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Daphniatox – Online monitoring of aquatic pollution and toxic substances
Chemosphere 167 (2017) 228e235
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Daphniatox e Online monitoring of aquatic pollution and toxic substances €der a, *, Gilmar S. Erzinger b Donat-P. Ha €hrendorf, Germany Emeritus from Friedrich-Alexander University, Department of Biology, Neue Str. 9, 91096, Mo Department of Medicine and Pharmacy, University of Joinville Region e UNIVILLE, Rua Paulo Malschitzki, 10 Campus - Industrial Zone, PO Box 246, Joinville, SC, CEP 89219-710, Brazil
a
b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Daphnia is an accepted bioassay organism and the biomonitoring protocol is standardized and established in many countries. The fully automatic Daphniatox warrants rapid standardized monitoring and provides high statistical significance of the data. Using 14 independent endpoints of motility, orientation, size and form indicate different classes of chemical toxicants and pollutants. The system is suitable for short- and long-term monitoring of fresh water and industrial, household and agricultural waste water. High sensitivity of the microcrustacean Daphnia and low initial costs and negligible running costs characterize the system.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 April 2016 Received in revised form 22 August 2016 Accepted 30 September 2016
The microcrustacean Daphnia is sensitive to many toxic substances and can be cultured easily. The Daphniatox instrument is based on computerized image analysis tracking swimming organisms in real time. The software evaluates 14 endpoints including motility, swimming velocity, orientation with respect to light and gravity as well as cell form and size. The system determines movement vectors of a large number of organisms to warrant high statistical significance and calculates mean values as well as standard deviation. Tests with K dichromate show that the toxin inhibits motility (EC50 0.75 mg/L), swimming velocity (EC50 0.70 mg/L) and even causes a significant decrease in length (16% at 4 mg/L) and changes the form of the animals, This bioassay can be used to monitor the toxicity of a large number of dissolved pollutants and toxic substances such as arsenic, dichromate and persistent organic pollutants. © 2016 Elsevier Ltd. All rights reserved.
Handling Editor: Jian-Ying Hu Keywords: Daphnia Daphniatox Aquatic ecosystems Biomonitoring Pollutants
* Corresponding author. €der), [email protected] E-mail addresses: [email protected] (D.-P. Ha (G.S. Erzinger). http://dx.doi.org/10.1016/j.chemosphere.2016.09.155 0045-6535/© 2016 Elsevier Ltd. All rights reserved.
€der, G.S. Erzinger / Chemosphere 167 (2017) 228e235 D.-P. Ha
1. Introduction Most of the Earth's water is not accessible for human consumption because it is either too salty or bound in the form of glaciers and snow. The small fraction of available fresh water compromises less than 1% of the global water reservoir (Gleick, 2014). But the need for fresh water for households, industry and agriculture has increased exponentially over the past few decades and further increases are predicted (Kasei et al., 2014). At the same time the limited resources are increasingly diminished by pollution from domestic, agricultural and industrial wastes (Jones, 2014). The main components of the growing number of pollutants include heavy metals, persistent organic pollutants (POP) as well as pharmaceuticals (Gleick, 2014). In addition, the excessive use of fertilizers in agriculture results in accumulation of nitrogen and phosphorous compounds which reach surface waters and ground water reservoirs by the runoff from the fields (Wu et al., 2014). Global climate change, which causes increasing temperatures, changing precipitation patterns and draught episodes as well as dropping ground water tables, is expected to deteriorate the conditions for fresh water availability (Elliott et al., 2014). In addition, the limited resources of fresh water for irrigation are bound to decrease agricultural productivity (Cotrufo, 2014; Kurukulasuriya and Rosenthal, 2013; Müller et al., 2014). About 780 million people in developing countries lack access to clean fresh water (WHO/UNICEF, 2012) and 2.2 billion fail to have safe sanitation (WHO, 2014). Each year an estimated 5 million people die from diseases related to polluted water resources such as diarrhoea and infections transmitted by water-borne parasites (http://www.who.int/topics/mortality/en/); many hundreds of thousands of premature deaths could be avoided by simple measures to improve fresh water quality for human consumption (Apte et al., 2015). High concentrations of heavy metals were found in rivers in Pakistan and India (Muhammad et al., 2011). In addition, organic pollutants as well as inorganic toxicants are accumulated in sediments and pose considerable long-term risks for the biota and humans (Kuku cka et al., 2015). Chlorophenol compounds derived from degradation of pesticides and chlorinated hydrocarbons (Karci, 2014) are considered to be among the most toxic pollutants in aquatic ecosystems because of their high toxicity, chemical stability and low degradability (Lindholm-Lehto et al., 2015; Wei et al., 2015). Personal care products and pharmaceuticals introduced into surface waters by household wastes are recycled and concentrated in rivers. The water of some rivers in heavily populated areas is recycled several times in the form of drinking water and household effluents so that substances like estrogens create problems such as s et al., 2014). In feminization in fish and amphibian species (Valde addition, chemicals such as antibiotics accumulate in crop plants when these waters are used for irrigation (Pan et al., 2014). Because of the increasing demand for clean fresh water, monitoring and assessment of both ground water and surface water reservoirs has a high priority. Chemical analyses are of limited value because they are time consuming, expensive and usually are restricted to a few classes of chemicals while the number of potentially toxic chemicals is excessive (Ginebreda et al., 2014). Often the toxicity of a certain chemical is underestimated or it may operate synergistically with other substances which is not detected in routine chemical analyses (Chen et al., 2015). Regulations vary between countries and upper limits for toxins may be altered over time and may not represent the effective threat for the biota or €der, 2013), Alternatively, wahuman consumers of fresh water (Ha ter quality can be monitored using bioassys. Early examples for this procedure include the deployment of fish in potentially polluted
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water. When they died or showed abnormal swimming behavior this was considered as a sign for the presence of a pollutant in the water. Many different organisms have been employed in bioassays such as bacteria, microorganisms, lower and higher plants as well as invertebrates and vertebrates (de Castro-Catal a et al., 2015; Hafner et al., 2015; Kottuparambil et al., 2014; Ma et al., 2014). Depending on the time scale for the evaluation different endpoints can be considered including mortality, motility and behavior, growth and reproduction as well as physiological parameters such as photosynthesis, protein biosynthsis and genetic alteration of aquatic organisms (Davies and Mazurek, 2014). While chemical analysis can identify a potentially toxic substance or at least a class of chemicals, bioassays do not provide this information. They indicate the presence of a toxin in the water which - above a certain concentration - can present a potential €der and health hazard for the biota or for human consumption (Ha Erzinger, 2015). The toxicity of a pollutant is often determined by the EC50 value which causes a 50% inhibition (Sebaugh, 2011) but any other value can be used instead. There are a number of commercially established bioassays. The Microtox test is based on the diminuation of the bioluminescence generated by genetically modified bacteria in the presence of toxic ndez-Pin ~ as et al., 2014). The biotest Lemnatox anmaterials (Ferna alyzes the growth of an aquatic higher plant under the influence of pollutants (Newman and van Valkenburg, 2013). A recently developed bioassay for municipal and industrial wastewater effluents is based on the delay of zoospore release from marginal disks cut from the thallus of the marine green alga Ulva pertusa under the influence of a toxin during a 96-h period (Han and Choi, 2005; Y.-J. Kim et al., 2014). Disadvantages of some commercial bioassays are the low sensitivity toward toxic chemicals and/or the long times required for analysis which can be on the order of several days. One approach is based on the on-line image analysis of motile photosynthetic microorganisms monitoring motility, orienation and cell shape fully automatically (Azizullah et al., 2013; Azizullah et al., 2011). This unicellular flagellate (Euglena) has also been employed to indicate the toxicity of phenolic substances (Kottuparambil et al., 2014). The microcrustacean Daphnia has been used as a sensitive organism to indicate the potential toxicity in water samples (Chen et al., 2012). There are a number of automatic bioassays on the market. However, many have serious limitations, such has high price tag, high costs of consumables, low sensitivity, long analysis time spans, complicated performance and large size. The aim of the current work is to describe a recently developed on-line, fully automatic image analysis system to monitor motility, organism shape and orientation of the water flea Daphnia. In contrast to the Ecotox bioassay, which uses a unicellular flagellate, Daphniatox employs a multicellular crustacean. The test is certified in many countries. The organism is as sensitive as the Ecotox, but differs with respect to the chemical nature of the toxicant. The Daphniatox test is also faster than the Ecotox test. The advantages of this test include high sensitivity of the organisms (Struewing et al., 2014) and the fast analysis of the selected 14 end points. While a bioassay by definition cannot identify the pollutant or toxic substance, the different endpoints show different sensitivities to varous groups of chemicals, which gives some indication to the nature of pollution. 2. Materials and methods 2.1. Organisms for the bioassay The water flea Daphnia is a genus of small (0.5e5 mm length) Cladoceran crustaceans found in fresh water bodies like streams, ponds and lakes. Culture conditions are simple, and they grow and
