USE OF CHLOROPHYLL FLUORESCENCE IN PHYTOPLANKTON … · 2018-09-14 · Zemri Khalida1, Boutiba zitouni2, Zemri mohammed3 & Popovic Radovan4 1University of SIDI BEL ABBES, Department

IJRRAS 27 (3) ● June 2016 www.arpapress.com/Volumes/Vol27Issue3/IJRRAS_27_3_03.pdf

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USE OF CHLOROPHYLL FLUORESCENCE IN PHYTOPLANKTON

"LEMNA GIBBA" AS BIOASSAYS TO DETECT THE TOXICITY OF

HERBICIDES "ATRAZINE"

Zemri Khalida1, Boutiba zitouni2, Zemri mohammed3 & Popovic Radovan4

1University of SIDI BEL ABBES, Department of Biology,

Laboratory of materials and systems reagents "LMSR" 2University of Oran, laboratory marine biology, Department of Environmental

3University of laval, Faculty of science and agriculture and food Sciences 4University of Quebec at Montreal, Department of Chemistry-TOXEN, C. P.,

Station Downtown, Montreal, QC H3C 3P8 Canada.

ABSTRACT

The activity of the photosynthetic apparatus represents a target for contaminants. In ecotoxicology, the easiest and

economical way which provides information on the presence and toxicity level of a contaminant that is bioassays

and biochemical analyzes. Bioassays represent a simple and economical approach but not sensitive and requires a

long followed by biochemical analyzes it time is faster and they have high sensitivity but it is costly and complex. In

this study, we used chlorophyll fluorescence to evaluate the effect of Atrazine on photosynthesis in Lemna Gibba

studying the mechanism of toxicity on the synthesis of chlorophyll and the transfer of electrons between

photosystem II and I. parallel, we tested the toxicity of Atrazine using a standardized bioassay fish. The results

obtained with experimental trials, show that Atrazine is very harmful to aquatic life, however, the most remarkable

observation relates to the use of chlorophyll fluorescence method which is still developing. This was used to assess

the degree of toxicity of the photosynthetic apparatus, while highlighting the site of action of this pollutant on the

mechanism of photosynthesis. Indeed, this new method is easy, reliable, and fast and can be used to develop

sensitive biomarkers to photosynthetic toxicity of xenobiotics.

Keywords: Bioassays, Chlorophylls Fluorescence, Photosynthesis, Pesticides, Atrazine, Lemna gibba,

Environmental toxicity, Bioassays of reproduction of the "Fathead minnows"

LIST OF THE ABBREVIATIONS

Fm for a maximum fluorescence in dark-adapted plant

Fm’ Fluorescence maximum for a plant adapted in the light

Fo 'Constant for Fo fluorescence in dark-adapted plant

Fo’ 'Fluorescence constant for a plant adapted in the light

Fv variable Fluorescence for a plant in dark-adapted

Fv’ variable fluorescence to a plant adapted to light

L A The actinic light

L M The modulated light

L S The light saturation

LHCI & LHCII: Light Harvesting Complexes of PSI and PSII

ML Modulated light

SL Saturating light

Ms Millisecond

NADPH: Nicotinamide adenine dinucleotide phosphate

O-J-I-D-P-S-M-T: Transitions of the rapid kinetics of fluorescence

P680 Reaction center of PSII

PAM Pulse Amplitude Modulation

PC Plastocyanine

PEA Plant Efficiency Analyser

Pheo Pheophytine a

PQ Plastoquinone

PQH2 Plastoquinol

PSI et PSII Photosystems I et II

IJRRAS 27 (3) ● June 2016 Zemri et al. ● Bioassays to Detect the Toxicity of Herbicides

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QA Primary electron acceptor of PSII

RL Red distant

'M Operational photochemical efficiency of PSII

M PSII photochemical efficiency

CF Index: Condition factor

GSI: Gonadosomatic

1. INTRODUCTION

All around the world, the industrialization, the urbanization and the profound transformation of the agriculture

drove, over the years, to a notable deterioration of the quality of the grounds, sediments and waters of lakes, rivers,

seas and oceans. With the aim of understanding better mechanisms implied in the degradation of the natural circles,

the search concerning the changes of the quality of the ecosystems became essential. The chemical or physical

analyses of the contaminated circles give an indication onto the present contaminants in the polluted circles, but do

not inform about their real effects on the ecosystems. On the other hand, the use of alive bodies as experimental

models, in the evaluation of the toxicity brings an ecological aspect. Indeed, the results obtained by means of

biological tests represent an integration of all the environmental factors.

