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A continuous-flow mini-diluter system for toxicity testing
Water Res. Vol 16, pp. 457 to 464, 1982 Printed m Great Britain. All rights reserved
0043-1354/82/040457-08103.00/0 Copyright O 1982 Pergamon Press Lid
A C O N T I N U O U S - F L O W MINI-DILUTER SYSTEM FOR
TOXICITY TESTING DUANE A. BENOIT,VINCE R. MATTSONand DIANE L. OLSOr~ U.S. Environmental Protection Agency, Environmental Research Laboratory-Duluth, 6201 Congdon Boulevard, Duluth, MN 55804, U.S.A. (Received July 1981)
Abstraei--A space saving portable mini-diluter exposure system for testing early life stages of fish and invertebrates has been developed and successfully used both in the laboratory and on-site with single chemicals and with complex effluents. This gravity-operated system can also be installed in a compact, vented enclosure to permit safe testing of hazardous volatile chemicals. The mini-diluter test system has several additional advantages over other widely used dosing systems in that it: (1) uses small volumes of complex effluents and/or single chemicals which reduces the problem of removing hazardous material from the test system's waste water; (2) works well with waste containing suspended solids; and (3) can maintain normal operation for up to 10 h if the effoent headbox supply pump fails and up to 4 h if the diluent water headbox supply pump fails.
INTRODUCTION Increased awareness of water pollution-related problems has prompted the recent development of many reliable and accurate toxicant dosing systems (Lemke et al., 1978; Peltier, 1978). Two of the oldest types of intermittent flow-through systems successfully used were a serial diluter and a proportional diluter described by Mount & Warner (1965) and Mount & Brungs (1967), respectively. Warner (1964), however, was the first to introduce a truly continuous-flow serial diluter (Fig. 1). The flow pattern diagrammed in this figure demonstrates the simplicity of the diluter in which toxicant from one source is continuously diluted by successive additions of water to produce lower and lower concentrations of toxicant. This water delivery system has several advantages over most other dosing systems in that it is gravity operated, does not require critical timing devices, is free of vacuum siphons and/or solenoid valves and once calibrated does not require further adjustment. It is also
easily adaptable to a wide variety of uses and applications with either single chemicals or complex effluents. Mean flow deviations are less than 2To at any dilution. Warner's continuous-flow serial diluter was used in exposure-related trout studies conducted by McKim" et al. (1970). This system, which worked exceedingly well, was essentially the same as shown in Fig. 1 except that a constant-drip Mariotte bottle, instead of a toxicant pump, was used to meter the chemical sol. ution. During the ensuing years, researchers at the U.S. EPA Environmental Research Laboratories at Duluth, MN and Corvallis, OR used, with increasing frequency, a modified version of the continuous-flow diluter: the modification consisted of a series of baffles, built into the diluter, to ensure adequate mixing of toxicant and dilution water prior to delivery to test tanks. These large scale, single-chemical exposure systems, recently described by Garton (1980), were designed to deliver more than 201. test water h-1 to each of 5 concentrations plus a control.
CONSTANT HEAD MANIFOLD
,l,
WATER
ill T.C. • I
T.C.=O.25
T.C. - 0 . 5
T.C.=0.125
T.C.=O.D65
TO EXPERIMENTAL TANKS
Fig. 1. Dilution principle used in Warner's original continuous-flow serial dilution apparatus (each arrow equals 1 volume of water per unit time). 457
458
DUANE A. BENOITet al.
Interest m developing the early life stage (ELS) fish toxicity test concept introduced by McKim (1977) led to the design of the compact continuous-flow minidiluter exposure system described in this paper. The system accurately delivers as little as 31. test water h - ~ to each of 5 concentrations plus a control. It can be used in the laboratory or in the field to test the effects of either single chemicals or complex effluents on young fish. The small ELS test apparatus takes very little space and requires only small volumes of test water: this latter point is a critical factor when there is a need to transport effluents to a laboratory test site at some distance. Using smaller volumes of test water also reduces filtration costs when one is required to remove hazardous test chemicals from the diluter waste water before discharge. The ELS test system described in this paper has been tested and evaluated both in the laboratory and on-site in a mobile trailer. To date. this apparatus has been used to conduct 33 ELS exposures to various toxicants, using the fathead minnow ( P i m e p h a l e s promelas) as the test animal. These tests have included * Manual for Construction of a Continuous-Flow MiniDiluter System for Toxicity Testing, by D. A. Benoit et at.. U.S, Environmental Protection Agency, Environmental Research Laboratory-Duluth, Duluth, MN 55804, U.S.A.
metal,s, pesticides, volatile organic compounds and treated complex effluents from metal plating, oil refinery and sewage treatment plants. The system has also recently been successfully used in tests conducted on macroinvertebrates (Spehar et al.. 1980; Anderson, Manuscript ). The following text contains a general description of the mini-diluter exposure system including principles of operation and evaluation. Limited space would not allow us to include detailed construction drawings and photographs. If the reader is interested in further details, complete fabrication instructions can be obtained from the Environmental Research Laboratory-Duluth*.
DESCRIPTION OF CONTINUOUS FLOW MINI-DILUTER
Figure 2 is a photograph of the mini-diluter. The diluter is constructed of double strength glass (3 mm thick) and mounted on a 1.9 cm thick plywood backboard (69 × 38 cm). It is divided into 4 major units as illustrated in the schematic drawing of Fig. 3: these units are the toxicant and water cell (TC and WC), dilution cell (DC); flow booster cell (FBC) and flow splitter cell (FSC).
Fig. 2. Continuous-flow mini-diluter for use with either single chemicals or complex effluents.
