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Actual standard and further development of an algal fluorescence bioassay
ECOTOXICOLOGY
AND
ENVIRONMENTAL
SAFETY
7,276-283
(1983)
Actual Standard and Further Development an Algal Fluorescence Bioassay’
of
CHRISTIAN SCHMIDT Institut fiir Wasserforschung, Gesellschaji mit Beschriinkter Haftung, 4600 Dortmund 1. Dortmund, Federal Republic of Germany Received June 9, 1982 A measurable result of nearly all influences which affect the primary processesof photosynthesis is a change of the fluorescence emission of a plant. This change of the fhrorescence emission due to substances which a&ct or block photosynthesis is well known since the work of Kautsky (1943). The fluorescence test used here, due to the optical characteristics of the photosynthetic pigments (chlorophyll fluorescence >660 nm), allows the measurement of the fluorescence of algae (S’cenedesmus sp./ChloreNa sp.). The measurement process is demonstrated. The results show the principal function of the bioassay and its sensitivity. Effects of the algae can be demonstrated at a very low pollutant concentration. The time from the dosage of a toxicant to a clear reaction of the algae is very short (5 min). The aim of the actual research is the determination of the sensitivity of the bioassay to a representative number of chemicals and possible interactions with physical parameters. A connection to a data system, e.g., computer registration of the fluorescence curve and an automated process control, will be the next step.
INTRODUCTION For many years, the Institute for Water Research (Institut fur Wasserforschung GmbH) at Dortmund has done research in the field of stream-quality monitoring. The aim of this research is an improved quality control of surface water, which is used for drinking water after certain purification steps. The drinking water produced by the Dortmund municipal water works (Dortmunder Stadtwerke AG) is only biologically treated by slow-sand filtration without any chemical purification steps. Thus, anything which could cause a damage of the biocoenosis in the slow-sand filter must be avoided (Fig. 1). To obtain information about the toxicity of substances which could have a negative influence on the algae and microorganisms on the filter, several bioassays were developed (Benecke, 1980; Benecke et al., 1982). The fluorescence bioassay, to which Gisela Benecke gave the idea some years ago, uses algae as test organisms (Scenedesmus quadricaudaJChlorella sp.). Algae normally have a representative sensitivity to a wide spectrum of pollutants and a relatively low one to matrix effects. The test is based on the registration of the spontaneous, variable fluorescence as one parameter of the algal physiological state. The characteristics of the test are high sensitivity, very quick results, and a possible automatization. MEASUREMENT OF FLUORESCENCE Figure 2 shows a schematic view of some photosynthetic reactions as they are known from many textbooks. We have put numbers in some places of the system ’ Presented at the International Symposium on Testing in Ecotoxicology-Methods tion, May 17-19, 1982, Munich, PRG. 0 147-65 13183 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any farm reserved.
276
and Their Evalua-
ALGAL
FINAL
FLUORESCENCE
BIOASSAY
277
TREATMENT Chbrinstiun
Deaciditication (NEDIf)
FIG.
1. Scheme of artificial groundwater recharge. The biologically active zones are the upper filter regions.
to show, where possible, that inhibitors can block photosynthesis. I will not sum up all possibilities, but from many papers, we know that especially antibiotics, carbamates, phenolic substances, and in general herbicides can retard the photosynthetic action. For our bioassay, it is of course not essential to know the exact place of inhibition, we only look for the effect. One very distinctive effect is the immediate change in the extent of the fluorescence emission of a plant. These changes refer to the fact that-in simple words-the acceptor molecules in the photosynthetic system are limited and a specific quantity of the excited electrons are emitted as fluorescence. Thus, any changes in the electron flux should result in a change of the spontaneous, variable fluorescence. We will see that these changes are measurable and reproducible. To measure fluorescence, we use the methods of fluorometry which have to be adapted to the optical characteristics of the photosynthetic pigments. Fluorescence can thus be measured above 680 nm; the algae are irridated with light of 450 nm. From Fig. 3, we notice several phases of the fluorescence curve: a first increase (I), a slight depression (2), a second increase (3), a maximum (4), and a slow decline (5). These very distinctive phases can be reproduced under two conditions: physiologically intact algae and a sufficient dark phase before the measurement. The principle of the measurement is demonstrated in Fig. 4. The algae are kept in a culture and continuously pumped with a peristaltic pump through the cell and
278 E.
CHRISTIAN
SCHMIDT Fl
(Volt) em 0
+ H202
I(
‘t H2° f 202+2Mv
Xl.4
1 /
@ ADP+p
-0.2
PI .I
\ e\\\ Fn I Fp \ \ \ -+ I \ e- \
e-
I \ I \ 4 ,eII*Cy
-a4
+
pelIQ a
i0.6
FIG. 2. Photosynthetic
reactions
with
possible
places of inhibition.
