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Determination of soils' and sediments' toxicity with Heterocypris incongruens assay

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EPM: Environmental Chemistry

Laboratory Instruction

Gdańsk, 2015/2016


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Passing requirements: it is obligatory to fully participate in the exercise, no late-coming will

be considered as an excuse. During the laboratory each student will catch 3-5 living

organisms (depending on Tutor’s instructions) and measure their length, these values will be

checked by Tutor. Error > 20% will be considered as failure to pass the laboratory. After the

classes students will receive “xls” file with toxicological data for real soil/sediment sample

and concluding on possible reason of ecotoxicological will have to be done as a part of

laboratory report. The individual report (in pdf form) must be given to the Tutor within 7 days

after the classes, also within 1 week the written tests will be performed individually for each

student. The theoretical definitions below are only introductory guidelines, more detailed

information will be presented at the beginning of laboratory classes. Delay in delivering the

report or writing the tests > 14 days will be considered as not passing the laboratory.

1. Theoretical background

Sources and environmental fate of pollutants

Environment is a very complex system divided into biotic (living) and abiotic (non-

living) part, among which there is a continuous exchange of matter and energy. These

processes should remain in balance called homeostasis. The sensitive balance between biotic

and abiotic part may be disrupt by chemicals introduced into environment, which are derived

from natural or anthropogenic sources. Through the years of evolution plant and animal

organisms have developed number of defense mechanisms against different natural pollution.

“Champions” in this race are plants, which have developed many defense mechanisms to

survive due to the lack of mobility. Changes under the influence of anthropogenic stress can

have negative effects on the organisms inhabiting a given environment. Every day water, soil

and air are “attacked” by hazardous chemicals coming from different industrial branches,

some of them undergo any legal regulations because these substances have not yet been

identified or studied. Fig. 1. shows the distribution of pollution in terms of their origin and

due to the legal regulation. Bold examples of sources of pollutants are classified as a natural

environment pollution group, as can be seen in Fig. 1., they are a minority among the

pollutants of anthropogenic origin. Virtually all industries cause environmental pollution, but

some of them are especially famous for significant impact. Among them one can mention:

petrochemical industry,

mining of precious metals and stones,

tanneries,

lead battery industry,

industrial discharges and/or municipal.

Other manifestations of human activity such as: motorization, housing, agriculture,

sewage and municipal wastes are also not without significance. Negative effects can be cause

by both organic and inorganic compounds, those that are synthesized by man deliberately or

formed as a by-product, such as: PAHs (polycyclic aromatic hydrocarbons), PCBs

(polychlorinated biphenyls), organochlorine pesticides OCPs, BPA (bisphenol A), PCDDs

(polychlorinated dibenzo - paradioxins), PCDFs (polychlorinated dibenzofurans), PBDEs

(polybrominated diphenyl ethers), NOx,(mixture of nitrogen oxides NO and NO2) SOx

(mixture of sulfur oxides SO2 and SO3) and heavy metals. Tab. 1. summarizes information

concerning compounds considered to be most environmentally burdensome. Part of these

hazardous compounds has been removed from use years ago, but as may be noted by

analyzing the data from Tab. 1. their content in the environment is still considerable.


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Chemical compounds characterized by toxic properties, that easily undergo

bioaccumulation processes, are resistant to biodegradation and have high transport potential

are called persistent organic pollutants (POPs). Due to lipophilic and hydrophobic nature of

POPs, they accumulate mainly in sediments and soils. Half - life of POPs may vary from

years or even decades in soils/sediments in contrast to several days half - life in atmosphere.

More than a decade ago, scientists recognized the problem which may create POPs therefore,

those compounds become a main topic of the Stockholm Convention on Persistent Organic

Pollutants signed in 2001.

Environmental pollution does not respect boundaries and under favorable conditions it

may be transmitted over long distances and “travel” throughout the biosphere. Study of the

Polar Regions show that pollutants may be transmitted over long distance. Polar Regions and

its specific environment are ideal to study transport of pollutants, because all the sources of

emission are distant urban areas.

