2. Introduction
2.1. Biodegradability testing
2.1.2. Tests on Inherent Biodegradability:
% degradation = O2. /
vii) Biochemical Oxygen Demand (EC C.5)
This test is applied to water-soluble organic compounds; however, it may be applied for volatile and low water soluble compounds by taking special measures and precautions.
A measured amount of the substance is dissolved or dispersed in a suitable medium, inoculated with micro-organisms, well-aerated, and incubated in the dark at a constant defined temperature.
The BOD is measured by determining the difference in dissolved oxygen content at the beginning and at the end of the test. The duration of the test must be at least five days and not more than 28 days.
A blank must be measured in parallel with the test sample.
2.1.2. Tests on Inherent Biodegradability:
i) Modified Semi-Continuous Activated Sludge (SCAS) Test (OECD 302A, ISO 9887)
This method was adapted from the Soap and Detergent Association semi-continuous activated sludge (SCAS) procedure for assessing the primary biodegradation of alkyl benzene sulphonate. It is used to evaluate the potential ultimate biodegradability of non-volatile, water soluble (at least 20 mg dissolved organic carbon/liter) organic substances under exposure to relatively high concentrations of microorganisms over a long time
15 period (at least 12 weeks). The microorganisms viability is maintained over this time period by daily addition of a settled sewage feed.
The test is applicable to water soluble and non-volatile organic chemicals that do not significantly adsorb within the test system, have negligible vapour pressure, are not lost by foaming from the test solution and are not inhibitory to bacteria at the test concentration.
In an aeration (SCAS) unit, activated sludge from a sewage treatment plant is placed, the test compound and settled domestic sewage are added, and the mixture is aerated for 23 hours. Then the aeration is stopped, the sludge is allowed to settle and the supernatant liquor is removed. The sludge remaining in the aeration chamber is then mixed with more aliquot of test compound and sewage and the cycle is repeated. Biodegradation is evaluated by the determination of the dissolved organic carbon content of the supernatant liquor. This value is compared with the one of the liquor obtained from a control tube dosed with settled sewage only.
The dissolved organic carbon results in the supernatant liquors of the test units and the control units are plotted against time.
% Degradation = Where,
OT = concentration of test compound as organic carbon added to the settled sewage at the start of the aeration period.
Ot = concentration of dissolved organic carbon found in the supernatant liquor of the test at the end of the aeration period.
Oc = concentration of dissolved organic carbon found in the supernatant liquor of the control.
The level of biodegradation is therefore the percentage elimination of organic carbon.
ii) Modified Zahn-Wellens/EMPA Test (OECD 302B, ISO 9888)
In this method, the measurement of DOC or COD is used to assess the ultimate biodegradability of the test substance.
16 The test substance, mineral nutrients and a relatively large amount of activated sludge in aqueous medium are mixed. The mixture is agitated and aerated in the dark or in diffuse light at 20-25°C for up to 28 days. Blank controls (without test substance) are run in parallel. Monitoring the biodegradation process is done by evaluating the DOC (or COD) in the filtered samples taken at daily or other time intervals. The biodegradation percentage at the sampling time is the ratio of eliminated DOC (or COD), corrected for the blank, after each time interval, to the initial DOC (or COD) value. The biodegradation percentage is plotted against time to give the biodegradation curve.
The chemical structure, water solubility and vapour pressure of the test substance should be known and its foaming properties also. The toxicity of the test substance to bacteria is advantageous for selecting the proper test concentrations and in understanding the results that show poor biodegradability. This test is usually executed only after the test substance fail to pass ready biodegradability test.
Chemicals which are soluble in water to at least 50 mg DOC/l, non-volatile, do not significantly adsorb within the test system, are not lost by foaming from the test solution and are not inhibitory to bacteria at the test concentration may be determined by this test.
[
] where:
Dt = percentage degradation at time t;
CA = concentration (mg/l) of DOC or COD in the test suspension measured after 3h ± 30 min of incubation;
Ct = mean concentration (mg/l) of DOC or COD in the test suspension at time t;
CBA = mean concentration (mg/l) of DOC or COD in the blanks measured after 3h ± 30 min of incubation;
CB = mean concentration (mg/l) of DOC or COD in the blanks at time t.
17 2.1.3. Simulations Tests:
Activated Sludge Simulation Tests: Coupled Units Test (OECD 303A, EC Activated Sludge Simulation Tests, ISO 11733)
This test is applicable only to the organic substances which are soluble in water, have negligible vapour pressure, and are not inhibitory to bacteria, under the test conditions.