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multiply at a fast rate characterizing these organisms as cost effective and easy to handle. The cultivation of the Cladoceran D. magna was performed according to ISO-6342 (ISO-6342, 2012). 50 Daphnia magna were kept in 5-L glass beakers containing 2 L of culture medium (M4 medium) (Erzinger et al., 2015). The medium was renewed and the offspring removed every three or four days. After 4 weeks breeding Daphnia were removed and replaced by neonatal organisms. The organisms were fed twice daily with the unicellular green alga Raphidocelis subcapitata (previously Selenastrum capricornutum) according to ISO-8692 (ISO-8692, 2012) using a peristaltic pump controlled by a timer to deliver 7 ml of algal suspension (at 3.6 107 cells/mL) per day. Daphnia is recommended as a suitable organism in aquatic bioassays because of their high sensitivity to many toxic chemicals (Chen et al., 2012), and the biotest is certified by several national and international agencies such as the German standard methods (DIN 38412 L-30) and the OECD test 202 to monitor fresh water, waste water and sludge toxicity (GSM, 1989). The organisms respond to many organic and inorganic pollutants (Knops et al., 2001) including carbamate, pyrethroids and organophosphorous pesticides (Ren et al., 2015). In order to prove the suitability of the bioassay we used increasing concentrations of K dichromate which is of ecological interest since it is used in tanning, galvanoplastic and to produce chrome-sulfuric acid. This chemical is highlighted as the reference to quantify the sensitivity of the organism and for comparison among laboratories. Endpoints in these tests are motility and mortality after a specified time of exposure (e.g. 24 or 48 h) (Y. Kim et al., 2008). Several Daphnia species, Chaoborus crystallinus and Mesocyclops leuckarti have been used to indicate the presence of the pesticide triphenyltin hydroxide and other novel anti-biofouling chemicals (Gergs et al., 2015). Pollution by detergents was monitored by quantifying the motility of D. magna (Pettersson et al., 2000; Uc-Peraza & Delgado-Blas). Another endpoint in bioassays employing Daphnia is mortality which was used to determine the toxicity of the fungicide metalaxyl (S. Chen and Liu, 2008). In addition, the heart beat pattern was determined in Daphnia exposed to toxic pollutants (Kiss et al., 2003) and the precision of phototactic orientation of the organism was found to deteriorate when the organisms were exposed to heavy metals (Zhou et al., 2008). The commercial instrument Daphnia Toximeter uses real-time image analysis to determine motility and orientation of the organism with respect to gravity but it does not monitor phototaxis (Netto, 2010). Chronic tests monitoring survival and reproduction require longer time scales (Naddy et al., 2007; Tong et al., 1996). 2.2. Hardware The hardware approach for the Daphniatox is straight forward: A monochromatic USB 3.0 camera (Point Grey, Blackfly BFLY-U3-13S2M-CS) with 1.3 mega pixel resolution (1288 964) equipped with a macro zoom objective (Computar 2.8e12 mm, 1:1.3 IR 1/3”) screwed into a CS mount is aimed at a 50-ml cell culture flask (Aldrich) containing about 100 organisms. Daphnia shows vertical migrations which are controlled by gravity, light and a circadian rhythm. Phases of upward movement alternate with those of downward movement (J Ringelberg, 1991; J. Ringelberg, 1999). In order to observe gravitaxis (orientation with respect of gravity) the flask is oriented vertically and the optical axis horizontally (Fig. 1). The culture flask is irradiated from behind with a diffuse white light beam so that the camera records dark organisms in front of a white background. The light level is controlled by the iris of the objective. Phototactic
orientation is induced by a blue LED light source impinging at 90 with respect to the vertical and the optical axis. In this set-up non-motile Daphnia drop out of the field of view and collect at the bottom. Therefore, in order to determine motility the optical axis is changed to vertical with the camera pointing downward. Non-motile or dead organisms sediment to the bottom of the flask but are in the view of the camera, and thus the percentage of motile cells can be determined correctly. Control measurements and analyses with organisms exposed to different concentrations of toxins are performed in sequence creating a result file for each measurement (see section 2.3).
2.3. Software for image analysis The Daphniatox software is based on the open source software Fiji derived from the open source ImageJ (Schindelin et al., 2012). Since this program package is only capable of processing recorded video (.avi) sequences, a software plugin, which allows direct input of the online video into the imaging software (Phase, Lübeck, Germany), has been implemented. The Daphniatox software is written in the Macro Language of ImageJ. A button allows starting the camera control to provide a live camera image. Another button facilitates the setup of the parameters for the analysis. This allows calibrating the system in terms of physical units (pixels per mm), selecting a certain size range for the organisms to be tracked (to avoid tracking of disturbing other objects such as the food organisms for Daphnia) and the velocity range in order to eliminate tracking drifting or sedimenting organisms. The number of tracked organisms and the maximal time per experiment are selected after which the tracking stops, whichever comes first. Another button in the task bar starts the tracking software which commences by taking a number of snapshots from the video input stream at regular time intervals and storing them in a stack of images together with the elapsed time in order to calculate the swimming velocity. The quality of these frames is improved by background subtraction and contrast enhancement to yield clear images of dark organisms in front of a bright background. A thresholding results in images with two levels of brightness: black for the background (0) and white for the organisms (255); this step inverts the image. After this pre-processing the stack is used to extract the movement vectors of the motile objects (up to 256 in parallel). The results from the vector analysis are stored, and the data from this analysis are displayed in a separate window in the form of a circular histogram showing the fraction of cells moving in sectors, the size of which the user has preselected in the settings. In addition, the number of tracks recorded, the mean velocity, the percentage of motile cells, the mean area and length of the organisms, the mean angular deviation from the vertical as well as the percentage of upward swimming organisms (in the case of a vertical movement) and the precision of orientation are indicated. After the analysis of tracks in one image stack has been finalized a new set of frames is taken from the life video and stored in a new stack and the analysis is repeated cyclically until a user-defined number of tracks has been determined or a predefined length of time has elapsed, whichever comes first. After each tracking cycle the angular histogram is updated with the additional tracked vectors. At the end the results can be stored together with a description of the details of the experiment, which the user provides, and - if selected - the raw data such as the areas of the individual organisms, the track coordinates and the angular distribution. The movement direction of each track is calculated as the angular deviation a from the 0 direction
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Fig. 1. Optical setup of the Daphniatox instrument with a Blackfly USB 3.0 camera equipped with a macro zoom lens and diffuse background monitoring light. Actinic blue light is provided by an LED impinging perpendicularly to the optical axis.
a ¼ tn1
y2 y1 x2 x1
where y2 and y1 are the end and beginning y-coordinates of each track, respectively, and likewise for the x coordinates (D.-P. H€ ader and Lebert, 2000). Since this calculation covers only the angles <90 the information, in which quadrant the organism moves, is used to determine all angles. The precision of orientation of the whole population is defined by the r-value, which is a statistical measure which runs between 0 (when the organisms move in random directions) and 1 (when all cells move in the same direction) (Batschelet, 1981).
r¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðSsin aÞ2 þ ðScos aÞ2 n
However this value does not indicate in which directions the organisms move. Therefore the mean movement angle of the population is calculated as q (Batschelet, 1981).
q ¼ arctan
Ssin a Scos a
In order to cover all four quadrants a correction has to be performed: if S sin a is negative then q is q þ360 and if S cos a is negative then q is q þ180 . The percentage of upward swimming cells (±90 around the vertical upward) is calculated. To determine phototactic orientation, the actinic light is impinging from 0 and the percentage of organisms swimming in the two quadrants toward the light (±90 ) is quantified. The swimming speed is calculated by analyzing the (meandering) total swimming path length divided by the time needed for this. Furthermore the direct speed is determined by dividing the distance from the beginning to the end of the vector by the time needed. The ratio of the direct path length divided by the actually covered path length gives the swimming directedness. The motility is determined as the percentage of moving organisms within the defined size range and which qualify for the predefined velocity range. The area of each object (which falls into the size limits) is determined and the mean value is calculated. The length of the outer boundary (perimeter), the circularity, roundness, solidity and aspect ratio (ratio of the major and minor axes of a particle's fitted ellipse) are calculated (see definitions below). In addition, the
Feret's diameter is determined which indicates the largest dimension of an object. This is achieved by turning the image stepwise until the largest dimension is found. The degree of turning gives the Feret angle, which is thus the angle of the longest axis with respect to the horizontal (Schindelin et al., 2012).
circularity ¼ 4p
roundness ¼
solidity ¼
area perimeter2 area
p major axis2
area convex area
For all parameters mean values and standard deviations are determined. The initial calibration (pixels per mm) allows calculating all size and velocity parameters in actual physical units (mm, mm2, mm/s). The results obtained with the Daphniatox system can be utilized to determine effect-concentration curves to obtain information on defined toxicity parameters like NOEC, LD and EC50 values (Ahmed €der, 2011). The system can be used for short-term as well as and Ha long-term (online) monitoring of pollution in all kinds of waters such as ground water, surface or wastewater as well as natural ecosystems.
3. Results The experiments for short-term (acute) tests were performed according to the standard NBR 12713 protocol (ABNT, 2003; EPA, 2002). Neonates of D. magna, 2e26 h old, were placed in K2Cr2O7 solutions (0, 0.125, 0.25, 0.5, 1, 2 or 4 mg/L) for a period of up to 48 h in 50-ml cell culture flasks (Flohr et al., 2005). Potassium dichromate was used as a toxin in the tests because this chemical is widely used in tanning, galvanoplastic, to produce chrome-sulfuric acid and to stain neutrons and nerves (Fisher-Scientific, 2007) and therefore is frequently found as a pollutant in water bodies. Potassium dichromate was defined by international institutions as the reference chemical to assess the sensitivity of the organism and to serve as a comparison among laboratories. The MAC value is 5 mg/ m3. Two flasks were used for each concentration and each flask was tracked three times. In order to determine the motility the
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organisms were tracked in flasks positioned horizontally on a light table. Since they did not undergo gravitactic orientation the motility vectors point in random directions which results in an rvalue of 0.02 (Fig. 2). The swimming directions of the tracks are binned in 60 sectors. The histogram is based on 101 tracks and shows the percentage of organisms swimming in each sector. 71% of the organisms were motile, swimming at a speed of 0.11 mm/s. The area was 0.19 mm2 and the length 0.82 mm 47.5% of the organisms swam in the two quadrants adjacent to 0 and q was 236.66 . Immediately after incubation in the potassium dichromate solutions all organisms remained motile, but after 4 h an increasing number of Daphnia became immobile at 2 and 4 mg/L (data not shown). After an incubation time of 24 h there were no motile organisms at concentrations 1 mg/L (Fig. 3). The motility (percentage of motile organisms) was more or less constant (ca. 85%) at the lower concentrations and in the control. Also the velocity was almost constant at concentrations <1 mg/L (Fig. 4). Even though the organisms were from the same batch, the length of them decreased with the concentration of potassium dichromate (Fig. 5a) while the area increased with lower concentrations (Fig. 5b) indicating a swelling of the organisms. At higher concentrations the area decreased. Since this indicates a change in the organisms’ form, the roundness (for definition see section 2.3) was determined for each population (Fig. 5c). In accordance with the above results, after an increase at the lowest concentrations this value decreased at higher concentrations. When exposed in a vertical cell growth chamber the organisms undergo gravitactic orientation swimming upward at times and downward at others (Fig. 6) while lateral movement does not occur as often. This histogram is based on 42 tracks. All organisms were motile swimming at a mean value of 0.14 mm/s 50% of all organisms swam upward. They had a length of 0.6 mm and an area of 0.1 mm2. The r-value was 0.12 and q was 190 , but these values are meaningless for a bimodal distribution. For each test 14 endpoints are quantified as shown in Table 1 as an example for the effects of 0.125 mg potassium dichromate after 24 h incubation. In addition to the percentage of motile organisms, the mean velocity, the area, length, roundedness and gravitactic orientation the following parameters are evaluated: the percentage of upward swimming organisms and the mean direction q. The circulatiry, the mean aspect ratio, the mean solidity as well as the perimeter in comparison to the length give further information on the form factor. The swimming velocity describes the speed of
Fig. 3. Effect-concentration curve of the motility (percentage of motile organisms, mean values ± SD, n ¼ 6) of D. magna after incubation in potassium dichromate for 24 h indicating the NOEC (¼0.5 mg/L, no observed effect concentration). EC50 (¼ 0.78 mg/L, concentration at which 50% inhibition occurs and LD (¼1 mg/L, lethal dose).
Fig. 4. Effect-concentration curve of the mean swimming velocity (mean values ± SD, n ¼ 6) of D. magna after incubation in potassium dichromate for 24 h indicating the NOEC (¼0.25 mg/L, no observed effect concentration). EC50 (¼0.68 mg/L, concentration at which 50% inhibition occurs and LD (¼1 mg/L, lethal dose).
movement between the beginning and end of the track. However the organisms may not follow a direct path but meander from the direct line. This is indicated by the mean directedness and the mean velocity which indicates the swimming speed along the actual path. For each concentration two independent samples were monitored and each test was repeated three times. For all parameters mean values and standard error are calculated. 4. Discussion
Fig. 2. Angular histogram of tracks of D. magna in a horizontally oriented swimming chamber binned in 60 sectors showing a random distribution.
As expected the percentage of motile organisms as well as the swimming velocity were affected by even small contentations of potassium dichromate. In contrast, changes in the length of the organism as well as the form factors were not expected since the organisms have a rigid exoskeleton. They would also not be detected by manual evaluation, but by the quantitative image analysis. These form and length changes can be explained by assuming that the organisms release or take up liquid under the pollutant stress. The results indicate that upon exposure to low concentrations of dichromate the organisms take up water and bulge which reduces the length, but increases the area, resulting in increased roundness. Modern bioassays using advanced technologies must be
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Fig. 5. Length (a), area (b) and roundness (c) of D. magna after incubation in potassium dichromate for 24 h. Mean values (n ¼ 6 ± SD).