NATURAL SOURCES ANTHROPOGENIC

SOURCES

REGULATED

POLLUTANTS

NON-REGULATED

POLLUTANS

NON-IDENTIFIED POLLUTANTAS NEW EMERGING POLLUTANTS

AREA SOURCE

POINT SOURCE

LINE SOURCE

ENVIRONMENTAL POLLUTION

VOLCANOES

WASTE WATER PLANTS

AGRICULTURE

RAIL TRACKS

ROADS

URBAN AREAS SEWERS

NON-POINT SOURCE

FOREST FIRES

CHIMNEYS OF INDUSTRIAL PLANTS

Fig. 1. Classification of environmental pollution due to the their sources and legal regulations


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Tab. 1. Information about groups of chemicals showing special environmental arduousness.

Group of

compounds

TOXICITY

Concentrations Sample type Toxic effect

∑ PAHs 0.69-32.73 [ng/m3] Air sample, Brazil,

Argentina

Genotoxicity, carcinogenesis,

teratogenicity, cytotoxicity,

mutagenic

∑ PCBs 0.0039-0.0365 [ng/g]

Soil and water

samples, river Chao,

China

Endocrine disrupting properties,

cancerogenesis, immunotoxicity,

hemotoxicity, infertility

∑ PBDEs 6.3-26050 [ng/L] River water samples,

river Aire, UK Endocrine disrupting properties

PBA 82-292 [ng/L] River samples,

Aisonas river, Greece


cancerogenesis

∑ PCDD 162-3450 [pg/g] Soil samples, Hong

Kong, China

Immunotoxicity, neurotoxicity,

hepatoxicity, mutagenic

cancerogenesis

∑ PCDF 3-213 [pg/g] Soil samples, Hong

Kong, China mutagenic, cancerogenesis

∑ OCPs 5-180 [ng/g] Soil samples,

Shouguang, China


immunotoxicity, neurotoxicity,

infertility, cancerogenesis

∑ NOx 110-150 [µg/m3] Seville metropolita,

Iberian Peninsula, Spain Acute toxicity, cytotoxicity,

cytostatic, genotoxicity

∑ SOx Causes lipid peroxidation processes in organ tissues

Heavy

metals

∑ Hg LOD-90 [mg/kg]

River s and lakes in

the world

Acute toxicity, nephrotoxicity,

cardiotoxicity, cytotoxicity

∑ Cu 1.33-924.54 [mg/kg]

∑ Cd LOD-81.79 [mg/kg]

∑ As LOD-83 [mg/kg]

∑ Pb LOD-5778.1

[mg/kg]

In the scientific terminology any movement of pollutants on a global scale is known as

long-range transportation. Pollutants may be transferred by different environmental

components like water and air or by living organisms. Water and air act as a transport

medium, however transport by living organisms it strongly depends on the migratory species.

In the environment chemical compounds undergo a number of processes depending on

their physicochemical properties. Hydrophilic substances remain dissolved in water and

hydrophobic substances accumulate in soil or/and sediment. Chemicals may be partially

bioaccumulated by living organisms. Bioaccumulation is a phenomenon in which living

organisms uptake substances from the environment, when consumption and the amount of

xenobiotic in different tissues become greater, than the ability of the body to its removal.

Bioaccumulation is a general term and applies to accumulation of pollutants from the soil,

water and air. More specific term is bioconcentration, which refers only to accumulation of

pollutants dissolved in water. Bioconcentration is usually defined by the ratio of chemical

concentration in organism to concentration in the water and is described by the

bioconcentration factor (BCF) shown in eq. (1).

(1) enviromentintoxinofionoConcentrat

organismintoxinofionConcentratBCF


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Another term that often appears in the literature is biomagnification. In this case,

accumulation of toxic substances increases along the food chain. Chemical substance,

depending on the physicochemical properties may be treated by the body in din two ways.

Hydrophilic compounds are filtered by the kidneys and excreted in the urine, and those

characterizing by lipophilic properties are metabolized by the liver, from where they are

excreted in the urine or faeces. Chemical compounds that have a high tendency to

bioaccumulate and bioconcentrate usually are characterized by high values of octanol-water

partition.