The presence of pollutants in the aquatic environment such as pesticides establishes a big ecological threat. Aware

of these problems, many governments in the world legislated by issuing physicochemical standards and toxicity

threshold for several chemical compounds and various releases. Thus, many methods of sewage treatment and

control of wastewater is carried out before their deportation to the natural environment.

In this thesis project, we studied the mechanism of toxicity of a pesticide Atrazine on some plant organisms and

aquatic animals.

- Atrazine is a weed-killer which is in abundance in streams because of its intense use for the weeding in agriculture

[16][17].

The present study will test the effect of exposure to an aquatic plant Lemna gibba to this herbicide is Atrazine to

evaluate photochemical alteration of photosystem II. We will try to measure the degree of toxicity of Atrazine on the

photosynthetic apparatus of the above-mentioned aquatic species and as the photosynthetic apparatus represents a

significant target to these pollutants, so we will also try to see if our results could help

us develop photosynthetic bioassays for the toxicity of heavy metals and pesticides.

2. THE CHLOROPHYLL FLUORESCENCE INDICATOR OF THE PHOTOSYNTHESIS

2.1. Introduction

The fluorescence of the chlorophyll has (Chl a) represent only a part of the energy got by the collection antennas of

the PSII which is not converted into chemical energy by the separation of load to the reactive center of the PSII.

When a molecule of (Chl a) receives a photon or from the energy of excitement passed on from a nearby molecule,

she becomes in an unstable excited state of high energy. This form of (Chl a) returns to a lower energy level (steady

state) in several ways: either by giving an electron to a pheophytin molecule in the reactive center (photochemistry)

or by the transfer of the excitation energy at a molecule, or by dissipating the energy as heat (excitement not

radioactive) or issuing (circulation) of a photon (fluorescence). (Figure 1)[10].

The fraction of energy dissipated by fluorescence mainly depends on the competition between the de-excitation

process by the photochemical route, therefore the transport of electrons from the PSII and PSI, plays an important

role in the dissipation level of fluorescence [2],[4].

This is why the fluorescence can be used to study the functioning of the transport of electrons and the mechanisms

of transfer of energy in the photosynthesis, because all the elementary processes which establish the photosynthesis

is strongly interdependent [25],[30].


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Figure 1: ways of waste of energy of the chlorophyll incited [30].

2.2. The fast kinetics and polyphasique of fluorescence

Kautsky and Hirsh (1931) discovered that green algae, beforehand adapted has the darkness and under illuminations

continue, presented a efficiency on fluorescence, which varied in the time. The variable fluorescence was then

known today under the noun "effect Kautsky ". The in vivo measurement of the fluorescence emission of Chl a

depends on the redox state of the primary electron acceptor of PSII, QA: the fluorescence rises when QA is reduced,

and decreases when the latter re-oxide. Many transitions have been identified on fluorescence kinetics (OIDPSMT),

each representing different states of PSII photochemical activity and its interaction with the PSI [5]. A new

transition, denoted by J, O and between I, was demonstrated when the time was observed on a logarithmic scale

[28], [30], [32]. (Figure 2,3).

Figure 2: Fluorescence induction curves showing the different transitions (OIDPSMT) on an increasing scale time [5].

Figure 3: curve of induction of fluorescence showing the transitions O-J-I-P, measured on a logarithmic time scale [20].

