A simple continuous-flow toxicity test system for microscopic life stages of aquatic organisms

This paper describes a flow-through toxicity testing system utilizing a new exposure chamber designed for microscopic organisms. Typically, flow-through testing is conducted with either a serial or proportional diluting system. While generally relying on gravity to drive water flow, as different amounts of toxicant and diluent are combined in glass or plexiglass mixing cells, they are almost always open to the surrounding atmosphere. In contrast, the system described here is based on a simple exposure design utilizing premixed stocks representing each exposure concentration. While the system is entirely closed to the surrounding atmosphere, for testing of volatile organic mixtures in addition to nonvolatile inorganic toxicants, the delivery manifold may be aerated for toxicants of low volatility and high BOD. The system incorporates flexible Teflon gas sampling bags for stock preparation and storage (thus avoiding need for a headspace), and digital unified-drive peristaltic pumps for controlled toxicant delivery. To reduce surface partitioning of toxicants, all system components are constructed of chemically inert materials (Teflon, glass and silicone).     

An aquatic respirometer for use in sublethal studies

A computer-controlled respirometer system which permits measurement of oxygen consumption of marine and freshwater organisms at regular intervals over a period of several days is described. The system enables assessment of changes in respiration during and after exposure to sublethal concentration of pollutants.     

A simple respirometer for measuring oxygen and nitrate consumption in bacterial cultures

A simple respirometer is described to measure simultaneously oxygen and nitrate concentrations. It proved to be an easy tool in denitrification studies. This was tested with Pseudomonas aeruginosa.     

A simple continuous-flow toxicant delivery system

This paper describes a simple continuous-flow toxicant delivery system for use in testing toxicity of dissolved or suspended compounds to aquatic organisms. Its advantages are simplicty, ease of construction, and capacity for high flow rates. The system consists of a glass headbox and diluter box, a toxicant pump, a framework for support, and a system of tubes to deliver a series of toxicant concentrations to the test aquaria. Methods are given for construction and for calibration of the system.     

A simple indoor artificial stream system designed to study the effects of toxicant pulses on aquatic organisms

Intermittent discharges of effluents are a common cause of reduced water quality. There have, however, been few studies of the effects of such discharges on the flora and fauna of receiving waters. A simple indoor artificial stream system designed to study the effects of toxicant pulses on aquatic organisms ranging from macroinvertebrates to small fish has been constructed and is described. A computer-controlled toxicant input system has been developed that is capable of generating and delivering toxicant pulses of varying shape, magnitude, amplitude and duration. Comparisons of predicted and actual toxicant concentrations have been carried out and hydraulic and toxicant conditions were found to be reproducible. Sample results are given of the effect of short-term pulses of cadmium on the amphipod Gammarus pulex. Possible future developments of the system are discussed.     

A simple gravity corer and continuous-flow adaptor for use in sediment/water exchange studies

This paper describes a gravity corer and continuous-flow adaptor for use in sediment/water exchange studies. The objectives in designing the apparatus were to provide efficient sampling of intact sediment cores from the range of sediment types that occur in Hartbeespoort Dam (South Africa), and to facilitate rapid experimental manipulation of sediment/water chemical gradients for measurement of potential exchange rates. Simple phosphate uptake/release experiments are used to demonstrate the use of the apparatus, and to establish the relationship between overlying water retention time and the time required to attain steady state conditions in the outflow. This study has demonstrated that: steady-state phosphate concentrations in the outflows from continuous-flow sediment/water systems are independent of the water volumes overlying the sediment cores; the time required to attain steady-state concentrations in the outflows is directly related to the water retention time in the reaction vessel; phosphate release rates from sediment cores decrease linearly with increasing phosphate concentration in the overlying water.     

A method for calculating effects, on aquatic organisms, of time varying concentrations

A method is presented for calculation of the effects, on aquatic organisms, of exposure to time variable concentrations of toxicants. The technique employs data from classical bioassay tests obtained using constant toxicant concentrations. Calculations of organism mortality as a result of exposure to time varying concentrations are compared to observed data providing a test of the proposed method. The calculation procedure employs available data to assess the duration and extent of carry over effects from individual exposures. Applications and limitations of the proposed method conclude the discussion.     

A continuous-flow mini-diluter system for toxicity testing

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 effluent headbox supply pump fails and up to 4 h if the diluent water headbox supply pump fails.     

Factors controlling biological availability and toxic effects of lead in aquatic organisms

The distribution of anthropogenic lead in the aquatic environment is governed by atmospheric input (leaded gasoline) and input from point sources (mining areas, smelters, factories producing chemicals and goods based on lead). The bioavailability of lead to organisms is a function of the primary and secondary speciation of lead. Dissolved chemical forms of lead are extremely toxic when present in high concentrations in an aquatic environment. Chronic toxicity occurs when lead is bioconcentrated in aquatic species over a period of time and when it is stored in internal organs. However, biomagnification has not been observed in the aquatic environment.     

