Melanie Voigt is grateful for a stipend from the Promotionskolleg of the Niederrhein University of Applied Sciences. The authors thank their institution for further financial support.

1. Sample Preparation: Solid Phase Extraction
<ol>
	<li>Collect about 1 L of water for the preparation of the samples.</li>
	<li>Filter the sample over a blue band filter with a pore size of 2 &#181;m to remove coarse particles.</li>
	<li>Equilibrate the SPE cartridge using 3 mL of methanol and 3 mL of ultrapure water.</li>
	<li>Apply the filtrate (1 L) onto the SPE cartridge and increase the flow velocity using a moderate vacuum, e.g., a diaphragm pump. 
	NOTE: Several SPE cartridges may be run in parallel.</li>
	<li>Wash the sample with 3 mL of ultrapure water.</li>
	<li>Elute the analytes from the cartridge sorbate with 3 mL of methanol.</li>
	<li>Concentrate the 3 mL eluate to dry using a rotary evaporator.</li>
	<li>Dissolve the residue in 1 mL of ultrapure water.</li>
	<li>Filter the solution through a syringe filter and store them in a vial for the non-targeted analysis by HPLC-ESI-Q-TOF-MS.</li>
</ol>
2. HPLC-ESI-Q-TOF-MS Method for Non-Targeted and Targeted Analysis, and MS/MS
<ol>
	<li>Transfer the vial to the HPLC-ESI-Q-TOF-MS autosampler.</li>
	<li>Set all relevant parameters (Table 1) for the HPLC-ESI-Q-TOF-MS. 
	NOTE: If a finite collision energy is used, i.e., collision energy (CE) &#8800; 0, ions will be fragmented. This mode corresponds to the targeted MS/MS method.</li>
	<li>Start the measurement.</li>
	<li>Analyze the resulting chromatograms and mass spectra.</li>
</ol>
3. UV Irradiation Experiments
<ol>
	<li>Dissolve the antibiotic compound, e.g., erythromycin (750 mg/L), in ultrapure water at 20 mg/L final concentration.</li>
	<li>Fill the 1 L photoreactor, wrapped in aluminum foil, with 750 mL of the solution.</li>
	<li>Introduce the lamp providing 15 W of power into the reactor.</li>
	<li>Apply the magnetic stirrer at 500 rpm.</li>
	<li>Adjust the pH value to the desired value 3-4, 6-7 or 8-9 by dropwise addition of HCl (0.1 M) or NH3 (0.1 M) if necessary. pH 6-7 is used as an example.</li>
	<li>Take 2 mL of the reaction solution as sample at time 0 using a syringe and transfer it into a 2 mL glass vial.</li>
	<li>Switch on the UV lamp and keep track of the elapsing time. 
	NOTE: Irradiation times of 10 min are often sufficient. If completeness of the photoreaction is desired, a second experiment series might need to be recorded using the results of the first series. 
	Caution: UV irradiation may lead to blindness.</li>
	<li>Draw a 2 mL sample from the solution every 30 s during the first 5 min. Then, take a sample every 60 s until the end of the experiment. Transfer the samples into 2 mL vials.</li>
	<li>Store the vials until HPLC-ESI-Q-TOF-MS analysis at -4 &#176;C.</li>
	<li>Analyze the 16 samples using HPLC-ESI-Q-TOF-MS with the methods described in step 2.</li>
</ol>
4. Kinetics Analysis
<ol>
	<li>Prepare a suitable software such as the curve-fitting toolbox of MATLAB R2016b.</li>
	<li>Fit the mass-area vs. time data of the photo-induced degradation of the parent antibiotic compound according to first order kinetics, see eq. 242,43 
	<img alt="Equation 1" src="/files/ftp_upload/57434/57434eq1.jpg" />&#160; &#160;(2) 
	The concentration <img alt="Equation 2" src="/files/ftp_upload/57434/57434eq2.jpg" /> refers to the initial concentration of the educt A, cA to the actual concentration over the reaction time t with the rate constant k1 from the first reaction step A to B.</li>
	<li>Fit the mass-area vs. time curves of the degradates using eq. 3 and 4, as they can be described as intermediates of a consecutive or subsequent follow-up reaction, i.e., product B or C according to the reaction model A &#8594;B &#8594; C &#8594; D. 