3Chl*

Light


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2.3. Parameters of the kinetics of fast fluorescence

2.3.1. The photochemical efficiency of the photosystem II

The illumination of a plant beforehand adapted to the darkness cause the increase of the signal of

fluorescence until the level Fm if the intensity of the light is sufficient to close all the reactive centers of the PSII. In

these conditions, the photochemical efficiency of the PSII (Fluorescence maximal variable / fluorescence - Fv / Fm)

can be calculated:

Fv/Fm = (Fm - Fo) / Fm [19]

This ratio is thus proportional in the quantum efficiency on the photochemistry of the PSII and demonstrates a high

degree of correlation with the quantum efficiency on the net photosynthesis of an intact sheet. It was demonstrated

that a reduction in Fv / Fm can be a good indication of the damage caused by stress environments such as the frost,

the drought, the weed-killers, the atmospheric pollutants and the UVB radiations [13].

2.4. The kinetics of the modulated fluorescence

The method for measuring the fast kinetics and multiphase fluorescence is imprecise to determine the level Fo which

causes a certain error on this value and the parameters that result. In addition, the measurement method does not

detail the components of fluorescence related to the dissipation of energy (photochemical and non-photochemical

quenching [7], [20]. The use of a modulated fluorimeter (PAM: Pulses Amplitude Modulation), introduced since

about fifteen years, allows an adequate determination of Fo as well as a better understanding of the relation between

the efficiency on fluorescence and the photosynthetic functions . The principle of this fluorimetre rests on the use of

three types of light sources. The constant fluorescence of a plant adapted to the darkness (Fo) is measured by using

the analytical modulated light (ML). The maximal level of fluorescence (Fm) is led (inferred) by a flash of

saturating light (LS) which causes (provokes) the reduction of all the QA. The change of the level of fluorescence

(F) occurs under continuous illumination with the Actinic light and which introduces the transport of electrons.

Simultaneously, the maximal level of the fluorescence (Fm) is obtained by using flashes of saturating light (SL)

period in every 40 seconds. In the still state of the transport of electron, the Actinic light is switched off and a light

of distant red (RL) is used to obtain the level Fo representing the fluorescence when all centers reactive of the PSII

are in a state "opened" for a sample adapted to the light [1],[3] (Figure4).

Figure 4: kinetics of modulated fluorescence obtained by the use of a fluorimeter PAM [20].

2.4.1 The parameters of the kinetics of modulated fluorescence

Four main parameters of fluorescence (Fv / Fm, F v / F' m, QP and QN) can be calculated from values which

represent very different transitions (Fo, Fm, F m, F o) on the kinetics of fluorescence modulated [6].


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2.4.2 The maximal photochemical efficiency of the photosystem II

When a plant placed in the darkness during a rather long period so that all the reactionary centers of the PSII are

"opened" (oxidized QA) is illuminated with a flash of saturating light the maximal level of fluorescence (Fm) is

reached. By using Fm and Fo, beforehand obtained under modulated light, the maximal photochemical efficiency of

the PSII (ΦM) can be calculated:

ΦM = (Fm – Fo) / Fm = Fv / Fm [19].

In these conditions ΦM represents the quantum efficiency on the transfer of the electrons of P680 to the primary

acceptor QA.

2.4.3 The operational photochemical efficiency of the photosystem II For a plant adapted to the light, the fraction of the reactive centers is in a "closed" state. In these conditions, the

photochemical efficiency of the PSII is thus proportional in the quantum yield on the transfer of energy of P680 to

QA when there is a balance in the processes of regulation of the transport of electrons between the PSII and the PSI.

The operational photochemical efficiency of the PSII (Φ’M) is equal in:

Φ’M = (F’m – F’o) / F’m [19].

3. THE BIOESSAYS

A bioassay is an experimental test using a biological organism which allows estimating the potential and the relative

effect of a chemical compound. He allows to compare the effects produced by alive bodies to those observed under

specific conditions (standards) at the same type of the organism. This type of the test is used for a long time in

pharmaceutical to test the effects of medicine. Today, the bioassays are many and diversified. We find the bioassais

measuring the degradation, the biotransformation, the bioaccumulation, the allergy, the toxicology, the kinetic

toxico the dynamic toxico,… [19], [33].