Fig. 6. Angular distribution histogram of D. magna swimming in a vertical cell culture flask showing positive (downward) and negative (upward) gravitactic orientation binned in 60 sectors.
evaluated by a number of characteristics. One of the important features is a short analysis time. Depending on the requested number of analyzed tracks a full measurement cycle in Daphniatox
can be completed in less than 2 min which is achieved by the online, real-time computerized image analysis. This is even faster than the ECOTOX system which requires about 6 min for a full measurement cycle (Azizullah et al., 2013). Because of the automated analysis human error-prone and subjective evaluation is avoided. Using high numbers of analyzed organism tracks warrants high precision and statistical significance. When evaluating longterm effects of potentially toxic substances the analysis time is equally short interrupted by incubation periods. As compared with other advanced automated bioassays the operational costs for Daphniatox are almost negligible. The only requirement is keeping a supply of the microcrustaceans and their food source which is simple and can be done at low costs. Operating the system is user friendly and due to the automated evaluation does not require extensive training of personnel. The organisms used for this bioassay are very sensitive to toxins. For motility, velocity and other determined parameters EC50 values of less than 2 mg/L have been found after an exposure of 2 h and even less after long-term exposure of 24 h. In comparison, the EC50 value for rats is 25 mg/kg after oral application of potassium dichromate and for rabbits 14 mg/kg after transdermal application (Gumbleton and Nicholls, 1988; Tandon, 1982). The other endpoints found in the raw data (for example see Table 1) confirm the main data shown in the results section. The data indicate that the organisms do not swim on straight paths but meander during the track. The percentage of upward moving cells indicates the degree of gravitactic orientation. The form parameters circularity, aspect ration and solidity as well as perimeter with respect to the organisms length confirm the data on roundedness.
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Table 1 Results for the 14 measured parameters indicting the effects of 0.125 mg/L after 24 h given as an example of the raw data obtained for each test. Each test is done triplicates and two parallel flasks are used for each concentration and exposure time. 3.11.2015 15:02:16 h 0.125 mg flask B 24 h exposure. test 3 83% motile Mean velocity ¼ 0.15 ± 0.06 mm/s Mean direct velocity ¼ 0.10 ± 0.05 mm/s Mean directedness ¼ 0.67 ± 0.25 41.82% organisms swam upward Theta ¼ 360.46 r-value ¼ 0.18 Mean area ¼ 0.16 ± 0.06 mm2 Mean perimeter ¼ 2.34 ± 0.86 mm Mean circularity ¼ 0.41 ± 0.19 Mean organism length ¼ 0.79 ± 0.31 mm Mean aspect ratio ¼ 2.68 ± 1.67 Mean roundness ¼ 0.48 ± 0.24 Mean solidity ¼ 0.73 ± 0.17
Funding This work was partially supported by the National Council of Technological and Scientific Development (CNPq).
Different endpoints selected for the evaluation of toxicity vary in their sensitivity toward different classes of toxic substances and pollutants. Using 14 different parameters in motility, orientation, size and form warrants a wide range of endpoints for evaluating toxicity. A similar number of parameters is evaluated by the Ecotox system which utilizes the motility, orientation and form parameters of the unicellular green flagellate Euglena gracilis (Azizullah et al., 2014). The sensitivity of Daphnia species and related genera to toxic substances has been found to be higher than that of the organisms utilized in other bioassays including algae (growth), fish (mortality) and bacteria (bioluminescence) (Ahmed and H€ ader, 2011). In contrast to chemical analysis biomonitoring systems are not designed to identify the chemical nature of a toxic pollutant. They rather indicate pollution above a critical level potentially posing a hazard for ecosystem safety or human health. If the level of pollution is found to exceed the accepted level chemical analysis is required to identify the chemical nature of the toxicant. One strong advantage is that, in contrast to pure chemical analysis, bioassays can indicate potential threats of combined polluting chemicals, which my act additively or synergistically. Often the toxicity of pollutants is augmented by solar radiation, especially in the UV-B range which is detected by bioassays (Zepp et al., 2011). Bioassays have been employed to indicate the toxicity of a wide range of pollutants such as heavy metals, herbicides, pesticides, fertilizers, detergents, wastewaters and many other organic and inorganic pollutants (Azizullah et al., 2014; Danilov and Ekelund, 2000). They have been used effectively to determine both shortand long-term ecotoxicity of pollutants (Ahmed, 2010; Azizullah et al., 2014). Daphniatox can be utilized as an early warning system for pollution monitoring surface and ground waters, municipal and industrial wastewaters as well as for controlling the efficiency of waste water treatments plants. The low initial and negligible running costs allow utilizing the system also in developing countries where poor funding and lack of environmental experts render efficient water quality monitoring difficult. Preliminary experiments have shown that the toxicity determined by Daphniatox correlates well with reported effects on human health indicating that it is a useful instrument to monitor drinking water, waste water and natural ecosystems. When the instrument enters industrial production the price tag is expected to be around 4000 V. 5. Conclusions Daphnia
is
an
accepted
bioassay
biomonitoring protocol is standardized and established in many countries (Asghari et al., 2012). The fully automatic, real-time computerized Daphniatox warrants rapid standardized monitoring eliminating error-prone human interpretation and provides high statistical significance of the data. Using 14 independent endpoints of motility, orientation, size and form of different sensitivity toward toxicants can be used to indicate different classes of chemical toxicants and pollutants. High sensitivity of the microcrustatian Daphnia and low initial costs and negligible running costs characterize the system as suitable for many tasks in short- and long-term monitoring of fresh and ground water resources as well as industrial, household and agricultural waste water.
organism
and
the
Acknowledgements The technical help of Aline Scheller and Lane Coutinho is gratefully acknowledged. References tica - Toxicidade cro ^nica - Me todo de ensaio com ABNT, 2003. Ecotoxicologia Aqua Ceriodaphnia spp. (Crustacea, Cladocera). Ahmed, H., 2010. Biomonitoring of Aquatic Ecosystems (PhD thesis). FriedrichAlexander-Universit€ at, Erlangen-Nürnberg. Ahmed, H., H€ ader, D.-P., 2011. Monitoring of waste water samples using the ECOTOX biosystem and the flagellate alga Euglena gracilis. J. Water, Air Soil Pollut. 216 (1e4), 547e560. http://dx.doi.org/10.1007/s11270-010-0552-4. Apte, J.S., Marshall, J.D., Cohen, A.J., Brauer, M., 2015. Addressing global mortality from ambient PM2. 5. Environ. Sci. Technol. 49 (13), 8057e8066. Asghari, S., Johari, S.A., Lee, J.H., Kim, Y.S., Jeon, Y.B., Choi, H.J., …, Yu, I.J., 2012. Toxicity of various silver nanoparticles compared to silver ions in Daphnia magna. J. Nanobiotechnol 10 (14), 1e14. €der, D.-P., 2011. Ecotoxicological evaluation of wasteAzizullah, A., Richter, P., Ha water samples from gadoon amazai industrial estate (GAIE), Swabi, Pakistan. Int. J. Environ. Sci. 1 (5), 959e976. €der, D.-P., 2013a. Gravitactic Azizullah, A., Murad, W., Adnan, M., Ullah, W., Ha orientation of Euglena gracilis - a sensitive endpoint for ecotoxicological assessment of water pollutants. Front. Environ. Sci. 1, 4. €der, D.-P., 2013b. Ecotoxicity evaluation Azizullah, A., Richter, P., Ullah, W., Ali, I., Ha of a liquid detergent using the automatic biotest ECOTOX. Ecotoxicology 22 (6), 1043e1052. Azizullah, A., Richter, P., H€ ader, D.-P., 2014. Effects of long-term exposure to industrial wastewater on photosynthetic performance of Euglena gracilis measured through chlorophyll fluorescence. J. Appl. Phycol. 27, 303e310. http:// dx.doi.org/10.1007/s10811-014-0332-4. Batschelet, E., 1981. Circular Statistics in Biology. Academic Press, London, New York. Chen, S., Liu, W., 2008. Toxicity of chiral pesticide Rac-metalaxyl and R-metalaxyl to Daphnia magna. Bull. Environ. Contam. Toxicol. 