back into the culture. The light source (150 W), flow-through cell, amplifier, and registrating oscilloscope are included in field B. Figure 5 demonstrates the measuring process. The algae culture is a normal chemostat which runs with a continuous input of a mineral solution and temperature and light regulation as a closed system. The algal density should be about 1.2 extinction units at 665 nm. Before reaching the cell, which is also cooled and aerated, the algae must pass a glass spiral, which is kept under dark. The optimal dark time
ALGAL
FLUORESCENCE
279
BIOASSAY
I TIME
FIG. 3. Normal
fluorescence
curve
with typical
l
phases.
for a sufficient fluorescence intensity is 20 min at least. In the flow-through cell, a stirrer prevents a settling of the algae. Before the measurement, stirrer and aeration are stopped. A shutter controls the light exposure time. The light passes a blue filter before reaching the cell. A red filter with a transmission maximum above 660 nm is placed on top of the cell, e.g., 90 degrees to the axis of the excitating light. A photomultiplier records the signal, which is then stored by an oscilloscope. The whole measurement needs 15 min. With this measuring device, it is now possible to obtain information about the effects of certain substances or substance combinations on the algae. Toxicants can be dosed directly into the cell and, after the measurement, the algae are pumped out of the system together with the pollutant. The cell is rinsed and clear for a new filling. Raw water, which has been filtered, can also be dosed into the cell. RESULTS
AND
DISCUSSION
Figures 6 and 7 show the results of a dosage of various substances. It can be seen that various reactions can be noticed, a possible result of a different mode of action of the pollutants. Nearly all curves show two effects: change of the fluorescence intensity and a leveling off of the curve’s slope, especially between phases 1 and 3. If the algae are exposed to a toxic substance for a relatively long time, a full depression of the fluorescence follows in some cases. Normally, we have a significant reaction of the algae after 5 min. A comparison to growth tests is not really correct. Until
~q$fqqfiiq FIG. 4. Scheme
of the measuring
system.
FIG. 5. Key
plan of the bioassay.
Note
water
that the fluorescence
23 raw
is registrated
24 mineral
chemostat
20 21 22
19
18
17
16
13 14 15
axis of the irridating
valve light.
rinsing water cooling system light source diaphragm shutter filter (425 nm) cell filter (690 nm) photomultiplier amplifier oscillograph overflow 12
HCL
outlet
11
outlet
system
10
9 pump
8
7 stirrer
4 light 5 cooling 6 frit
25 regulator not in the optical
solution
cleaner control
3 chemostat
1 air 2 air
Normal algal fluorescence
0.005
curve
mg/L HgC12
Fluorescence after 1 min.
0.007
mg/L
decrease N2
KCN
Tern erature
increase
(10 El C)
0.003
mg/L Carbaryl
FIG. 6. Normal fluorescence curve photographed from the oscilloscope. Fluorescence curves after dosage of a toxicant. Several curves on the photographs. Normal curve on the left 5, 10, 15, 20, and 25 min after dosage. The exception was a temperature increase, normal curve on the right.
0.033
Atrazine
mg/L
3.1 mg/L 2-Chlorotoluene
0.024
mg/L
0.0014
mg/L
Prometryne
Linuron
mg/L 1,3-Dinitrobenzene
Brook-water (Dortmund) contaminated with a green substance (Uranin?)
0.034
FIG. 7. Fluorescence curves after dosage of a toxicant; for legend see Fig. 6.
282
CHRISTIAN
SCHMIDT
TABLE
1
SENSITIVITY OF THE FLUORESCENCE TEST (m&l)
Atrazine HgCl2 cuso4 C4NW2 KCN Monolinuron 1,3-Dinitrobenzene 2-Chlorotoluene
Fluorescence
Growth ’
0.0325 0.005 0.003 0.007 0.007 0.014 0.034 3.1
0.003 0.005 0.03 0.07 0.07 0.14 0.17 31.0
’ Bringmann and Kiihn ( 1975).
now, we have looked at very quick effects. Growth tests almost measure chronical effects when green algae are used. In Table 1, the sensitivity of our test is compared to the growth test according to Bringmann and Kuhn (1975). We took their concentrations and, in most cases, the sensitivity of the fluorescence method is better. We have seen that we can record an immediate effect on algal photosynthesis with very low concentrations of toxic substances. Fortunately, our program is sponsored by the Minister of Research and Technology, which enables us to carry on in the development of this test. The program will run until 1984. I will complete my paper with some short remarks on this program. An advantage of the algae that we used in the test is their simple culturing. But a stable algae concentration is one of the problems that has to be solved. Thus, we will automate the culture by continuous density and chlorophyll measurements. An algae screening could also be useful. The alga we will use next is Scenedesmus subspicutus (Berlin). A next step in the program will be the test of reference chemicals proposed by Scheele (1980) and a comparison with two other bioassays: the Phormidium-inhibition test and the growth test according to Bringmann and Kuhn.
