A toxicity warning monitor using the weakly electric fish, Gnathonemus petersi

Water Res. Vol. 18. No. 10, pp. 1285-1290. t984 Printed in Great Britain.All rights reserved

0043-135484S3.00+0.00 Copyright ~: 1984Pergamon Press Ltd

A TOXICITY W A R N I N G MONITOR USING THE WEAKLY ELECTRIC FISH, GNA T H O N E M U S PETERSI* WALTER GELLER Limnological Institute, University of Konstanz. D-7750 Konstanz, Federal Republic of Germany (Received August 1983) Abstract--A technically simple fish monitor was developed using the weakly electric fish, Gnathonemus petersi, as the test organism. The test system consists of a plexiglass test chamber (21., 27C) with a flow-through of 4--5 times the chamber volume per hour. The spontaneous electric organ discharges (EOD) are recorded as activity signals. EODs, which have a potential of about 500 mV, last 0.3 ms and they can be converted to computer-compatible signals by a simple amplifier. The EOD-activity is analyzed by a low-cost desk-top computer. Two different modes of analysis which represent two levels of response sensitivity, are possible either alternatively or simultaneously. The toxicity response of Gnathonemus petersi was measured in experiments with the toxicants Hg z°, Cu-", NaAsO: and CN-. The sensitivity to acutely toxic concentrations was found to be as high as that of trout or minnows. Key words--water quality, aquatic biology, water pollution, drinking water, toxicity, fish. electric fish. Gnathonemus, biomonitoring, monitors

INTRODUCTION Early warning monitors for the detection of hazardous acute toxicity in water should be automatically working systems with short response times. Various types of biological monitors using aquatic organisms like fish or invertebrates have been developed. Cairns and van der Schalie (1980) gave a comprehensive review of the known test-systems and discussed the basic problems and limits of biological early warning monitors. Characteristic elements of such monitors are a flow-through system with an experimental chamber containing the test organism under controlled conditions and a recording system which is able to measure a physiological or behavioral activity of the organism, to convert it into an electronic signal and to discriminate the "normal" and the "abnormal" state as effected by a toxicant. Some biomonitors record the respiration activity by measuring the oxygen concentration in the test chamber continuously or in regular intervals. This parameter can be used in very different test organisms, in bacteria (Axt, 1973), algae (GeIler and Mfickle, 1977), invertebrates (Maki et al., 1973) and fish (Maki, 1980). Several monitors evaluate the general locomotor or the rheotactic behavior of fish (Besch et al., 1977; Poels, 1977; Lindahl et al., 1977). In these systems different fish species can be used. A fish monitor of very high sensitivity uses the opercular rhythm as an indicator for the fish breathing activity (Morgan and Kuhn, 1974). In all these

*The development and standardization of the system was supported by the Kuratorium ffir Wasserwirtschaft, Bonn.

systems the measured parameters have to be transduced to the data analysis system by means of complex mechanic sensors or highly sensitive electrodes. These monitors are expensive, furthermore they have to been handled very carefully in order to avoid technical defects. This paper presents a new type of fish monitor which uses the weakly electric fish Gnathonemus petersi as the test organism. The characteristic electric organ discharges (EOD) of this fish make it possible to use a very simple, low-cost monitor thus avoiding the disadvantages mentioned above. The Gnathonemus monitor was developed for the survey of drinking water supplies in combination with two additional new monitors which used different types of organisms: Haematococcus and Daphnia (Geller and M/ickle, 1977). The fish monitor was improved to the present form by the addition of a microcomputer data analysis system.