Determination of the substance toxicity to the micro-organisms contributes in the interpretation of the results with low values and in the selection of appropriate test concentrations.
The simulation tests aim at evaluating the extent and rate of biodegradation and the fate of chemicals in a designed laboratory system to represent either the aerobic treatment stage of STP or certain environmental compartments, such as fresh or marine surface water.
Two continuously operated test units are used. Each unit consists of an aeration vessel, a separator and a sludge recirculation. The system is designed to determine the removal and the primary and/or ultimate biodegradation of water-soluble organics by monitoring the changes in DOC and/or COD. One unit is used as control and the other as test item treatment.
The test units are run in parallel under the same conditions. Generally, the mean hydraulic retention time is 6 h, the mean sludge age is 6 to 10 days and the test duration should not exceed twelve weeks after adding the test item. The addition of the test substance is at a concentration of between 10 mg/l DOC and 20 mg/l DOC to only one of the units.
Running and sampling are classified into three main periods:
Stabilization Period: to allow the system to reach a steady state of efficient DOC removal. At least one sample is taken per week.
Running-In Period: starts from the addition time of test substance till its removal reaches a plateau. Three samplings per week are taken.
Evaluation Period: A three weeks period starts when the plateau phase is reached. If the substance shows little or no degradation in the first six weeks, the evaluation period is performed in the following three weeks. Around 15 valid values in the plateau phase are necessary for the test results evaluation.
18 The difference between the concentrations of DOC/COD in the effluent from the test and control units is compared with the added test substance concentration to calculate the elimination of the test substance.
The percentage of removal is plotted versus time. From the elimination curve, the conclusions about the test substance removal process can be drawn.
2.2 Ecotoxicity testing
It is a well-known paradigm in ecotoxicology that bioassays (and test organisms) show different sensitivity to different contaminants. A bioassay will not be equally sensitive to all toxic compounds, its applicability should always be determined for a given purpose. Toxicity assessments are generally based on a battery of tests, where the bioassays represent (1) different trophic levels; (2) exposure routes and (3) might have different sensitivity. Such a minimum battery should include a primary producer (generally an alga), a zooplankton organism (primary consumer) and a fish (secondary consumer). It is also recommended to include a bacterial bioassay.
2.2.1. Algal inhibition test
This test determines the effects of a substance on the unicellular green algal species growth and it takes 72 hours, so the test can measure the effects over several generations.
In this test, the fast-growing green alga species are preferred to be used such as Selenastrum capricornutum or Scenedesmus subspicatus, which are put into the test vessels in a known density. Every 24h the density is measured, and after 72h the test is terminated. Growth is expressed as specific growth rate (r), and the effect as inhibition relative to control growth. The algae density is evaluated by using fluorescence measurements and is confirmed by cell counts.
A cellular fluorescence capacity (CFC) index can be used as another endpoint (Thompson, 1997). The algal fluorescence before (F) and after (Fd) the addition of the toxicant is used to calculate it. The index is expressed as a fraction [(Fd-f)/Fd].
The ISO 8692:2004 standard identifies a growth inhibition of unicellular green algae method applicable for the water soluble substances. This method was modified as
19 described in ISO 14442 and ISO 5667-16, so, the poorly soluble organic and inorganic materials, heavy metals, volatile compounds and waste water can be tested. ISO 10253:2006 describes a method for evaluating the growth inhibition of the unicellular marine algae Skeletonema costatum and Phaeodactylum tricornutum by substances and mixtures in the sea water.
Algae can be employed in on-line water quality testing. The Algae Toximeter is based on the measurements of chlorophyll fluorescence (i.e. toxicants react with the chloroplasts of green algae causing photosynthetic inhibition).
2.2.2. Daphnia-based toxicity testing
Daphnids are the most widely used zooplankton as test organisms. They represent the primary consumers in the aquatic ecosystem. Daphnia-based toxicity test is an acute toxicity test, where the exposing period is 24-48 hours. ISO 6341:1996 standard employs Daphnia magna and the test end-point is the motility inhibition (practically mortality).
Daphnids are specific in the way that they can show other symptoms which are relatively easy to evaluate and give an earlier warning of the toxic effect. Many Sub-lethal Daphnia tests were developed. Some were based on measuring the changes in the heart beat rates (Baylor, 1942), another on grazing intensity (Lotocka et al., 2001), moulting disruption (Rohrlack et al., 2004), inhibition of feeding activity (Ács et al., 2009), and life-history traits such as somatic (individual) growth (Burks et al., 2000), time to first reproduction, number of newborns (Lürling and van der Grinten, 2003). Lürling (2003) used population growth as the measure of toxic effect.