81, 531e534. Chen, L., Fu, X., Zhang, G., Zeng, Y., Ren, Z., 2012. Influences of temperature, pH and turbidity on the behavioral responses of Daphnia magna and Japanese Medaka (Oryzias latipes) in the biomonitor. Procedia Environ. Sci. 13, 80e86. Chen, C., Wang, Y., Qian, Y., Zhao, X., Wang, Q., 2015. The synergistic toxicity of the multiple chemical mixtures: implications for risk assessment in the terrestrial environment. Environ. Int. 77, 95e105. Cotrufo, M.F., 2014. Global change and terrestrial ecosystems, introduction. In: Friedman, B. (Ed.), Glob. Environ. Change 291e296. Danilov, R., Ekelund, N., 2000. Applicability of Growth Rate, Cell Shape, and Motility of Euglena Gracilis as Physiological Parameters for Bioassessment at Lower Concentrations of Toxic Substances: an Experimental Approach. Mid Sweden €rno € sand, Sweden. University, John Wiley & Sons, Inc, Ha Davies, J.C., Mazurek, J., 2014. Pollution Control in United States: Evaluating the System. Routledge. , N., Kuzmanovic, M., Roig, N., Sierra, J., Ginebreda, A., Barcelo , D., de Castro-Catala …, Schuhmacher, M., 2015. Ecotoxicity of sediments in rivers: invertebrate community, toxicity bioassays and the toxic unit approach as complementary assessment tools. Sci. Total Environ. 540, 2097e2396. Elliott, J., Deryng, D., Müller, C., Frieler, K., Konzmann, M., Gerten, D., …, Best, N., 2014. Constraints and potentials of future irrigation water availability on agricultural production under climate change. Proc. Natl. Acad. Sci. 111 (9), 3239e3244. EPA, 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and
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Environ. Earth Sci. 4 (20), 84e96. Kim, Y., Jung, J., Oh, S., Choi, K., 2008. Aquatic toxicity of cartap and cypermethrin to different life stages of Daphnia magna and Oryzias latipes. J. Environ. Sci. Health 43 (1), 56e64. Kim, Y.-J., Han, Y.-S., Kim, E., Jung, J., Kim, S.-H., Yoo, S.-J., …, Han, T., 2014. Application of the Ulva pertusa bioassay for a toxicity identification evaluation and reduction of effluent from a wastewater treatment plant. Front. Environ. Sci. http://dx.doi.org/10.3389/fenvs.2014.00057. ts, N., Szalay, T., 2003. Evaluation of some alternative guidelines for risk Kiss, I., Kova assessment of various habitats. Toxicol. Lett. 140e141, 411e417. Knops, M., Altenburger, R., Segner, H., 2001. Alterations of physiological energetics, growth and reproduction of Daphnia magna under toxicant stress. Aquat. Toxicol. 53, 79e90. Kottuparambil, S., Kim, Y.-J., Choi, H., Kim, M.-S., Park, A., Park, J., …, Han, T., 2014. A rapid phenol toxicity test based on photosynthesis and movement of the freshwater flagellate, Euglena agilis Carter. Aquat. Toxicol. 155, 9e14. bov Kuku cka, P., Audy, O., Kohoutek, J., Holt, E., Kala a, T., Holoubek, I., Kl anov a, J., 2015. Source identification, spatio-temporal distribution and ecological risk of
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Daphniatox e Online monitoring of aquatic pollution and toxic substances €der a, *, Gilmar S. Erzinger b Donat-P. Ha €hrendorf, Germany Emeritus from Friedrich-Alexander University, Department of Biology, Neue Str. 9, 91096, Mo Department of Medicine and Pharmacy, University of Joinville Region e UNIVILLE, Rua Paulo Malschitzki, 10 Campus - Industrial Zone, PO Box 246, Joinville, SC, CEP 89219-710, Brazil
a
b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Daphnia is an accepted bioassay organism and the biomonitoring protocol is standardized and established in many countries. The fully automatic Daphniatox warrants rapid standardized monitoring and provides high statistical significance of the data. Using 14 independent endpoints of motility, orientation, size and form indicate different classes of chemical toxicants and pollutants. The system is suitable for short- and long-term monitoring of fresh water and industrial, household and agricultural waste water. High sensitivity of the microcrustacean Daphnia and low initial costs and negligible running costs characterize the system.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 April 2016 Received in revised form 22 August 2016 Accepted 30 September 2016
The microcrustacean Daphnia is sensitive to many toxic substances and can be cultured easily. The Daphniatox instrument is based on computerized image analysis tracking swimming organisms in real time. The software evaluates 14 endpoints including motility, swimming velocity, orientation with respect to light and gravity as well as cell form and size. The system determines movement vectors of a large number of organisms to warrant high statistical significance and calculates mean values as well as standard deviation. Tests with K dichromate show that the toxin inhibits motility (EC50 0.75 mg/L), swimming velocity (EC50 0.70 mg/L) and even causes a significant decrease in length (16% at 4 mg/L) and changes the form of the animals, This bioassay can be used to monitor the toxicity of a large number of dissolved pollutants and toxic substances such as arsenic, dichromate and persistent organic pollutants. © 2016 Elsevier Ltd. All rights reserved.
Handling Editor: Jian-Ying Hu Keywords: Daphnia Daphniatox Aquatic ecosystems Biomonitoring Pollutants
* Corresponding author. €der), [email protected] E-mail addresses: [email protected] (D.-P. Ha (G.S. Erzinger). http://dx.doi.org/10.1016/j.chemosphere.2016.09.155 0045-6535/© 2016 Elsevier Ltd. All rights reserved.
€der, G.S. Erzinger / Chemosphere 167 (2017) 228e235 D.-P. Ha
1. Introduction Most of the Earth's water is not accessible for human consumption because it is either too salty or bound in the form of glaciers and snow. The small fraction of available fresh water compromises less than 1% of the global water reservoir (Gleick, 2014). But the need for fresh water for households, industry and agriculture has increased exponentially over the past few decades and further increases are predicted (Kasei et al., 2014). At the same time the limited resources are increasingly diminished by pollution from domestic, agricultural and industrial wastes (Jones, 2014). The main components of the growing number of pollutants include heavy metals, persistent organic pollutants (POP) as well as pharmaceuticals (Gleick, 2014). In addition, the excessive use of fertilizers in agriculture results in accumulation of nitrogen and phosphorous compounds which reach surface waters and ground water reservoirs by the runoff from the fields (Wu et al., 2014). Global climate change, which causes increasing temperatures, changing precipitation patterns and draught episodes as well as dropping ground water tables, is expected to deteriorate the conditions for fresh water availability (Elliott et al., 2014). In addition, the limited resources of fresh water for irrigation are bound to decrease agricultural productivity (Cotrufo, 2014; Kurukulasuriya and Rosenthal, 2013; Müller et al., 2014). About 780 million people in developing countries lack access to clean fresh water (WHO/UNICEF, 2012) and 2.2 billion fail to have safe sanitation (WHO, 2014). Each year an estimated 5 million people die from diseases related to polluted water resources such as diarrhoea and infections transmitted by water-borne parasites (http://www.who.int/topics/mortality/en/); many hundreds of thousands of premature deaths could be avoided by simple measures to improve fresh water quality for human consumption (Apte et al., 2015). High concentrations of heavy metals were found in rivers in Pakistan and India (Muhammad et al., 2011). In addition, organic pollutants as well as inorganic toxicants are accumulated in sediments and pose considerable long-term risks for the biota and humans (Kuku cka et al., 2015). Chlorophenol compounds derived from degradation of pesticides and chlorinated hydrocarbons (Karci, 2014) are considered to be among the most toxic pollutants in aquatic ecosystems because of their high toxicity, chemical stability and low degradability (Lindholm-Lehto et al., 2015; Wei et al., 2015). Personal care products and pharmaceuticals introduced into surface waters by household wastes are recycled and concentrated in rivers. The water of some rivers in heavily populated areas is recycled several times in the form of drinking water and household effluents so that substances like estrogens create problems such as s et al., 2014). In feminization in fish and amphibian species (Valde addition, chemicals such as antibiotics accumulate in crop plants when these waters are used for irrigation (Pan et al., 2014). Because of the increasing demand for clean fresh water, monitoring and assessment of both ground water and surface water reservoirs has a high priority. Chemical analyses are of limited value because they are time consuming, expensive and usually are restricted to a few classes of chemicals while the number of potentially toxic chemicals is excessive (Ginebreda et al., 2014). Often the toxicity of a certain chemical is underestimated or it may operate synergistically with other substances which is not detected in routine chemical analyses (Chen et al., 2015). Regulations vary between countries and upper limits for toxins may be altered over time and may not represent the effective threat for the biota or €der, 2013), Alternatively, wahuman consumers of fresh water (Ha ter quality can be monitored using bioassys. Early examples for this procedure include the deployment of fish in potentially polluted