	<img alt="Equation 3" src="/files/ftp_upload/57434/57434eq3.jpg" />&#160; &#160;(3) 
	<img alt="Equation 4" src="/files/ftp_upload/57434/57434eq4v5.jpg" />&#160; &#160;(4) 
	The concentrations cB and cC refer to the intermediates B and C; and k2,k3 to the corresponding rate constants B to C, C to D.</li>
	<li>Use eq. 5 to fit the data, if the irradiation time was not sufficient to observe the degradation of a photo-product. This degradate can be treated as final product D with the concentration CD to obtain rate constants. 
	<img alt="Equation 6" src="/files/ftp_upload/57434/57434eq6.jpg" />&#160; &#160;(5)
	<ol>
		<li>Calculate the concentration of B using eq. 6 instead of eq. 3, if the reaction ends with B. If C is the final product, calculate the concentration of C according to eq. 7 instead of eq. 4. 
		<img alt="Equation 7" src="/files/ftp_upload/57434/57434eq7.jpg" />&#160; &#160;(6) 
		<img alt="Equation 8" src="/files/ftp_upload/57434/57434eq8.jpg" />&#160; &#160;(7)</li>
	</ol>
	</li>
	<li>Use eq. 8 for the determination of the half-lives t1/2. 
	<img alt="Equation 10" src="/files/ftp_upload/57434/57434eq10.jpg" />&#160; &#160;(8)</li>
</ol>

The authors declare no competing financial interests.

The example of a non-targeted analysis presented in this report demonstrated the identification of pharmaceuticals in surface water using HPLC-ESI-Q-TOF-MS, MS/MS and comparison with reference standards as the final proof. The strength of non-targeted analysis using TOF-MS is based on the detection of all ions present at a given retention time and the high mass accuracy which leads to the prediction of the tentative molecular formula. As an alternative to a TOF mass spectrometer, the application of an orbital ion trap has been described for contaminant analysis in water44. The molecular formula prediction was used as the starting point to quickly select reference standards. The application of the targeted MS method of the Q-TOF-MS instrument allowed the detection of specific compounds, since only pre-selected ions pass the quadrupole filter. In general targeted analysis is performed using triple quadrupole mass spectrometer also in water analysis45. To compensate for the deviation from the theoretical mass due to instrumental imperfections, a chromatographic comparison with a reference standard might be performed. The targeted MS/MS method may also be chosen for identification analysis. Here, ions are selected, fragmented and their fragments detected. Since MS/MS is less sensitive than MS, the concentration of the pharmaceuticals in the investigated water samples was too low to yield meaningful fragments. However, if fragments are detected, compounds can be identified with higher confidence. The insufficient sensitivity might be overcome by concentrating a larger initial water sample volume. In addition, the measurement should be carried out as soon as possible after sampling because of potential biodegradation46,47,48,49. Otherwise, samples should be stored at -20 &#176;C to exclude compound degradation or reaction.
Sometimes the same m/z values appear at different retention times. This may be due to that the isomers require different analytical techniques. It can also occur that no compounds might be detected at all, which does not necessarily prove their absence. They might just not form ions or occur below the limit of detection. The type of water also exercises an influence on the presence of pharmaceuticals. Pharmaceuticals rarely enter source water and groundwater as compared to sewage water and effluents from wastewater treatment plants48,50,51,52,53.
For the degradation experiments, the irradiation source should be characterized in advance, since the photon flux or photon fluence rate of the lamp contributes significantly to the degradation and the mechanism of degradation. For initial attempts, a VUV/UVC lamp, probably a low-pressure mercury lamp is sufficient. In general, the addition of hydrogen peroxide, H2O2, accelerates the degradation27,36,37,54. When a different lamp, e.g., a UVA lamp, is used, the formation of hydroxyl radicals should be ensured, e.g., through the addition of titanium dioxide 23,24,30,31. For many compounds, such as erythromycin, OH radicals rather than photo-reactivity of the pharmaceutical itself27are the degradation-inducing species.
For the determination of the kinetic parameters, the area of the signals in the mass-detected chromatograms, representing concentration, is plotted versus irradiation time. To fit the data, it is advisable to use suitable software. Here, the curve fitting tool of MATLAB was used, which allowed to quickly compute and fit the data with the correct equations. The kinetic of the intermediates is determined by more complex equations. The quality parameters for the fit, i.e., R2 and RMSE, were readily obtained as well.
This study demonstrated the analysis of river water to detect and identify pharmaceutical pollutants and the photodegradation of erythromycin in ultrapure water. In environmental waters, such as surface water, different degradation velocities and rate constants would be obtained due to light absorbing substances, such as humins. According to the authors&#39; experience, degradation often takes place more slowly, but sometimes at comparable rates41,56.