A continuous-flow mini-diluter system for toxicity testing Toxicant and water cell
The unit containing the constant head toxicant cell (TC) and water cell (WC) measures 7.6cm wide x 54.6cm long × 5.1cm high. Each cell is separated by a vertical glass partition between the emergency outlet (EO) standpipes (Fig. 3t. Float valves (FV) which connect to headboxes maintain a constant depth of toxicant or diluent water in each cell and can be adjusted up or down to change head pressure. Emergency outlet drains in each cell are set slightly above the normal water depth. Water from both cells passes through a 20 mesh stainless-steel screen for large particle filtration before it reaches the capillary flow tubes shown in Fig. 3, (T-1 and W-1 through W-5). Capillary flow tubes are initially adjusted up or down in each cell to obtain desired flow rates. Diluter flow rates can also be changed by using different length or diameter flow tubes. Dilution cell
The second unit of the mini-diluter is a dilution cell (DC) which measures 3.8cm wide x 37.5cm long x 5.1cm high. This cell contains a series of baffles to ensure adequate mixing as the toxicant is continuously diluted by successive additions of diluent water. Principles of operation are diagrammed with arrows shown in Fig. 3. Flow tube T-I (2 mm, i.d.) delivers toxicant solution (100ml rain-1) to the
459
left end of the d~lution cell. Half of this volume, at the rate of 5 0 m l m i n -1, flows out the C-1 flow tube (1.5 mm, i.d.) and is the highest test concentration. The other half of the volume, 50 ml m i n - t, is diluted by one-half when it mixes with 50 ml of diluent water per rain from the W,1 flow tube (1.5 mm, i.d.). Half of this combined volume of I00 ml min-1 flows out the C-2 flow tube to become the second highest exposure concentration, and the remainder is again diluted by one-half from the next addition of diluent water from the W-2 flow tube. This flow pattern continues until the last dilution is made. The extra volume of test water flows over a baffle and into a water outlet (WO). Water used for the control tank flows through the diluter exactly the same way as the test water. Flow tube W-5 (2 mm, i.d.) delivers control water ( 1 0 0 m l m i n -~) to the right end of the dilution cell tFig. 3). Half of this volume, 50 ml rain- t, flows out the C-6 flow tube (1.5 mm, i.d.), and the remainder flows over a baffle and into a water outlet. Flow booster cell
Test water concentrations (C-1 through C-6) flow from the dilution cell to individual flow booster cells (FBC) which measure 2.5 cm wide x 4.5 cm long x 5.4 cm high (Fig. 3). Each of these cells contain one standpipe siphon similar to those described by Benoit & Puglisi (1973). Approximately every 30 s
FV WC i
i
2x / /
Ix
Ix
Ix
Ix
®
2x
DC ;-I'm'
C-2'm'
C-3'm'
C-4'~"
1
1
1
EO
C-5"t~""E3" "m'C-6
1
1 FBC
FSC
-
i
ABCD
2
ABCD
3
ABCD
4
ABCD
5
ABCD
s
ABCD
Fig. 3. Schematic drawing and flow pattern of continuous-flow mini-diluter. (C) concentration flow tube; (DCI dilution cell; (EO) emergency outlet: (FBC) flow booster cell: (FSC) flow splitter cell: (FV) float valve: (T) toxicafit flow tube; (TC) toxicant cell: (Wt water flow tube: (WC) water cell; (WO) water outlet: (IX) one volume; (2XL two volumes.
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DUAnE A. BENOIt et at.
each cell rapidly empties its content to a flow splitter cell located directly below. As the name implies these flow booster cells are used to increase flow to the flow splitter cells. The rapid filling of each flow splitting cell is necessary to obtain a precise split of the test water through each flow splitting delivery tube.
certain lots of neoprene stoppers are acutely toxic to fathead minnow larvae. IBroderius. personal communication)*.
Flow splitter cell
Figure 4 shows a portable early life stage exposure system. The slotted angle iron support frame measures 66cm wide × 102cm long × 74cm high and sits on a 61 × 9t × 66cm high table. With minor alterations, the same system can be used for testing either complex effluents or single chemicals.
Flow splitter cells (FSC) measure 2.5cm wide × 5.7cm long × 4.8cm high (Fig. 3). Each cell contains four tubes (1.5 mm, i.d.) cut equally in length and adjusted to the same height. Test water received from each flow booster cell is divided into four equal volumes, thereby creating four replicate concentrations at each treatment level.
Diluter evaluation Daily flow rate measurements through each of the 12 diluter flow tubes over a 5 week period resulted in an overall mean standard deviation of + 1.3°o. During a 5 week trial test with dissolved NaC1 the mean variability of each concentration ranged from 2 to 7° o. In this test, 5 concentrations (0.02-0.3°o) were measured daily by conductivity. Variation within each flow splitter cell ranged from 2 to 8°o. During four ELS fathead minnow toxicity tests with hexachlorobutadiene, 1,2-dichloropropane. 1,3-dichloropropane and 1,2-dichloroethane, comparisons of chemical concentrations within each group of four replicates, sampled simultaneously, showed that concentrations were within 90°o of each other and ranged from 85 to 98°0 (Benoit et al., 19821. Float valves are quite sensitive and once they are adjusted to the desired height they will maintain a constant depth of water within +2 mm. If headbox pressure changes, the valves will automatically readjust themselves to maintain the same head pressure m the toxicant and water cell. If either float valve sticks open, excess water will flow out the emergency standpipe and the diluter will continue to operate normally. The mini-diluter will not operate smoothly until the glass flow tubes in each cell wet-up. The system, therefore, should be started at least 24 h before a test ~s begun. As long as the head pressure remains similar in the toxicant and water cell, no further adjustments are necessary for proper diluter operation during a test except routine cleaning of float valve and flow tube orifices with pipe cleaners. Some test chemicals or effluents can cause fungus to gro~ very rapidly, in which case diluter flow tubes may have to be cleaned daily. With practice, however, one can complete this task in about 5 rain. All batches of neoprene stoppers should be checked for toxicity prior to their use in the diluter and exposure chambers. Recent static tests have shown that * S. J. Broderius. U.S. Environmental Protection Agency. Duluth. MN 55804. U.S.A.