CONNECTION
OF TM BIOASSAY WITH A DATA SYSTEM
FIG. 8. Connection of the bioassay to a data system.
ALGAL
FLUORESCENCE
BIOASSAY
283
If the sensitivity to a representative number of chemicals is determined, the interaction between toxicants and physical parameters and a dosage of raw water can be analyzed. A general aim of this program is the connection of the fluorescence bioassay and parts of the Phormidium test to a data system. Figure 8 shows the principal key plan for a data system. The computer will have three main functions: 1. Process control, e.g., light on/off, activating the dosage apparatus. 2. Registration of certain parameters such as oxygen concentration, temperature, and fluorescence signal. 3. Data storage and mathematical evaluation of the fluorescence curves by automated integration, slope control, and comparison to previous curves. The installation of that system is under work at the moment; the problem is, of course, the writing of the software. We hope that it will function by the beginning of the next year. ACKNOWLEDGMENTS I thank the Umweltbundesamt and the Minister for Research and Technology for supporting this research (No. 037292).
REFERENCES BENECKE, G. (1980). Verijffentlichungen des Instituts fur Wasserforschung und der Hydrologischen Abteilung der Dortmunder Stadtwerke AC. 33, l- 124. BENECKE, G., FALKE, W., AND SCHMIDT, C. (1982). Bull. Environ. Contam. Toxicol. 28, 385-395. BRINGMANN, G., AND R. KUHN (1975). GWF-Wasser/Abwasser 117(9), 410-413. KAUTSKY, H. et al. (1943). Biochem. Zeitschrts 274, 423-434. SCHEELE, B. (1980). Chemosphere 9, 293-309.
AND
ENVIRONMENTAL
SAFETY
7,276-283
(1983)
Actual Standard and Further Development an Algal Fluorescence Bioassay’
of
CHRISTIAN SCHMIDT Institut fiir Wasserforschung, Gesellschaji mit Beschriinkter Haftung, 4600 Dortmund 1. Dortmund, Federal Republic of Germany Received June 9, 1982 A measurable result of nearly all influences which affect the primary processesof photosynthesis is a change of the fluorescence emission of a plant. This change of the fhrorescence emission due to substances which a&ct or block photosynthesis is well known since the work of Kautsky (1943). The fluorescence test used here, due to the optical characteristics of the photosynthetic pigments (chlorophyll fluorescence >660 nm), allows the measurement of the fluorescence of algae (S’cenedesmus sp./ChloreNa sp.). The measurement process is demonstrated. The results show the principal function of the bioassay and its sensitivity. Effects of the algae can be demonstrated at a very low pollutant concentration. The time from the dosage of a toxicant to a clear reaction of the algae is very short (5 min). The aim of the actual research is the determination of the sensitivity of the bioassay to a representative number of chemicals and possible interactions with physical parameters. A connection to a data system, e.g., computer registration of the fluorescence curve and an automated process control, will be the next step.
INTRODUCTION For many years, the Institute for Water Research (Institut fur Wasserforschung GmbH) at Dortmund has done research in the field of stream-quality monitoring. The aim of this research is an improved quality control of surface water, which is used for drinking water after certain purification steps. The drinking water produced by the Dortmund municipal water works (Dortmunder Stadtwerke AG) is only biologically treated by slow-sand filtration without any chemical purification steps. Thus, anything which could cause a damage of the biocoenosis in the slow-sand filter must be avoided (Fig. 1). To obtain information about the toxicity of substances which could have a negative influence on the algae and microorganisms on the filter, several bioassays were developed (Benecke, 1980; Benecke et al., 1982). The fluorescence bioassay, to which Gisela Benecke gave the idea some years ago, uses algae as test organisms (Scenedesmus quadricaudaJChlorella sp.). Algae normally have a representative sensitivity to a wide spectrum of pollutants and a relatively low one to matrix effects. The test is based on the registration of the spontaneous, variable fluorescence as one parameter of the algal physiological state. The characteristics of the test are high sensitivity, very quick results, and a possible automatization. MEASUREMENT OF FLUORESCENCE Figure 2 shows a schematic view of some photosynthetic reactions as they are known from many textbooks. We have put numbers in some places of the system ’ Presented at the International Symposium on Testing in Ecotoxicology-Methods tion, May 17-19, 1982, Munich, PRG. 0 147-65 13183 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any farm reserved.
