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Maria Droba, Magorzata Dugan,
Maciej Balawejder, Radosaw Jzefczyk, Anna Pasternakiewicz
Current problems of environmental monitoring
UNIVERSITY OF RZESZOW Faculty of Biology and Agriculture
Department of Chemistry and Food Toxicology Rzeszow 2013
2
3
Ta publikacja zostaa wydana przy pomocy finansowej Unii Europejskiej, w ramach Programu
Wsppracy Transgranicznej Polska Biaoru Ukraina 2007-2013. Odpowiedzialno za zawarto
tej publikacji ley wycznie po stronie Uniwersytetu Rzeszowskiego i nie moe by w adnym
wypadku traktowane jako odzwierciedlenie stanowiska Unii Europejskiej.
"This publication was co-financed by the European Union within the framework of the Cross-border
Cooperation Programme Poland-Belarus-Ukraine 2007-2013. Responsibility for the content of this
publication lies solely with the University of Rzeszow and can not under any circumstances be
considered to reflect the position of the European Union in any way.
Publikacja powstaa w ramach projektu Integracja naukowa pogranicza polsko-ukraiskiego w
zakresie monitoringu i detoksykacji substancji szkodliwych w rodowisku i jest dystrybuowana
bezpatnie.
Free copy edited and published within the project Scientific integration of the Polish Ukrainian
borderland area in the field of monitoring and detoxification of harmful substances in
environment
Wydawca publikacji: Uniwersytet Rzeszowki
Al. Rejtana 16 C 35-959 Rzeszw
Tel. (17)
Publisher:
University of Rzeszow
Rejtana Street 16C
35-959 Rzeszow
Nakad 15 egz./Circulation 15
Copyright by Biuro Projektu Integracja naukowa obszaru pogranicza polsko-ukraiskiego w zakresie
monitoringu i detoksykacji substancji szkodliwych w rodowisku
Copyright by Project Office Scientific integration of the Polish Ukrainian borderland area in the field
of monitoring and detoxification of harmful substances in environment
All rights reserved
4
INTRODUCTION
Environmental monitoring can be defined as the systematic sampling of air, water, soil,
and biota in order to observe and study the environment, as well as to derive knowledge from
this process. Monitoring can be conducted for a number of purposes, including to establish
environmental baselines, trends, and cumulative effects, to inform policy design and decision-
making, and to assess the effects of anthropogenic influences, or to conduct an inventory of
natural resources.
Environmental monitoring programs can vary significantly in the scale of their spatial and
temporal boundaries. In Poland, the State Environmental Monitoring (PM) constitutes the
source of environmental information being the outcome of the measurements and assessments
of the state of the environment, as well as the analysis focusing on the impact of various
anthropogenic factors. It is addressed to society, to government and local government
administrations as well as to international institutions.
Environmental monitoring techniques evaluate contaminants and the rate at which they
will dissipate. Monitoring measures physical or chemical properties of the media suspected of
contamination (soil, water, gases, tissues). Over time, environmental monitoring establishes
a trend from multiple measurements of the same parameter. The monitoring systems include:
air quality, water quality, soil and land quality, nature, noise, electromagnetic field and
ionizing radiation monitoring subsystems.
The data obtained by common chemical and physicochemical methods becomes a source
of information about the state of specific environmental compartments and the processes
within. However, chemical analysis techniques usually can be used under laboratory
conditions only and that introduces additional delay between sampling and sample analysis.
Moreover, they do not evaluate the effect of interaction among toxic substances, such as
synergism or antagonism. For these reasons, in recent years, there has been intense
development of bioanalytical techniques that employ live organisms as indicators
(bioindicators). Ecotoxicological tests (biotests) are commonly used as one of the tools in
integrated monitoring of the environment. In this case, the environmental data and the
information on the impact of pollutants on live organisms are obtained from environmental
samples which have been analyzed with both chemical techniques and biotests.
5
1. CHEMICAL ANALYSIS OF THE ENVIRONMENTAL STATE
1.1. Components of environment
The two major classifications of environment are:
Physical environment - external physical factors like air, water, and land; this is also
called the Abiotic Environment,
Living environment - all living organisms around us (plants, animals, and
microorganisms); this is also called the Biotic Environment.
Earths environment can be further subdivided into the following four segments:
Lithosphere
Hydrosphere
Atmosphere
Biosphere.
The names of the four spheres are derived from the Greek words for stone (litho), air
(atmo), water (hydro), and life (bio). The last sphere is the region occupied by living
organisms such as plants, animals, fungi. They are temporary accumulators (e.g. lead) and
sources of pollutants (natural forest burning) in a very complex set of relationships with the
atmosphere, hydrosphere and lithosphere (Fig.1).
Fig. 1. Relationships between various components of environment
Environmental monitoring can be conducted on biotic and abiotic components of any of
these spheres, and can be helpful in detecting baseline patterns and patterns of change in the
inter- and intra-process relationships between and within these spheres. The sampling of air,
water, and soil through environmental monitoring can produce data that can be used to
understand the state and composition of the environment and its processes.
The atmosphere is a mixture of nitrogen (78%), oxygen (21%), and traces (remaining 1%)
of carbon dioxide, argon, water vapor and other components. Although the atmosphere is
approximately 1,100 km high, the stratosphere (10 to 50 km) and the troposphere (less than
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10 km) are the main atmospheric interactors of the biosphere. The atmosphere is a prime
mean for the spatial diffusion of pollutants and a temporary mean of their accumulation until
they precipitate. A number of sources produce these chemical compounds but the major man-
made source is the burning of fossil fuel. Indoor air pollution is caused by cigarette smoking,
the use of certain construction materials, cleaning products, and home furnishings. Outdoor
gaseous pollutants come from volcanoes, fires, and industry, and in some areas may be
substantial.
Particulate matter (PM) is present in the atmosphere as both a primary and a secondary air
pollutant. Primary PM is released into the atmosphere directly from a source, such as ash in
the flue gas emitted from a coal-fired furnace. Particle pollution is made up of a number of
components, including acids (such as nitrates and sulfates), organic chemicals, metals, and
soil or dust particles. Secondary PM is produced in the atmosphere in the form of ammonium
nitrate and ammonium sulfate. Most of the secondary PM is the respirable fraction known as
PM 2.5 which is very small particulate matter having a size of 2.5 m or less. The size of
particles is directly linked to their potential for causing health problems. Particles that are
10 micrometers in diameter (PM 10) or smaller generally pass through the throat and nose and
enter the lungs. Once inhaled, these particles can affect the heart and lungs and cause serious
health effects.
Radon and environmental tobacco smoke (ETS) are the two indoor air pollutants of
greatest concern from a health perspective. Radon is a naturally occurring gas that is odorless,
colorless, and radioactive. Environmental tobacco smoke (ETS) is the smoke emitted from the
burning of a cigarette, pipe, or cigar, and smoke inhaled by a smoker. It is a complex mix of
more than 4,000 chemical compounds, containing many known or suspected carcinogens and
toxic agents, including particles, carbon monoxide, and formaldehyde.
The hydrosphere is the accumulation of water in all its states (solid, liquid and gas) and
the elements dissolved it in (sodium, magnesium, calcium, chloride and sulphate). Water
covers around 71% of the earth's surface (Fig. 2) and is an important accumulator of
pollutants and a significant vector of diffusion.
There are many specific causes of water pollution, but it is more important to understand
the two broad categories of water pollution point and nonpoint sources. An example of
a point source of water pollution is a pipe from an industrial facility discharging effluent
directly into a river, whereas a nonpoint-source of water pollution is when fertilizer from
a farm field is carried into a stream by rain (i.e. run-off). Nonpoint sources are much more
difficult to monitor and control, and today they account for the majority of contaminants in
7
streams and lakes.
Fig. 2. Distribution of global water [http://ygraph.com/chart/2410]
Water pollution takes many forms:
oxygen depletion - caused by the release of biodegradable matter into the water and the
natural process of breaking this down uses the oxygen in the water;
nutrients - such as phosphorus and nitrogen are essential to plant growth, too many
nutrients in the water encourage the growth of weeds and algae (algae bloom),
chemical - adding unwanted chemicals to the water is done through the accidental
spillage of substances into water (for example oil when an oil tank ruptures or a ship
sinks), waste from factories or industry, and through pesticides running off fields into
water; chemicals in water are poisonous and harmful to wildlife as well as making the
water too polluted to drink,
suspended matter - the tiny particles of matter (not soluble in water) stay in the water
and eventually fall to the bottom, causing long term problems due to an imbalance in
the natural infrastructure of the water,
biological - some viruses and bacteria are waterborne and they can cause serious
diseases in people in direct contact with this contaminated water.