DESCRIPTION OF PORTABLE EXPOSURE
SYSTEM
Complex effluent test ~ystem Two identical pair of stainless-steel headboxes are used with the system, one pair to supply diluent water for the diluter water cell and the other pair to provide effluent water for the diluter toxicant cell. Each pair sits on top of the support frame and consists of two interconnected headboxes A and B which are gently aerated to avoid temperature stratification (Fig. 41. Headbox A (30.5 +_ 30.5 _ 40.6 cm high) contains a float valve and standpipe drain. If ample quantities of either diluent or effluent water are available, they can be continuously circulated throughout the headbox and out the standpipe. If either supply is limited, float valves can be adjusted so there is no waste overflow. These headboxes also act as a settling basin for large debris and can temper the water several degrees before it is heated to the desired test temperature. Water subsequently flows by gravity from headbox A through a 20 mesh stainless-steel screen for large particle filtration and into headbox B which is insulated and measures 30.5 ,x 45.7 × 40.6 cm high. The water is then warmed with a 500 W stainless-steel immersion heater and passed through another 20 mesh screen in headbox B before it flows through the diluter to the exposure chambers. Styrafoam covers for either headbox A or B may be used to increase insulation or to reduce the loss of volatile chemicals or effluents (Fig. 4). Glass exposure chambers are designed to accommodate 15 test fish per chamber and measure 7 cm wide × 18.8cm long ~ 9.2cm high with a water depth of 4.5 cm, Separate delivery tubes distribute the test water from the diluter to each replicate exposure chamber. Delivery tubes are positioned by stratified random assignment, Flow rate to each chamber containing 500ml of test water, averages 12.5 ml minwith one chamber volume displacement every 40 min. A white background can be used under the chambers to help make the test fish more visible, Eggs are incubated in screened 140 meshl bottom glass jars (120 ml)which slowly oscillate up and down 13.5 cm) in the test water. The glass jars are powered by a 2 rev min rocker arm assembly IMount, 1968). One 61cm long fluorescent tube over the test chambers provides an average light intensity of 20-60 lumens, and photoperiod is controlled by an automatic timer.
A continuous-flow mini-diluter system for toxicit3 testing
461
Single chemical test system
Portable system eraluation
In a single chemical test. the headboxes shown in Fig. 4 contain only diluent water which subsequently flows to the diluter water cell and toxiant cell. Predetermined volumes of the test chemical are then delivered to the toxicant cell by an F M I ®* metering pump and thoroughly mixed with diluent water using a small magnetic stirrer (Figs 2 and 3t. Float valves do not have to be readjusted when single chemicals are added directly to the toxicant cell because the valve will automatically readjust itself to maintain the same constant head.
Comparative fathead minnow ELS feeding density tests with clean water were conducted between the small 500 ml chambers described in this report and larger 6000ml chambers described and used by Benoit & Holcombe 11978j. This study was conducted to determine if the smaller chambers with lower flow rates were suitable for normal growth and survival of young fathead minnows. Subsamples of preserved brine shrimp nauplii were counted on a Sedgewick Rafter counting chamber to determine approximate numbers of live shrimp fed. Results of the test, shown in Table 1, demonstrated that although the three feeding densities greatly affected growth, there was no significant [P = 0.05j difference in growth or survival between either size of chamber at each feeding density. [P = 0.05, analysis of variance with Dunnett's testl.
*Fluid Metering. Inc.. Oyster Bay. NY 11771, U.S.A. The U.S. Environmental Protection Agency neither recommends nor endorses any commercial product: trade names are used only for identification.
Fig. 4. Portable early life stage exposure system, (A) headbox with float valve: IB) insulated headbox with immersion heater. Water flows by gravity from A to B through an interconnecting pipe. w,R. 16 4-~G
462
DUANE A. BENOIT et al.
Table t. Survival and growth of 4-week-old fathead minnows fed live brine shrimp nauplii at various densities in two different size chambers with different flow rates. All tests were conducted at 25 + 1 C Chamber size and flow rate
Feeding density
Survival (o,,)
Weight (mg)
500 ml 12.5 ml min- ~
4000* 12,000 20.000
95+ 95 80
24 + 10.+ 77 _ 20 94 ___28
6000 ml 47mlmin -~
4000 12.000 20,000
97 83 92
23 _ 10 68 + 17 100 _ 24
* Number of live brine shrimp fed 3 times daily t Each entry represents the mean of four groups of 15 fish each. :~ Mean + SD,
Oxygen concentrations in the 500 ml chambers during 32 day fathead minnow ELS toxicity tests have consistently been measured at levels /> 75"0 saturation except during tests with certain complex effluents which exhibit a high BOD. The effects of low dissolved oxygen on the fathead minnow have been demonstrated by Brungs (197l) at 60°,, saturation and lower. If the test water is dark or turbid, exposure chambers can individually be removed from the system and placed over a fluorescent light box to observe the test fish or to count mortalities. A small beaker can be used to catch the flow of test water during this time. A unique feature of the system described in this report is that it uses a total of only 241. test water h - t. If the main supply of effluent water is tempor-
Fig. 5. Stationary vented early life stage exposure ,;}stem
A continuous-flow mini-diluter system for toxicity testing ariiy interrupted, the headbox holding capacity and location is such that the system can continue in normal operation for up to 10h. If the diluent water supply is interrupted the system will operate for up to 4 h. An alarm system can easily be installed to alert the investigator before the headboxes run dry. If the problem cannot be corrected in time, additional water can be added to the headboxes by hand. With such low volumes, it is also easy to screen out troublesome debris in the headboxes before it reaches the small diluter flow tubes. Since the test system described in this report uses only 100 ml of toxicant m i n - J , a 2101. polyethylene drum with a submersible pump can be conveniently used to hold a 35 h supply of complex effluent. If necessary, the drum can also be used to premix daily effluent samples before the diluted test effluent is pumped to the headboxes. Overall height of the entire system including headboxes measures only 183 cm which makes the system ideally suitable for use in almost any size laboratory room or mobile trailer. When using the portable system in a trailer the table must be securely attached to the floor and headboxes should be removed from the support frame during transportation. All other parts including diluter and exposure chambers can remain attached to the support frame which is bolted to the table top. The portable system (excluding table and headboxes) weighs approx. 75 kg: and can easily be moved from one facility to another by removing the headboxes, detaching the frame f/'om the table, and transporting it in a vehicle. Break-down and set-up time usually requires about 2 h. Complete fabrication costs of the portable system averages approx. $800-900. Over half of this initial cost is for immersion heaters and stainless steel headboxes.