De Mott and Dhawale (1995) studied and used in vitro protein phosphatase activity inhibition as a biochemical end-point.
Daphnia can be used in on-line water quality testing. The Daphnia Toximeter is based on the movement pattern of daphnids. These movements are recorded and the live images are analyzed online. Any behavioral changes are immediately estimated.
20 2.2.3. Fish-based toxicity tests
a) Tests based on mortality as an endpoint
These tests are often used to determine the substance acute lethal toxicity to fish. Zebra fish and rainbow trout are the most preferred test species. The fishes are exposed to different concentrations of the toxic sample for a period of 96 hours. Mortalities are recorded at 24-hour intervals. There are three types of mortality tests can be used:
1. Static test: test solution remains unchanged during the test period.
2. Semi-static test: in which a regular batch of test solution is renewed after long periods (e.g. 24 hours).
3. Flow-through test: in which the test solution is renewed regularly in the test chambers.
ISO 7346/1, /2 and /3 -Water Quality -Determination of the acute lethal toxicity of substances to a fresh water fish (Brachydanio rerio Hamilton-Buchanan -Teleostei, Cyprinidae) test protocols are used in the EU.
b) Tests based on sub-lethal endpoints
Behavioural endpoints in fish are easily noted. The earliest automated systems were based on rheotaxis (Besch et al., 1976). When a toxicant damages the nerve system of the fish, they will sweep away by the current. Following the fish movement can be recorded and converted into x,y coordinate data (Vogl et al., 1999; Beauvais et al., 2000), and transformed into relevant endpoints which include velocity, swimming activity, water column position, angular change, or total distance travelled (Little and Finger, 1990;
Steinberg et al., 1995). van der Schalie et al., (1988) and Diamond et al., (1990) studied the changes in fish ventilator response pattern. U.S. Army Center for Environmental Health Research (USACEHR has developed a system to detect and record the ventilatory movements for continuous automatic evaluation (Sarabun et al., 1999). various test designs have been developed based on detecting during embryonic/larval development abnormalities as well (e.g. Oberemm et al., 1997; Palíková et al., 2007).
Fish can be used in on-line water quality testing. The Fish Toximeter is based on the changes in the fish behaviour. The measurements of speed, swimming activity such as turns or circular motions and number of active fish are used in the analysis.
21 2.2.4. Vibrio fischeri bioluminescence inhibition assay
Vibrio fischeri is a marine, bioluminescent bacterium. Bioluminescence is a natural phenomenon in which visible light is generated by an organism as a result of a chemical reaction. These reactions can be reconstructed outside the organisms from which they originate, thereby enabling exploitation of this natural process. There are diverse types of organisms that display bioluminescence: bacteria, protozoa, fungi, sponges, crustaceans, insects, fish, squid, jellyfish, and lower plants. Bioluminescent organisms occur in a variety of habitats, particularly the deep sea, where light is employed for functions including defence, reproduction and feeding. The enzymes involved in the luminescent (lux) system, including luciferase, as well as the corresponding lux genes, have been most extensively studied from the marine bacteria in the Vibrio and Photobacterium genera and from terrestrial bacteria in the Xenorhabdus genus. It has been found that the light-emitting reactions are quite distinct for different organisms with the only common component being molecular oxygen.
V. fischeri can be found in small amounts in the ocean and in large amount in isolated areas such as the light organs of a squid, Euprymna scolopes with which it has a symbiotic relationship (Brovko, 2010). When the squid is young, it draws in free-living bacteria from the ocean into its light organ. Here they are provided with all of the nutrients that they need to survive. Light emittance is activated only within the squid, as in the ocean cell density is app. 102 cells/ml, and this low concentration of cells is not enough to cause the luminescence genes to be activated. Cell density-dependent control of gene expression of lux genes is activated by autoinduction that involves the coupling of a transcriptional activator protein with a signal molecule (autoinducer) that is released by the bacteria into its surrounding environment (this „communication” is called quorum-sensing1). When in the light organ of a squid, the cell concentration is about 1010 cells/ml, and the autoinducer causes the bacteria to emit light. The squid is even capable of controlling light emittance: during the day, it keeps the bacteria at lower concentrations
1 The regulation of density-dependent behaviour by means of quorum sensing is widespread in bacteria. It can be used for example by opportunistic/infectous bacteria such as Pseudomonas aeruginosa to overcome the host’s immune system: bacteria grow to a certain concentration without any warning sign and they become aggressive only when reaching a critical mass.
22 by expelling some of them into the ocean during regular intervals. At night, as the squid is night-feeder, the bacteria are „allowed in”.