In Ecotoxicology, the bioassais consist in exposing unicellular bodies or pluricellular in various concentrations of

toxic samples. These Biotest measure the answer of these bodies to the lethal acute effects or to the subacute or

chronic effects (behavior, reproduction, growth) after an exposure in particular concentrations of chemicals,

effluents, locates or atmospheric emanations. These measures are realized in checked conditions (temperature,

humidity, light) [16].

3.1 Selection criteria of a bioassay

So that a battery (bio essay) is considered effective, he has to answer several criteria:

1) be simple;

2) offer a speed in the acquisition of the data;

3) present a sensibility to an outfit of pollutants;

4) allow a reproducibility between the trees (essays);

5) have a representative to obtain an acceptable simulation of the body or the studied environment (middle);

6) least expensive [30].

4. MATERIAL AND METHODS

4.1 Choice of pollutants

1-2 Atrazine [2-chloro-4-(éthylamine)-6-(isopropylamine)-s-triazine] : it is the active substance of a phytosanitary

product (or produced phytopharmaceutique, or pesticide), that presents a herbicidal effect and which belongs to the

chemical family of tyrosine (characterized by a cycle - triazine)

(Figure 5) (http://fr.wikipedia.org/).

Figure 5: Chemical formula of Atrazine (http://fr.wikipedia.org/).

http://fr.wikipedia.org/

http://fr.wikipedia.org/


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The main ecotoxic effects of Atrazine is not only due of its direct toxicity, but also its effects of endocrine

disruptor by affecting the action of a female hormone in the male (oestrogen) [32].

Experimental studies to rats, to which we had administered of the Atrazine, showed an increase of the tumors of the

mammary glands and the womb, as well as the leukaemia and the lymphomas. Indeed, according to the studies made

on rats, Atrazine would possibly be carcinogenic for the human being [30],[32].

4.2. Biological materials

The aquatic plant Lemna gibba is considered the most sensitive to herbicides. Lemna Gibba is a species of

duckweed, a plant of the Araceae family and subfamily Lemnoideae (formerly a family of Lemnaceae), It is a

freshwater aquatic plant small, which forms a green carpet covering the stagnant water bodies. It floats on the water

surface and to measure the diameter of 05mm. It has a single root, which hang in the water. Found in a large mass of

water still or slow moving, duckweed can also occur in the rocks and sludge [11] (Figure 6).

Figure 6: Lemna gibba [21]

4.3 Culture of the solutions mothers.

Plants 7 days reaching a physiological state of the exponential growth were used for the treatments of the herbicide

Atrazine [2-chloro-4- (ethylamine) -6- (isopropylamine) -s-triazine]. Plants were introduced into Petri dishes of 6 cm

diameter containing 10 ml of the treatment environment Bonner-Devirian with a pH of 7.3 [12]. In each Petri dish, 5

plants were exposed to different concentrations of the target herbicide pass 0.5mg / l to 10 mg / l. For 24 hours, the

temperature and lighting during treatments are those of the growth conditions (at a temperature of 21°C under

continuous light of 75 µmol.m-2 S-1)[18].

4.4 Measurement of chlorophyll fluorescence kinetics

The apparatus used in this study is the fluorometer PEA and PAM fluorometer.

The PEA system (Plant Efficiency analysis) has a light source composed of 6 LEDs emitting in the red with a

maximum at 650 nm. Its data acquisition system is capable of recording the fluorescence signal every 10 μseconde

for measuring the fast kinetics of fluorescence. A dark adaptation of the sample long enough to allow full re-

oxidation of the photosynthetic apparatus is necessary to calculate the maximum efficiency of the maximum activity

of the activity of PSII and the proportion of absorbed energy to the photochemical reaction (Figures 7).

Figure 7: (a) Photo of a fluorometer PEA designed by Hansatech [24]

(b) Schematic representation of the PEA consists of a source; a photo detector and filters to select the excitation wavelength and

measurement [24].