463
or special filters used to remove hazardous test chemicals from the diluter water before discharge. This system is ideally suited for use in testing hazardous volatile chemicals and was designed to protect the investigator from possible harmful exposures to toxic fumes. Negative pressure created on the inside of the enclosure enables one to safely service the system and take care of the te~t fish through small sliding glass doors. During tests conducted at our laboratory the enclosure was vented through the laboratory air exhaust system which drew an average of 0.7 m a min-1 through the enclosure (approx. one air volume every 2 min). Air samples taken with one 30 x 30cm sliding glass door open have shown measurable quantities of volatile test chemicals inside the enclosure but no detectable concentrations were found outside. Another feature of the enclosure is if the diluter leaks or overflows, the spilled test water can be diverted directly to the system's drain lines and will not flood the room. Due to the fiberglass or epoxy paint, the enclosure bottom is also water tight and can hold up to 100 liters if a leak should occur in some other part of the system. An alarm can be installed in the enclosure base to warn the investigator of major leaks. The accumulated test water in the base can then be drained off through a discharge valve and if necessary passed through a filter. Fabrication costs of the complete enclosure averages approx. $200-300.
Acknowledgements--We thank B. Riedel for photography and daily assistance and G. Endicott, R. Syrett, L. Herman and G. Lien for their assistance with the vented enclosure.
REFERENCES
DESCRIPTION OF STATIONARY VENTED EXPOSURE SYSTEM Figure 5 shows a compact stationary vented exposure system containing the same basic equipment as described for the portable system except that the metal support frame is not used and the headboxes are hung from overhead. The vented plywood enclosure is sealed with fiberglass or epoxy paint on the inside and measures 76 cm wide × 120 cm long with a height of 112 cm over the exposure chambers and 159 cm over the diluter. Both apparatus and test fish can easily be observed through viewing windows located on the sides and top (Fig. 5). One 5 cm hole located near the bottom of each side allows a continuous-flow of air to be drawn through the enclosure and out a 10 ¢m exhaust vent located over the diluter. Exhaust air can be purified through a charcoal filter if necessary. Ample space is available in the bottom of the enclosure to install apparatus such as chemical saturators (Veith & Comstock, 1975; Gingerich et al., 1979), stock bottles, metering pumps,
Anderson R. L. Effect of pydrin on non-target aquatic invertebrates. Environmental Research Laboratory, Duluth, MN 55804, U.S.A. (Manuscript). Benoit D. A. & Holcombc G. W. (19"/8) Toxic effects of zinc on fathead minnows Pimephales proraelas in soft water. J. fish Biol. 13, 701-705. Benoit D. A. & Puglisi F. A. (1973) A simplified flowsplitting chamber and siphon for proportional diluters. Water Res. 7, 1915-1916. Bcnoit D. A., Puglisi F. A. & Olson D. L. (1982) A fathead minnow (Pimephales promelas) early life stage toxicity test method evaluation and exposure to four organic chemicals. J. envir. Pollut. In press. Brungs W. A. (1971) Chronic effects of low dissolved oxygen concentrations on the fathead minnow. J. Fish Res. Bd Can. 28, 1119-1123. Garton R. R. 0950) A simple continuous-flow toxicant delivery system. Water Res. 14, 227-230. Gingcrich W. H., Seim W. K. & Schonbord R. D. (1979) An apparatus for the continuous generation of stock solutions of hydrophobic chemicals. Bull. era'Jr, contain. Toxic. 23, 685-689. Lemke A. E., Brungs W. A. & Halligan B. J. 0978) Manual for construction and operation of toxicity-testing proportional diluters. USEPA, Ecology Research Series
EPA-600/3-78-072.
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DUANE A. BENOIT et al.
McKim J. M. (1977) Evaluation of tests with early life stages of fish for predicting long-term toxicity. J. Fish. Res. Bd Can. 34, 1148-1154. McKim J. M.. Christensen G. M. & Hunt E. P. (1970) Changes in the blood of brook trout Salvelinusfontinalis after short-term and long-term exposure to copper. J. Fish. Res. Bd Can. 27, 1883-1889. Mount D. I. (1968)Chronic toxicity of copper to fathead minnows. Water Res. 2, 215-223. Mount D. I. & Brungs W. A. (1967) A simplified dosing apparatus for fish toxicology studies. Water Res. 1, 21-29. Mount D. I. & Warner R. E. (1965) A serial dilution apparatus for continuous delivery of various concentrations of materials in water. U.S. Department of Health, Education and Welfare, Health Services Publication 999-WP-23.
Peltler W. (1978) Methods for measuring the acute toxicity of effluents to aquatic organisms. USEPA. Ecology Research Series EPA-600/4-78-012. Spehar R. L.. Tanner D. K. & Gibson J. H. (1980) The effects of ke[thane and pydrin on early life stages of lathead minnows (Pimephales promelas) and amphipods (Hyallela azteca). Presented at the American Society for Testing and Materials 5th Annual Proceedings of Aquatic Toxicology. Veith G. D. & Comstock V. M. (1975) Apparatus for continuously saturating water with hydrophoblc organic chemicals. J. Fish. Res. Bd Can. 32, 1849-1851. Warner R. E. (1964) Toxicant-induced behavioral and histological pathology: a quantitative study of sublethal toxication in the aquatic environment. U.S. Public Health Service. Contract PH 66-63-72.
