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Real-time Effluent Biomonitoring: The Toxicity Early Warning System (TEWS)

C.Sereres (Environmental Engineering), Dr. W. Keeler (Physics), and Dr.P.Lee (Biology) Lakehead University, Thunder Bay, ON, CAN

Introduction
The traditional monitoring of industrial effluent quality has relied heavily on two laboratory-based components. First of which is physical-chemical measurements to determine the total concentration of a particular analyte in the effluent. The second is gathering biological data in the form of a static non-renewable acute bioassay. With industrial effluents increasing in frequency, intensity and severity, increased research in new technologies has revealed that traditional effluent monitoring has the following limitations: (1) complicated, expensive and time consuming; (2) cannot measure chemical compounds below analytical detection limits; (3) does not present data regarding additive, subtractive, or synergistic toxic effects; (4) mainly use a grab sample, which neglects to provide a representative picture of the extent of contamination; (5) early detection of poor effluent conditions (e.g. result of accidental spill) is minimal; (6) use mortality as a test endpoint and disregard the concept of ecological death. Lakehead University has developed a new biomonitoring system that utilizes rainbow trout (Oncorhynchus mykiss) bevahiour to rapidly detect acute levels of toxicity in industrial effluent: The Toxicity Early Warning System (TEWS). With full implication and success of the TEWS, the tools will be available to conserve and protect our water resources from the byproducts of industrial activities.

In Situ Design
Located at final outfall pipe Complete flow through system
Fish are acclimatized on site Physical-chemical & biological parameters assessed in real-time Line conditioner prevents unwanted electrical noise System components cleaned
regularly after each test

(1) (2) (3) (4)

Cough Frequency has wide potential application as a bioassay tool because : a wide array of chemical substances generate behavioral responses the behavioral responses are rapid and sensitive the level of behavioral response occurs in proportion to the toxicant concentration The cough is predictive of long-term adverse effects at levels near the maximum acceptable toxicant concentration (MATC) Coughing below displayed at 2548 gl-1 zinc

Chilling tank volume is 200 L Chamber flow rate is 200ml/m System is kept at 15 oC +/- 2oC
Figure 4: TEWS In Situ Flowchart Figure 5: TEWS pulp mill operations

Figure 12: Shows cough frequency to 5 different zinc concentrations

Figure 13: Approximately a 25 second recording

.0400 sec/div

Behavioural Response
Whole Body Movement Fin Movement Breathing

In Lab Results
Complex Effluents
Aerated 4419.454545 8.403363636 1254.504545 7.669090909 n/a 2.001363636 0.201454545 24.70181818 786.8727273 0.107313667 0.0025 0.071660101 0.00025 42.9626777 0.000833437 0.007047344 0.003916354 0.008024767 0.28426816 191.7544001 122.3398569 0.037211335 0.025 783.2704801 0.053722559 0.015 11.81642515 0.025 0.71862321 n/a 0.00556428 0.007651625 0.865547121 38.27819513 Non-Aerated 5873.416667 7.766416667 1911.810833 26.23076923 n/a 0.0025 0.005 25.38256154 879.3923077 0.067384615 0.0025 3.5 0.324123077 0.00025 86.43 0.000976923 0.005423077 0.008769231 0.009930769 5.306846154 222.5384615 131.8846154 0.217961538 0.025 631.3076923 0.066023077 0.0549 13.54307692 0.025 1.334538462 n/a 0.004825 0.009333333 0.616307692 62.34162879 Lake Superior 115.39 7.36 41.55 Ontario Guidelines 8.5

Real-time Monitoring

In Situ Results

Note : From peak to peak is one breath Which is the sh opening and closing its gills!

Table 2. Values for all parameters in both landfill leachate lagoons have reference data from Lake Superior and the Ontario Water Quality Guidelines. All parameters except conductivity and pH are in units of mg l-1
Analysis Conductivity mS/cm pH Alkalinity mg/L Br NO2-N NO3-N PO4-P SO4 Cl Al As B Ba Be Ca Cd Co Cr Cu Fe K Mg Mn Mo Na Ni Pb S Se Sr Ti V Zn T Phosphorus T Nitrogen

Non-Aerated Lagoon LC50=13.397 95% CL(10.808-16.607)

Quantify fish behavioral responses to reference toxicants and know complex effluents Implement TEWS in situ to monitor industrial effluent quality in real-time

