuable information about pollution sources. The groups with
potential in heavy metals monitoring are: Plecoptera, Ephemeroptera, Trichoptera,
Hemiptera, Coleoptera, Odonata, Diptera, Crustacea, Gastropoda, Bivalvia, Tricladi,
Hirudinea, Oligochaeta (Arimoro et al., 2009; Testi et al., 2012; Moldovan et al., 2013).
The disturbance in their populations can be caused by the metal toxicity. It is very
important that the metal concentration from their bodies to be correlated with population
density. There are disadvantages because many of them are relatively small and more effort
is required to obtain the biomass for metal analysis. Another disadvantage is the locomotion
capacity and the capacity to tolerate in time the toxic effects of the pollutant (Arimoro et
al., 2009; Testi et al., 2012; Moldovan et al., 2013). Bivalves and other species with low
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Analele Științifice ale Universității „Alexandru Ioan Cuza” din Iași, s. Biologie animală, Tom LX, 2014
capacity of locomotion are the best candidates; if they are big enough we can do analyzes
on different organs. The metals accumulate in organisms by time for the chronic pollution.
Another variable is the biological life cycle. In the acute pollution, the species with a faster
biological cycle are the best because pollutants can enter very fast in the cells and create
damages.
In the case of vertebrates, the juveniles are the best indicators in the acute
poisoning with heavy metals. For instance, the tadpoles that absorb very fast the lead into
their bodies (Strungaru et al., 2012) and the mortality of the young fish which is the first
indicator of the high contamination and disturbance of the environment (Hoffman et al.,
2002). The abnormal developing and the behavior of the young fish are symptoms for
heavy metals contamination. The accumulation of the metals must be studied for different
organs in order to set the target organ of the toxicant from environment (Fig. 3) because
there is the possibility for the toxic metal to be localized inside an organ only.
The water birds are indicators in chronic exposure for heavy metals in freshwater
environments. The high juvenile’s mortality and food source contamination are huge
problems. The main in vivo analyses for water birds contamination with heavy metals are
for blood, feather and excrement. This method does not provide significant results all the
time and there is the possibility that the birds were accidentally contaminated during
migration. The analysis of the internal organs (liver, kidneys, spleen and bones) are the
most important to prove the contamination (Binkowski et al., 2013).
Figure 3. The metal pathway from environment in a predator and to the target organs.
In case of mammals, the blood samples and urine are used to observe the level of
contamination with heavy metals. The otter (Lutra lutra B.) can be a good indicator
because is a predator species. The behavior is a preliminary indicator in case of metal
contamination. In humans, especially in the mining areas, there were recorded many cases
of exposure to cadmium and lead that caused new types of diseases, damages to the body
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Ștefan-Adrian Strungaru et al.
and abnormal child development (Andujar et al., 2010; Menai et al., 2012; Faramawi et al.,
2012; Sun et al., 2014; Kim et al., 2013; Inaba et al., 2005).
All the results and conclusions should be integrated with education, health,
strategies and future investments to reduce the anthropogenic pressure caused by pollution
(Fig. 4) on freshwater environments.
Figure 4. Results from freshwater monitoring studies of the heavy metals pollution integrated to
reduce the anthropogenic pressure.
Conclusions
This short overview presented a few groups of invertebrates and vertebrates
capable to react in the process of heavy metals pollution of the freshwater ecosystems. In
case of invertebrates, population densities must be correlated with the metal from
environment and from their bodies. For vertebrates must be identified the target organs
where each studied metal proves its negative effects. It will be a problem in the future with
freshwater resource if the people will not react with strategies for reducing the
anthropogenic pressure. The education, health, investments and resources management
integrated with the data provided by indicators is the key in the protection of the freshwater
ecosystems.
Acknowledgments
This work was supported by the strategic grant POSDRU/159/1.5/S/133391,
Project “Doctoral and Post-doctoral programs of excellence for highly qualified human
resources training for research in the field of Life sciences, Environment and Earth
Science” cofinanced by the European Social Fund within the Sectorial Operational Program
Human Resources Development 2007-2013.