0043-1354/82/040457-08103.00/0 Copyright O 1982 Pergamon Press Lid
A C O N T I N U O U S - F L O W MINI-DILUTER SYSTEM FOR
TOXICITY TESTING DUANE A. BENOIT,VINCE R. MATTSONand DIANE L. OLSOr~ U.S. Environmental Protection Agency, Environmental Research Laboratory-Duluth, 6201 Congdon Boulevard, Duluth, MN 55804, U.S.A. (Received July 1981)
Abstraei--A space saving portable mini-diluter exposure system for testing early life stages of fish and invertebrates has been developed and successfully used both in the laboratory and on-site with single chemicals and with complex effluents. This gravity-operated system can also be installed in a compact, vented enclosure to permit safe testing of hazardous volatile chemicals. The mini-diluter test system has several additional advantages over other widely used dosing systems in that it: (1) uses small volumes of complex effluents and/or single chemicals which reduces the problem of removing hazardous material from the test system's waste water; (2) works well with waste containing suspended solids; and (3) can maintain normal operation for up to 10 h if the effoent headbox supply pump fails and up to 4 h if the diluent water headbox supply pump fails.
INTRODUCTION Increased awareness of water pollution-related problems has prompted the recent development of many reliable and accurate toxicant dosing systems (Lemke et al., 1978; Peltier, 1978). Two of the oldest types of intermittent flow-through systems successfully used were a serial diluter and a proportional diluter described by Mount & Warner (1965) and Mount & Brungs (1967), respectively. Warner (1964), however, was the first to introduce a truly continuous-flow serial diluter (Fig. 1). The flow pattern diagrammed in this figure demonstrates the simplicity of the diluter in which toxicant from one source is continuously diluted by successive additions of water to produce lower and lower concentrations of toxicant. This water delivery system has several advantages over most other dosing systems in that it is gravity operated, does not require critical timing devices, is free of vacuum siphons and/or solenoid valves and once calibrated does not require further adjustment. It is also
easily adaptable to a wide variety of uses and applications with either single chemicals or complex effluents. Mean flow deviations are less than 2To at any dilution. Warner's continuous-flow serial diluter was used in exposure-related trout studies conducted by McKim" et al. (1970). This system, which worked exceedingly well, was essentially the same as shown in Fig. 1 except that a constant-drip Mariotte bottle, instead of a toxicant pump, was used to meter the chemical sol. ution. During the ensuing years, researchers at the U.S. EPA Environmental Research Laboratories at Duluth, MN and Corvallis, OR used, with increasing frequency, a modified version of the continuous-flow diluter: the modification consisted of a series of baffles, built into the diluter, to ensure adequate mixing of toxicant and dilution water prior to delivery to test tanks. These large scale, single-chemical exposure systems, recently described by Garton (1980), were designed to deliver more than 201. test water h-1 to each of 5 concentrations plus a control.
CONSTANT HEAD MANIFOLD
,l,
WATER
ill T.C. • I
T.C.=O.25
T.C. - 0 . 5
T.C.=0.125
T.C.=O.D65
TO EXPERIMENTAL TANKS
Fig. 1. Dilution principle used in Warner's original continuous-flow serial dilution apparatus (each arrow equals 1 volume of water per unit time). 457
458
DUANE A. BENOITet al.
Interest m developing the early life stage (ELS) fish toxicity test concept introduced by McKim (1977) led to the design of the compact continuous-flow minidiluter exposure system described in this paper. The system accurately delivers as little as 31. test water h - ~ to each of 5 concentrations plus a control. It can be used in the laboratory or in the field to test the effects of either single chemicals or complex effluents on young fish. The small ELS test apparatus takes very little space and requires only small volumes of test water: this latter point is a critical factor when there is a need to transport effluents to a laboratory test site at some distance. Using smaller volumes of test water also reduces filtration costs when one is required to remove hazardous test chemicals from the diluter waste water before discharge. The ELS test system described in this paper has been tested and evaluated both in the laboratory and on-site in a mobile trailer. To date. this apparatus has been used to conduct 33 ELS exposures to various toxicants, using the fathead minnow ( P i m e p h a l e s promelas) as the test animal. These tests have included * Manual for Construction of a Continuous-Flow MiniDiluter System for Toxicity Testing, by D. A. Benoit et at.. U.S, Environmental Protection Agency, Environmental Research Laboratory-Duluth, Duluth, MN 55804, U.S.A.
metal,s, pesticides, volatile organic compounds and treated complex effluents from metal plating, oil refinery and sewage treatment plants. The system has also recently been successfully used in tests conducted on macroinvertebrates (Spehar et al.. 1980; Anderson, Manuscript ). The following text contains a general description of the mini-diluter exposure system including principles of operation and evaluation. Limited space would not allow us to include detailed construction drawings and photographs. If the reader is interested in further details, complete fabrication instructions can be obtained from the Environmental Research Laboratory-Duluth*.
DESCRIPTION OF CONTINUOUS FLOW MINI-DILUTER
Figure 2 is a photograph of the mini-diluter. The diluter is constructed of double strength glass (3 mm thick) and mounted on a 1.9 cm thick plywood backboard (69 × 38 cm). It is divided into 4 major units as illustrated in the schematic drawing of Fig. 3: these units are the toxicant and water cell (TC and WC), dilution cell (DC); flow booster cell (FBC) and flow splitter cell (FSC).
Fig. 2. Continuous-flow mini-diluter for use with either single chemicals or complex effluents.