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water. When they died or showed abnormal swimming behavior this was considered as a sign for the presence of a pollutant in the water. Many different organisms have been employed in bioassays such as bacteria, microorganisms, lower and higher plants as well as invertebrates and vertebrates (de Castro-Catal a et al., 2015; Hafner et al., 2015; Kottuparambil et al., 2014; Ma et al., 2014). Depending on the time scale for the evaluation different endpoints can be considered including mortality, motility and behavior, growth and reproduction as well as physiological parameters such as photosynthesis, protein biosynthsis and genetic alteration of aquatic organisms (Davies and Mazurek, 2014). While chemical analysis can identify a potentially toxic substance or at least a class of chemicals, bioassays do not provide this information. They indicate the presence of a toxin in the water which - above a certain concentration - can present a potential €der and health hazard for the biota or for human consumption (Ha Erzinger, 2015). The toxicity of a pollutant is often determined by the EC50 value which causes a 50% inhibition (Sebaugh, 2011) but any other value can be used instead. There are a number of commercially established bioassays. The Microtox test is based on the diminuation of the bioluminescence generated by genetically modified bacteria in the presence of toxic ndez-Pin ~ as et al., 2014). The biotest Lemnatox anmaterials (Ferna alyzes the growth of an aquatic higher plant under the influence of pollutants (Newman and van Valkenburg, 2013). A recently developed bioassay for municipal and industrial wastewater effluents is based on the delay of zoospore release from marginal disks cut from the thallus of the marine green alga Ulva pertusa under the influence of a toxin during a 96-h period (Han and Choi, 2005; Y.-J. Kim et al., 2014). Disadvantages of some commercial bioassays are the low sensitivity toward toxic chemicals and/or the long times required for analysis which can be on the order of several days. One approach is based on the on-line image analysis of motile photosynthetic microorganisms monitoring motility, orienation and cell shape fully automatically (Azizullah et al., 2013; Azizullah et al., 2011). This unicellular flagellate (Euglena) has also been employed to indicate the toxicity of phenolic substances (Kottuparambil et al., 2014). The microcrustacean Daphnia has been used as a sensitive organism to indicate the potential toxicity in water samples (Chen et al., 2012). There are a number of automatic bioassays on the market. However, many have serious limitations, such has high price tag, high costs of consumables, low sensitivity, long analysis time spans, complicated performance and large size. The aim of the current work is to describe a recently developed on-line, fully automatic image analysis system to monitor motility, organism shape and orientation of the water flea Daphnia. In contrast to the Ecotox bioassay, which uses a unicellular flagellate, Daphniatox employs a multicellular crustacean. The test is certified in many countries. The organism is as sensitive as the Ecotox, but differs with respect to the chemical nature of the toxicant. The Daphniatox test is also faster than the Ecotox test. The advantages of this test include high sensitivity of the organisms (Struewing et al., 2014) and the fast analysis of the selected 14 end points. While a bioassay by definition cannot identify the pollutant or toxic substance, the different endpoints show different sensitivities to varous groups of chemicals, which gives some indication to the nature of pollution. 2. Materials and methods 2.1. Organisms for the bioassay The water flea Daphnia is a genus of small (0.5e5 mm length) Cladoceran crustaceans found in fresh water bodies like streams, ponds and lakes. Culture conditions are simple, and they grow and
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multiply at a fast rate characterizing these organisms as cost effective and easy to handle. The cultivation of the Cladoceran D. magna was performed according to ISO-6342 (ISO-6342, 2012). 50 Daphnia magna were kept in 5-L glass beakers containing 2 L of culture medium (M4 medium) (Erzinger et al., 2015). The medium was renewed and the offspring removed every three or four days. After 4 weeks breeding Daphnia were removed and replaced by neonatal organisms. The organisms were fed twice daily with the unicellular green alga Raphidocelis subcapitata (previously Selenastrum capricornutum) according to ISO-8692 (ISO-8692, 2012) using a peristaltic pump controlled by a timer to deliver 7 ml of algal suspension (at 3.6 107 cells/mL) per day. Daphnia is recommended as a suitable organism in aquatic bioassays because of their high sensitivity to many toxic chemicals (Chen et al., 2012), and the biotest is certified by several national and international agencies such as the German standard methods (DIN 38412 L-30) and the OECD test 202 to monitor fresh water, waste water and sludge toxicity (GSM, 1989). The organisms respond to many organic and inorganic pollutants (Knops et al., 2001) including carbamate, pyrethroids and organophosphorous pesticides (Ren et al., 2015). In order to prove the suitability of the bioassay we used increasing concentrations of K dichromate which is of ecological interest since it is used in tanning, galvanoplastic and to produce chrome-sulfuric acid. This chemical is highlighted as the reference to quantify the sensitivity of the organism and for comparison among laboratories. Endpoints in these tests are motility and mortality after a specified time of exposure (e.g. 24 or 48 h) (Y. Kim et al., 2008). Several Daphnia species, Chaoborus crystallinus and Mesocyclops leuckarti have been used to indicate the presence of the pesticide triphenyltin hydroxide and other novel anti-biofouling chemicals (Gergs et al., 2015). Pollution by detergents was monitored by quantifying the motility of D. magna (Pettersson et al., 2000; Uc-Peraza & Delgado-Blas). Another endpoint in bioassays employing Daphnia is mortality which was used to determine the toxicity of the fungicide metalaxyl (S. Chen and Liu, 2008). In addition, the heart beat pattern was determined in Daphnia exposed to toxic pollutants (Kiss et al., 2003) and the precision of phototactic orientation of the organism was found to deteriorate when the organisms were exposed to heavy metals (Zhou et al., 2008). The commercial instrument Daphnia Toximeter uses real-time image analysis to determine motility and orientation of the organism with respect to gravity but it does not monitor phototaxis (Netto, 2010). Chronic tests monitoring survival and reproduction require longer time scales (Naddy et al., 2007; Tong et al., 1996). 2.2. Hardware The hardware approach for the Daphniatox is straight forward: A monochromatic USB 3.0 camera (Point Grey, Blackfly BFLY-U3-13S2M-CS) with 1.3 mega pixel resolution (1288 964) equipped with a macro zoom objective (Computar 2.8e12 mm, 1:1.3 IR 1/3”) screwed into a CS mount is aimed at a 50-ml cell culture flask (Aldrich) containing about 100 organisms. Daphnia shows vertical migrations which are controlled by gravity, light and a circadian rhythm. Phases of upward movement alternate with those of downward movement (J Ringelberg, 1991; J. Ringelberg, 1999). In order to observe gravitaxis (orientation with respect of gravity) the flask is oriented vertically and the optical axis horizontally (Fig. 1). The culture flask is irradiated from behind with a diffuse white light beam so that the camera records dark organisms in front of a white background. The light level is controlled by the iris of the objective. Phototactic
orientation is induced by a blue LED light source impinging at 90 with respect to the vertical and the optical axis. In this set-up non-motile Daphnia drop out of the field of view and collect at the bottom. Therefore, in order to determine motility the optical axis is changed to vertical with the camera pointing downward. Non-motile or dead organisms sediment to the bottom of the flask but are in the view of the camera, and thus the percentage of motile cells can be determined correctly. Control measurements and analyses with organisms exposed to different concentrations of toxins are performed in sequence creating a result file for each measurement (see section 2.3).