The lithosphere is the thin crust between the mantle and the atmosphere, and it contains
rocks, minerals, and soils. Soil is a complex mixture of eroded rock, mineral nutrients,
decaying organic matter, water, air, and billions of living organisms (Fig. 3). Main chemical
soil constituents are oxygen (47%), silicon (28%), aluminum (8%), iron (5%), calcium (4%),
sodium (3%), potassium (3%) and magnesium (2%) in a crystalline state.
Fig. 3. Soils main components [http://www.physicalgeography.net/fundamentals/10t.html]
Soils support a number of inorganic and organic chemical reactions. Many of these
reactions are dependent on some particular soil chemical properties. One of the most
8
important chemical properties influencing reactions in a soil is pH. Soil pH is primarily
controlled by the concentration of free hydrogen ions in the soil matrix. Soil fertility is
directly influenced by pH through the solubility of many nutrients. At a pH lower than 5.5,
many nutrients become very soluble and are readily leached from the soil profile whereas at
high pH, nutrients become insoluble and unavailable for plants. Maximum soil fertility occurs
in the range 6.0 to 7.2.
Soil pollution is the result of toxic chemicals, solvents, salts, microorganisms and other
harmful substances introduced to the top layers of soil as a result of dumping garbage, waste,
and other toxins. Manmade land pollution comes as accidental disasters, brownfields, waste
management and landfills, pesticides and agricultural practices, clear cutting, urban
development and energy production. Soil pollution decreases the fertility of the soil by
reducing its mineral content, making it difficult for plants to survive or thrive. Erosion, or loss
of fertile topsoil is another problem that many areas must face due to deforestation, which is
the cutting down of trees for urban and agricultural purposes.
Soil contamination affects an ecosystems equilibrium, and whenever any imbalances
occur, the ecosystem restores itself through biogeochemical cycles to the equilibrium state;
this may take a few days or many years. Elements carried through the biogeochemical cycles
are stored in their natural reservoirs, and are released to organisms in small consumable
amounts. For example, through the nitrogen cycle and with the help of the nitrogen fixing
bacteria, green plants are able to utilize nitrogen in bits even though it is abundant in the
atmosphere. Since the biogeochemical cycles pass through different spheres, the flow of
elements is regulated because each sphere has a particular medium and the rate at which
elements flow is determined by the viscosity and density of the medium.
1.2. The most dangerous environmental pollutants
1.2.1. Gaseous pollutants
The most common gaseous pollutants are: carbon dioxide and monoxide (CO and CO2),
hydrocarbons (PAHs, CFC), nitrogen oxides (NOx), sulfur oxides (SO2). They belong to
primary air pollutants (Fig. 4) and are directly emitted from an emission source. Secondary air
pollutants are those that are formed by reactions between the primary air pollutants and
normal atmospheric constituents, sometimes by utilizing sunlight energy (sulfuric acid, nitric
acid, nitrogen dioxide, ozone (O3), formaldehyde, peroxyacetyl nitrate (PAN), ammonium
nitrate and ammonium sulfate).
9
Fig. 4. Primary and secondary air pollutants
[http://science.kennesaw.edu/~jdirnber/scienceII/OutlineAir/OutlineAirNotes.html]
There are several main types of air pollution and well-known effects of pollution which
are commonly discussed. These include smog, acid rain (Fig. 5), the greenhouse effect, and
"holes" in the ozone layer. Each of these problems has serious implications for our health and
well-being as well as for the whole environment.
Fig. 5. The effect of gaseous pollutants on the environment [PhysicalGeography.net]
1.2.2. Inorganic pollutants
Inorganic chemical pollutants are naturally found in the environment but due to human
development, these pollutants are often concentrated and released into the environment. The
primary inorganic pollutants are mainly divided into two categories: heavy metals and
nutrients. The list below highlights just a few of the inorganic pollutants:
Arsenic (as Arsenite)
Lead
Copper
Chlorine
Cyanide
Nitrate
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Cadmium
Mercury
Chromium
Ammonia
Phosphate
Inorganic pollutants - such as hydrochloric acid, sodium chloride, and sodium carbonate -
change the acidity, salinity, or alkalinity of the water, making it undrinkable or unsuitable for
the support of animal and plant life. These effects can result in dire consequences for higher
mammals and humans.
Heavy metals
Among the many heavy metals released from various products and processes, cadmium,
lead and mercury are of great concern to human health because of their toxicity and their
potential to cause harmful effects at low concentrations and to bioaccumulate. Living
organisms possess diverse sensitivity to heavy metals (Tab.1).
Table 1. Sensitivity of living organisms to heavy metals [Rajmer, 1997]
Metal Plant Animal Human
Cd Cu Hg Ni Pb Zn
Sensitivity low mean high
Significant progress has been made in reducing emissions to air of these metals in Europe,
with 1995 emissions being about 50 % of 1990 levels and decreasing further to 40 % by 1999.
Although controlling diffuse emissions of cadmium and mercury remains problematic (e.g.
batteries), point source emissions of these metals have declined as a result of improvements in
sectors such as wastewater treatment, incinerators and the metals sector. Factors contributing
to this include large decreases of lead emissions from the transport sector following the
introduction of unleaded petrol in the early 1990s, continuing move away from the use of
lignite in the eastern European energy sector, and the introduction of improved pollution
abatement technologies across a range of industrial and waste treatment sectors.
Cadmium is often discussed in relation with food safety issues. It readily accumulates in
crops, especially in acidic soils with low binding capacity. Exposure risk due to soil ingestion
11
is less critical, although recent investigations indicate that exposure through indoor dust (soil
related) is as high as exposure due to consumption of homegrown vegetables. In slightly
acidic soils with low binding capacity, there is a high risk of leaching to the subsoil and the
groundwater. In living organisms, cadmium affects renal, pulmonary, skeletal, testicular and
nervous systems, and disrupts zinc-dependent enzymes.
Although emissions from lead to soil are decreasing, it is still a substance of concern
given the amounts of lead stored especially in organic layers of topsoils. Critical levels in soil
(based on total concentrations) for long-term exposure are often exceeded in urban areas,
although bioavailability may be low. Lead inhibits hemoglobin synthesis (anemia); substitutes
for calcium, reducing cellular functions (e.g. ATP production); is stored in bones; and
moreover, organic lead compounds affect brain function and may cause lung cancer.
Zinc is an essential element for life but it is also very toxic for micro-organisms leading to
a decreased decomposition of organic matter. Ecological risk assessment is difficult for this
element since generic safe levels derived from toxicity tests are below the levels to sustain life
in less sensitive organisms and even lower than background levels. Experience with the
assessment of ecological impacts may improve if zinc is monitored in soil in conjunction with
biodiversity indicators and assessments of soil decomposition processes. Also, zinc
deficiencies related to crop production could be identified.
Mercury is very toxic, mainly accumulates in soils and sediments, but can be transformed
into mobile fractions (e.g. methyl mercury). Hg is prioritized in many environmental
programs at EU level and also national level. Unfortunately, it is difficult and expensive to
measure. Mercury vapors and organomercury enter the central nervous system, affecting the
brain and nerve cells, causing sensory disturbance, reduced field vision and ataxia, and
impairing speech, hearing and mental functions.
1.2.3. Organic environmental pollutants
A list of organic pollutants includes:
hydrocarbons (aromatic, PAHs),
halogenated hydrocarbons (PCBs, dioxins, CFC),
pesticides (insecticides, herbicides, fungicides, rodenticides).
All of these substances are highly lethal to animals, and many can be readily absorbed
through the skin. These contaminants are the most differentiated in their chemical structure
(Fig. 6).
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Fig. 6. Different structures of organic pollutants [de Mello- Farias et al, 2011; DOI: 10.5772/24355]
The majority of organic pollutants belong to the group of Persistent Organic Pollutants
(POPs). They persist in the environment, bioaccumulate through the food web, and pose a risk
of causing adverse effects to human health and the environment (PAHs, PCBs, dioxins,
DDT). With the evidence of long-range transport of these substances to regions where they
have never been used or produced and the consequent threats they pose to the environment of
the whole globe, the international community has now, at several occasions, called for urgent
global actions to reduce and eliminate releases of these chemicals.
Polycyclic aromatic hydrocarbons (PAHs)
PAHs (which are known for their strong toxic properties) are composed of carbon and
hydrogen atoms arranged in the form of fused benzene rings (linear, cluster or angular
arrangement). This group contains a number of known carcinogens; the most dangerous one
among them is benzo(a)pirene - BaP (Fig. 6). The widespread occurrence of PAHs is largely
due to their formation and release in all processes of incomplete combustion of organic
materials. The last century of industrial development caused a significant increase of PAH
concentrations in the natural environment. Research shows that air contributes 3-20% of total
human exposure to PAHs and comes in second position (after food) as a source of these
13
pollutants for humans. Cigarette smoke can significantly contribute to potential PAH doses
via inhalation (over 50% of total exposure). The ambient standards regarding exposure to
PAHs refer usually to BaP; in Poland, its permissible concentration in the air as a daily
exposure is 5 ng/m3 and as a year exposure - 1 ng/m3.