The worldwide problem of pharmaceuticals, especially antibiotics, in the aquatic environment and the resulting hazards still continue to grow1. Due to the variety and diversity of chemicals, metabolites, and degradates thereof, non-targeted analysis will become the most important analytical weapon for their discovery in the environment57. For the effective elimination, novel stages in wastewater treatment plants will need to be designed based on advanced oxidation processes, which UV irradiation might be part of.

Pharmaceuticals are regularly found in the aquatic environment1,2,3,4,5. An important source are effluents from wastewater treatment plants (WWTP)6,7,8,9. The occurrence of pharmaceuticals in the throughout the water cycle has been studied exemplarily in the Turia River Basin10. Among others, antibiotics represent a particular hazardous class of drugs, since they often pass the biological stage of WWTPs unaltered and may cause bacterial resistances in the environment11,12,13. Macrolides constitute a class of antibiotic drugs that are applied both in human and in veterinary medicine. Their representatives were found in concentration up to 1 &#181;g/L in effluents14,15,16,17,18,19. One of them is erythromycin (Ery)20,21. In waters, erythromycin is often accompanied by anhydroerythromycin A (Ery A - H2O), a dehydrate22,23. Water elimination from erythromycin is due to acid instability. The ratio of erythromycin vs. anhydroerythromycin depends on the pH24,25,26,27.
Chemically, macrolides contain a macrocylic lactone to which various sugar moieties are attached, e.g., desosamine, cladinose or mycaminose. Since macrolides are chemically modified natural products from fermentation processes, they often exist as mixtures. The species termed A, B, C, etc., differ in the sugar substituents. The sugar moieties and their position at the lactone are responsible for the mode of action of macrolides28,29. In order to minimize environmental hazard, it is desirable to completely mineralize the pharmaceuticals before entering the aquatic environment27,30,31,32.
The first part of this study deals with the detection of pharmaceutical in surface waters, which is important for monitoring both effluents and open waters. To search for a variety of unidentified substances in the microgram range in different matrices, non-targeted analysis is the method of choice20,33,34,35. In particular, high-performance liquid chromatography (HPLC) electrospray ionization quadrupole time of flight mass spectrometry (HPLC-ESI-Q-TOF-MS) has been proved of extraordinary value due to its specificity and sensitivity. After the identification of the substance, sensitivity can further be extended by using the targeted MS approach with the quadrupole operated in select mode and the collision energy within the collision cell set to zero. Hence, ions arrive non-fragmented at the TOF detector.
The second focus of this work is the elimination of erythromycin. For the elimination of the pharmaceuticals, so-called advanced oxidation processes (AOPs) are used, e.g., started by irradiation with UV light36,37,38. Essential for the degradation is the formation of hydroxyl radicals from water by VUV / UVC irradiation following eq. 1.
H2O + h&#957;(&#60;200 nm) &#8594; H2O* &#8594; H. + .OH&#160; &#160; &#160; &#160; (1)
Hydroxyl radicals possess a high oxidation potential of 2.8 V, which positively contributes to the degradation of the substances36,37.
Here, the degradation of erythromycin using vacuum UV/UVC-irradiation in water is described taking the influences of the pH into account. The formation of even more hazardous products is believed to be a disadvantage of using AOPs39,40. Thus, it is important to irradiate until complete mineralization of the pharmaceuticals. To better estimate the irradiation time, the kinetic model of the reaction, the reaction rate constants and the half-lives are determined both for the initial drug and for its photodegradates. To this purpose, concentration-time (c-t) plot were derived from HPLC-ESI-Q-TOF-MS measurements and compared to chemical kinetics models using MATLAB. The kinetics of the degradation proceeded according to first-order, and the photodegradates were described as intermediate products of a consecutive or subsequent follow-up reaction27,41.