A continuous-flow mini-diluter system for toxicity testing Toxicant and water cell
The unit containing the constant head toxicant cell (TC) and water cell (WC) measures 7.6cm wide x 54.6cm long × 5.1cm high. Each cell is separated by a vertical glass partition between the emergency outlet (EO) standpipes (Fig. 3t. Float valves (FV) which connect to headboxes maintain a constant depth of toxicant or diluent water in each cell and can be adjusted up or down to change head pressure. Emergency outlet drains in each cell are set slightly above the normal water depth. Water from both cells passes through a 20 mesh stainless-steel screen for large particle filtration before it reaches the capillary flow tubes shown in Fig. 3, (T-1 and W-1 through W-5). Capillary flow tubes are initially adjusted up or down in each cell to obtain desired flow rates. Diluter flow rates can also be changed by using different length or diameter flow tubes. Dilution cell
The second unit of the mini-diluter is a dilution cell (DC) which measures 3.8cm wide x 37.5cm long x 5.1cm high. This cell contains a series of baffles to ensure adequate mixing as the toxicant is continuously diluted by successive additions of diluent water. Principles of operation are diagrammed with arrows shown in Fig. 3. Flow tube T-I (2 mm, i.d.) delivers toxicant solution (100ml rain-1) to the
459
left end of the d~lution cell. Half of this volume, at the rate of 5 0 m l m i n -1, flows out the C-1 flow tube (1.5 mm, i.d.) and is the highest test concentration. The other half of the volume, 50 ml m i n - t, is diluted by one-half when it mixes with 50 ml of diluent water per rain from the W,1 flow tube (1.5 mm, i.d.). Half of this combined volume of I00 ml min-1 flows out the C-2 flow tube to become the second highest exposure concentration, and the remainder is again diluted by one-half from the next addition of diluent water from the W-2 flow tube. This flow pattern continues until the last dilution is made. The extra volume of test water flows over a baffle and into a water outlet (WO). Water used for the control tank flows through the diluter exactly the same way as the test water. Flow tube W-5 (2 mm, i.d.) delivers control water ( 1 0 0 m l m i n -~) to the right end of the dilution cell tFig. 3). Half of this volume, 50 ml rain- t, flows out the C-6 flow tube (1.5 mm, i.d.), and the remainder flows over a baffle and into a water outlet. Flow booster cell
Test water concentrations (C-1 through C-6) flow from the dilution cell to individual flow booster cells (FBC) which measure 2.5 cm wide x 4.5 cm long x 5.4 cm high (Fig. 3). Each of these cells contain one standpipe siphon similar to those described by Benoit & Puglisi (1973). Approximately every 30 s
FV WC i
i
2x / /
Ix
Ix
Ix
Ix
®
2x
DC ;-I'm'
C-2'm'
C-3'm'
C-4'~"
1
1
1
EO
C-5"t~""E3" "m'C-6
1
1 FBC
FSC
-
i
ABCD
2
ABCD
3
ABCD
4
ABCD
5
ABCD
s
ABCD
Fig. 3. Schematic drawing and flow pattern of continuous-flow mini-diluter. (C) concentration flow tube; (DCI dilution cell; (EO) emergency outlet: (FBC) flow booster cell: (FSC) flow splitter cell: (FV) float valve: (T) toxicafit flow tube; (TC) toxicant cell: (Wt water flow tube: (WC) water cell; (WO) water outlet: (IX) one volume; (2XL two volumes.
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DUAnE A. BENOIt et at.
each cell rapidly empties its content to a flow splitter cell located directly below. As the name implies these flow booster cells are used to increase flow to the flow splitter cells. The rapid filling of each flow splitting cell is necessary to obtain a precise split of the test water through each flow splitting delivery tube.
certain lots of neoprene stoppers are acutely toxic to fathead minnow larvae. IBroderius. personal communication)*.
Flow splitter cell
Figure 4 shows a portable early life stage exposure system. The slotted angle iron support frame measures 66cm wide × 102cm long × 74cm high and sits on a 61 × 9t × 66cm high table. With minor alterations, the same system can be used for testing either complex effluents or single chemicals.
Flow splitter cells (FSC) measure 2.5cm wide × 5.7cm long × 4.8cm high (Fig. 3). Each cell contains four tubes (1.5 mm, i.d.) cut equally in length and adjusted to the same height. Test water received from each flow booster cell is divided into four equal volumes, thereby creating four replicate concentrations at each treatment level.
Diluter evaluation Daily flow rate measurements through each of the 12 diluter flow tubes over a 5 week period resulted in an overall mean standard deviation of + 1.3°o. During a 5 week trial test with dissolved NaC1 the mean variability of each concentration ranged from 2 to 7° o. In this test, 5 concentrations (0.02-0.3°o) were measured daily by conductivity. Variation within each flow splitter cell ranged from 2 to 8°o. During four ELS fathead minnow toxicity tests with hexachlorobutadiene, 1,2-dichloropropane. 1,3-dichloropropane and 1,2-dichloroethane, comparisons of chemical concentrations within each group of four replicates, sampled simultaneously, showed that concentrations were within 90°o of each other and ranged from 85 to 98°0 (Benoit et al., 19821. Float valves are quite sensitive and once they are adjusted to the desired height they will maintain a constant depth of water within +2 mm. If headbox pressure changes, the valves will automatically readjust themselves to maintain the same head pressure m the toxicant and water cell. If either float valve sticks open, excess water will flow out the emergency standpipe and the diluter will continue to operate normally. The mini-diluter will not operate smoothly until the glass flow tubes in each cell wet-up. The system, therefore, should be started at least 24 h before a test ~s begun. As long as the head pressure remains similar in the toxicant and water cell, no further adjustments are necessary for proper diluter operation during a test except routine cleaning of float valve and flow tube orifices with pipe cleaners. Some test chemicals or effluents can cause fungus to gro~ very rapidly, in which case diluter flow tubes may have to be cleaned daily. With practice, however, one can complete this task in about 5 rain. All batches of neoprene stoppers should be checked for toxicity prior to their use in the diluter and exposure chambers. Recent static tests have shown that * S. J. Broderius. U.S. Environmental Protection Agency. Duluth. MN 55804. U.S.A.