Fig. 2. shows the pathways of xenobiotics entrance from environment to the

different levels of the food chain together with an indication of bioaccumulation,

bioconcentration and biomagnification. People as the last link in the food chain are

particularly vulnerable to the adverse effects of accumulated toxins.

The most common challenges and problems of modern analytics are associated with

the step of sample preparation, which can take up to 80% of the time spent on analysis and

determination. Other issues that need to be taken into account (solved) leading indications are:

• the amount of analyte which must be determined in the sample,

• complex matrix sample (presence of interfering substances);

• analytes occurring at various concentration levels;

• the number and cost of reagents consumed by a single analysis;

• time-consuming multi-step sample preparation procedure.

In order to check the usefulness of the analytical method, it needs to be validated and

optimized by determining parameters such as accuracy, sensitivity, reproducibility, simplicity,

cost effectiveness, flexibility and speed however, none of the parameters do not contribute to

reducing the environmental burden of the method. At this point should be considered

targeting not only “dry” validation parameters but also the principles of green chemistry. The

recognition of green analytical chemistry as part of both green chemistry and analytical

chemistry seems obvious therefore.

XENOBIOTIC IN ENVIRONMENT

SOIL WATER AIR

PLANTS

HUMANS

BIO

MA

GN

IFIC

ATI

ON

ALO

NG

TH

E

FOO

D C

HA

IN

BIO

CO

NC

ENTR

ATI

ON

ANIMALS

BIO

AC

CU

MU

LATI

ON

IN T

ISSU

ES O

F

LIV

ING

OR

GA

NIS

MS

Fig. 2. Environmental fate of pollutants


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Not always determination of continuously decreasing concentration levels makes

sense because one have to consider environmental samples as a mixture of different chemical

compounds, which not remain neutral to each other. There are three fundamental interactions,

which can occur between chemicals:

additivity occurs, when the total effect, which can produce chemical compounds

present in sample is a sum of the effects of compounds separately.

synergism is potentiating of the effects of the substance, the total effect is greater

than the effects produced by the individual compounds alone.

anatgonism means decreasing of total effect, the total effect is smaller than the

effects caused by the individual compounds alone.

Interactions between the compounds may result in decrease or increase in toxicity

against the exposed organisms inhabiting a given ecosystem therefore, substances even at low

levels of the concentration can cause adverse reactions. In such cases classical analytical

analysis made even by applying principles of green chemistry is not enough, chemical

determination should be supplemented by tools from biological field.

Biological environmental monitoring can be carried out by a number of techniques and

methods and with the use of appropriate tools (see Fig. 3).

Biomarkers: each reaction to xenotoxins

provide by organism.

Distinguished by:

Biomarkers of susceptibility

Biomarkers of exposure

Biomarkers of effect

BIOMONITORING BIOANALYTICA

EVALUATION OF THE STATE OF THE

ENVIROMENT

Observation of changes in biota in ecosystems:

saprobic index;

biotic index.

Bioindicators, biomonitors:

observation of indicator organisms.

Bioassays: biological assays to

determine biological activity of the

sample.

Biosensors: device for determining

analytes, consisting of a biological and

transducer

Fig. 3. Tools of biological methods of estimating the state of the environment


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In situ biomonitoring involves observation of bioindicators, species of organisms

having a narrow range of ecological tolerance for xenobiotics, such organisms are called

stenobionts. Changes induced in the function, behavior or in the whole population of

bioandicators may indicate degradation of the ecosystem. Further information can be provide

by biomonitors, which not only reflect the quality of the environment but also levels of

concentration of given pollutants, at this point one can mention the famous lichen scale,

where the existence and diversity of species indicative quality of given ecosystem.