<table border="0" cellpadding="0" cellspacing="0" style="width:944px;" width="942">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Methanol for liquid chromatography LiChrosolv</td>
			<td style="width:199px;">Merck</td>
			<td style="width:179px;">1060181000</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">formic acid</td>
			<td style="width:199px;">Fluka</td>
			<td style="width:179px;">94318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">HCl</td>
			<td style="width:199px;">Riedel-de Haen</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">NH3</td>
			<td style="width:199px;">Riedel-de Haen</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Simplicity 185 Water Purification System</td>
			<td style="width:199px;">EMD Millipore</td>
			<td style="width:179px;"></td>
			<td>for producing MilliQ-water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Erythromycin</td>
			<td style="width:199px;">BioChemica AppliChem</td>
			<td>A2275,0005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Filter Rotilabo-filter, Typ 113A</td>
			<td style="width:199px;">Roth</td>
			<td style="width:179px;">AP78.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">SPE-Cartridges Oasis HLB 3cc (60mg)</td>
			<td style="width:199px;">Waters</td>
			<td style="width:179px;">WAT094226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">BAKER SPE-12G</td>
			<td>J.T. Baker</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">membrane pump PC3001 VarioPro&#160;</td>
			<td style="width:199px;">Vacuubrand</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">rotary evaporator; Laborota 4000 efficient</td>
			<td style="width:199px;">Heidolph Instruments</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">syringe, 2 mL</td>
			<td style="width:199px;">Terumo</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Nylon Syringe Filters Target2</td>
			<td>Thermo Scientific</td>
			<td style="width:179px;">10301345</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">C-18 CoreShell column 50 mm x 2.1 mm dimensions, 2.6 &#956;m particle size</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">HPLC 1200</td>
			<td style="width:199px;">Agilent</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">ESI-Q-ToF-MS 6530</td>
			<td style="width:199px;">Agilent</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">photoreactor, UV Labor Reactor System 3</td>
			<td>Peschl Utraviolet GmbH</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">VUV/UVC-lamp, TNN 15/32, 15 W</td>
			<td style="width:199px;">Heraeus</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">pH-meter, pHenomenal pH 1100L</td>
			<td style="width:199px;">vwr</td>
			<td>662-1657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">magnetic stirrer</td>
			<td style="width:199px;">Heidolph Instruments</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">MassHunter Workstation B.06.00</td>
			<td style="width:199px;">Agilent</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">MATLAB R2016b</td>
			<td style="width:199px;">Mathworks</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of pharmaceuticals in the aquatic environment using hplc-esi-q-tof-ms and elimination of erythromycin through photo-induced degradation

Monitoring pharmaceuticals throughout the water cycle is becoming increasingly important for the aquatic environment and eventually for human health. Targeted and non-targeted analysis are today's means of choice. Although targeted analysis usually conducted with the help of a triple quadrupole mass spectrometer may be more sensitive, only compounds previously selected can be identified. The most powerful non-targeted analysis is performed through time of flight mass spectrometers (TOF-MS) extended by a quadrupole mass analyzer (Q), as used in this study. Preceded by solid phase extraction and high-performance liquid chromatography (HPLC), the non-targeted approach allows to detect all ionizable substances with high sensitivity and selectivity. Taking full advantage of the Q-TOF-MS instrument, tandem mass spectrometry (MS/MS) experiments accelerate and facilitate the identification while a targeted MS method enhances the sensitivity but relies on reference standards for identification purposes. The identification of four pharmaceuticals from Rhine river water is demonstrated. The Rhine river originates in Tomasee, Graub&#252;nden, Switzerland and flows into the North Sea, near Southern Bight, The Netherlands. Its length amounts to 1232.7 km. Since it is of prime interest to effectively eliminate pharmaceuticals from the water cycle, the effect UV-C irradiation is demonstrated on a laboratory scale. This method allows fast degradation of pharmaceuticals, which is exemplarily shown for the macrolide antibiotic erythromycin. Using the above HPLC-Q-TOF-MS method, concentration-time diagrams are obtained for the parent drug and their photodegradation products. After establishing the equations for first-order sequential reactions, computational fitting allows the determination of kinetic parameters, which might help to predict irradiation times and conditions when potentially considered as fourth stage within wastewater treatment.

As result of the solid phase extraction, a yellowish to dark green solution was obtained in all cases, which indicated the presence of chlorophyll-containing substances (Figure 1). Pharmaceuticals contained in this water sample would not lead to visible coloration since their concentration and their absorbance would generally be too low. Instead, the occurrence of pharmaceuticals needs to be analyzed using HPLC and high-resolution mass spectrometry.
In non-targeted analysis, a HPLC-ESI-Q-TOF-MS was used because of its outstanding mass accuracy allowing to obtain the accurate mass for each compound ion. The mass-detected chromatogram of the performed analysis was represented as a base peak chromatogram (BPC), which displays the most intense peak of each mass spectrum recorded in the course the chromatographic separation. The example shown in Figure 2 presents the BPC of a water sample from the river Rhine.