Polychlorinated biphenyls (PCBs)
It is a large family (209) of compounds (Fig. 6) that are very stable and that are used as
closed system and heat transfer fluids (transformers, capacitors, fluorescent light ballasts,
etc.), as plasticizers, as hydraulic fluids and lubricants and as form base for pesticides. PCBs
are persistent and show significant bioaccumulation and low acute toxicity. They strongly
accumulate in the food chain and significant levels of them have been found in marine
species, particularly mammals (even 5-10 mg/kg) and sea birds. They are carcinogenic and
capable of damaging the liver, the nervous system and the reproductive system in adults.
Daily intake of PCB with food amounts to 5-100 g in developed countries. When PCBs are
burned, even more toxic dioxins are formed, so they are called dioxin-like compounds.
Dioxins
It is a class of super-toxic chemicals formed as a by-product of the manufacture,
moulding, or burning of organic chemicals and plastics that contain chlorine; they have been
never produced. This group includes polychlorinated dibenzo-p-dioxin - PCDD (Fig. 6) and
polychlorinated dibenzofuran (PCDF). The most known and toxic is 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD). They are virtually indestructible (they are stable at
1000oC in oxygen conditions and destroyed by UV light only), cumulated in fatty tissues and
excreted by the body extremely slowly. They cause serious health effects even at levels as low
as a few parts per trillion (10-14). They bind to cell receptors and disrupt hormone functions
in the body and they also affect gene functions (named as Endocrine disruptors, EDs).
Because dioxins refer to such a broad class of compounds that vary widely in toxicity, the
concept of toxic equivalence (TEQ) has been developed to facilitate risk assessment and
regulatory control. The uncertainty and variability in the dose-response relationship of dioxins
in terms of their toxicity, as well as the ability of dioxins to bioaccumulate mean that the
tolerable daily intake (TDI) of dioxins has been set very low by the WHO to 10 pg/kg body
weight per day.
Pesticides
Pesticide contamination is a particular problem in rural agricultural areas where pesticide
use is heavy and drinking water supplies come directly from groundwater or surface water.
Pesticides can migrate via water into the food chain as well, ultimately being consumed by
14
humans or animals in food.
Pesticides can be divided according chemical structure as:
Organochlorines - DDT, HCH, Methoxychlor, Aldrin, Endosulfan,
Organophosphates - Malathion, Parathion, Diazinon, Chlorpyrifos, Glyphosate (Roundup),
Carbamate Esters - Carbofuran, Aldicarb,
Phenoxy Esters - 2,4-D, Silvex, 2,4,5-T.
Some of these pesticides will persist for long periods of time in the environment. DDT
was a pesticide (with a half-life of over 3-10 years) that was in use for a long time around the
world (and is still being used in parts of the world for mosquito control), but has been banned
in the US since 1972. Yet we still find DDT in our environment, sometimes at very high
levels. Pesticide in soils undergo degradation and its transformation products (TPs) can be
present at higher levels in the soil than the parent pesticide itself. Generally, pesticide TPs
could show lower toxicity to biota than the parent compounds. However, sometimes TPs are
more toxic, so they represent a greater risk to the environment than the parent molecules. In
terms of general human health effects, pesticides can affect and damage the nervous system,
cause liver damage, damage DNA and cause a variety of cancers, cause reproductive and
endocrine damage and cause other acutely toxic or chronic effects. There is a pressing need
for pesticides determination in food and environment, but pesticide analysis is a very complex
problem, including proper sample preparation, selective and sensitive chromatographic
detection and determination of pesticide residues, and the application of biological
(immunoassays-and biosensors-based) methods.
1.3. Measurement of chosen pollutants in water and air
1.3.1. Evaluation of water contaminants using VISOCOLOR colorimetric test kits
The visual colorimetric analytical systems VISOCOLOR
and VISOCOLOR ECO
(MACHEREY-NAGEL GmbH & Co.KG, Germany) have been successfully applied in water
analysis for many years. During assessment, the determined substance, which is not visible or
cannot be measured directly, is converted into a coloured compound by addition of a suitable
reagent. If it is done according to instructions, the resulting colour intensity is proportional to
the concentration of the determined substance. For evaluation of the VISOCOLOR test kits a
so-called comparator is used, whereas VISOCOLOR
ECO tests are evaluated with a colour
scale. For determination of more precise values, the same analytical preparation is measured
with the filter photometer PF-11 (or PF-12) (MACHEREY-NAGEL). In particular,
NANOCOLOR
reagent sets are ready-to-use reagent preparations of high precision and
15
selectivity for photometric analysis.
The most important advantages of these kits are: economic pricing, convenient handling,
higher accuracy and sensitivity, pictographic instructions, reagent bottles with clear dosing
instructions, availability of refill packs, visual evaluation.
The set of VISOCOLOR ECO test kits include:
Aluminium Copper Manganese Silica Ammonium
Cyanide Nickel Sulphate Calcium Cyanuric acid
Nitrate Sulphide Carbonate hardness DEHA
Nitrite Sulphite Chloride Fluoride
Oxygen Swimming pool Chlorine Hardness (total)
pH Zinc Chlorine dioxide Hydrazine
Phosphate Chromium (VI) Iron Potassium
[http://www.mn-net.com/tabid/4648/Default.aspx]
Procedure for Visocolor test NITRATE 50
In a weakly acidic medium, nitrate ions are reduced to nitrite ions, which react with sulphanile
acid and l- naphthylamine to form red azo-dye. Nitrites disturb sampling (the same reaction);
therefore, when they are found in water, they should be removed using amidosulphonic acid.
Range: 1 to 40 mg NO3/l (0.2-9.0 mg/l NO3-N)
Filter:4 Reaction time: 5 min
Assay:
1. Rinse test tube 14mm ID several times with sample and fill up to the ring mark (10 ml)
2. Add 10 drops Nitrate-1 and shake test tube
3. Add 1 small, level measuring spoon Nitrate-2, close test tube and shake vigorously for
15-30s, wait 5 min.
4. Using PF-11 photometer perform measurement.
General procedure for VISOCOLOR tests
switch on photometer
with key M call up VISOCOLOR
with key rounded ARROW call up the test NITRATE 50 with range NO3,
press key NULL ZERO
adjust filter wheel to 4, place clean round glass cell with blank value (untreated sample) into the photometer and press
key NULL ZERO
after 2s, place clean round glass cell with sample solution into the photometer, press key M and read the result in mg/l NO3
1.3.2. Determination of nitrates in water by spectrophotometric method
(according to PN-82/C-04576/08)
Nitrates (V) and (III), i.e. nitrates and nitrites are the most frequent chemical pollution of
water, and they seriously affect human health. The presence of nitrates in water is the
consequence of the use of mineral fertilizers in agriculture, sewage draining to waters, but
also natural nitrification processes. Nitrates (NO3-) are not dangerous to people; however, they
16
can transform into much worse nitrites (NO2-) in acid environment. Nitrites are precursors of
carcinogenic nitrosamines. Nitrates are especially dangerous for young infants because they
cause methaemoglobinaemia, and in adults indirectly lead to an increase in the incidence of
stomach and intestines cancer.
There are three main diet sources of nitrates (V): crops and vegetables (up to 80%), meat
products (10%) and drinking water (10%). Various plants have different nitrates accumulation
capacity, and the biggest amount of nitrates is assimilated by beet root (150-5960 mg
NO3/kg), lettuce (382-3520 mg NO3/kg), and radish (261- 1186 mg NO3/kg). Nitrates are
used in the food processing industry as effective preservatives preventing bacteria
colonization. They positively influence qualitative features of a product (preservation of pink
colour of meat products, flavouring in pickled meat). Surface waters usually comprise a small
amount of nitrates (0-1 mg N-NO3), while underground waters may contain a significant
amount of nitrates depending on soil pollution and geological conditions. The highest
concentration of nitrates in drinking water is found in shallow domestic wells located in rural
areas and on the outskirts of towns, depending on the country region, from 30 to 85% of these
water intakes contain water with an excessive amount of nitrates (the acceptable levels in
Poland are 50 mg/l NO3 and 0.1 mg/l NO2).
ASSAY PROCEDURE
The complex, formed by nitration of salicylic acid under highly acidic conditions, absorbs
maximally at 410 nm in basic (pH>12) solutions. Absorbance of the chromophore is directly
proportional to the amount of nitrate-N present. Ammonium, nitrite and chloride ions do not
interfere.