276
and Their Evalua-
ALGAL
FINAL
FLUORESCENCE
BIOASSAY
277
TREATMENT Chbrinstiun
Deaciditication (NEDIf)
FIG.
1. Scheme of artificial groundwater recharge. The biologically active zones are the upper filter regions.
to show, where possible, that inhibitors can block photosynthesis. I will not sum up all possibilities, but from many papers, we know that especially antibiotics, carbamates, phenolic substances, and in general herbicides can retard the photosynthetic action. For our bioassay, it is of course not essential to know the exact place of inhibition, we only look for the effect. One very distinctive effect is the immediate change in the extent of the fluorescence emission of a plant. These changes refer to the fact that-in simple words-the acceptor molecules in the photosynthetic system are limited and a specific quantity of the excited electrons are emitted as fluorescence. Thus, any changes in the electron flux should result in a change of the spontaneous, variable fluorescence. We will see that these changes are measurable and reproducible. To measure fluorescence, we use the methods of fluorometry which have to be adapted to the optical characteristics of the photosynthetic pigments. Fluorescence can thus be measured above 680 nm; the algae are irridated with light of 450 nm. From Fig. 3, we notice several phases of the fluorescence curve: a first increase (I), a slight depression (2), a second increase (3), a maximum (4), and a slow decline (5). These very distinctive phases can be reproduced under two conditions: physiologically intact algae and a sufficient dark phase before the measurement. The principle of the measurement is demonstrated in Fig. 4. The algae are kept in a culture and continuously pumped with a peristaltic pump through the cell and
278 E.
CHRISTIAN
SCHMIDT Fl
(Volt) em 0
+ H202
I(
‘t H2° f 202+2Mv
Xl.4
1 /
@ ADP+p
-0.2
PI .I
\ e\\\ Fn I Fp \ \ \ -+ I \ e- \
e-
I \ I \ 4 ,eII*Cy
-a4
+
pelIQ a
i0.6
FIG. 2. Photosynthetic
reactions
with
possible
places of inhibition.
back into the culture. The light source (150 W), flow-through cell, amplifier, and registrating oscilloscope are included in field B. Figure 5 demonstrates the measuring process. The algae culture is a normal chemostat which runs with a continuous input of a mineral solution and temperature and light regulation as a closed system. The algal density should be about 1.2 extinction units at 665 nm. Before reaching the cell, which is also cooled and aerated, the algae must pass a glass spiral, which is kept under dark. The optimal dark time
ALGAL
FLUORESCENCE
279
BIOASSAY
I TIME
FIG. 3. Normal
fluorescence
curve
with typical
l
phases.
for a sufficient fluorescence intensity is 20 min at least. In the flow-through cell, a stirrer prevents a settling of the algae. Before the measurement, stirrer and aeration are stopped. A shutter controls the light exposure time. The light passes a blue filter before reaching the cell. A red filter with a transmission maximum above 660 nm is placed on top of the cell, e.g., 90 degrees to the axis of the excitating light. A photomultiplier records the signal, which is then stored by an oscilloscope. The whole measurement needs 15 min. With this measuring device, it is now possible to obtain information about the effects of certain substances or substance combinations on the algae. Toxicants can be dosed directly into the cell and, after the measurement, the algae are pumped out of the system together with the pollutant. The cell is rinsed and clear for a new filling. Raw water, which has been filtered, can also be dosed into the cell. RESULTS
AND
DISCUSSION
Figures 6 and 7 show the results of a dosage of various substances. It can be seen that various reactions can be noticed, a possible result of a different mode of action of the pollutants. Nearly all curves show two effects: change of the fluorescence intensity and a leveling off of the curve’s slope, especially between phases 1 and 3. If the algae are exposed to a toxic substance for a relatively long time, a full depression of the fluorescence follows in some cases. Normally, we have a significant reaction of the algae after 5 min. A comparison to growth tests is not really correct. Until
~q$fqqfiiq FIG. 4. Scheme
of the measuring
system.
FIG. 5. Key
plan of the bioassay.