THE MONITOR FISH Gnathonemus petersi (Mormyridae) inhabits the fresh waters of tropical Africa. All Mormyrids are warm water fish and have electric organs situated at the tail base. The electric discharges produced are used for communication and orientation. Gnathonemus petersi can be acquired from a commercial aquarium. Considerable numbers of fish (approx. 15 in a 200 l.-aquarium) can be kept for periods longer than a year in an aquarium with tap water which was aerated for several days prior to use. Clay tubes serve as hiding places. The natural food consists primarily of insect larvae (Okedi, 1971). In the aquarium good

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200 300 ms Interval length

Fig. 1. Frequency spectrum of interval lengths between consecutive electric organ discharges during diurnal activity cycle (redrawn after Moiler, 1970).

long term food sources were Enchytraea earthworms and deep frozen chironomid larvae. THE ELECTRIC ORGAN DISCHARGE ACTIVITY

Gnathonemus continuously shows a spontaneous activity of its electric organ. The duration of single discharges is approx. 0.3 ms and the potential, as conducted through the water at a distance of 10-20 cm, is about 500 mV. The EODs are produced at irregular time intervals. Their sequences can be evaluated in two ways: as average frequencies of EOD numbers per unit time (Fig. 3, Reinhardt 1978) or as frequency class spectra of the variable time intervals between the consecutive EODs (Fig. 1). The average frequencies can be measured easily by a simple pulse counter. The normal activity level measured as a 10 min-average results from individually variable EODs ranging from 4 to 15 Hz (Moiler, 1970) although during short periods (s) single high bursts of activity can exceed 50 Hz (Kramer, 1976). The EOD frequency changes during the diurnal cycle according to the general activity: during the day

Gnathonemus has a low activity level with phases of immobility, but it is very active during the night. In daytime the average EOD frequency is between 8-10 Hz with a minimum of~ Hz, whereas at night the range is 10-15 Hz with a minimum of 7 Hz (Moiler, 1970). Comparable results were found in the experimental series of Reinhardt (1978): the normal activity never was lower than 150 EOD rain- ~. Using a computer the additional information resuiting from the irregularities in the EOD pattern can be evaluated by statistical analysis of the frequency spectra of EOD intervals. Different spectra produced by the fish during a diurnal cycle are shown in Fig. I: the EOD intervals range from 10 to 250ms during the night. In daytime during periods of immobility, longer intervals up to 300 ms occurred. Similar results were reported by several authors (Moiler and Bauer, 1973; Bell et al., 1974). Belbenoit (1972) found 95% of all observed EOD intervals between 40-220 ms during phases of immobility. When fish were swimming fast the intervals became shorter (20-140 ms). The densest EOD pattern was observed when a fish approached an unknown object with EOD intervals of 18-60 ms. When two fish are kept together in one aquarium they use their electric activity for communication. A cessation of the EOD activity was observed during seconds and perhaps up to minutes in this situation (Moiler and Bauer, 1973; Szabo et aL, 1973). The interacting fish behave like "listener" and "talker" alternatively or depending on dominancy (Bell et al., 1974; Kramer and Bauer, 1976). When fish were kept alone in individual test chambers no cessation of EOD activity due to antagonistic behavior was found and EOD intervals longer than 250 ms hardly occurred. T H E MONITOR SYSTEM

The Gnathonemus monitor system (Fig. 2) consists of a glass vessel for temperature control (25-27°C) and aeration of test water which is connected to the test chamber with one fish by a membrane pump

Early warning monitor using the weakly electric fish, Gnathonemus +5 V

for transformation to S V-square pulses

Fig. 2. Gnathonemus monitor system with test chamber, signal amplifier and computer for data analysis. Test water supply with temperature control, aeration and pump not shown.