The PAM fluorometer (Pulse Amplitude Modulation) uses a data acquisition system; called FMS (fluorometer

monitory system); for analyzing the kinetics of fluorescence and photosynthetic parameters to calculate. This

detection system measures the total emission of chlorophyll fluorescence of the photosynthetic apparatus and


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provides information on the use of the light energy absorbed by the photochemical reactions and non

photochemical. Under physiological conditions of temperature, 95% of the fluorescence measured by the FMS from

chlorophyll molecules associated with photosystem II. The FMS uses a modulated excitation light (594 nm)

resulting in a series of short flash (1.8 microseconds) and low current (<0.05 .mu.mol photons m-2;. S-1). This

modulated light is synchronized with the electronic method and allows detection and correction of interference of

background noise on the measurement of the fluorescence signal (Ogren and Baker, 1985). Actinic light PFD

between 0 and 3000 micromoles of photons m-2; s-1) is emitted by a halogen lamp and its intensity can vary from

180 to 18000 .mu.mol of photons. m-2; s-1). It is possible to use a source LED (Light emitting diode) that emits in

the far red to 735 n, to preferentially excite photosystem I (Figures 8).

Figure 8: (a) Schematic representation of MAP consists of a modulated light source (1); actinic (2); saturating (3) and in the far-

red (4); the photodetector (D); connected with a data acquisition system (A); is synchronized with the modulated light. Use the

filters to select the excitation wavelength and measurement

(b)Photo of a PAM fluorometer designed by Hansatech [24].

4.5 Reproduction test in "Fathead minnow"

The "Ball head Minnow" (Fathead minnow for Anglophones) whose scientific name is Pimephales promelas is a

freshwater fish of North American temperate zones. Recently introduced into Europe, it belongs to the genus

Pimephales and Family Cyprinidae.

The test of "Minnows" lasts one year, is a follow-up whole reproductive cycle from egg, larva, juvenile to adult fish,

in our study we reduced the cycle, was tested fertilization, egg laying, hatching and a few days of survival of the

larvae for three days, so the cycle lasted only four weeks.

We used four dilutions Atrazine 0.1, 1.0, 3.0, 10mg / l and control. These concentrations are chosen as the lower

threshold than the EC50 and IC50 (50% inhibitory concentration or physiological parameters, concentration causing

a 50% effect) of Atrazine to fish. Each dilution requires four aquariums and aquarium each contains two males and

four females. During the 4 weeks of the test period are the weekly measurements of the oxygen concentration,

temperature, water hardness and pH (these parameters are measured to ensure that the experience takes place under

strict conditions and according to the standards prescribed in the method of bioassay). The counting of eggs laid,

monitoring eggs, hatching and surveillance of larvae do every day[16], [17].

5. RESULTS AND DISCUSSION

5.1 Test of Atrazine at Lemna gibba

5.1.1 The measure of the fast kinetics

By observing the curve of the rapid kinetics and multiphase (OPJI) (Figure7), we find that the performance of Fo is

constant in all dilutions, so there is no alteration of pigment-protein complexes associated with PSII.

When the excitation energy is high at the PSII reaction center, this high reaction triggered by the charge separation

reaction of the water photolysis (electron donor), so the primary electron acceptor (QA ) is reduced causing an

increase in fluorescence until the transition J. This indicates the efficiency of electron transport through QA. The

results show a gradual decrease to the five concentrations of Atrazine. This demonstrates that there is an alteration in

the transport of electrons through the primary acceptor which is QA (Figure 9).

The transition (JIP) determines the speed of the reduction plastoquinones basin. At this level, we see that the

fluorescence decreases each time the concentration of Atrazine increases (Figure 7). This phenomenon shows that


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the active transport system of electrons between the PSII and PSI begins to be altered. There are various

types of herbicides that inhibit the photosynthetic activity by different types of action, Atrazine reacts directly on the

electron transport by binding to plastoquinone that is a transport protein of the photosynthesis system, this reaction

inhibits electron transport. This is confirmed in our results. The Fv / Fm is representing the quantum efficiency of

the transfer of electrons from reaction center P680 in the primary electron acceptor QA. It has a slight decrease

gradually with respect to the concentration of Atrazine, so the transfer of electrons from reaction center P680 and

primary acceptor QA is slightly affected (Figure 9).

Figure 9: Induction of fluorescence affected by different concentrations of Atrazine Lemna gibba obtained by the use of a

fluorimeter PEA.