Reagents
0.5% salicylic acid
0.5% NaOH
conc. H2SO4 alkaline potassium-sodium tartrate solution
Standards
Stock solution 0.7216 g/l KNO3
Working stock solution: 0.0434 mg NO3 per mL
1. 10 mL of stock solution with 2-3 drops of NaOH solution and 20 mL of salicylate are
evaporated in an evaporating dish using boiling water bath.
2. After cooling down, add 1 mL of concentrated H2SO4, so in that way the entire residue is
dehumidified and allowed to stand for 10 minutes.
17
3. Add 30 mL of distilled (DI) water and quantitatively transfer it to a 100 mL volumetric
flask and make up to 100 mL with DI water.
Calibration curve
1. To a series of test volumetric flask (50 mL), pipette carefully 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0
and 5.0 mL of working stock solution.
2. Add 7 mL of potassium-sodium tartrate to each flask and make up to 50 mL with DI
water. Mix thoroughly.
3. Measure the absorbance at 436 nm against a blank prepared in the same way (without
nitrates) using EPOLL 20 photometer.
4. Prepare a calibration curve by plotting the corrected absorbance (y axis) vs. milligrams of
NO3 (x axis). Determine the slope and intercept for the calibration curve.
Water sample assay
1. 20 mL of water sample with 2-3 drops of NaOH solution and 1 mL of salicylate are
evaporated in an evaporating dish using boiling water bath.
2. After cooling down, add 1 mL of concentrated H2SO4, so in that way the entire residue is
dehumidified and allowed to stand for 10 minutes.
3. Add 20 mL of distilled water and 7 mL of potassium-sodium tartrate and quantitatively
transfer it to a 50 mL volumetric flask, make up to 50 mL with DI water and mix
thoroughly.
4. Measure absorbance at 436 nm.
Calculations
The nitrate content be determined from a standard curve and converted into 1 liter of water
(x50). Compare result to the Polish acceptable level of nitrates in drinking water is
50 mg/dm3.
1.3.3. Determination of formaldehyde migration from wood panels
Formaldehyde (HCHO) is an important pollutant of indoor air, and because of its
chemical and toxic characteristics an individualized evaluation is recommended. Everyone is
exposed to small amounts of formaldehyde in the air, some foods, and products, including
composite wood products. Release from formaldehyde-based resins in which it is present as
a residue and/or through their hydrolysis and decomposition by heat (e.g. during the
manufacture of wood products, textiles, synthetic vitreous insulation products, and plastics).
In general, the use of phenol-formaldehyde resins results in much lower emissions of
formaldehyde than those of urea- based resins. It enters the atmosphere also as a result of the
combustion of any fuel energy, fuel propellants waste. It is also a component of tobacco
smoke - a 1 m3 of smoke is about 40 cm
3 of formaldehyde.
18
Formaldehyde is used widely by various industries to manufacture a range of building
materials and numerous household products. It is contained in resins used to manufacture
some composite wood products (e.g. hardwood plywood, particleboard and medium-density
fiberboard). It is found (as an ingredient or impurity) in some cosmetics/personal hygiene
products, such as some soaps, shampoos, hair preparations, deodorants, sunscreens, dry skin
lotions, and mouthwashes, mascara and other types of products, such as eye makeup, nail
creams, vaginal deodorants, and shaving creams.
At room temperature, formaldehyde is a colorless, flammable gas that has a distinct, pungent
smell. Formaldehyde is commonly produced as an aqueous solution called formalin, which
usually contains about 37% formaldehyde and 12-15% methanol. Methanol is added to
formalin to slow polymerization that eventually leads to precipitation as paraformaldehyde.
Due to its capability for protein denaturation, formaldehyde is the most commonly used
fixative. It serves to stabilize the fine structural details of cells and tissue in biological
examinations.
The primary way of exposure is by breathing air containing it. Formaldehyde can cause
irritation of the skin, eyes, nose, and throat. High levels of exposure may cause some types of
cancers. The maximum concentration of formaldehyde NDS = 0,5 mg/m3.
There are different methods for detecting formaldehyde in air, all of them based on taking
samples for later analysis, with active fixing or by diffusion. The traditional methods are
based on obtaining a sample by bubbling air through distilled water or a solution of 1%
sodium bisulphate at 5C, and then analysing it with spectrofluorometric methods. While the
sample is stored, it should also be kept at 5C. SO2 and the components of tobacco smoke can
create interference.
ASSAY PROCEDURE
In the monitor, formaldehyde vapors are absorbed on bisulfite impregnated paper and
desorbed with formaldehyde-free distilled water. Aliquots are reacted with chromotropic acid
in the presence of sulfuric acid to form a purple monocationic chromogen. The absorbance of
the colored solution is read in a spectrophotometer at 580 nanometers (nm) and is
proportional to the amount of formaldehyde in the solution. In this experiment, the chipboard
shavings are extracted with water within 1 hour.
Reagents
1% chromotropic acid solution: dissolve 0.5 g of 4,5-dihydroxy-2,7-naphthalenedisulfonic
acid disodium salt dihydrate in 50 mL of distilled water. Make a fresh solution each day.
Stock formaldehyde solution - 25 L of 37% formaldehyde is diluted to 100 mL with distilled
19
water and mix thoroughly.
Working formaldehyde solution: Transfer a 5 mL aliquot of the stock solution to a 50 mL
volumetric flash and make up to 50 mL with DI water. This solution is used to prepare the
calibration curve each milliliter is equivalent to 10 micrograms of formaldehyde.
Calibration curve
1. To a series of test tubes, carefully add 0.0, 0.1, 0.2, 0.3, 0.5, 0.7, 0.8, and 1.0 mL of
working stock solution of formaldehyde.
2. Adjust the volumes to 1 mL with distilled water.
3. Develop the color: add 0.2 mL of chromotropic acid solution to each sample and mix
well. After 3 min., carefully add 5 mL of concentrated sulfuric acid slowly while mixing
(use a glass stick).
CAUTION: Take proper safety precautions such as goggles, gloves and apron when
handling concentrated sulfuric acid.
4. Allow the samples to cool to room temperature (for 20 min) and measure the absorbance
at 585 nm using EPOLL 20 photometer against reagent blank (without formaldehyde).
5. Prepare a calibration curve by plotting the corrected absorbance (y axis) vs. micrograms
of formaldehyde (x axis).
6. Determine the slope and intercept for the calibration curve.
Sample analysis
Particle board shavings are leached with water for 24 hours (the ratio 5 g per 250 mL of
distilled water). Before analysis, filtrate the extract and prepare four dilutions of extract: 10-,
20-, 50- and 100- fold. Transfer a 1 mL aliquot of each dilution to a test tube for color
development exactly as described above.
Calculations
The formaldehyde content should be determined from a standard curve and converted into
1 kg of shavings. Assuming that all of the formaldehyde emitted by 1 kg of chipboard goes
into the room with a capacity of 30 cubic meters, calculate the concentration of formaldehyde
in the room air as mg/m3. Compare the result to the exposure limit in the air which amounts to
0.5 mg/m3.
20
2. ENVIRONMENTAL ANALYTICS
Analytics is a typical example of a scientific discipline that uses achievements both in the
scope of basic research, as well as those used in a variety of other disciplines. Said disciplines
include:
various fields of chemistry (especially physical chemistry and biochemistry),
physics,
computer science,
electronics, automatics and robotics,
materials engineering,
biology,
instrumentation (science about building and use of measuring and control instruments),
chemometrics.
Whereas in environmental analytics, the most often used methods are those known to
analytical chemistry, mainly:
Spectroscopy analytical methods, particularly:
Molecular spectroscopy, including spectrophotometry UV-VIS and IR,
Atom spectrometry, including atomic absorption spectrometry AAS and atomic
emission spectrometry AES,
Mass spectrometry,
Electrochemical analytical methods,
Chromatographic analytical methods.
Many pollutants of anthropogenic origin are organic compounds. In analytics of this type
of pollutants, the most widely used methods are chromatographic methods.
Exotoxicological concerns and the desire to reduce description thoroughness of the state
of the environment constitute great challenges for analysts in terms of the necessity to assay
concentration of a wide variety of analytes in samples with a complex (and sometimes also
variable) matrix composition. Two approaches can be distinguished for assay of analytes
present in test samples at low content levels:
1. Utilization of more sensitive, selective or even specific detectors for chromatography.
2. Introduction of an additional stage to the analytic procedure: isolation and/or
enrichment of analytes before the final assay stage. The additional stage ensures:
simplification of the matrix (as a result of transferring analytes from the sample to
a proper solvent or gas feed stream) and removal of at least some of the interfering
substances (interferents) from the test sample before the final assay stage. This also
21
ensures an increase of analyte concentration in the sample to a higher level than the
detection limit of the method and/or the instrument being used.