An automated batch reactor for the preparation of artificial waters for toxicity tests on aquatic organisms

The use of artificially reconstituted aqueous solutions for experiments on freshwater organisms has the advantage that standard conditions are reproducible and can be predetermined. A batch reactor which automatically produces a reconstituted water from five salt solutions and deionised water is described. Suitable hardness was achieved by dissolution of a CaCO₃ suspension using CO₂ gas, and the final solution was brought to equilibrium with the atmosphere by aeration through glass sinters. Results to date show the equipment to be efficient and the water to be of stable quality and suitable for experimental purposes. The reactor is capable of producing a range of different waters and has proved reliable in operation.     

A continuously recording polarographic respirometer and its use in oil biodegradation studies

A continuously recording flow respirometer based on an oxygen electrode is described. It was designed to give long-term respiration measurements without loss of the carbonate-bicarbonate buffering character of sea water, and without the build up of waste products as in closed systems. An experiment on the microbial degradation of oil is described, together with investigations of a photochemical oxidation discovered fortuitously during similar experiments. The relationship between nutrient supply and bio-oxidation rate is discussed.     

An apparatus for the preparation of varying concentrations of chemicals for toxicity tests with aquatic organisms

An apparatus is described for the preparation of varying concentrations of chemicals for use in toxicity tests with aquatic organisms. Results of performance tests are given.Although ideally continuous monitoring of chemical concentrations is desirable, for many chemicals no such continuous monitoring devices exist. The extreme reliability and accuracy of the apparatus' operation and the use of inert construction materials reduces the requirement for monitoring to a minimum to ensure satisfactory preparation of variations in chemical concentration required.     

A titrimetric respirometer measuring the nitrifiable nitrogen in wastewater using insensor-experiment

Measurement of nitrifiable nitrogen contained in wastewater by combining the existing respirometric and titrimetric principles is reported. During an in-sensor-experiment using nitrifying activated sludge, both the dissolved oxygen (DO) and pH in the mixed liquor were measured, and the pH was controlled at a set-point through titration of base or acid. A combination of the oxygen uptake rate (OUR), which was obtained from the measured DO signal, and the titration data allowed calculation of the nitrifiable nitrogen and the short-term biological oxygen demand (BOD) of the wastewater sample that was initially added to the sludge. The calculation was based solely on stoichiometric relationships. The approach was preliminarily tested with two types of wastewaters using a prototype sensor. Good correlation was obtained.     

Simple, continuous-flow systems for dissolving the water-soluble components of crude oil into sea-water for acute or chronic exposure of marine organisms

A continuous flow apparatus, referred to as a solubilizer, was constructed to dissolve the water-soluble fraction (WSF) of crude oil into water for short and long-term experiments with marine organisms. The basic solubilizer system consists of an oil reservoir, oil pump, modified glass bottle, and oil waste reservoir. This system dissolves the water-soluble components of crude oil without the loss of the more volatile compounds and without the formation of emulsions or oil droplets. Four solubilizer systems (two continuous flow and two recirculating) were evaluated for their efficiency in extracting the water-soluble components from Cook Inlet crude oil by analyzing for six monoaromatics over a 96-h period of continuous operation. When a solubilizer system was started, a stable concentration of the monocyclic aromatics in the water-soluble fraction was reached within 24 h. The water quality of the WSF effluent was not altered markedly in the continuous (open) flow systems, but there was a substantial reduction in dissolved oxygen in the recirculated (closed) systems. A single solubilizer system (open flow) with a 3 ml min⁻¹ oil flow and a 1 l min⁻¹ sea-water flow generated approx. 1.4 ppm of the total monoaromatics, which includes benzene, toluene, ethylbenzene and the three xylene isomers. This concentration varied depending on physical parameters and the chemical composition of the crude oil and water. With only slight modifications of the basic continuous flow system, a higher concentration (> 3.4 ppm total monoaromatics) of the water-soluble fraction of crude oil was achieved. By recirculating the water supply, concentrations of 6.7–11 ppm total monoaromatics in the WSF were produced. The stability, reproducibility, and reliability of the four systems were demonstrated in several experiments. Researchers working in aquatic systems with crude oil can well appreciate a simple system that is easily cleaned, relatively maintenance-free, and which produces stable, reproducible concentrations of the WSF over extended periods of time. The increasing demands for ecological studies on aquatic organisms with crude oil, a difficult mixture of chemicals with which to work, precipitated the development of these solubilizer systems.     