Environmental samples can also be collected and analyzed ex situ using biosensors or

bioassays. Biosensor is used for the determination of analytes, and it is a hybrid combination

of biological and electronic (transducer) parts. The biological element (tissue, cell receptor,

enzyme, antibodies, nucleic acids or microorganisms, organelles) produce biological response

to analyte or group of analytes. Response of biological part converted to an electrical signal,

which is a measure of the amount of the substance in the sample for example glucose

biosensors. Uniqueness of biosensors lies in the fact they can operate both in situ and ex situ,

and results can be obtained in real time. It seems that biomarkers can be the next step in the

ecotoxicology study. Observation of changes occurring at the cellular level whether it is the

emergence of specific chemical compounds, metabolites, enzymes or proteins gives

opportunity to rapid response. Bioassays do not contain any electronic parts and often visual

assessment is sufficient therefore, any equipment is required. Bioassays and its possible

contribution as green analytical methods, as an alternative to classical analytical methods will

be developed further in the chapter.

Toxicity, toxicology, ecotoxicology

In people's minds the word toxicity or toxin produces virtually negative associations. It

is difficult to disagree with the author of the book The poison paradox: chemicals as friends

and foes, that we live in a world where people do not have to worry no more about search for

food, drink and shelter therefore, reign something that can be called “poison’s paranoia”. On

the Internet, on television, in the press can be found various information about harmfulness of

all kinds’ chemical compounds often not verify. However, this does not mean, that the case of

environmental pollution should be trivialized, as the proverb says: “prevention is better than

cure” or rather in this case better to prevent than to overcome the effects of ecological

catastrophe.

Phillippus von Hohenheim (better known as Paracelsus) is considered to be the father

of toxicology and in the 16th

century he formulated his famous words “All things are poison

and nothing (is) without poison; only the dose makes that a thing is no poison” which can be

understood that the dose “makes”a toxin. Nowadays as a toxin are considered chemicals that

quickly and in small doses, cause the death of the organism and according to the definition

adopted from the United States for a poison are considered substances with lethal dose of 50

mg or less per kilogram of body weight.

There is no universal definition of toxicity, but it can be considered that toxicity is a

feature of chemical compounds that cause disturbance in the physiology of the organism,

which can result even in death. This definition may apply to the whole organism but also to

individual organs, tissues and cells. Toxicology as scientific discipline deals with the study

of toxic properties of chemical substances against living organisms. Living organisms absorb

both negative and positive effects of chemicals. The basis of toxicological studies is the dose-

response dependence however, the observed effect is a component of many factors like

species and individual differences, age and gender.

Chemicals present in the environment can affect the organisms in two ways: some

of those compounds present at high concentration levels produce immediate toxic effects, i.e.

acute toxicity, which can even lead to death of the organism but most of the environmental


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Fig. 4. Long-term effects can be caused by xenobioatics present in the environment.

pollutants occurs at low concentration levels, and can cause long-term exposure consequences

such as carcinogenesis, endocrine disruption or DNA damage (see Fig. 4.). Ecotoxicology

derives from toxicology, but there are differences between these two domains of science.

Toxicology focuses on living organisms and the effects of xenobiotics thereon, mainly on

uptake, propagation and metabolism of “poisons” in their systems. Ecotoxicology also deals

with the fate of chemical substances but in the context of their distribution in the air, water,

soil and sediments, as well on particular levels of the trophic chain. Moreover, ecotoxicology

takes into account potential chemical and biological transformation of chemicals and focuses

on contamination effects on the entire ecosystem, from the molecular and cellular level to the

organisms. Biotests (experimental biological assay intended to demonstrate the presence of

toxic substances in the environment or to establish the harmfulness thereof by estimating the

effects on a living organism (compared to control trial)) is the key tool for the assessment of

the biotoxicity.

The term “ecotoxicity testing” should be understood as an assessment of the effect of

substances or their mixtures and physical parameters on living organisms – the effect can be

favorable or negative. This statement is substantiated by observations made during this

study: some of the samples have had a favorable effect on the biotest organisms – such

phenomenon is called hormesis (test organisms were growing better in contact with the

sample that the control population). A favorable or negative effect of agents on living

organisms may appear shortly (acute response) or over a long time (chronic response) after

the first contact of the test organism with the sample tested. Therefore, it is recommended to

use different organisms in the tests (genetically modified bacteria that react to exposure after

several minutes – acute response, and crustaceans or higher plants, wherewith validated

procedures last several days – chronic response). Accordingly, a battery of biotests must be

prepared (a set of biotests comprising organisms that represent different trophic levels).