2.1. Use of chromatographic methods in pesticide monitoring
2.1.1. Basics of GC chromatography
Chromatography is currently one of the most widespread instrumental methods in
chemical analysis, mainly because of its capability to detect the analyzed substance and assay
its quantity even at very low concentration and in presence of many other substances.
Moreover, chromatography as an analytical method, when combined with other analytical
techniques (e.g. mass spectrometry), allows to solve most analytical problems. It is a method
of separating homogenous mixtures in which the separated components undergo partitioning
between two phases: one of them is the stationary phase, the other - mobile phase (Fig. 7).
Fig. 7. Mechanism of the chromatographic process. Substances marked as triangles and circles are
characterized by varied retention factors k
The stationary phase can be a solid, a liquid on a carrier, or a gel; and the mobile phase
a gas or a liquid. If the mobile phase is a gas, then this type of chromatography is called gas
chromatography (GC), whereas if the mobile phase is a liquid, then it is called liquid
chromatography (LC).
If the stationary phase in gas chromatography is:
a solid (adsorbent), then we are dealing with adsorption chromatography,
a liquid that resides on a solid carrier in the form of a homogenous layer, then we are
dealing with partition chromatography.
The mobile phase moves inside the column, whereas the stationary phase resides on the
columns inner walls. Chemical compounds with larger affinity for stationary phase are
selectively stopped by it and move much more slowly along the column. Chemical
compounds with smaller affinity for stationary phase move much faster along the column and,
22
as a result, they leave the column, i.e. the eluate from the column, in the first instance.
Separation balance between the phases is of a dynamic character, i.e. substance particles
move continuously from mobile phase to stationary phase and back.
The phenomena determining the separation process are diverse in character; even so, some
of the most often used processes include adsorption and separation of substances between two
non-mixing liquids. Due to the nature of the phenomena occurring during chromatographic
separation, chromatographic methods can be classified into adsorption chromatography or
separation chromatography. In the first case, separation of the mixture is conditioned by
a variable adsorption affinity of mixture components toward the surface of the stationary
phase called the adsorbent. In the case of separation chromatography, separation of mixture
components is based on the differences in separation factor values of each of the mixture
components between two non-mixing phases. One of the most often used chromatographic
methods in environmental monitoring includes gas chromatography and high-performance
liquid chromatography.
In the case of gas chromatography, the liquid sample evaporates in the dispenser and,
through the carrier gas stream, it is fed into the column where sample components are
separated and are then moved to the detector where they generate an electrical charge.
Voltage signals are read with a multimeter and the data is sent to a PC computer. Using the
right software, it is possible to download the data from a measure card.
Whereas in the case of high-performance liquid chromatography, the pump from the tank
(or tanks) sucks in the mobile phase and pumps it through a dispenser into the
chromatographic column. The column is sometimes placed in a thermostat. The sample is
injected by a injector through the top of the chromatographic column, after that mixture
components undergo separation in the column and, upon exiting, are detected by the detector.
The detectors electric signal, after boosting, is saved on recording paper or recorded with an
integrator or a computer in the form of a chromatographic peak. Flow of liquid through the
system can be controlled with a manometer and a flow meter. With certain apparatus, it is
possible to collect the separated components in a fraction collector.
GC-MS method
Gas chromatography is one of the few chromatographic methods that allows for analysis of
a complex mixture and performing of quantitative assay of its components as part of a single
process. In this way, it is possible to separate and identify mixtures composed of more than
a few hundred components. However, this chromatographic method can only separate those
23
substances that, during chromatographic conditions, are in gaseous or vapor form. Therefore,
they must be gaseous, liquid or solid substances whose boiling point or sublimation point
does not exceed 400C. Despite the fact that gas chromatography is the ideal method for
substance separation, it does not provide clear information about the consistence of the
mixture being separated. The perfect solution to this problem turned out to be combining gas
chromatography with other instrumental analysis techniques. The widest use was found when
linking gas chromatography with mass spectrometry (GC-MS). This combination is of
particular significance in environmental analysis because mass spectrometry (MS) is one of
the most efficient methods of qualitative and structural analysis of organic compounds. In this
method, the gas chromatographer separates mixture components and inserts into the mass
spectrometer pure compounds of the right volatility and with a speed adjusted to the speed of
processes taking place inside the mass spectrometer (Fig. 8). In gas chromatography, the two
most often used column types are: packed columns and capillary columns. Capillary columns
achieve considerably higher separation efficiency than packed columns. Gas chromatography
uses such adsorbents as: carbon adsorbents, silica gel, molecular sieves and porous polymers.
Among inorganic adsorbents the most widely used are molecular sieves, and among organic
ones - porous polymers.
Fig. 8. Basic elements of the GC-MS system
Detectors used in GC chromatography
Substances separated in the chromatographic column are detected by the detector upon their
exit from the column. The detector, in response to the presence of the analyzed substance in
carrier gas, reacts by generating an electrical signal. A good detector should possess the
following traits:
good sensitivity,
good detection of analyzed substances,
wide linearity range of indication,
stability of indications and low noise level,
24
selectivity of indications at low cost.
In gas chromatographs, there are many various types of detectors. The most important
ones include:
Thermal conductivity detector (TCD),
Flame ionization detector (FID),
Flame photometric detector (FPD),
Electron capture detector (ECD),
Mass spectrometer (MS).
The principle of operation of the most important ones in environmental analysis has been
discussed below.
Flame Ionization Detector (FID)
Principle of operation: Compounds are burned in a hydrogen and air flame. Compounds
containing carbon produce ions that are collected on the collector (collector electrode). The
number of ions collected by the electrode is recorded and in this way the signal is produced.
Selectivity: Compounds containing the C-H bond. Weak response to organic compounds
lacking hydrogen (e.g. hexachlorobenzene).
Sensitivity 0.1 10 ng
Linear range: 105 107
Gases: For combustion - hydrogen and air; additional gas (make-up) - helium or nitrogen
Temperature: 250-300C; 400-450C for analyses conducted at a higher temperature.
FID is a destructive, mass detector. The number of ions produced in the flame is countable.
Ions generate the detectors signal. Analytes, which exhibit the largest number of carbons at
low oxidization level, generate the strongest signal.
Fig. 9. FID detector scheme
Electron Capture Detector (ECD)
Principle of operation: Electrons are delivered from the radioactive 63
Ni placed in the
detector cell, in which the electricity is being generated. Electronegative compounds capture
electrons, causing reduction of electricity. Reduction of electricity, when measured indirectly,
is background signal.
25
Selectivity: chlorides, nitrates and conjugated carbonyl groups.
Sensitivity: 0.1-10 pg (chlorinated compounds), 1-100 pg (nitrates); 0.1-2 ng (carbonyl compounds)
Balance range: 103-10
4
Gases: Nitrogen or argon / methane
Fig. 10. ECD detector design scheme
Mass Spectrometer (MS)
Principle of operation: Vacuum is present in the detector. Compounds are bombarded with
gas electrons (EI) or particles (CI) and then fragmented into ions with characteristic charges.
Produced ions are focused and accelerated in the mass filter. Mass filter selectiveness lets
through all ions of specific mass into an electron multiplier. All ions of specific mass are
detected. Next, the mass filter lets through the next mass that distinguishes itself from the
mass of other ions.
The mass filter gradually searches through the specified mass range a few times a second.
Each time the total number of ions is calculated. With each search, the intensity or number of
ions is graphed as function of time on the chromatogram (called Total Ion Chromatograph -
TIC). Each scanning provides a mass spectrum that shows various masses of ions as the
function of their intensity or number.
Selectiveness: All compounds providing fragments in the selected mass range. It is possible to
use full search - scan or only selected ion monitoring (SIM).
Sensitivity: 1-10 ng (full search); 1 - 10 pg (selected ion monitoring, SIM)
Linear range: 105-10
6
Gases: none
Temperature: 250-300C (transfer line); 150-250C (source)
Fig. 11. MS detector scheme
26
Chromatogram analysis
The effect of chromatographic separation is graphed in the form of an elution
(chromatogram) curve. The chromatogram presents a graph of dependence of detector
indications on the time or volume of mobile phase.
The chromatogram provides the following information:
qualitative - on the basis of retention time on the chromatogram, it is possible, e.g., to
draw conclusions about the type of the separated substance. On the basis of the number
of peaks - about the number of components in a mixture, assuming that the used
chromatographic system ensured the separation of all mixture components and that the
detector is sensitive to all of the components,
quantitative - by measuring peak height or calculating the integral on the
chromatographic peak surface, it is possible to draw conclusions about the
concentration or mass of analytes in the injected sample.