DESCRIPTION OF PORTABLE EXPOSURE
SYSTEM
Complex effluent test ~ystem Two identical pair of stainless-steel headboxes are used with the system, one pair to supply diluent water for the diluter water cell and the other pair to provide effluent water for the diluter toxicant cell. Each pair sits on top of the support frame and consists of two interconnected headboxes A and B which are gently aerated to avoid temperature stratification (Fig. 41. Headbox A (30.5 +_ 30.5 _ 40.6 cm high) contains a float valve and standpipe drain. If ample quantities of either diluent or effluent water are available, they can be continuously circulated throughout the headbox and out the standpipe. If either supply is limited, float valves can be adjusted so there is no waste overflow. These headboxes also act as a settling basin for large debris and can temper the water several degrees before it is heated to the desired test temperature. Water subsequently flows by gravity from headbox A through a 20 mesh stainless-steel screen for large particle filtration and into headbox B which is insulated and measures 30.5 ,x 45.7 × 40.6 cm high. The water is then warmed with a 500 W stainless-steel immersion heater and passed through another 20 mesh screen in headbox B before it flows through the diluter to the exposure chambers. Styrafoam covers for either headbox A or B may be used to increase insulation or to reduce the loss of volatile chemicals or effluents (Fig. 4). Glass exposure chambers are designed to accommodate 15 test fish per chamber and measure 7 cm wide × 18.8cm long ~ 9.2cm high with a water depth of 4.5 cm, Separate delivery tubes distribute the test water from the diluter to each replicate exposure chamber. Delivery tubes are positioned by stratified random assignment, Flow rate to each chamber containing 500ml of test water, averages 12.5 ml minwith one chamber volume displacement every 40 min. A white background can be used under the chambers to help make the test fish more visible, Eggs are incubated in screened 140 meshl bottom glass jars (120 ml)which slowly oscillate up and down 13.5 cm) in the test water. The glass jars are powered by a 2 rev min rocker arm assembly IMount, 1968). One 61cm long fluorescent tube over the test chambers provides an average light intensity of 20-60 lumens, and photoperiod is controlled by an automatic timer.
A continuous-flow mini-diluter system for toxicit3 testing
461
Single chemical test system
Portable system eraluation
In a single chemical test. the headboxes shown in Fig. 4 contain only diluent water which subsequently flows to the diluter water cell and toxiant cell. Predetermined volumes of the test chemical are then delivered to the toxicant cell by an F M I ®* metering pump and thoroughly mixed with diluent water using a small magnetic stirrer (Figs 2 and 3t. Float valves do not have to be readjusted when single chemicals are added directly to the toxicant cell because the valve will automatically readjust itself to maintain the same constant head.
Comparative fathead minnow ELS feeding density tests with clean water were conducted between the small 500 ml chambers described in this report and larger 6000ml chambers described and used by Benoit & Holcombe 11978j. This study was conducted to determine if the smaller chambers with lower flow rates were suitable for normal growth and survival of young fathead minnows. Subsamples of preserved brine shrimp nauplii were counted on a Sedgewick Rafter counting chamber to determine approximate numbers of live shrimp fed. Results of the test, shown in Table 1, demonstrated that although the three feeding densities greatly affected growth, there was no significant [P = 0.05j difference in growth or survival between either size of chamber at each feeding density. [P = 0.05, analysis of variance with Dunnett's testl.
*Fluid Metering. Inc.. Oyster Bay. NY 11771, U.S.A. The U.S. Environmental Protection Agency neither recommends nor endorses any commercial product: trade names are used only for identification.
Fig. 4. Portable early life stage exposure system, (A) headbox with float valve: IB) insulated headbox with immersion heater. Water flows by gravity from A to B through an interconnecting pipe. w,R. 16 4-~G
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DUANE A. BENOIT et al.
Table t. Survival and growth of 4-week-old fathead minnows fed live brine shrimp nauplii at various densities in two different size chambers with different flow rates. All tests were conducted at 25 + 1 C Chamber size and flow rate
Feeding density
Survival (o,,)
Weight (mg)
500 ml 12.5 ml min- ~
4000* 12,000 20.000
95+ 95 80
24 + 10.+ 77 _ 20 94 ___28
6000 ml 47mlmin -~
4000 12.000 20,000
97 83 92
23 _ 10 68 + 17 100 _ 24
* Number of live brine shrimp fed 3 times daily t Each entry represents the mean of four groups of 15 fish each. :~ Mean + SD,
Oxygen concentrations in the 500 ml chambers during 32 day fathead minnow ELS toxicity tests have consistently been measured at levels /> 75"0 saturation except during tests with certain complex effluents which exhibit a high BOD. The effects of low dissolved oxygen on the fathead minnow have been demonstrated by Brungs (197l) at 60°,, saturation and lower. If the test water is dark or turbid, exposure chambers can individually be removed from the system and placed over a fluorescent light box to observe the test fish or to count mortalities. A small beaker can be used to catch the flow of test water during this time. A unique feature of the system described in this report is that it uses a total of only 241. test water h - t. If the main supply of effluent water is tempor-
Fig. 5. Stationary vented early life stage exposure ,;}stem
A continuous-flow mini-diluter system for toxicity testing ariiy interrupted, the headbox holding capacity and location is such that the system can continue in normal operation for up to 10h. If the diluent water supply is interrupted the system will operate for up to 4 h. An alarm system can easily be installed to alert the investigator before the headboxes run dry. If the problem cannot be corrected in time, additional water can be added to the headboxes by hand. With such low volumes, it is also easy to screen out troublesome debris in the headboxes before it reaches the small diluter flow tubes. Since the test system described in this report uses only 100 ml of toxicant m i n - J , a 2101. polyethylene drum with a submersible pump can be conveniently used to hold a 35 h supply of complex effluent. If necessary, the drum can also be used to premix daily effluent samples before the diluted test effluent is pumped to the headboxes. Overall height of the entire system including headboxes measures only 183 cm which makes the system ideally suitable for use in almost any size laboratory room or mobile trailer. When using the portable system in a trailer the table must be securely attached to the floor and headboxes should be removed from the support frame during transportation. All other parts including diluter and exposure chambers can remain attached to the support frame which is bolted to the table top. The portable system (excluding table and headboxes) weighs approx. 75 kg: and can easily be moved from one facility to another by removing the headboxes, detaching the frame f/'om the table, and transporting it in a vehicle. Break-down and set-up time usually requires about 2 h. Complete fabrication costs of the portable system averages approx. $800-900. Over half of this initial cost is for immersion heaters and stainless steel headboxes.