2.3. Software for image analysis The Daphniatox software is based on the open source software Fiji derived from the open source ImageJ (Schindelin et al., 2012). Since this program package is only capable of processing recorded video (.avi) sequences, a software plugin, which allows direct input of the online video into the imaging software (Phase, Lübeck, Germany), has been implemented. The Daphniatox software is written in the Macro Language of ImageJ. A button allows starting the camera control to provide a live camera image. Another button facilitates the setup of the parameters for the analysis. This allows calibrating the system in terms of physical units (pixels per mm), selecting a certain size range for the organisms to be tracked (to avoid tracking of disturbing other objects such as the food organisms for Daphnia) and the velocity range in order to eliminate tracking drifting or sedimenting organisms. The number of tracked organisms and the maximal time per experiment are selected after which the tracking stops, whichever comes first. Another button in the task bar starts the tracking software which commences by taking a number of snapshots from the video input stream at regular time intervals and storing them in a stack of images together with the elapsed time in order to calculate the swimming velocity. The quality of these frames is improved by background subtraction and contrast enhancement to yield clear images of dark organisms in front of a bright background. A thresholding results in images with two levels of brightness: black for the background (0) and white for the organisms (255); this step inverts the image. After this pre-processing the stack is used to extract the movement vectors of the motile objects (up to 256 in parallel). The results from the vector analysis are stored, and the data from this analysis are displayed in a separate window in the form of a circular histogram showing the fraction of cells moving in sectors, the size of which the user has preselected in the settings. In addition, the number of tracks recorded, the mean velocity, the percentage of motile cells, the mean area and length of the organisms, the mean angular deviation from the vertical as well as the percentage of upward swimming organisms (in the case of a vertical movement) and the precision of orientation are indicated. After the analysis of tracks in one image stack has been finalized a new set of frames is taken from the life video and stored in a new stack and the analysis is repeated cyclically until a user-defined number of tracks has been determined or a predefined length of time has elapsed, whichever comes first. After each tracking cycle the angular histogram is updated with the additional tracked vectors. At the end the results can be stored together with a description of the details of the experiment, which the user provides, and - if selected - the raw data such as the areas of the individual organisms, the track coordinates and the angular distribution. The movement direction of each track is calculated as the angular deviation a from the 0 direction
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Fig. 1. Optical setup of the Daphniatox instrument with a Blackfly USB 3.0 camera equipped with a macro zoom lens and diffuse background monitoring light. Actinic blue light is provided by an LED impinging perpendicularly to the optical axis.
a ¼ tn1
y2 y1 x2 x1
where y2 and y1 are the end and beginning y-coordinates of each track, respectively, and likewise for the x coordinates (D.-P. H€ ader and Lebert, 2000). Since this calculation covers only the angles <90 the information, in which quadrant the organism moves, is used to determine all angles. The precision of orientation of the whole population is defined by the r-value, which is a statistical measure which runs between 0 (when the organisms move in random directions) and 1 (when all cells move in the same direction) (Batschelet, 1981).
r¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðSsin aÞ2 þ ðScos aÞ2 n
However this value does not indicate in which directions the organisms move. Therefore the mean movement angle of the population is calculated as q (Batschelet, 1981).
q ¼ arctan
Ssin a Scos a
In order to cover all four quadrants a correction has to be performed: if S sin a is negative then q is q þ360 and if S cos a is negative then q is q þ180 . The percentage of upward swimming cells (±90 around the vertical upward) is calculated. To determine phototactic orientation, the actinic light is impinging from 0 and the percentage of organisms swimming in the two quadrants toward the light (±90 ) is quantified. The swimming speed is calculated by analyzing the (meandering) total swimming path length divided by the time needed for this. Furthermore the direct speed is determined by dividing the distance from the beginning to the end of the vector by the time needed. The ratio of the direct path length divided by the actually covered path length gives the swimming directedness. The motility is determined as the percentage of moving organisms within the defined size range and which qualify for the predefined velocity range. The area of each object (which falls into the size limits) is determined and the mean value is calculated. The length of the outer boundary (perimeter), the circularity, roundness, solidity and aspect ratio (ratio of the major and minor axes of a particle's fitted ellipse) are calculated (see definitions below). In addition, the
Feret's diameter is determined which indicates the largest dimension of an object. This is achieved by turning the image stepwise until the largest dimension is found. The degree of turning gives the Feret angle, which is thus the angle of the longest axis with respect to the horizontal (Schindelin et al., 2012).
circularity ¼ 4p
roundness ¼
solidity ¼
area perimeter2 area
p major axis2
area convex area
For all parameters mean values and standard deviations are determined. The initial calibration (pixels per mm) allows calculating all size and velocity parameters in actual physical units (mm, mm2, mm/s). The results obtained with the Daphniatox system can be utilized to determine effect-concentration curves to obtain information on defined toxicity parameters like NOEC, LD and EC50 values (Ahmed €der, 2011). The system can be used for short-term as well as and Ha long-term (online) monitoring of pollution in all kinds of waters such as ground water, surface or wastewater as well as natural ecosystems.
3. Results The experiments for short-term (acute) tests were performed according to the standard NBR 12713 protocol (ABNT, 2003; EPA, 2002). Neonates of D. magna, 2e26 h old, were placed in K2Cr2O7 solutions (0, 0.125, 0.25, 0.5, 1, 2 or 4 mg/L) for a period of up to 48 h in 50-ml cell culture flasks (Flohr et al., 2005). Potassium dichromate was used as a toxin in the tests because this chemical is widely used in tanning, galvanoplastic, to produce chrome-sulfuric acid and to stain neutrons and nerves (Fisher-Scientific, 2007) and therefore is frequently found as a pollutant in water bodies. Potassium dichromate was defined by international institutions as the reference chemical to assess the sensitivity of the organism and to serve as a comparison among laboratories. The MAC value is 5 mg/ m3. Two flasks were used for each concentration and each flask was tracked three times. In order to determine the motility the
232
€der, G.S. Erzinger / Chemosphere 167 (2017) 228e235 D.-P. Ha
organisms were tracked in flasks positioned horizontally on a light table. Since they did not undergo gravitactic orientation the motility vectors point in random directions which results in an rvalue of 0.02 (Fig. 2). The swimming directions of the tracks are binned in 60 sectors. The histogram is based on 101 tracks and shows the percentage of organisms swimming in each sector. 71% of the organisms were motile, swimming at a speed of 0.11 mm/s. The area was 0.19 mm2 and the length 0.82 mm 47.5% of the organisms swam in the two quadrants adjacent to 0 and q was 236.66 . Immediately after incubation in the potassium dichromate solutions all organisms remained motile, but after 4 h an increasing number of Daphnia became immobile at 2 and 4 mg/L (data not shown). After an incubation time of 24 h there were no motile organisms at concentrations 1 mg/L (Fig. 3). The motility (percentage of motile organisms) was more or less constant (ca. 85%) at the lower concentrations and in the control. Also the velocity was almost constant at concentrations <1 mg/L (Fig. 4). Even though the organisms were from the same batch, the length of them decreased with the concentration of potassium dichromate (Fig. 5a) while the area increased with lower concentrations (Fig. 5b) indicating a swelling of the organisms. At higher concentrations the area decreased. Since this indicates a change in the organisms’ form, the roundness (for definition see section 2.3) was determined for each population (Fig. 5c). In accordance with the above results, after an increase at the lowest concentrations this value decreased at higher concentrations. When exposed in a vertical cell growth chamber the organisms undergo gravitactic orientation swimming upward at times and downward at others (Fig. 6) while lateral movement does not occur as often. This histogram is based on 42 tracks. All organisms were motile swimming at a mean value of 0.14 mm/s 50% of all organisms swam upward. They had a length of 0.6 mm and an area of 0.1 mm2. The r-value was 0.12 and q was 190 , but these values are meaningless for a bimodal distribution. For each test 14 endpoints are quantified as shown in Table 1 as an example for the effects of 0.125 mg potassium dichromate after 24 h incubation. In addition to the percentage of motile organisms, the mean velocity, the area, length, roundedness and gravitactic orientation the following parameters are evaluated: the percentage of upward swimming organisms and the mean direction q. The circulatiry, the mean aspect ratio, the mean solidity as well as the perimeter in comparison to the length give further information on the form factor. The swimming velocity describes the speed of
Fig. 3. Effect-concentration curve of the motility (percentage of motile organisms, mean values ± SD, n ¼ 6) of D. magna after incubation in potassium dichromate for 24 h indicating the NOEC (¼0.5 mg/L, no observed effect concentration). EC50 (¼ 0.78 mg/L, concentration at which 50% inhibition occurs and LD (¼1 mg/L, lethal dose).