The ideal chromatogram contains non-overlapping, closely spaced peaks. Overlapping
peaks are called co-eluting peaks. Retention time and peak size are very important as they
allow for identification and estimation of the number of analyzed compounds present in the
sample. The size of obtained peaks is proportional to the amount of the component in the
sample. The larger peaks are observed when the concentration of a given component
increases.
Fig. 12. Sample chromatogram of a pesticide mixture
2.1.2. Assay of presence and concentration of pesticides in the soil
Pesticides occur in nature in very small concentrations. In most cases - both in assay of
individual compounds, as well as when determining overall or individual parameters - it is
necessary to isolate organic compounds from the complex and troublesome matrix and enrich
them before final assay. Isolation and enrichment is necessary because of the imperfection of
current analytical methods. This is because in many cases they are not sensitive enough to
conduct final assay directly on the obtained sample and to assay trace components. During
27
isolation and enrichment, the concentration ratio of a micro-component to macro-components
increases. The basic activities during this part of analysis are: isolation of analyte from the
matrix (most often - extraction), fractioning or combination of these activities, whereby the
manner of isolation is determined by the matrix (complexity, composition and concentration),
selectiveness and sensitivity required by the analytical method and by analytical goals. This
method must characterize itself with simplicity of execution, ensure the separation of micro-
components from the matrix and should conform with the final assay method.
Methods of extraction from wet samples
For extraction, solvents mixing with water are used, e.g. acetone, acetonitrile, or a mixture
of solvents (polar/nonpolar), e.g. acetone/hexane, methanol/hexane. Extraction from wet
samples does not involve preliminary drying. After main extraction is completed, the solvent
is replaced (as a result of liquid-liquid extraction) with a nonpolar one, e.g. hexane or
dichloromethane, together with removing the water from the extract. Prior to this stage, water
is added to make it easier for analytes to move to the nonpolar layer. It is a toilsome stage,
conducted in a separatory funnel. Solvent replacement can also occur by enriching the analyte
in an extraction column to stationary phase (after diluting the extract with water), which is
followed by drying the column and elution with a nonpolar solvent.
Extraction from wet samples can be done in two ways. The first one is liquid extraction by
shaking or supported by ultrasounds (sonication). The process is conducted in multiple stages
(more than 3 times) due to the very low efficiency of the first stage of extraction. The
advantage of this method is the use of simple apparatus. The second way consists of
extraction by use of a Soxhlet apparatus. It is a procedure that is less toilsome compared to
liquid extraction by shaking or by sonication. The drawbacks include the possibility of a large
scattering of salvage values caused by the channeling effect in the extraction thimble, and also
the long time of extraction (6-48 h).
Methods of extraction from dry samples
For extraction from dry samples, it is possible to use methods used in extraction from wet
samples, i.e. liquid extraction by shaking or supported by ultrasounds and also extraction by
use of a Soxhlet extractor. In such cases, what is used is a mixture of a polar solvent (must be
removed during the course of the procedure) and a nonpolar solvent (e.g. 20% of acetone in
hexane produces an azeotrope containing 56% acetone which can removed during distillation)
or such solvents as: dichloromethane (CH2Cl2), hexane, ethyl acetate/cyclohexane, toluene/methanol,
28
2-propanol, and also CH2Cl2, acetone-hexane, cyclohexane and methanol-CH2Cl2. It is usually not
necessary to conduct the stage of liquid-liquid extraction in order to remove water.
Extraction from dry samples can be carried out:
by using a fluid in a supercritical state. This process is conducted by using high-
compressed gases (most often CO2) in the critical temperature range. This method
combines two properties of a liquid as a solvent with behavior of gases in flow. In the
extraction process, modificators are added to the extraction fluid (methanol, ethanol and
others) in order to increase the polarity of the fluid in the supercritical state. This
method is often combined with sorption on solid sorbents, which has a few advantages,
e.g. it requires small amounts of solvents, it has a short extraction time (ca. 1.5 h.), ease
of automation and high selectiveness. As this method of extraction is commonly used, it
limits the high cost of equipment and servicing.
by using microwaves. The process occurs by taking advantage of the phenomenon of
microwave energy absorption by chemical compound molecules. Energy proportional to
the dielectric constant value causes the rotation of dipoles in the electric field (as a rule,
devices are used that generate radiation at a frequency of 2.45 GHz). For extraction,
bombs are used that are made of teflon, quartz or composite materials. As solvents, the
most often used are CH2Cl2 or acetone/hexane. Extraction can be carried out in one of
two ways. The first way consists of using a solvent that absorbs microwaves (of a high
dielectric constant). Microwave radiation causes the solvent to be heated to a
temperature higher than the boiling point thanks to the high pressure present inside the
bomb. The heated solvent allows for a quick extraction of analytes from the matrix.
This way of conducting the process is used for extraction of policyclic aromatic
hydrocarbons, organochlorine pesticides, polichlorinated biphenyls from soils and
bottom residues. The second way of conducting extraction consists of using a solvent
that does not absorb microwaves (with a small dielectric constant). The sample and
solvent can be placed in a closed or open tank. Under the influence of radiation activity,
the solvent does not undergo heating as it does not absorb microwave energy. The
sample, usually containing water or other compounds with a high dielectric constant,
absorbs microwaves and releases heat to a cold solvent, selected in a way that ensures
sample dissolution. It is a method more gentle in comparison to the previous one; it can
be used for extracting thermolabile compounds, e.g. organochloride compounds.
Extraction with use of microwaves allows for quick and effective acquisition of analytes
from the matrix (time of extraction lasts ca. 15-30 minutes).
29
as accelerated extraction with a solvent. It occurs at an increased temperature (up to
200C) and pressure (up to 20 MPa), ensuring the liquid state of the solvent in given
conditions. After this stage, usually lasting only a few minutes, the extraction bowl is
washed with a pure solvent and then leached with nitrogen. The whole process lasts
several minutes. Its advantages include small use of solvent, possibility of automation,
simplicity of execution and high repeatability. Its disadvantages include degradation of
thermolabile compounds and loss of easily volatile compounds. This method found use
for extraction of organochloride herbicides and pesticides from the soil.
Extract enrichment
The next stage of sample preparation is the evaporation (and/or replacement of) the
solvent. Among the many known methods of extract enrichment, one can distinguish:
evaporation of the solvent in a vacuum evaporator. It is a quick and commonly used
method, though not without its drawbacks, such as, e.g. the possibility of losing analytes
as a result of co-distillation (distilling analytes together with the solvent) and
overheating of sample, and also adsorption of analytes on the instruments walls;
evaporation of the solvent in the Kudemy-Danish apparatus. Thanks to this technique, it
is possible to reduce the volume to 1 cm3 and, compared with evaporation in a vacuum
evaporator, to achieve higher analyte retrieval rate;
evaporation of the solvent in a gas stream (nitrogen or air). The advantages of this
method include simple equipment and low cost. The disadvantage is the danger of
losing the sample as a result of nebulization. With this method, it is possible to
evaporate high-boiling solutions. A gas of very high purity is necessary for evaporation.
2.2. Assay of MCPA remains in the soil
2.2.1. Qualitative assay with the SPME-GC method
Performing the assay:
1. Place 200 mg of sample soil in the flue. Thermostate the sample at 40C.
2. Carefully place the needle over the sample by puncturing the rubber septum, and then
remove the fibre.
3. Conduct exposition for 30 minutes, then slide in the fibre and remove the needle from over
30
the sample.
Fig. 13. Device for microextraction: a) general scheme of the instrument for sample acquisition,
b) scheme of a needle end during puncturing through the rubber membrane, c) scheme of
needle end during sample acquisition (sorption) or desorption of analytes in a chromatograph
dispenser.
1-adsorption fiber, possibly covered with a layer of sorption liquid, 2-steel wire, 3-
microsyringe needle, 4-microsyringe body, 5-microsyringe piston, 6-bowl with test sample
(closed with a cork with a rubber membrane), 7-magnetic stirrer
4. Chromatographic analysis:
Part I: Preparation of GC/MS acquisition method
GC/MS methods can be created and edited with the help of the Method Builder application.
Open the *.mth file and, according to the lecturers instructions, write the defined conditions
in which the GC-MS analyses will be conducted.
Part II: Performing the analyses:
1. Load the *.mth program into the computer steering the GC-MS system, according to the
lecturers instructions, in order to conduct GC-MS analysis. Next, when the apparatus shows
readiness (glowing diode Ready on the chromatograph), perform an injection according to the
lecturers instructions.
Once the analysis is complete, write down the analysis number displayed on the monitor and
save the file containing the analysis results onto a storage device.
Data processing:
1. Load the file with GC-MS analysis results. Using the MS Data Review application, conduct
qualitative analysis and write down the analysis results in tabular form.