The light output of luminescent microorganisms which emit light as a normal consequence of respiration is read by a luminometer. Chemicals or chemical mixtures, which are toxic to the bacteria, cause changes in some cellular structures or functions such as the electron transport system, cytoplasmic constituents or the cell membrane (Fig.
1), resulting in a reduction in light output proportional to the strength of the toxin (Fig.
2). As bioluminescence of V. fischeri is directly linked to respiratory activity, it provides a good indicator of the metabolic status and has been found to be well correlated with in vivo toxicity tests using higher organisms (e.g. Kaiser et al., 1994).
Fig. 1: The biochemical mechanism of bioluminescence.
Bioluminescence
100
50
15 min 30 min
0
a b c d e
Control
The most toxic sample/
dilution
Samples/dilutions 50 % light reduction
Time (min)
Fig.2: Luminescence inhibition is roughly proportional to the concentration of the toxic compound
23 This principle is used by several commercially available systems as the Microtox, LumisTox, BioTox or ToxAlert. Most experiments were conducted using the Microtox version. It is the most referred in the literature. Numerous authors studied and compared the toxicity values obtained using the Microtox ® system (for a wide range of organic and inorganic chemicals, … etc) with values obtained using other live organisms such as fish, crustaceans, and algae. These studies proved an excellent correlation (Farré and Baceló, 2003). Microtox® offers great sensitivity, cost effective, accuracy and is time saving (Curtis et al., 1982; Gutiérrez, 2002; Dalzell, 2002; Wang, 2002).
One main benefit of the test is that it requires very short exposure: maximum exposure is 30 minutes but an indicative value is given even after 5 minutes. This fact and the easy-to-perform nature of the test make it suitable to use when a very high number of samples are to be processed.
Many field portable devices are based on the measurement of biolumescence inhibition either using Vibrio fischeri or another test organism2. Commercially available for example the ToxScreen system developed by CheckLight Co. On-line versions of the bioluminescent bactera test are also available such as the TOXcontrol On-line Biomonitor developed by microLAN On-line Biomonitoring Systems.
Despite its advantages, the Vibrio fischeri bioluminescence inhibition assay is still rather controversial in ecotoxicology. Some authors question its ecological relevance, emphasizing that Vibrio fischeri, being a marine bacterium, cannot be used to reflect the behaviour of terrestrial or freshwater organisms. New trends in ecotoxicology might help to resolve this problem. The mechanism of bioluminescence is already well-known (e.g.
Nunes-Halldorson and Duran, 2003), purified luciferase enzyme was made available already in the 1950’s (Mc Elroy and Green, 1955). Bioluminescent reporter bacteria can be genetically engineered by placing a lux gene under the control of an inducible promoter, making originally non-luminescent organisms capable to show bioluminescence. The lux system consists of five genes: luxA, luxB, luxC, luxD and luxE.
LuxAB bioreporters contain only the luxA and luxB genes and due to methodological
2 Another system uses the natural bioluminescence of the microscopic marine dinoflagellate algal Pyrocystis lunula. The principle, trade-named Lumitox, was developed into a portable, hand-held instrument (TOX-BOX), in response to a solicitation by the United States Army for a rapid field toxicity test for water supplies. As dinoflagellates are eukaryotic, they might be better models for human risk assessments. Also, it can detect the presence of toxins in the ppb range.
24 constraints not discussed here are not as widely used as luxCDABE reporters. Ben-Israel et al. (1998) used the luxCDABE bioluminescence genes of the Vibrio fischeri lux system as a reporter system for different stress promoters of Escherichia coli, making qualitative and quantitative analysis possible. Riether et al. (2001) constructed two plasmids in which the metal-inducible zntA and copA promoters from Escherichia coli were fused to a promoterless Vibrio fischeri luxCDABE operon and studied the specific response given by heavy metals induction. They found that in optimized assay conditions, metals could be detected at threshold concentrations ranging from nanomolar to micromolar. These tests are referred to as “bioreporters” or “biosensors”: living microbial cells have been genetically engineered to produce a measurable signal. By „transplanting luminescence” to such organisms which are native and common in our environment ecological relevance of the test is ensured. In addition to E. coli, Ralstonia eutropha and Pseudomonas sp. are used as recipient organisms3. A Ralstonia eutropha AE2515 strain was produced for detecting Ni2+ and Co2+ (Tibazarwa, 2001), and a Pseudomonas fluorescens DF57 strain for bioavailable copper indication (Tom-Petersen et al., 2001).
Another novelty is the increased specificity of the bacterial test. Specificity is
Another novelty is the increased specificity of the bacterial test. Specificity is