Moreover, if both solid and liquid samples are planned to be tested, it must be proposed to use

biotest organisms than live in open bodies of water and at the sediment-liquid interface. This

XENOBIOTICS

Cyto

toxicity

Mutagenocity

Ph

yto

toxi

city

Endocrine disrupting properties


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would provide a more in-depth insight into the effect of the tested samples on the test

organisms and, by the same, on the environment. If a sample is found to have a negative

effect on living organisms, ecotoxicological studies are expected to determine whether the

involved substances are adsorbed or absorbed by the matrix of a solid sample. Absorbed

substances are known to be easily extractable even with such mild extractants as water.

Therefore, solid samples have been extracted or diluted and doped with a sediment of

reference. Samples tested under this project were diversified in terms of consistency: liquid

and solid samples extracted with water, as well as dual phase samples. The science that deals with the study of xenobiotics in the environment is

ecotoxicology.

Ecotoxicology is a separate branch of toxicology with the area of interest oriented to:

the distribution and fate of xenotoxins in the biosphere,

fate and transport in living organisms including the chemical transformation

xenobiotics,

exposure of ecosystems as a result of the effects of pollution impacts.

Ecotoxicology covers the whole of “life cycle” of the substance toxic on biosphere by controlling

their movements ecosystems and between them.

Bioassays

Bioanalytics is a fast-growing branch of science, it began to take shape in the early

twentieth century, and since that time it began to be successfully used to monitor and evaluate

the quality of the environment. Initially for those purposes were used previously mentioned

bioindicators or biomonitors, subsequently scientists started to used bioassays. Bioassays not

only have the potential to determine the toxicity of the sample as well as qualitative and

quantitative evaluation, because there are bioassays specifically aimed at detection of the

specific groups of compounds. First described bioassay was based on marine bacteria Vibrio

fischeri and developed in 70’s, shortly thereafter were introduced tests based on animal and

plant species. Bioassays are currently available in kit form, which contains all of the

necessary reagents and accessories. Production of toxkits started in Belgium, test organisms

are supplied by producers in the cryptobiotic form i.e. in the form of cysts, crustaceans in the

egg cysts form, plants in the form of seeds and algae in the fluid carrier immobilized. In order

to perform the test organisms should hatch from the cryptobiotic form under appropriate

conditions according to the manufacturer. The most common tests are carried out on fish, uses

more than 150 species used as bioindicators by leading US agencies such as the USE PA (US

Environmental Protection Agency), APHA (American Public Health Association), but fish

farming requires a specialized laboratory. Among the invertebrates and crustaceans due

widespread occurrence in the environment, the most commonly used are Daphnia magna with

Daphnia pulex (DAPHTOXKIT F™), which play a very important role in the trophic chain

bridging the gap between producers and consumers of higher orders. The test organisms must

meet a number of requirements such as: they should be widely and easily available throughout

the year in large quantities, a little different in terms of genetics and not undergoing disease or

parasites, should be sensitive to a wide range of toxins and the observed response to the toxin

must be distinctive and reproducible.

Studies carried out for toxicity are to establish and define:

the risk of the immediate effects of exposure to the agent or agents;

determine whether there is a risk of long-term effects of exposure to a specific agent or

agents such as mutagenicity, genotoxicity, cytotoxicity, and others;

the dose or concentration causing toxic effect of a factor or substance.


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EC – effective concentration – the concentration of a substance (or of a mixture) that

induces observable changes in test organisms, e.g. immobilization, inhibition of biochemical

processes or growth. Test result is expressed as concentration that affects given physiological

process by 50% after a specific time of exposure – EC50 in relation to control one.

IC – inhibitory concentration (inhibiting the growth, chlorophyll generation, etc.) – the

concentration of a substance (or of a mixture) that in sub-lethal tests (where the organisms are

not killed) inhibits physical and biological activity of test organism by a specific percent in

relation to control one (e.g. IC25).

