Daphnia-based toxicity testing - Ecotoxicity testing

2. Introduction

2.2. Ecotoxicity testing

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

15 min 30 min

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 achieved by the controlled expression of the lux operon genes. In this controlled expression a transcriptional regulatory protein takes part which specifically recognizes the target analyte. The target analyte triggers an increased bioluminescence even at very low concentration. (Please note that this mechanism is just the opposite of the standard Vibrio fischeri test where toxicity induces a decrease in bioluminescence!)

Up to now, a wide range of bacterial biosensors are available, developed and tested for a given heavy metal, such as cadmium and lead (e.g. Tauirainen et al., 1998), mercury (e.g. Ivask et al., 2001, 2002, Ivask and Bernaus, 2004), zinc and chromium (Ivask et al., 2002), as well as nickel (e.g. Tibazarwa et al., 2001). Many of them have

3 Genetically modified luminescent bacteria are not only produced for ecotoxicity testing but also for detecting a targeted microbe marked with bacterial lux genes in a mixed culture for example in bioremediation systems. For example, when special degraders are introduced into the system, their interaction with the indigenous microorganisms can be followed (Xing et al., 2000).

25 proven applicable in field conditions (e.g. Kahru et al., 2005). Specific biosensors are already commercially available in a kit form such as the BIOMET^®.

The Vibrio fischeri bioluminescence inhibition test was originally developed for wastewater toxicity testing but has gained ground in water and sediment toxicity testing as well. For water analysis, the following standards apply:

 ISO/CD 11348-2: Water quality -- Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) -- Part 2: Method using liquid-dried bacteria

 ISO/CD 11348-3: Water quality -- Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) -- Part 3: Method using freeze-dried bacteria

As V. fischeri is a marine bacterium, the conventional protocol uses aqueous samples (in compliance with ISO 11348-3). The test has been widely used for assessing wastewater and activated sludge toxicity (Bailey and Beney, 2000; Gutiérrez et al., 2002;

Manusadzianas et al., 2003; Ren, S., 2004; Barbusiński, 2005), monitoring pesticides ecotoxicity in water (Köck et al., 2010), assessing insecticides degradation and degradation intermediates toxicity (Šojić et al., 2012), and is often incorporated in national regulations.

However, turbidity and colour of the sample might reduce the light output recorded by the luminometer due to physical eﬀects, creating the potential for false-positives (Campisi et al., 2005). Wastewater samples are very often turbid and/or coloured, which might result in overestimating their toxicity if they are tested unfiltered.

Filtering can be an option, in fact, ISO 11348-3 prescribes that samples should be filtered if turbid. However, toxicity can be underestimated if the toxic eﬀect depends mostly on particle-bound compounds (Harkey and Young, 2000).

Lappalainen et al. (1999, 2001) presented a novel protocol for testing the toxicity of solid and/or coloured samples. Here luminescence intensity is evaluated in a kinetic mode. As the bacterial suspension is injected to the sample, the luminous intensity

26 increases, to a peak (maximum) within 30 s (that is why the system is called Flash). The results are expressed as the ratio of luminescence at 30s normalized to the peak value.

The method has several advantages. First, the light output in the sample is assessed independently from the control, taking peak luminescence measured during the first 30s interval and comparing it to the value recorded after the present exposure time.

The protocol has been standardised, the ISO standard (ISO 21338:2010: Water quality - Kinetic determination of the inhibitory effects of sediment, other solids and coloured samples on the light emission of Vibrio fischeri /kinetic luminescent bacteria test/) was issued recently, in 2010. The test has been successfully used for testing contaminated soils or sediments and compost toxicity (Pollumaa et al., 2000, 2004; Degli-Innocenti et al., 2001; Heinlaan et al., 2007; Karlsson et al., 2010), nanoparticle toxicity (Mortimer et al., 2008; Farré et al., 2009) and for assessing the potential ecological hazard of the red mud accident of Devecser, Hungary (Gelencsér et al., 2011). However, no data are available yet on its efficacy in wastewater/whole effluent toxicity testing.

Expression of ecotoxicity:

The test end-point (which was defined as a quantifiable biological response such as mortality, growth inhibition, enzymatic inhibition, etc.) is measured at a range of effluent concentrations to develop a concentration-response curve. This curve is typically sigmoidal (Fig. 3), but is linearized by data transformation methods, e.g. probit transform.

From the resulting linearized concentration-response curve, a point estimate effect concentration can be calculated (Fig. 4), most often expressed as EC₅₀⁴. The EC₅₀ of a graded dose response curve represents the concentration of a compound where 50% of its maximal effect is observed or represents the concentration of a compound where 50% of the population exhibits a response. The EC₅₀ generally substitutes LC where the half lethal concentration is estimated, supposing that the effect observed is mortality. In addition, the term IC₅₀ can be used which is a measure of a compound's inhibition (50%

inhibition).