5.1.2 The measure of the kinetics of modulated fluorescence.

The technique of fluorescence modulated (PAM) allows a better understanding of the mechanisms of waste of the

energy in the photosystems.

The use of modulated light, Actinic and saturating allows the analysis of the fluorescence of the chlorophyll has in

various stadiums of oxidations of the carriers of electron associated to the PSII and PSI. After the adaptation of

seaweeds to the darkness, we submit them to a light modulated by low intensity. The absorbed energy is insufficient

to lead in a separation of load in the reactive center (P680) which is in an open state (oxidized). Thus, this modulated

light is going to allow the measure of the fluorescence of which is not still constant and depends on the state of the

transfer of energy between the antennas collector of light and the reactive centers of the PSII.

In our results (Figure 10), Fo was not influenced by the treatment with Atrazine, thus a 24h exposure, concentrations

of atrazine used does not affect the light harvesting antennae of PSII, it is the same finding that we had using the

PEA. The flashes of lightning of saturating light allow to determine the maximal yield) on fluorescence Fm, when

the PSII reaction centers are closed, so the QA is reduced. We noticed Fm decreases as the concentration of Atrazine

increases (Figure 10), it proves that the Atrazine affects electron transport system through the QA.

As regards the Fv / Fm representing the quantum efficiency of electron transport of the reaction center in the

primary acceptor QA (for a suitable alga in the dark), the results show a decrease with increasing concentration of

Atrazine (Figure 11).

The illumination of algae by actinic light (light energy under which photosynthesis operates normally) can analyze

the fluorescence kinetics related to electron transport between PSI and PSII. Sending saturating flashes of light

provide the performance of fluorescence Fm’ where the PSII reaction centers will close [15], [24], [25]. We

observed a small decrease Fm’ with increasing concentration of Atrazine, which explains a slight alteration of the

electron transport system.

The actinic light is extinguished and a far-red light is used for the fluorescence yield Fo’ which is the reaction center

PSII to the open state when the plant is adapted to light. At this level, a proportion of PSII reaction centers are

closed in these light conditions, the photochemical efficiency of PSII becomes proportional to the quantum

efficiency of energy transfer between the P680 and QA steady state transport of electrons. From the values Fo ', Fm'

0

1

2

3

4

5

6

0.01 0.1 1 10 100 1000 10000

Flu

ore

sce

nce

(R

.U.)

Time (ms)

Lemna gibba exposed to atrazine for 24 hours

Control

0,5 ppm

1 ppm

2 ppm

3 ppm

5 ppm


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that represent distinct transitions in the modulated fluorescence kinetics, we can calculate a significant

parameter which is the ΦM’ representing operational photochemical efficiency of PSII. [28],[29] [33].

ΦM’ = Fm’-Fo’/Fm’ = Fv’ /Fm’ [16].

We find that the ratio Fv '/ Fm' decreased by increasing the concentration of Atrazine to 10 ppm, this means that the

balance in the process of regulating the transport of electrons between PSII and PSI does not work naturally (Figure

12).

- If we wanted to compare the toxic effect of several weed-killers, the photosynthetic parameters are little sensitive

and it is not easy to fix a relation enter the degree of toxicity of various experimented weeds-killers. The calculation

of the index of toxicity Fi from the fast kinetics and polyphonic avoids us this difficulty. The index of toxicity Fi is

quantitatively very sensitive and helps to compare well the toxic effect of a weed-killer with various concentrations.

We used this indication Fi to estimate the toxic effect of Atrazine at various concentrations. The figure 13 shows

that fee increases, according to the increase of the concentration of Atrazine. Thus more the concentration in

Atrazine increases more the index of toxicity Fi is raised [27].


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Figure 10: Kinetics of modulated fluorescence of Lemna Gibba treated by the Atrazine exposure of 24 hours, obtained

by using a PAM Fluorometer.

Figure 11: The effect of Atrazine with various concentrations on the report Fv / Fm at Lemna gibba obtained by the use of a

fluorimeter PAM.

Figure 12: The effect of Atrazine with various concentrations on the report Fv’ /Fm’ at Lemna gibba obtained by the use of a

fluorimeter PAM.