LC – lethal concentration – the concentration of a substance (or of a mixture) that is lethal to

a specified number (expressed as %) of population members after a specified time of

exposure, e.g. LC50, LC100 in relation to control one.

LOEC – lowest observed effect concentration – the lowest concentration of a substance (or

of a mixture) which (in a specified time of exposure) has an effect causing changes in test

organisms in relation to control ones.

NOEC – no observed effect concentration – the highest concentration of a substance (or of

a mixture) which (in a specified time of exposure) has no effects causing changes in test

organisms in relation to control ones.

chronic toxicity – adverse effects on test organisms from exposure to a chemical compound

(or to a mixture with relatively low concentrations) over a long period of time – normally,

1/10 of a life cycle until the first offspring generation is produced. Sublethal concentrations

are used in model tests. Changes in physiological activity, e.g. alimentary, reproductive

functions, genetic disorders and organ disturbances are observed and assessed.

acute toxicity – adverse effects on test organisms from exposure to a chemical compound (or

to a mixture with relatively high concentrations) that may lead to disturbances of

physiological activity and death after a short time of exposure.

2. Experimental procedure

Toxicity tests of solid and liquid samples include the determination of chronic toxicity

based on a “direct contact” test – Ostracodtoxkit FTM

(MicroBioTests Inc., Nazareth,

Belgium). Toxicity assay is based on two effects: inhibition of growth in tested organisms and

the determination of their mortality after contact with the sample. The procedure is shown in

Fig. 5. Specimens of Heterocypris incongruens hatched from cysts are selected for testing.

Fifty two hours before the beginning of the test, the cysts are placed on Petri dishes with 10

cm3 of a standard medium prepared using salt (NaHCO3, CaSO4, MgSO4, KCl) solutions

supplied with the testing set. The cysts are incubated at 25 °C in permanent light of 3000–

4000 lux. Freshly hatched specimens of Heterocypris incongruens are measured and

transferred with glass micro-pipette to multi-well test plates filled with a solution of living

algae and the sediment sample being tested. Subsequently, the plates are incubated in

darkness for 6 days at 25 °C. After that time the length of live ostracods is measured and

compared with that established at the beginning of the test. Moreover, dead animals are

counted in each well. Growth inhibition and mortality [%] are calculated by comparison with

specimens living in the culture of reference.


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Figure 5. Schematic presentation of Heterocypris incongruens sub-chronic toxicity

determination procedure.

Hatching of dormant Heterocypris incongruens cysts on Petri dish (48h, 25°C, continuous illumination

of 3000-4000 lux)

Pre-feeding for 4h of freshly hatched organisms after 48h (1vial of Spirulina powder)

Length measurement of freshly hatched Ostracods under microscope (15 organisms)

Preparing the test plates

CONTROL SAMPLES

Adding 1cm3 of reference sediment to

each well. Transfer of algal food

(Selenastrum capricornatum sp) suspension

to each well (2cm3). Transfer of distilled

water (2 cm3).

SAMPLES

Adding 1cm3 of reference sediment to each

well. Transfer of algal food (Selenastrum

capricornatum sp) suspension to each well

(2cm3). Transfer of samples (2 cm

3).

Transfer of 10 Heterocypris incongruens organisms to each well (under the microscope)

Sealing each plate with Parafilm® and covering with the lid

Running the test (25°C, darkness, 6 days)

Mortality scoring for test and control organisms and their growth measurement (Lugol solution

fixation, under microscope)

Data treatment and analysis


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3. Concluding remarks

As stated at the beginning, during the laboratory each student will catch 3-5 living

organisms (depending on Tutor’s instructions) and measure their length, these values will be

checked by Tutor. Error > 20% will be considered as failure to pass the laboratory. After the

classes students will receive “xls” file with toxicological data for real soil/sediment sample

and concluding on possible reason of ecotoxicological will have to be done as a part of

laboratory report. The individual report (in pdf form) must be given to the Tutor within 7 days

after the classes, also within 1 week the written tests will be performed individually for each

student. More detailed information will be presented at the beginning of laboratory classes.

Delay in delivering report or passing the test > 14 days will be considered as not passing the

laboratory.