Qualitative analysis is possible thanks to the NIST08 library, which is an integral part of the
system. On the displayed chromatogram, select the first peak using the left mouse button in
order to increase its size. Next, click on the tab Chromatogram/Set Spectra and select 3.
Under the chromatograph, there will be a MS spectrum, i.e. a mean of the three measure
31
points.
Click with the right mouse button on the spectrum and select Library Search Spectrum A. It
will display the library search result.
Increase the size of the search result window. The fourth column describes the probability that
the experiment spectrum matches the spectrum that is present in the library. Repeat the search
procedure (after closing the library window by clicking on the gray X button) in a different
place on the peak, so that the result has the highest probability factor.
2.2.2. Quantitative assay with GC-MS method
1. Inject the calibration solution containing the amount of analyte that corresponds with the
100mg/kg concentration in soil.
2. Conduct analyte extraction from the test sample according to the instruction:
For the extraction of MCPA 2-ethylhexyl ester from soil, 10g of soil samples were treated
with 10 ml acetone for organic phase extraction for 1 hour. The extract was separated by
filtration through a filter of medium density from Alchem, and residues were extracted
again with 5 ml of acetone for 1 hour. Then, after the second filtration both extracts were
mixed together. Obtained extract was placed in a separator flask and 50 ml of distilled
32
H2O, 3 ml of saturated NaCl solution and 7 ml of methylene chloride were added.
Mixture was shaken for 5 minutes. After separation of phases, another 7 ml portion of
fresh methylene chloride was added to the aqueous layer, and the shaking process was
repeated. The organic phases were combined and evaporated in a vacuum evaporator at
60C at pressure of 0.4 bar. The residue was dissolved in 2 ml of acetone and dried over
anhydrous sodium sulfate (VI). The resulting solution was injected to GC-MS analysis.
Linear response of detector was examined by analysis of several standards with different
concentration. Calibration curves from the peak areas were linear with high coefficient of
more than 0.99 at the range from 0-0.1 mg/kg for MCPA 2-ethylhexyl ester. Repeat the
analysis three times.
3. Conduct integration of peak surface areas (after prior identification, see point: Data
processing) in calibration injection and determine the slope of calibration curve.
4. Conduct integration of peak surface areas and correlate them with calibration injection in
order to determine analyte concentration in the sample. Provide the result as an arithmetic
mean of the three measures standard deviations.
33
3. BIOMONITORING
The idea of using live organisms, or their populations, to record and evaluate certain
characteristic traits of the environment is based on the notion that there exists equilibrium
between environmental factors and living requirements of various live organisms species. The
historical data confirm the relation between certain plant species and the presence of ores in
the ground, soil fertility evaluation, or later on between evaluation of air pollution and the
presence of lichen. Nowadays, use of living organisms for these purposes is widespread and
routine, and the latest trends in researching the quality of natural environment only widen
their use. This is related with avoiding costly laboratory techniques of chemical analysis and
relying on simple tests (which are possible thanks to advanced biotechnology) conducted in
the field for specific indicator species. On the other hand, there is a growing number of
analyses being conducted in an increasingly shorter time. This has led to increased
significance of quick miniaturized toxicity tests called microbiotests, or alternative tests.
Microbiotests work on the basis of single-celled organisms or small multi-celled ones,
which, as a result of contact with liquid sample, exhibit a specific response. Due to their
numerous advantages, alternative tests are used most often in the form of biotest battery based
on using organisms belonging to various trophic levels. The increase in significance of these
kinds of tests is caused by such parameters as elimination of the need for maintaining a lab
culture, low cost per sample analysis, short response time, low requirements regarding the
sample volume and laboratory space, possibility of field use. The role of these kinds of tests
will certainly continue to increase.
3.1. Biotests
Biotest (gr. bios - life + lat. testari - to testify) can be defined as an experimental
biological trail that aims to show the presence of toxic substances in the environment or
discover their harmfulness through quantitative estimation of the influence of a given
substance on a living organism (on the basis of comparison with the control sample). There
are three main methods of conducting research through biotesting:
toxicity tests realized in laboratory conditions during which the toxic substance is
artificially introduced into clean water or residue,
toxicity tests conducted in laboratory conditions on the basis of obtained real samples
(water, soil, residues),
test conducted in situ, with use of populations living in natural conditions.
Biotests used in analytical practice can also be classified by organism type, constituting
34
the tests active element. Most often used are plants, bacteria and animal organisms.
One of the more common tests utilizing bacteria uses the bioluminescence of sea bacteria
Vibrio fisheri in order to evaluate the level of pollution in water, bottom residues and soils. It
is a simple, easy to use test and also serves as a basis of operation of automatic analyzers.
In the case of toxicity tests that use plants (phytotests) as an active element, these plants
are usually algae (chlorophyta, cyanophyta, diatoms), aquatic lemna and rooted aquatic and
land macrophytes (plant and its seeds). They represent organisms of particular significance for
their natural habitats: they provide oxygen, ensure course of organic substances, control water
quality and equilibrium of soil and bottom residues. Vascular plants are used mainly in testing
against pesticides, rarely against heavy metals. Much wider use was found for algae as
bioindicators in situ in controlling water quality, due to their ability of accumulating chemical
compounds and heavy metals, such as copper, manganese, nickel. The most often used plants
are microalgae, belonging to chlorophyta, of species Selenastrum capricornutum and
Scenedesmus quadricauda. Algae are immobilized on special medium or assayed in flow
cytometers. Plant organisms are used much more rarely than animal ones and, e.g. according
to AQUIRE data base, they constitute only 10% of conducted acute toxicity tests.
Among animal organisms used for environment evaluation, the dominating ones are land
animals, and among those - invertebrates. Common application of invertebrates was found for
three ecotoxicological tests - namely earthworms, snails and bees. Bees are used as
representative land invertebrates, mainly for evaluating danger caused by pesticides. Tests on
earthworms are widely used and standardized. OECD recommendations on this matter have
the best methodical background and in some countries are a legal requirement prior to
introducing new chemical compounds into the environment. This usually includes
toxicological tests of acute toxicity. Use of earthworms for toxicological tests has a few
advantages, including: the animals are widespread and common in the environment, they have
a short life cycle, they are characterized by a high reproduction rate and they are easy to
cultivate in laboratory conditions. These tests use model soil. The recommended species of
earthworms for acute toxicity testing is, according to OECD recommendations, Eisenia
foetida. Specimens used for tests should be adult, i.e. have clitellum, specimen weight should
be within 300-600g for an earthworm. This test determines the acute toxicity level of
substances entering an earthworms body. After comparing the results from a toxicity test that
uses artificial soil with tests conducted on natural soil, it can be observed that toxicity in
artificial soil is much larger compared to toxicity in natural soils. This difference stems from
the fact that chemical substances in artificial soil are much more available for earthworms.
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The methodical problem of the standard OECD test is that during the experiment, animals do
not receive food and their body mass tends to become reduced. The latest tests include
feeding animals and/or the time of experiment is lengthened to make reproduction possible.
Reproduction rate can easily be determined by counting the number of laid cocoons. Influence
on reproduction is detected with lower concentrations of xenobiotic. A characteristic trait of
annelids and snails, useful in ecotoxicology, is the presence of metallothionein - proteins
binding metals which cause metals to accumulate in an animals body above the background
level. This trait is commonly used for bioindication as heavy metal contamination in an
earthworms body is easily determined by chemical analytics. Moreover, this trait has become
the basis for a test measuring the organisms condition with a simple parameter - neutral red
retention time in lysosomes of coelomic cells.
Tests on springtails are not as popular as tests on earthworms, but much research has been
conducted by using these invertebrates for evaluating the influence of contaminated soil on
beneficial soil arthropods. The species that is mainly used for these tests is Folsomia candida.
Another invertebrate that is commonly used in laboratories are onsicus. Just as is the case
with springtails, there is no ready-made normative assay for them. The disadvantage of using
this animal species for testing is their slow growth rate and long reproduction time in
comparison to earthworms.
In aquatic systems, the animals that are most commonly used for bioindication are
bivalvia Mytilus edulis, which is reflected in the widely developed monitoring programs of
supraregional significance. It is related to their particular role as biofiltrators in the
environment, resulting in accumulation of heavy metals, PAHs, PCBs, and others. Mytilus
edulis can be a source of research samples for most chemical analytical techniques used in
laboratories, many markers are used for them. Another aquatic organism useful toxicity
testing is daphnia Damphnia magna. The reproductive test carried out with daphnias is
commonly known, but it does not have such wide significance as is the case with bivalvias.
For residue evaluation, use is made of carinaria Rhephoxynius abronius. The most often
measured parameter is survival rate.