Electric fish warning monitor

on-line and the computer progr~-m reads the status of the input memory repeatedly by use of a "'waif'-loop until a non-zero result is obtained by an arriving fish EOD. As soon as the pulse is recorded the time interval between the consecutive pulses is measured. Because of the refractory period of the computer the maximum frequency which can be analyzed by the system is limited to 300 EODs m i n - ' .

ensuring constant water flow. The fish EODs are converted by an amplifier to computer compatible standard signals which are recorded and analyzed every 10 rain by a desk-top computer. The test chamber (2 I.) is made of a plexiglass tube of 10cm diameter and a length of 25cm which is suitable for the use of juvenile fish of about 10 cm body length. The test chamber was supplied with 8-10 1. of water per hour resulting in an exchange of 90°0 of the chamber volume within 60rain. All sensitivity values and response times given in this paper are calibrated to this flow speed, because the response time of the monitor is not only dependent on the reaction time of the test fish, but also on the water renewal time. Since the fish is sensitive to electric irritations, direct contact between the test water and metal surfaces such as thermostats or other electric instruments should be avoided. The plexiglass tube of the test chamber is closed on both sides by plexiglass covers fitting exactly into the tube by rubber gaskets. The water flows horizontally through the central openings from one side cover to the other. A standpipe on the top of the tube serves for air escape and as an access for feeding. The sensor electrodes are fixed on the inside surfaces of the side covers. They consist of wire grids made of stainless steel which are connected to a 3-step amplifier converting the fish EOD pulses to TTLcompatible 5 V square signals. Since all signals are uniform, only "yes/no" information regarding the emission of a pulse is conveyed. The signals are input 11

16

I

EOD FREQUENCY ANALYSIS AND ALARM THRESHOLDS

The EOD activity has to be analyzed by the monitor system in a way that the "normal" behavior can be clearly distinguished from an "abnormal" activity caused by physiological damage. According to the present results and to data from the literature, limits of the "normal" activity range can be classified into three modes of analysis using data records of 10min length: average EOD frequency below 150min -r (1), EOD intervals shorter than 200ms comprising less than 80% of the total number of intervals (2) and EOD intervals shorter than 300 ms less than 90% of the total number [mode (3)1. Figure 3 shows a toxicity experiment comparing these evaluation modes of 10-min records. The toxicant Hg -'÷ was not given continuously, but in concentration waves in order to simulate practical monitoring conditions (Fig. 3A). The monitor response in evaluation mode (I) is shown in Fig. 3(B). Frequencies less than 150 rain -t

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Fig. 3. Gnathonemus test: evaluation of reactions to the toxicant Hg-'+. (A) Pulse doses of Hg'+ given for 4 (200/ag I-~), 3 and 1.5 h (500,ug 1-t each) intermitted by dilution with clean water. (B) 10-min records of electric organ discharges (EOD) evaluated in mode (I), as average frequency per minute, arrows indicate alarm responses (frequency below 150 EODsmin-t). (C) 10-rain records of EOD-activity analyzed for interval lengths. Solid circles represent evaluation of the signals in mode (2). alarm threshold: decrease of proportion of intervals < 200 ms below 80~ of total interval numbers. Line represents evaluation mode (3), alarm threshold: decrease of intervals < 300 ms below a percentage of 90%.

V~'ALTER GELLER

1288

only appeared during toxin application periods or a few hours thereafter (see arrows). The first alarm response was followed by a period of recovery when the toxicant was diluted by clean water. The second response 24 h later led to a continuously lowered activity due to physiological damage. The evaluation mode (1) is shown for comparison because the values for the characteristic responses to intoxications as given in this study and by Reinhardt (1978) have been measured using this evaluation mode. This mode, however, can be disturbed if a fish is small enough to take a position at right angles to the long axis of the experimental chamber thus preventing proper reception of the EOD signals. This type of interference can be avoided by using the evaluation modes (2) or (3) which are not sensitive to pauses of EOD activity or its reception. Modes (2) and (3) of EOD activity analysis both evaluate the variability in EOD interval durations. A comparison of the two modes is given Fig. 3(c), setting the alarm limit to a minimun of 80% for intervals < 200 ms, an alarm response was monitored only 2 h after the application of 500 t~g I -~ Hg -'~. In mode (3), with the limitation of intervals < 300 ms to at least 90,°/, the system responded with an alarm

warning after 4 h. The mode (2) represents a highly sensitive way of evaluation with fast response to toxicants, false alarms, however, may occur. Mode (3) by contrast is less sensitive, but better secured against false alarms. The computer program (Appendix) functions in the high sensitivity mode (2). The program can be alternatively changed to the lowsensitivity mode (3). The most effective evaluation of the EOD activity of Gnathonemus is simultaneous use of both modes, combining an "'attention signal" (2) and an "'alarm warning" (3).