Research Objectives Research Methods

3.44 1.4 0.031 0.005 0.011 0.002 13.66 0.001 0.01 0.002 0.002 0.051 0.52 2.88

0.002 0.075 0.1 0.2

X-Axis: 0.8 s/div


Figure 6: Displaying an ideal fish breathing pattern

X-Axis: 0.8 s/div


Figure 7: Full body movements and fin movements of a fish

0.0002 0.0009 0.0089 0.005 0.3

Aerated Lagoon LC50= 10.153 95% Cl (8.520-12.099

0.04 1.58 0.002 0.025


5-Very High 4-High 3-Medium 2-Low 1-Very Low

Figure 8

Determining Response Values


2.662 Breaths per second Start of the test has very low movement Majority of movement

Size of Fish: 4.229 g

Breaths Per Second: 2.662

Depth of Breath: 0.065V

0.01

0.1

0.01 0.002 0.0031 0.5053

0.006 0.02 0.03

Employs fish behaviour sensors to detects the presence of acute toxic substances Fish are confined in flow-through test chamber which are exposed first to dechlorinated water (6 hour baseline), then are exposed to effluent (6 hour) Significant changes in ventilatory behavior and locomotion activities are detected through noninvasive electrodes in each tank Types of responses include: changes in fish whole body movement, fin movement, increased ventilatory frequency, and increased cough frequency, all of which help indicate signs of stress Fish Signals are amplified, filtered, compared to reference chamber and interfaced to a computer where WinDaqXL software is used to analyze waveforms. Computer based algorithms continuously monitor and assess each fishs reaction to the effluent
Power Source Floating Switch Bridge Circuit Testing Cell
X-Axis: Hz

Degradation of Breath

Figure 14A and 14B showing dose response curves

Control 6.25
Moderate movement

12.5 25 50

I n c r e a s i n g T o x i c i t y

X-Axis: 66.4 s/div

Figure 9: Windowed Fast Fourier Transform (FFT) showing how we can calculate breaths per second for Figure 4.

Analyzing Waveforms
Functional Generator

Figure 10: A compressed one hour fish test in baseline water showing how we can calculate overall movement

100
Figure 15: Display of how the aerated lagoon effluent degrades fish breath Table 3. Showing breath parameters

Toxic Fish Response Levels


Level I: Cough frequency increases significantly
without a change in breath rate or depth. Indicates adverse long-term (chronic) effects

Aerated Effluent Conc.

Breath Rate Breath (Breaths per sec) Depth (Volts)

Whole body Cough Rate movement Deviation (%) (%)

Figure 17: Shows that both biological and physical-chemical measurements are needed to produce valid real-time warning

In Lab Design
Stainless Steel Electrodes

Control 6.25 12.5 25 50 100

2.515 3.20 2.490 2.002 1.367 .366

0.185 0.079 0.033 0.032 0.019 0.005

28.732 7.483 3.623 1.882 2.167 0.227

0 150 800 1100 650 100

Note: Rainbow Trout survived less than an hour in both 100% lagoon effluents

Biological
Table 4

Physical-Chemical
4/7/08 4/11/08 6/2/08 O.mykiss 96 hr
LC50

Summary of Chemical Analysis performed by LUIL-ICP (Fullscan)

DataQ Instrument

Level 2: Large increase in cough frequency with


moderate increases in breath rate and/or decrease in amplitude. Indicates sublethal to lethal effects

Units: mg l-1 Total Aluminum

4/4/08 4/6/08 1.132 0.9031

0.713

0.407

0.1108 0.161 to 0.310


EPA ECOTOX DATABASE

Level 3: Large and rapid increases in cough


WinDaqXL Software Oscilloscope Personal Computer
Figure 11: Shows technical components of TEWS

frequency, breath rate, and/or decrease in amplitude. Indicates lethal (acute) effects

Level 4: Cough frequency, breath rate, and


amplitude greatly reduced or erratic. Response indicates death is approaching

Figure 16: Shows how fish recover after being taken out of 100 aerated lagoon effluent and put back into dechlorinated water

Based on the following results, a TEWS Level Three toxic fish response occurred in early spring .Management was notified directly and corrective action followed.

Fish inside testing cell

In Lab Results
Single Toxicants
Response Type
Cough Rate, Ventilatory Rate Cough Rate, Ventilation Ra t e Cough Rate NR NR

Inow from head tank


Table 1 Zinc (!g l-1)
2548 1274 637 315 161

Figure 1: Shows both the test and reference chamber

Figure 3: Picture of a baseline test with dechlorinated water

Rainbow Trout used are between 2-5 g System Temperature is 15 oC +/- 2oC Flow rate of the system is 200ml/m Acclimatized for 24 hour before test Toxicant is recirculated by a pump

Figure 2: Schematic of TEWS lab setup

Dechlorinated water parameters Temperature (oC): 14.3- 16.8 pH: 7.15- 7.90 Dissolved oxygen (mg l-1): 9.0- 10.5 Conductivity (S): 109-118 Harness (mg l-1 as CaCO3): 47-52

Fraction of Rainbow Trout 96-h LC50


0.98 0.49 0.25 0.12 0.062

Time to first Response (h)


<1 hour 3 hour s 2 hours 3 hour s 5 hour s NR NR

Percent Change
Increase 420% Increase 24 % Increase 340% Increase 21 % Increase 26 0 % NR NR

Allows for real-time response to potentially toxic events year round Prevents industry from failing monthly government regulated effluent tests Economical viable system to set-up and run Data acquisition system and user interface is easy to collect and analyze data The TEWS system is a portable self-contained chamber

Advantages of the TEWS

North-western Ontario needs TEWS technology in order to protect its pristine environment

96-hour LC50 of zinc found to be 2600 gl-1

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