Figure 13: The effect of Atrazine with various concentrations on the index Fi at Lemna gibba obtained by the use of a fluorimeter

PAM.

0.0

0.2

0.4

0.6

0.8

1.0

0 0.5 1 2 3 5 10

Fv/F

M

Atrazine concentration (ppm)

Lemna gibba exposed to Atrazine during 24 hours

0

40

80

120

0 0.5 1 2 3 5

Fv

'/F

m'



0

100

200

300

400

500

600

700

800

0 0.5 1 2 3 5 10

Fi




95

5.2 Bioassay of reproduction in the "Fathead minnow" exposed to Atrazine Exposure "Fathead minnow" Atrazine shows us that after the exposure, there was a significant decrease in the

number of eggs laid, this is perceivable especially at the highest concentration . From this, it appears that atrazine

disrupts the activity of reproduction in "Fathead minnow" from 10 mg / l (Figure14).

Figure 14: Curve of reproduction at the "Fathead minnow" exposed to Atrazine.

5.2 .2.Indication of sexual maturity to fishes «Fathead minnow "exposed to Atrazine

5.2.2.1 Caclul of the Condition Factor "CF"

At the end of reproduction test "Fathead minnow" exposed to Atrazine, we made the dissection of the 121 fish that

were used for this test, by measuring weights and lengths for each fish all confused sex. Using the morpho- metric

and weight data obtained on the fish sampled, we proceed to the evaluation of the CF condition factor targeted fish.

Formula: CF % = (fish weight (g)

(fish length (cm)/10 ^ 3∗ 𝟏𝟎𝟎 [16].

The results that we present in Figure 15 show the absence of a significant difference in male CF specimens for

various Atrazine concentrations used in this experiment. In contrast, in female individuals, we note a small decrease

in the value of the CF as and as the dose Atrazine increases in volume, but this is remarkable primarily for the

concentration of 10 mg / l.

Furthermore, note that these results are in perfect conformity with the results for egg laying where it was noticed a

significant drop in the amount of the number of eggs laid after exposure to 10 mg / l of Atrazine (Figure 14). We can

advance the hypothesis that the toxicity of Atrazine significantly and negatively influences the FC report, suggesting

that the negative impact of the contaminant Atrazine has a harmful effect on the physiological activity of

reproduction in fish female sex and it is likely that a relationship exists with fish reproduction "fathead minnow"

The more the female fish is weakened over time, the greater its ability to reproduce and significantly slowed.

0

2000

4000

6000

8000

10000

12000

14000

16000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Nu

mb

er o

f eg

gs

hu

nged

Days

Reproduction of "fathead minnow ": Pre-exposure and exposure of the herbicide Atrazine

Contrôle

0.1mg/l

1mg/l

3mg/l

10mg/lPre-Exposure Atrazine Exposure


96

Figure 15: Evaluation of the Condition Factor (CF) in specimens ♂ and ♀ fish "Fathead minnow " after exposure to different

concentrations of Atrazine.

5.2.2.2 Evaluation of the Gonadosomatic index (GSI)

After the sacrifice of the fish sampled, measures lengths and weight of fish " Fathead minnow " are

performed later, we take the male and female gonads that we weigh, in order to calculate the Index gonadosomatic

according IGS.

Formula: IGS % = (Weight of gonads (g)

(weight of fish (g)∗ 𝟏𝟎𝟎 [16]

In Figure 16 below, we note that the IGS% of male fish, showed no remarkable difference; by cons, in female fish, a

significant decline is seen at the highest concentration of 10 mg / l of Atrazine, where IGS index equal 5% compared

to an average of about 16% for other concentrations are including those of the controls (control) (Figure 16).

Consequently, we can say that this report represents a coefficient of maturity confirms that the toxicity of Atrazine

to a direct negative effect on the reproductive system of female fish "Fathead minnow".

Figure 16: Evaluation of the Gonadosomatic index (GSI) in specimens ♂ and ♀ des fish "Fathead minnow Minnows" after

exposure to different concentrations of Atrazine.