Fig. 4. Effect-concentration curve of the mean swimming velocity (mean values ± SD, n ¼ 6) of D. magna after incubation in potassium dichromate for 24 h indicating the NOEC (¼0.25 mg/L, no observed effect concentration). EC50 (¼0.68 mg/L, concentration at which 50% inhibition occurs and LD (¼1 mg/L, lethal dose).
movement between the beginning and end of the track. However the organisms may not follow a direct path but meander from the direct line. This is indicated by the mean directedness and the mean velocity which indicates the swimming speed along the actual path. For each concentration two independent samples were monitored and each test was repeated three times. For all parameters mean values and standard error are calculated. 4. Discussion
Fig. 2. Angular histogram of tracks of D. magna in a horizontally oriented swimming chamber binned in 60 sectors showing a random distribution.
As expected the percentage of motile organisms as well as the swimming velocity were affected by even small contentations of potassium dichromate. In contrast, changes in the length of the organism as well as the form factors were not expected since the organisms have a rigid exoskeleton. They would also not be detected by manual evaluation, but by the quantitative image analysis. These form and length changes can be explained by assuming that the organisms release or take up liquid under the pollutant stress. The results indicate that upon exposure to low concentrations of dichromate the organisms take up water and bulge which reduces the length, but increases the area, resulting in increased roundness. Modern bioassays using advanced technologies must be
€der, G.S. Erzinger / Chemosphere 167 (2017) 228e235 D.-P. Ha
233
Fig. 5. Length (a), area (b) and roundness (c) of D. magna after incubation in potassium dichromate for 24 h. Mean values (n ¼ 6 ± SD).
Fig. 6. Angular distribution histogram of D. magna swimming in a vertical cell culture flask showing positive (downward) and negative (upward) gravitactic orientation binned in 60 sectors.
evaluated by a number of characteristics. One of the important features is a short analysis time. Depending on the requested number of analyzed tracks a full measurement cycle in Daphniatox
can be completed in less than 2 min which is achieved by the online, real-time computerized image analysis. This is even faster than the ECOTOX system which requires about 6 min for a full measurement cycle (Azizullah et al., 2013). Because of the automated analysis human error-prone and subjective evaluation is avoided. Using high numbers of analyzed organism tracks warrants high precision and statistical significance. When evaluating longterm effects of potentially toxic substances the analysis time is equally short interrupted by incubation periods. As compared with other advanced automated bioassays the operational costs for Daphniatox are almost negligible. The only requirement is keeping a supply of the microcrustaceans and their food source which is simple and can be done at low costs. Operating the system is user friendly and due to the automated evaluation does not require extensive training of personnel. The organisms used for this bioassay are very sensitive to toxins. For motility, velocity and other determined parameters EC50 values of less than 2 mg/L have been found after an exposure of 2 h and even less after long-term exposure of 24 h. In comparison, the EC50 value for rats is 25 mg/kg after oral application of potassium dichromate and for rabbits 14 mg/kg after transdermal application (Gumbleton and Nicholls, 1988; Tandon, 1982). The other endpoints found in the raw data (for example see Table 1) confirm the main data shown in the results section. The data indicate that the organisms do not swim on straight paths but meander during the track. The percentage of upward moving cells indicates the degree of gravitactic orientation. The form parameters circularity, aspect ration and solidity as well as perimeter with respect to the organisms length confirm the data on roundedness.
€der, G.S. Erzinger / Chemosphere 167 (2017) 228e235 D.-P. Ha
234
Table 1 Results for the 14 measured parameters indicting the effects of 0.125 mg/L after 24 h given as an example of the raw data obtained for each test. Each test is done triplicates and two parallel flasks are used for each concentration and exposure time. 3.11.2015 15:02:16 h 0.125 mg flask B 24 h exposure. test 3 83% motile Mean velocity ¼ 0.15 ± 0.06 mm/s Mean direct velocity ¼ 0.10 ± 0.05 mm/s Mean directedness ¼ 0.67 ± 0.25 41.82% organisms swam upward Theta ¼ 360.46 r-value ¼ 0.18 Mean area ¼ 0.16 ± 0.06 mm2 Mean perimeter ¼ 2.34 ± 0.86 mm Mean circularity ¼ 0.41 ± 0.19 Mean organism length ¼ 0.79 ± 0.31 mm Mean aspect ratio ¼ 2.68 ± 1.67 Mean roundness ¼ 0.48 ± 0.24 Mean solidity ¼ 0.73 ± 0.17
Funding This work was partially supported by the National Council of Technological and Scientific Development (CNPq).
Different endpoints selected for the evaluation of toxicity vary in their sensitivity toward different classes of toxic substances and pollutants. Using 14 different parameters in motility, orientation, size and form warrants a wide range of endpoints for evaluating toxicity. A similar number of parameters is evaluated by the Ecotox system which utilizes the motility, orientation and form parameters of the unicellular green flagellate Euglena gracilis (Azizullah et al., 2014). The sensitivity of Daphnia species and related genera to toxic substances has been found to be higher than that of the organisms utilized in other bioassays including algae (growth), fish (mortality) and bacteria (bioluminescence) (Ahmed and H€ ader, 2011). In contrast to chemical analysis biomonitoring systems are not designed to identify the chemical nature of a toxic pollutant. They rather indicate pollution above a critical level potentially posing a hazard for ecosystem safety or human health. If the level of pollution is found to exceed the accepted level chemical analysis is required to identify the chemical nature of the toxicant. One strong advantage is that, in contrast to pure chemical analysis, bioassays can indicate potential threats of combined polluting chemicals, which my act additively or synergistically. Often the toxicity of pollutants is augmented by solar radiation, especially in the UV-B range which is detected by bioassays (Zepp et al., 2011). Bioassays have been employed to indicate the toxicity of a wide range of pollutants such as heavy metals, herbicides, pesticides, fertilizers, detergents, wastewaters and many other organic and inorganic pollutants (Azizullah et al., 2014; Danilov and Ekelund, 2000). They have been used effectively to determine both shortand long-term ecotoxicity of pollutants (Ahmed, 2010; Azizullah et al., 2014). Daphniatox can be utilized as an early warning system for pollution monitoring surface and ground waters, municipal and industrial wastewaters as well as for controlling the efficiency of waste water treatments plants. The low initial and negligible running costs allow utilizing the system also in developing countries where poor funding and lack of environmental experts render efficient water quality monitoring difficult. Preliminary experiments have shown that the toxicity determined by Daphniatox correlates well with reported effects on human health indicating that it is a useful instrument to monitor drinking water, waste water and natural ecosystems. When the instrument enters industrial production the price tag is expected to be around 4000 V. 5. Conclusions Daphnia
is
an
accepted
bioassay
biomonitoring protocol is standardized and established in many countries (Asghari et al., 2012). The fully automatic, real-time computerized Daphniatox warrants rapid standardized monitoring eliminating error-prone human interpretation and provides high statistical significance of the data. Using 14 independent endpoints of motility, orientation, size and form of different sensitivity toward toxicants can be used to indicate different classes of chemical toxicants and pollutants. High sensitivity of the microcrustatian Daphnia and low initial costs and negligible running costs characterize the system as suitable for many tasks in short- and long-term monitoring of fresh and ground water resources as well as industrial, household and agricultural waste water.
organism
and
the
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