Note
water
that the fluorescence
23 raw
is registrated
24 mineral
chemostat
20 21 22
19
18
17
16
13 14 15
axis of the irridating
valve light.
rinsing water cooling system light source diaphragm shutter filter (425 nm) cell filter (690 nm) photomultiplier amplifier oscillograph overflow 12
HCL
outlet
11
outlet
system
10
9 pump
8
7 stirrer
4 light 5 cooling 6 frit
25 regulator not in the optical
solution
cleaner control
3 chemostat
1 air 2 air
Normal algal fluorescence
0.005
curve
mg/L HgC12
Fluorescence after 1 min.
0.007
mg/L
decrease N2
KCN
Tern erature
increase
(10 El C)
0.003
mg/L Carbaryl
FIG. 6. Normal fluorescence curve photographed from the oscilloscope. Fluorescence curves after dosage of a toxicant. Several curves on the photographs. Normal curve on the left 5, 10, 15, 20, and 25 min after dosage. The exception was a temperature increase, normal curve on the right.
0.033
Atrazine
mg/L
3.1 mg/L 2-Chlorotoluene
0.024
mg/L
0.0014
mg/L
Prometryne
Linuron
mg/L 1,3-Dinitrobenzene
Brook-water (Dortmund) contaminated with a green substance (Uranin?)
0.034
FIG. 7. Fluorescence curves after dosage of a toxicant; for legend see Fig. 6.
282
CHRISTIAN
SCHMIDT
TABLE
1
SENSITIVITY OF THE FLUORESCENCE TEST (m&l)
Atrazine HgCl2 cuso4 C4NW2 KCN Monolinuron 1,3-Dinitrobenzene 2-Chlorotoluene
Fluorescence
Growth ’
0.0325 0.005 0.003 0.007 0.007 0.014 0.034 3.1
0.003 0.005 0.03 0.07 0.07 0.14 0.17 31.0
’ Bringmann and Kiihn ( 1975).
now, we have looked at very quick effects. Growth tests almost measure chronical effects when green algae are used. In Table 1, the sensitivity of our test is compared to the growth test according to Bringmann and Kuhn (1975). We took their concentrations and, in most cases, the sensitivity of the fluorescence method is better. We have seen that we can record an immediate effect on algal photosynthesis with very low concentrations of toxic substances. Fortunately, our program is sponsored by the Minister of Research and Technology, which enables us to carry on in the development of this test. The program will run until 1984. I will complete my paper with some short remarks on this program. An advantage of the algae that we used in the test is their simple culturing. But a stable algae concentration is one of the problems that has to be solved. Thus, we will automate the culture by continuous density and chlorophyll measurements. An algae screening could also be useful. The alga we will use next is Scenedesmus subspicutus (Berlin). A next step in the program will be the test of reference chemicals proposed by Scheele (1980) and a comparison with two other bioassays: the Phormidium-inhibition test and the growth test according to Bringmann and Kuhn.
CONNECTION
OF TM BIOASSAY WITH A DATA SYSTEM
FIG. 8. Connection of the bioassay to a data system.
ALGAL
FLUORESCENCE
BIOASSAY
283
If the sensitivity to a representative number of chemicals is determined, the interaction between toxicants and physical parameters and a dosage of raw water can be analyzed. A general aim of this program is the connection of the fluorescence bioassay and parts of the Phormidium test to a data system. Figure 8 shows the principal key plan for a data system. The computer will have three main functions: 1. Process control, e.g., light on/off, activating the dosage apparatus. 2. Registration of certain parameters such as oxygen concentration, temperature, and fluorescence signal. 3. Data storage and mathematical evaluation of the fluorescence curves by automated integration, slope control, and comparison to previous curves. The installation of that system is under work at the moment; the problem is, of course, the writing of the software. We hope that it will function by the beginning of the next year. ACKNOWLEDGMENTS I thank the Umweltbundesamt and the Minister for Research and Technology for supporting this research (No. 037292).
REFERENCES BENECKE, G. (1980). Verijffentlichungen des Instituts fur Wasserforschung und der Hydrologischen Abteilung der Dortmunder Stadtwerke AC. 33, l- 124. BENECKE, G., FALKE, W., AND SCHMIDT, C. (1982). Bull. Environ. Contam. Toxicol. 28, 385-395. BRINGMANN, G., AND R. KUHN (1975). GWF-Wasser/Abwasser 117(9), 410-413. KAUTSKY, H. et al. (1943). Biochem. Zeitschrts 274, 423-434. SCHEELE, B. (1980). Chemosphere 9, 293-309.