6. CONCLUSION

The overall objective of this work is focused on the use of chlorophyll fluorescence as a device to detect the

presence of pesticides. Plant chlorophyll fluorescence provides information on the physiological condition of the

plant. It has been used in the past mainly for the basic research of photosynthesis. Currently she studies the relations

existing between the basic information obtained from photosynthetic processes and the effects of pollutants on

plants. This technique can be used as the presence of environmental stress indicator [34].

Our present work was carried out with this in mind and allowed us to make the following observation:

CF ♀

CF♂

0.00

0.50

1.00

1.50

2.00

contrôle0,1mg1mg3mg10mg

Effect of Atrazine on the CF ratio ♂ and ♀ of "Fathead minnow "

CF ♀CF♂

Atrazine concentration

GSI, %♂

GSI, %♀

0.00

5.00

10.00

15.00

20.00

contrôle0,1mg1mg3mg10mg

Effect of Atrazine on the GSI report in ♂ and ♀ of "fathead minnows"

GSI, %♂

GSI, %♀

Atrazine concentration


97

1- Through the toxicity testing of a organonitrogen herbicide (Atrazine), we demonstrated the harmful effects

to aquatic species. The test we have chosen is a bioassay used officially by major laboratories accredited and

affiliated to the Governments of Environment Canada, the reproduction test "of fathead minnow Minnows."

2- We measured chlorophyll fluorescence in the plant Lemna gibba exposed to Atrazine same pollutant, according to

our results, we find that Atrazine affects the photosynthetic apparatus of the plant Lemna gibba:

a) Atrazine does not affect the light harvesting antennae, since Fo fluorescence remains constant for all

concentrations.

b) The level of fluorescence during the transition (JIP) decreases, so alteration of the transport system of electrons

between the PSII and PSI is noticed. Atrazine therefore reacts directly on the electron transport.

c) The Fv / Fm decreases with increasing concentration of Atrazine, due to malfunctions reaching the proper

functioning of PSII.

When one wants to compare the toxic effect of many herbicides, photosynthetic parameters are insensitive and it

becomes difficult to correlate the herbicides tested, and the degree of their inhibitory effects. The calculation of the

toxicity index Fi from the rapid kinetics and multiphase fluorescence has filled this gap. Therefore, this index Fi is

quantitatively very sensitive and can well compare the effects of a herbicide tested at different concentrations. In our

study the Fi increases with increasing the concentration of Atrazine.

3- Based on experimental and bibliographic research, we compared the results obtained from the use accredited

bioassay recognized reliability with the new bioassay "chlorophyll fluorescence measurement" which is still in

development. In conclusion, we can say that the use of chlorophyll fluorescence as bioassay is a very reliable

method that can detect a presence of environmental stress directly on a chlorophyll species (algae or higher plants).

The final test result shows precisely how is the toxic effect of a pollutant on the photosynthesis system.

7. GENERAL CONCLUSION

Bioassays currently used, each of these difficulties, the most encountered are the long term, the need for a significant

number of qualified staff and without neglecting the equipment and expensive reagent. In contrast, the bioassay in

development, that of chlorophyll fluorescence; we may save such obstacles as to realize, he asks only a fluorometer

Portable and we can do our tests directly on site and get immediate results.

This bioassay will not ban other reliable bioassays like the one we used in the present study, because each has its

specificity. However, the bioassay based on the chlorophyll fluorescence has many advantages:

- It gives quick results;

- It is non-destructive and requires very small and inexpensive equipment.

In the end, the expertise gained during these extensive investigations enabled us to acquire important knowledge on

the use of chlorophyll fluorescence signal to detect the effects of environmental stress.

8. ACKNOWLEDGMENTS

My deepest thanks are dedicated to the late Professor. Radovan Popovic, TOXEN laboratory director and all the

team toxicology research laboratory at the University UQAM in Montreal.

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Documents

USE OF CHLOROPHYLL FLUORESCENCE IN PHYTOPLANKTON … · 2018-09-14 · Zemri Khalida1, Boutiba zitouni2, Zemri mohammed3 & Popovic Radovan4 1University of SIDI BEL ABBES, Department