463
or special filters used to remove hazardous test chemicals from the diluter water before discharge. This system is ideally suited for use in testing hazardous volatile chemicals and was designed to protect the investigator from possible harmful exposures to toxic fumes. Negative pressure created on the inside of the enclosure enables one to safely service the system and take care of the te~t fish through small sliding glass doors. During tests conducted at our laboratory the enclosure was vented through the laboratory air exhaust system which drew an average of 0.7 m a min-1 through the enclosure (approx. one air volume every 2 min). Air samples taken with one 30 x 30cm sliding glass door open have shown measurable quantities of volatile test chemicals inside the enclosure but no detectable concentrations were found outside. Another feature of the enclosure is if the diluter leaks or overflows, the spilled test water can be diverted directly to the system's drain lines and will not flood the room. Due to the fiberglass or epoxy paint, the enclosure bottom is also water tight and can hold up to 100 liters if a leak should occur in some other part of the system. An alarm can be installed in the enclosure base to warn the investigator of major leaks. The accumulated test water in the base can then be drained off through a discharge valve and if necessary passed through a filter. Fabrication costs of the complete enclosure averages approx. $200-300.
Acknowledgements--We thank B. Riedel for photography and daily assistance and G. Endicott, R. Syrett, L. Herman and G. Lien for their assistance with the vented enclosure.
REFERENCES
DESCRIPTION OF STATIONARY VENTED EXPOSURE SYSTEM Figure 5 shows a compact stationary vented exposure system containing the same basic equipment as described for the portable system except that the metal support frame is not used and the headboxes are hung from overhead. The vented plywood enclosure is sealed with fiberglass or epoxy paint on the inside and measures 76 cm wide × 120 cm long with a height of 112 cm over the exposure chambers and 159 cm over the diluter. Both apparatus and test fish can easily be observed through viewing windows located on the sides and top (Fig. 5). One 5 cm hole located near the bottom of each side allows a continuous-flow of air to be drawn through the enclosure and out a 10 ¢m exhaust vent located over the diluter. Exhaust air can be purified through a charcoal filter if necessary. Ample space is available in the bottom of the enclosure to install apparatus such as chemical saturators (Veith & Comstock, 1975; Gingerich et al., 1979), stock bottles, metering pumps,
Anderson R. L. Effect of pydrin on non-target aquatic invertebrates. Environmental Research Laboratory, Duluth, MN 55804, U.S.A. (Manuscript). Benoit D. A. & Holcombc G. W. (19"/8) Toxic effects of zinc on fathead minnows Pimephales proraelas in soft water. J. fish Biol. 13, 701-705. Benoit D. A. & Puglisi F. A. (1973) A simplified flowsplitting chamber and siphon for proportional diluters. Water Res. 7, 1915-1916. Bcnoit D. A., Puglisi F. A. & Olson D. L. (1982) A fathead minnow (Pimephales promelas) early life stage toxicity test method evaluation and exposure to four organic chemicals. J. envir. Pollut. In press. Brungs W. A. (1971) Chronic effects of low dissolved oxygen concentrations on the fathead minnow. J. Fish Res. Bd Can. 28, 1119-1123. Garton R. R. 0950) A simple continuous-flow toxicant delivery system. Water Res. 14, 227-230. Gingcrich W. H., Seim W. K. & Schonbord R. D. (1979) An apparatus for the continuous generation of stock solutions of hydrophobic chemicals. Bull. era'Jr, contain. Toxic. 23, 685-689. Lemke A. E., Brungs W. A. & Halligan B. J. 0978) Manual for construction and operation of toxicity-testing proportional diluters. USEPA, Ecology Research Series
EPA-600/3-78-072.
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DUANE A. BENOIT et al.
McKim J. M. (1977) Evaluation of tests with early life stages of fish for predicting long-term toxicity. J. Fish. Res. Bd Can. 34, 1148-1154. McKim J. M.. Christensen G. M. & Hunt E. P. (1970) Changes in the blood of brook trout Salvelinusfontinalis after short-term and long-term exposure to copper. J. Fish. Res. Bd Can. 27, 1883-1889. Mount D. I. (1968)Chronic toxicity of copper to fathead minnows. Water Res. 2, 215-223. Mount D. I. & Brungs W. A. (1967) A simplified dosing apparatus for fish toxicology studies. Water Res. 1, 21-29. Mount D. I. & Warner R. E. (1965) A serial dilution apparatus for continuous delivery of various concentrations of materials in water. U.S. Department of Health, Education and Welfare, Health Services Publication 999-WP-23.
Peltler W. (1978) Methods for measuring the acute toxicity of effluents to aquatic organisms. USEPA. Ecology Research Series EPA-600/4-78-012. Spehar R. L.. Tanner D. K. & Gibson J. H. (1980) The effects of ke[thane and pydrin on early life stages of lathead minnows (Pimephales promelas) and amphipods (Hyallela azteca). Presented at the American Society for Testing and Materials 5th Annual Proceedings of Aquatic Toxicology. Veith G. D. & Comstock V. M. (1975) Apparatus for continuously saturating water with hydrophoblc organic chemicals. J. Fish. Res. Bd Can. 32, 1849-1851. Warner R. E. (1964) Toxicant-induced behavioral and histological pathology: a quantitative study of sublethal toxication in the aquatic environment. U.S. Public Health Service. Contract PH 66-63-72.