SENSITIVITY OF THE G N A T H O N E M U S MONITOR

Toxicity experiments were carried out with solutions of Hg -'+, Cu :~, C N - and As (NaAsO2) in lakewater of pH 8 and conductivity of 295/~mho cm -E using only a pulse counter. The EOD activity was analyzed in the evaluation mode (1). The results were obtained using juvenile fish between 5 and 20 g freshweight. The results of this study are incorporated in Table 1. Concentrations of toxicants affecting monitor response times of 4 h are given, since the 4-h period is considered as the minimum

Table 1. Response times of Gnathonemus monitor system to different concentrations of four toxicants as compared to data from the literature on other fish species used in warning monitors or in standard toxicity tests Toxin Hg" ~

C u-' "~

CN-

NaAsOz

(mg I -~)

Response (h)

0. 100 0.500 0.010 0.005 0.300-0.500 0.500 0.100

20 4 24 24 48 48 (72 h survival)

0.500 0.120 0.050 0.048 0.250-2.000 0.800

4 24 24 24 48 48

0.100 0.140 0.090 0.050 0.010 0.010 0.100 12 (mg As 1-i) 18 41 58 98 12 18 98 195 953 35 103

4 2.5 27 24 24 24 (72 h survival) 28 24 II 8 5 36 36 6 4 1 48 48

Test species

Author

Gnathonemus petersi

( 1)

Micropterus salmoides Barbus holubi Leuciscus idus melanotus

(2)

Lebistes reticuluta Gnathonemus petersi Sarotherodon mossambicus Barbus holubi Micropterus salmoides Leuciscus idus mehmotus

(31 (4) (5) ( 1) (21

(31 (41

Gnathonemus petersi Salmo gairdneri

( I) (61

Sarotherodon mossambicus Micropterus salmoides Barbus holubi Lebistes reticulata

(21 (5)

Gnathonemus petersi

(7)

Salmo trutta

(8) (6)

Phoxinus phoxinus

Salmo gairdneri Leuciscus idus melanotus

(9) (4)

(I) This study, decrease of EOD-frequency below 150 min -* as averaged during l0 rain-record; 12) Morgan 0977) increased opercular rhythm; (3) Mann (1976) standard toxicity fish test FRG, range L C _ 0 - L C _ ~ ; (4) Knie (1978) L C ~ ; (5) Jones (1975) no reaction observed; (6) Liebmann (1958) loss of swimming ability; (7) Reinhardt (1978) EOD-frequency below 150min-t; (8) Bauer (1961) death; (9) Crosby and Tucker (1966) LC_~.

Electric fish warning monitor passage time of water through ~ater treatment plants. For all toxicants tested the concentrations which effected 4-h alarm responses were one to three orders of magnitude below the acute toxicity threshold for humans (acute lethal dose per 2 I. water). A comparison of results obtained by Gnathonemus with data from the literature on reactions of other fish species in monitor systems and in standard toxicity tests (Table 1) indicate that some of the standard test species, Leuciscus idus or Lebistes reticulata, only register toxic effects after a time lag of days or are inadequate in detecting toxicant concentrations which are monitored by the Gnathonemus system within a few hours. Gnathonemus petersi apparently is a very sensitive fish species reacting to inorganic toxicants within response times as short as those found in very sensitive cold water species like trout or minnows. The Gnathonenms monitor has not been tested using organic pesticides. In the system losses of volatile substances due to temperating and aeration of the water have to be considered. Solutions of most of the pesticides applied, however, cannot be affected by short-time warming up to 27~C (Melnikov, 1971). Until now the Gnathonemus monitor has been used for drinking water control only. It is not known whether the system is suitable for other purposes such as effluent water control. Gnathonemus can be kept in the range from "'soft" to " h a r d " fresh waters. The natural habitat of the species makes sure Gnathonemus, by contrast to cold water species of comparable sensitivity, can live in turbid water under low oxygen concentrations.