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3.2. Measure of neutral red retention time as a test showing the influence of soil
pollutants on earthworm Eisenia fetida
Dyeing with neutral red is one of the dye tests that assesses survival rate, has a long
tradition of use and consists of using small concentrations of dye selectively collected in
certain cell parts as lysosomes or vacuoles. This test is derived from mammal cell cultivation,
later on it was adjusted for ecotoxicological research in aquatic systems on fish and molluscs
(mainly Mytilus edulis). Transfers on earthworms were done by Svendsen and Weeks in 1996
when they used that to evaluate soil contamination after the fire in a plastic factory in
Thetford. It was a very good move as it paved the way for a very useful discipline - soil
contamination testing. The choice of test organism was also highly appropriate as earthworms
have, for many decades, been considered the perfect indicator organism of soil state, a fact
that was reflected, among others, during toxicity tests. The above-mentioned fire caused the
pyrolysis of plastic and formed a mixture of many toxic organic compounds and of released
heavy metals such as Pb, Sb, Zn, Cd, Cr contaminating the environment, including the soil.
The wide range of pollutants formed in the soil required the use of the right biomarker that
would indicate the integrated effect of influence of all pyrolysis products. This is why the use
of a non-specific biomarker was justified, such as measuring the integrity of lysosomal
membranes. The first test organisms used for research were earthworms living in
contaminated soil (Lumbricus castaneus). Samples were taken 0; 20; 60; 140 and 200 meters
away from exposure area and also from 3 kilometers away for use as control area. The
earthworms were transported together with soil samples to the laboratory where neutral red
retention time was determined for coelomocytes, i.e. cells acting as leukocytes taken from an
earthworms coelom. Results were compared with heavy metal content in animal bodies and
in soil, achieving high correlation. Use of coelomocytes was another excellent choice -
coelomocytes, cells suspended in coelomic fluid filling the space between the intestine and
the skin-muscle sac of an earthworm, are the basic element of an animals resistance system.
In natural conditions, earthworms are exposed to losing coelomic fluid together with
coelomocytes and dissolved substances, as it is ejected through pores in the back part of the
body in specimens irritated by chemical or mechanical factors. In experimental conditions,
such ejection of coelomocytes can be provoked by ethanol activity or a weak electric current
(5V). Coelomocytes can respond to environment contamination with intra-cellular changes,
including the number, size and shape of cells. Not without significance is also the ability to
easily take these cells for research without killing the animal.
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Release of the publication describing this research was the beginning of a cycle of works
dedicated to use of neutral red retention of coelomocytes in earthworms of various species for
measuring response to the presence of heavy metals, polyaromatic hydrocarbons and
pesticides. Exposure was conducted in the laboratory and in the field. Use of this test, which
is based on measuring neutral red retention time, has become commonplace and was
described in review work regarding aquatic as well as land systems.
When researching the influence of soil contaminants on earthworms by use of the neutral
red test, the natural environment can provide samples of earthworms that are representative
for the given area. Another method is field exposure of a chosen earthworm species in
specially constructed field-mesocosms that prevent the animals from escaping or laboratory
exposure of earthworms to soil acquired from the field. In each case, the test (acquiring
coelomocytes and determining neutral red retention time) must be conducted in the
laboratory.
In order to present the method for determining neutral red retention time, one can use
model conditions in which the chosen test organism is Eisenia fetida, a species that has a long
tradition of use in ecotoxicology.
Use of this test is related with conducting other observations, such as:
counting animals pre- and post-exposure, visual evaluation of their condition,
weighing animals pre- and post-exposure (despite the fact that individual specimens are
not assayed, it is possible to obtain information describing the sets, such as mean SD),
counting coelomic cells ejected by research specimens,
counting cocoons post-exposure
CONDUCTING THE EXERCISE
Experimental material
Adult, clitellum-possesing specimens of earthworm Eisenia fetida from the university
culture are exposed to contaminated soil taken from the vicinity of the bunker used for storing
pesticide and also to non-contaminated soil (control).
Exposure of earthworms to researched soil
Conduct toxicity tests using a photoperiod of 12L:12D. For exposure of earthworms to
contaminated soil, use plastic food containers 19x13x8 with a perforated covering. Before
putting earthworms into the container, wash them under running water, dry them on filtration
paper and weigh them. Next, put 10 Eisenia fetida each per container containing 600 g of
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researched soil. Place food on research soil each week in the form of 6 g of over fermented
horse manure, wetted in 20 ml of water. Sprinkle the soils surface with clean drinking water.
After 21 days of exposure:
count surviving specimens, evaluate their physical condition,
count cocoons laid by earthworms and observe occurrence of young specimens,
weigh each specimen.
Take coelomocytes from specimens that survived exposure in order to determine neutral
red retention time and counting of amebocytes and eleocytes.
Releasing coelomocytes
Before releasing coelomocytes, wash earthworms under running water, unmoisten on
crepe paper, and then weigh them. In order to acquire coelomocytes from earthworms, use the
non-invasive method which consists of ejecting coelomic fluid through body pores when
irritated with an electric current from a battery of 4 V voltage. Place clean earthworms on
a Petri dish containing 3 ml of solution for coelomocyte suspension, prepared from PBS and
2 g/L EDTA. Such a prepared solution allows for long review of coelomocyte preparations
under the microscope and prevents cells from gluing together into aggregates. An earthworm
must be irritated with an electric current a few times throughout the span of 1 minute in order
to fully complete ejection. Immediately after acquiring coelomocytes, transfer them onto
microscope slides for neutral red testing and to Brkers hemocytometer.
Counting eleocytes and amebocytes
Use Brkers chamber to count the number of coelomic cells. Introduce into the chamber,
using a pipette, 10 l of solution containing coelomocytes. The grid can be viewed under the
microscope in 10 large squares, each with a side of 0.2 mm. Count cells lying inside the
square with an edge of 1/5 mm, and also on upper and left border lines, do not count cells
present on bottom and right border lines to avoid counting the same cells twice. It might be
practical to make photos of the preparation and later use the photos for counting at a suitable
time. Count eleocytes and amebocytes separately.
Calculate the number of amebocytes and eleocytes acquired from individual specimens
according to the formula:
Number of cells from a single specimen = a 250 3 1000
where: a - average number of cells found in a single square;
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250, 1000 - conversion for 1 ml of volume;
3 - volume of solution used for suspending coelomocytes on a Petris dish, onto which
the coelomic fluid was ejected [ml].
Fig. 14. Grid from a Brkers hemocytometer with visible coelomic cells: amebocytes (1) and
eleocytes (2); source: own work
Conducting the lysosomal membrane integrity test in an earthworms coelomocytes
Preparing the neutral red dye solution:
Following the methodology prepared by: Svendsen et al. 1996; Weeks and Svendsen
1996, Weeks and Svendsen 1997 with minor modifications: dissolve 20 mg of neutral red
(SIGMA) in 1 ml of dimethylsiloxane (DMSO), then dissolve 10 l of the resultant solution
in 2.5 ml of solution for coelomocyte suspension, which will create a working solution of
neutral red at a concentration of 80 g/ml.
Preparing microscope preparation
Using a semi-automatic pipette, take from the Petris dish a representative sample of 10 l
of solution containing coelomocytes and transfer it onto microscope slide. After 30 seconds,
during which the coelomocytes undergo sedimentation, add 10 l of working solution of
neutral red onto the slide, achieving the final dye concentration of 40g/ml, next put on cover
slip. Transfer the preparation to the dessicator containing water in order to protect the
preparation from drying. Prepare further preparations in the same way. At this stage, the dye
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is absorbed by the lysosomes of the coelomocytes.
Determining retention time
Take out microscope preparations from the hydrostatic chamber and view them under the
optical microscope using 640x zoom, do this repeatedly (in intervals, every few minutes). The
microscopes field of view will show two types of cells: darker, more visible and numerous
ones - eleocytes and light, staining ones - amebocytes. When viewing the preparations, choose
a few spots in which amebocytes are well visible. Count the cells that were observed to have
released dye from lysosomes to cytozole and those not stained. Stop observations when the
number of cells with stained cytozole amount to 50% in relation to non-stained cells. The time
from the moment of adding the working dye onto the microscope preparation until the
moment of terminating observation is treated as the neutral red retention time in the
lysosomes of the coelomocytes. Observations can be made more efficient by taking photos of
the preparation for later counting at a suitable time. Below are example photos of
coelomocytes during test.
Fig. 15. Coelomocytes Eisenia fetida during the neutral red test. The green circle shows non-stained
amebocytes, black cells are eleocytes (optical microscope 640x); source: own work
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Fig. 16. Coelomocytes Eisenia fetida during the neutral red test. Red circles show amebocytes with
non-stained cytosoles, dark cells are eleocytes (optical microscope 640x); source: own work
Read the retention time for each preparation for each of the studied specimens. Calculate
mean SD.
Put in tabular form all obtained data for the control sample and