4 A free software developed for probit calculation can be downloaded from: András, EPA

27 Fig.3 Sigmoidal concentration-response curve

Concentration measures typically follow an S-shaped curve, increasing rapidly over a relatively small change in concentration. The point at which the effectiveness slows with increasing concentration is the IC50. This can be determined mathematically by derivation of the best-fit line. However, it is more easily observed from a graph and estimated rather than through complex calculus equations.

Fig. 4 Determination of point estimates from a linearized concentration-response curve.

[Agonist, M]

Response Percent of Control

Concentration Response (percent mortality)

28 The toxic compound or sample can also be characterised by NOEC and LOEC values (NOEC is the highest concentration of toxicant in which the values for the observed responses are not statistically significantly different and LOEC is the lowest concentration of toxicant in which the values for the observed responses are statistically significantly different from the controls).

2.3. Wastewaters of high organic (biodegradable) matter content

In this thesis, the ecotoxicity of different types of wastewater and their degradation under different conditions were assessed, but, they were of high organic (biodegradable) matter content in common.

Municipal wastewater used came from municipal wastewater treatment plant.Municipal wastewater is consisting mainly of water (99.9%) and relatively small concentrations of inorganic and suspended and dissolved organic solids. Organic matters are carbohydrates, lignin, proteins and their decomposition products, fats, soaps and synthetic detergents, several natural and synthetic organics from the process industries, anddisease-causing pathogens. The inorganic substances may include a number of potentially toxic elements such as arsenic, cadmium, chromium, copper, lead, mercury, zinc, etc. Even if they are not at toxic-level concentrations to humans, they might be at phytotoxic levels, which would limit their agricultural use or at toxic levels to animals or to any other ecosystem compartment.

Apart from municipal wastewater, an industrial and an agricultural wastewater were tested. Agricultural wastewater used came from liquid manure. Ammonia concentration is posing the highest threat to water quality. The running Hungarian National Environmental Program (2003-2008, number 132/2003; X.11. OGy) is emphasizing that the liquid manure produced by agriculture in huge quantities can cause serious environmental risks. Reduction of the environmental impacts of liquid manure can be done by building up disposal ponds with up-to-date technological protection.

Natural formations (compact sand layers or rock) can be considered as technological protection, as well.

29 Animal urine and manure (including polluted straw) are classified into the second waste chapter of EWC (Code: 02 01 06, from harmonized EC rule, under 16/2001.

(VII.18.) KÖM degree). This rule applies to the selectively collected but untreated (in situ) liquid manure as well. In order to prevent and reduce the nitrate pollution in water, the 49/2001. (IV.3.) order controls the professional agricultural activity including the rules of organic manure harmless deposition and disposal. The main goal is to prevent and reduce the nitrate pollution in natural waters, as well as guaranteeing the optimal substrate supply for plants and keeping the soil productivity. Liquid manure can be used as natural fertilizer, under very strict conditions. The nitrogen content of the manure has to be utilized by plants without any nitrogen emission to natural waters.

Our basic aims were to test how the degradation processes, especially biodegradation, affect the toxicity and the organic matter content of different liquid manures. During degradation processes not only concentration of the chemicals (and therefore exposure) will change but also, photo-degradable, hydrolytically unstable, oxidizable and biodegradable substances in addition may form such breakdown products which can be even more toxic than the parent substance was.

Industrial wastewaters were represented by paper mill effluent. Pulp and paper industry is one of the most polluted industries in the world (Thompson et al., 2001;

Sumathi and Hung, 2006). Two main steps are included in the production process:

pulping and bleaching. Pulping is the first step and source of the most pollution of this industry. In which, wood chips (raw material) are treated to remove lignin and improve fibers for papermaking. Bleaching is the last step of the process, which aims to whiten and brighten the pulp. These processes are very energy and water intensive in terms of the fresh water utilization (Pokhrel and Viraraghavan, 2004).

The wastewater generated from this industry include high concentration of chemicals such as, sodium carbonate, sodium hydroxide, bisulfites, sodium sulfide, elemental chlorine or chlorine dioxide, calcium oxide, hydrochloric acid, toxic components, … etc. (Sumathi and Hung, 2006). But, the major problems are the high organic content (20-110 kg COD/air dried ton paper), wood debris, soluble wood materials, adsorbable organic halide (AOX), dark brown coloration, toxic pollutants, etc.

In document Szennyvizek ökotoxikológiai változása a biológiai tisztítási folyamat során (Pldal 26-0)