REFERENCES

Axt G. (1973) Kontinuierliche Toxizit/itsmessungen mit Bakterien (Toximeter). Gewdss. Wass. Abwass. 10, 297-306. Bauer K. (1961) Studien fiber Nebenwirkungen yon Pflanzenschutzmitteln auf Fische und Fischn/ihrtiere. Mitt. biol. Bunddnst. 105, 1-72. Belbenoit P. (1972) Relations entre la motricit6 et la d6charge 61ectrique chez les Mormyridae (Teleostei). J. Physiol. (Paris) 65, 197A. Bell C, C., Myers J. P. and Russell C. J. (1974) Electric organ discharge patterns during dominance related behavioral displays in Gnathonemus petersi (Mormyridae). J. comp. Physiol. 92, 201-228. Besch W. K., Kemball A., Meyer-Waarden K. and Scharf B. (1977) A biological monitor system employing rheotaxis of fish. In Biological Monitoring of Water and Effluent Quality (Edited by Cairns J. Jr, Dickson K. L. and Westlake G. F.), pp. 56--74, STP 607. American Society for Testing and Materials, Philadelphia, PA. Cairns J. Jr and van der Schalie W. H. (1980) Biological monitoring. Part l---early warning systems. Water Res. 14, 1179-1196. Crosby D. G. and Tucker R. K. (1966) Toxicity of aquatic herbicides to Daphnia rnagna. Science 154, 289-291. Geller W. and M~ckle H. (1977) I~berwachung von Rohund Trinkwasser. II. Kontinuierlicher Biotest zur