A method to measure total antioxidant capacity against peroxyl radicals in aquatic organisms: Application to evaluate microcystins toxicity

Determination of total antioxidant capacity, instead of the measurements of limited number of antioxidants, is very important for the understanding of how antioxidants interact with reactive oxygen species (ROS). Several techniques already exist with this propose, although some of them are extremely time-consuming. A new methodology is proposed, based on the detection of ROS by fluorometry (ex/em: 485/520 nm) employing 2′,7′-dichlorofluorescein diacetate (H₂DCF-DA) as substrate. Supernatant of homogenized samples from different organs (gill, muscle, liver, and brain) of the teleost fish Jenynsia multidentata (Anaplebidae) were exposed to peroxyl radicals generated by thermal (35 °C) decomposition of 2,2′-azobis (2 methylpropionamidine) dihydrochloride (ABAP, 4 mM). Different protein concentrations (0.5, 1, 2 and 8 mg/ml) were assayed to get the best signal and curve fitting of fluorescence data over time (30 min). Total antioxidant capacity against peroxyl radicals was estimated as the difference in ROS area with and without ABAP, relative to the fluorescence registered without ABAP. For application of this methodology, J. multidentata specimens were exposed for 24 h to microcystins, cyanotoxins known to induce oxidative stress. Almost all organs showed a lower antioxidant capacity (p < 0.05) in samples with 8 mg proteins/ml, when compared to protein content of 1-2 mg/ml. In liver samples, higher (p < 0.05) free iron content was determined in samples with 8 mg proteins/ml. Sensitivity test employing GSH spiked in homogenized samples showed the protocol efficiency in detecting total antioxidant capacity. In the test with microcystins a dose-dependent decrease (p < 0.05) of antioxidant competence in gills and brain and an inverse result with liver samples were observed. The use of antioxidant defenses was efficient in avoiding oxidative damage, as the content of oxidized proteins was not altered. Data obtained show the potential of this new methodology to be used in ecotoxicological studies.     

An evaluation of mathematical models for the effects of pH and temperature on ammonia toxicity to aquatic organisms

Available data on the pH and temperature dependence of ammonia toxicity to aquatic organisms were examined and their agreement with various models was evaluated. A model which considers alteration of the relative concentration of un-ionized ammonia at the gill surface failed to adequately describe either pH or temperature dependence. A model that assumes that un-ionized ammonia and ammonium ion are jointly toxic was strongly supported by the data on pH dependence, but could not explain observed temperature dependence. Temperature dependence can be described empirically by a simple log-linear model. The effects of pH and temperature were generally found to be qualitatively and quantitatively similar among fish species.     

Comparison of effectiveness of different ashing auxiliaries for determination of phosphorus in natural waters,aquatic organisms and sediments by ignition method

An improved method for the determination of phosphorus in natural waters, aquatic organisms and sediments by ignition method is proposed. The recoveries of phosphorus (P) from selected inorganic and organic P-containing compound standards after ignition with different auxiliaries, such as MgSO(4), Mg(NO(3))(2), MgCl(2), Mg(Ac)(2) and CaCl(2), were compared. We found that the phosphorus from most compound standards could not be completely recovered when these compounds were ignited (450-500 degrees C) with the MgSO(4) as auxiliary and the baked residue was extracted with 0.2 mol x l(-1) HCl for 30 min at 80 degrees C or at room temperature. P recoveries, for example, were poor, less than 85%, if pyrophosphate and metaphosphate were ignited with the addition of MgSO(4) prior to the extraction of the baked residue with 0.2 mol x l(-1) HCl at 80 degrees C for 30 min. In contrast, MgCl(2), Mg(Ac)(2) and CaCl(2), as well as Mg(NO(3))(2), could all yield complete P recoveries at routine ashing temperatures (450-500 degrees C). The results demonstrate that MgCl(2) is a more effective auxiliary agent for the determination of phosphorus in natural waters, aquatic organisms and sediments by ignition method than MgSO(4) which is commonly used.     

Modifications to electrolytic respirometer systems for precise determination of BOD exertion kinetics in receiving waters

Modifications to an electrolytic respirometric system are described which facilitate precise measurements of BOD progressions for low substrate concentration (BOD₅ < 20 g m^?3) as found in natural waters receiving organic enrichment. Samples may be incubated at any desired temperature or dissolved oxygen concentration with automatic hourly monitoring of the BOD progression. Subsamples may be withdrawn at any time without interference to the measurement. The system is independent of atmospheric temperature and pressure fluctuations and has a detection sensitivity of 0.05 mg of oxygen. Nomographs are presented to enable the maximum dissolved oxygen deficit, and the “apparent lag” time to be estimated. CO₂ removal, which may result in an extended lag phase, need not be used for BOD values below 50 g m^?3 provided adequate buffering is supplied.     

A pressurized proportional diluter for aquatic toxicological studies

A half-liter proportional diluter using positive pressure was designed and tested. Its primary siphons are activated by a pulse of pressure instead of by a partial vacuum as in conventional diluters. This method eliminates the need for venturi tubes, vacuum manifolds, and individual water blocks. Pressure can be supplied by air pumps, air cylinders, or air compressor lines. The diluter's advantages are ease of construction, calibration, compact size, and rapid cleaning. Unit cost is approximately $250.00.