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Analele Științifice ale Universității „Alexandru Ioan Cuza” din Iași, s. Biologie animală, Tom LX, 2014
References
Andujar, P., Bensefa-Colas, L, Descatha, A., 2010. Acute and chronic cadmium poisoning. Revue de Medecine
Interne, 31: 107-115.
Arimoro, F.O., Ikomi, R.B., 2009. Ecological integrity of upper Warri River, Niger Delta using aquatic insects as
bioindicators. Ecological Indicators, 9: 455-461.
Binkowski, Ł.J., Sawick-Kapusta, K., Szarek, J., Strzyżewska, Felsmann, M., 2013. Histopathology of liver and
kineys of wild living Mallards Anas platyrhynchos and Coots Fulica atra with considerable
concentrations of lead and cadmium. Science of Total Environment, 450-451: 326-333.
Biziuk, M., Beyer, A., Zukowska, J., 2010. Preservation and storage of water samples. Analytical Measurements in
Aquatic Environments, 19-39.
Faramawi, M.F., Liu, Y. , Caffrey, J.L., Lin, Y.-S., Gandhi, S., Singh, K.P., 2012. The association between urinary
cadmium and frontal T wave axis deviation in the US adults. International Journal of Hygiene and
Environmental Health, 215: 406-410.
Hoffman, J.D., Rattner, A.B., Burton, G.A., Jr., Cairns, J., 2002. Handbook of ecotoxicology, Second edition. CRC
Press.
Inaba, T., Kobayashi, E., Suwazono, Y., Uetani, M., Oishi, M.N., Nogawa, K., 2005. Estimation of cumulative
cadmium intake causing Itai-itai disease. Toxicology Letters, 159: 192-201.
Kim, S., Arora, M., Fernandez, C., Landero, J., Caruso, J., 2013. Lead, mercury, and cadmium exposure and
attention deficit hyperactivity disorder in children. Environmental Research, 126: 105-110.
L’vov, B.V., 2005. Fifty Years of Atomic Absorption Spectrometry. Journal of Analytical Chemistry, 60(4): 382392.
Marcovecchio, J.E., Botté, S.E., Freije, R.H., 2007. Heavy metals, major metals, trace elements. In Handbook of
water analysis, second edition, 275-311.
Menai, M., Heude, B., Slama, R., Forhan, A., Sahuquillo, J., Charles, M.-A., Yazbeck, C., 2012. Association
between maternal blood cadmium during pregnancy and birth weight and the risk of fetal growth
restriction: The EDEN mother-child cohort study. Reproductive Toxicology, 34: 622-627.
Moldovan, O.T., Meleg, I.N., Levei, E., Terente M., 2013. A simple method for assessing biotic indicators and
predicting biodiversity in the hyporheic zone of a river polluted with metals. Ecological Indicators, 24:
412-420.
Neumann, M., Baumeister, J., Liess, M., Schulz, R., 2003. An expert system to estimate the pesticide
contamination of small streams using benthic macroinvertebrates as bioindicators. II. The knowledge
base of LIMPACT. Ecological Indicators, 2: 391-401.
Strungaru, Ş.-A., Jitar, O., Plăvan, G., Nicoară, M., 2012. Lead accumulation in the bodies of Rana tadpoles
(Anura: Ranidae). Analele Științifice ale Universității „Alexandru Ioan Cuza” din Iași, s. Biologie
animală, LVIII: 93-98.
Sun, H., Chen, W., Wang, D., Jin, Y., Chen, X., Xu, Y., 2014. The effects of prenatal exposure to low-level
cadmium, lead and selenium on birth outcomes. Chemosphere,108: 33-39.
Testi A., Fanelli G., Crosti R., Castigliani V., D’Angeli D., 2012. Characterizing river habitat quality using plant
and animal bioindicators: A case study of Tirino River (Abruzzo Region, Central Italy). Ecological
Indicators, 20: 24-33.
- 127 -