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Toxizit/itsfiberwachung yon Trinkwasser. DVGWSchr!/~enreihe Wasser 14, 173-1S6. Jones W. E. (1975) Detection of pollutants by fish tests. War. Treat. Exam. 24, 132-139. Knie J. (19781 Der dynamische Daphnientest---ein automatischer Biomonitor zur I~rberv,achung yon Gewassern und Abw/i.ssern. Wasser Boden 12, 310-312. Kramer B. 11976) Flight-associated discharge pattern in a weakly electric fish, Gnathonemus petersi IMormyridae. Teleostei). Behaviour 49, 88-95. Kramer B. and Bauer R. (1976) Agonistic behaviour and electric signalling in a Mormyrid fish, Gnathonemus petersi. Behav. Ecol. SociobioL !, 45-61. Liebmann H. (1958) Handbuch der Frischwasser- und Abwasserbiologie. Vol. II, Oldenbourg. Mfinchen. Lindahl P. E., Olofson S. and Schwanbom E. (1977) Rotary-flow technique for testing fitness of fish. In Biological Monitoring of Water and Effluent Quality (Edited by Cairns J. Jr, Dickson K, L. and Westlake G. F.), pp. 75-84, STP 607. American Society for Testing and Materials, Philadelphia. PA. Maki A. W. (1980) Monitoring aberrant respiratory activity of bluegill as a predictor of chronic fish toxicity values of surfactants. In Second Conference on Aquatic Toxicology and Hazard Eval,~ation. American Society for Testing and Materials, Philadelphia, PA. Maki A. W., Stewart K. W. and Silvey J. (1973) The effects of dibrom on respiratory activity of the stonefly, Hydroperla croshyi, hellgrammite. Corvdalis cornutus, and the golden shiner, Notem~onus chrvsoleucas. Trans. Fish. Soc. 102, 806--815. Mann H. (1976) Fischtest mit Goldorfen zur vergleichenden Prfifung der akuten Toxizitfit yon Wasserinhaltsstoffen und Abwfissern--Praktischc Erfahrungen aus drei Ringtesten. Z. Wass. Ahwass. Forsch. 9, 103-109. Melnikov E. (1971) Chemistry of Pesticides. Springer, New York. Moiler P. (1970)"'Communication" in weakly electric fish, Gnathonemus niger (Mormyridae). I. Variation in electric organ discharge (EOD) frequency elicited by controlled electric stimuli. Anim. Behav. 18, 768-786. Moiler P. and Bauer R. (1973) "'Communication" in weakly electric fish, Gnathonemus petersi (Mormyridae). If. Interaction of electric organ discharge activities of two fish. Anita. Behav. 21, 501-512. Morgan W. S. G. (1977) An electronic system to monitor the effects of changes in water quality on fish opercular rhythms. In Biological Monitoring of Water and Effluent Quali O' (Edited by Cairns J. Jr, Dickson K. k. and Westlake G. F.), pp. 38-55, STP 607. American Society for Testing and Materials, Philadelphia, PA. Morgan W. S. G. and Kuhn P. C. (1974) A method to monitor the effects of toxicants upon breathing rate of largemouth brass (Micropterus salmoides Lacepede). Water Res. 8, 67-77. Okedi J. (1971) The food and feeding habits of the small Mormyrid fishes of Lake Victoria, East Africa. Afr. J. trop. Hydrobiol. Fish. I, 1-12. Poels C. L. M. (1977) An automatic system for rapid detection of acute high concentrations of toxic substances in surface water using trout. In Biological Monitoring of Water and Effl,wnt Quality (Edited by Cairns J. Jr, Dickson K. L. and Westlake G. F.), pp. 85--95, STP 607. American Society for Testing and Materials, Philadelphia, PA. Reinhardt H. D. (1978) Untersuchungen zu Funktion und Empfindlichkeit eines Biotestsyztems ffir die automatischkontinuierliche Wasseriiberwachung mit Gnathonemus petersi und Daphnia pulex. Dipl. thesis, UniversitS.t Freiburg, FRG. Szabo T., Bauer R. and Moiler P. (1973) Elektrische Sinneswahrnehmungen und Verhalten bei elektrischen Fischen. Naturwissenschaften 60, 10-18.

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WALTER GELLER APPENDIX

Program Comments: Basic Program for CBM Desk-Top computer Line 8: Input of printer address number Line 13: Sets counter to 0 and clock to actual time Line 14: Wait loop until on-line input of fish EOD signal Line 15: Control of 10rain recording intervals Line 16: Control of alarm condition: alarm if 20 or more 5~ Line 16: of EOD inte~'als are longer than 0.2 s Line 18: --20: Alarm output on printer At 23.59.59 the last record period of a diurnal cycle is finished, followed by new start at 00.00.00. 1 2 3 4 8 9 11 12 13 14 15 16 17 18 19 20 21 22 23

REM ELECTRIC FISH MONITOR ALARM REM GNATHONEMUS TEST ACCORDING TO GELLER 1980 REM LIMNOLOGISCHES INSTITUT/UNIV. KONSTANZ REM PROGRAMMER J. KLEINER INPUT "INPUT : PRINTER ADDRESS NUMBER"; DR% OPEN 4,DR% PRINT "INPUT: ACTUAL TIME ( H H M M S S ) " : INPUT TIS POKE59468, PEEK(59468)OR1 N =0:L=0:T=TI A=TI:WAIT59469,2:E=TI:R = PEEK(59457);N = N + 1 : I F A B S ( E - A ) ) = 1 2 T H E N L = L+ 1 IFABS(TI - T) < = 36000THEN14 I F ( L - N/5)>0THENTS=TI$:GOTO18 GOTO13 PRINTS4, "ALARM FISH-INTOXICATION'" PRINTS4, "TIME:";T$ PRINT~4,1NT(Lxl00/N);"PER CENT OF EOD INTERVALS LONGER THAN 0.2 S'" GOTO 13 CLOSE 4 END