The BPC contained more than twenty-five peaks reflecting different m/z values, hence different compounds, seven of which were marked in the BPC. Since the substances were unknown a priori, the first step to their identification usually consists of deriving the molecular formula. This is accomplished through accurate mass and isotopic pattern provided by the TOF detection, although the isotopic pattern may not be observed in all cases due to low sample concentrations in environmental samples. With the help of public database, such as Pharmaceuticals in the Environment by the German Environment Agency (UBA) containing approximately 630 compounds, a preliminary identification of a small group of candidates is often successful. For a final proof, either comparison to commercially available reference standards may be performed or MS/MS fragmentation patterns may be considered (Figure 3).
In this work, comparison to standards with respect to retention time accounted for the identification of pharmaceuticals very often found in German surface waters. These substances include metoprolol, a &#946;-blocker, carbamazepine, an analgesic, and the macrolide antibiotic erythromycin A and its derivate anhydroerythromycin A. Erythromycin serves as example further investigated in this study. The studied Rhine river sample had a pH of 7.6 and an average temperature of 16.5 &#176;C. At this pH, anhydroerythromycin would also be expected to be present in the water sample. For the detailed analysis, the extracted ion chromatograms (EICs) of the water sample were compared with the reference standards (Figure 4).
The comparison shows good agreement between the retention time for metoprolol, carbamazepine and anhydroerythromycin and the observed analytes. The EIC of the reference standard anhydroerythromycin displayed two peaks, hence two compounds where dehydration had occurred at two distinct sites of erythromycin. Yet, only one anhydroerythromycin isomer was identified in the Rhine river sample. Erythromycin itself was only present in traces. Therefore, no MS/MS spectrum could be obtained. The accurate masses for the antibiotic and its dehydrate are given in Table 2. Using EIC, thus m/z value and retention time, metoprolol, carbamazepin, erythromycin and anhydroerythromycin could be identified in the Rhine river sample.
With respect to the aquatic environment, it is important to prevent pharmaceuticals from passing through wastewater treatment plants and entering surface waters. In the quest for an efficient elimination, UV-C irradiation experiments at different pH values were carried out for erythromycin as example. Concentration-time (c-t) diagrams were recorded using mass-area vs. time plots derived from EICs. The degradation was described according to equation 2. Erythromycin consists of erythromycin A and B and anhydroerythromycin A, with two isomers of the latter. The c-t curves of erythromycin A and their computational fits are shown in Figure 5. At pH 7,accelerated degradation was observed. This applies to all four compounds studied, data not shown. As a consequence, photo-induced degradation of erythromycin should be carried out around neutral pH. In the case of the Rhine river sample, pH adjustment was not required.
Photodegradates of the pharmaceuticals were also identified at all three pH values. An overview of these photodegradates with their corresponding structure proposals is given in Table 3. For the kinetic analysis of the photodegradates, the product with m/z = 720 serves as an example. Photodegradates can often be described as reaction intermediates. Therefore, the photodegradates were described in terms of aconsecutive and subsequent follow-up reaction. The decision between the resulting types of intermediates is based on the goodness of the fit computed with suitable software, where the coefficient of determination (R2) and the residual mean-squared error (RMSE) were taken as the criteria. Due to the fact that erythromycin is acid-instable, degradates as would occur upon irradiation were present prior to irradiation. The resulting effect on equations 3 and 4 was a finite starting concentration. Hence, a factor was added to the equations. Figure 6 shows experimental data and fits computed according to equation 3 and 4.
This example of an intermediate demonstrated the concentration increase with a sigmoidal rise followed by an exponential decay. This is indicative for a subsequent follow-up reaction intermediate. A consecutive reaction intermediate does not show the sigmoidal increase. Statistical quality parameters also indicated the slightly superior agreement of fit according to the subsequent follow-up reaction model. The coefficient of determination R2 of the consecutive reaction was 0.9898 and thus lower than that of the subsequent follow-up reaction being 0.9976. Therefore, the examined photoproduct was interpreted as intermediate of a subsequent follow-up reaction. The k-values resulted from the computational fit as well, the half-life was calculated following equation 5. All relevant kinetic parameters are collected in Table 3.
The fastest degradation was observed at pH 7, followed by pH 9, while the slowest degradation was found for pH 3 (Figure 5). This finding also applied to the formation and degradation of the photoproducts. Three photodegradates were observed. Their m/z values were 750.46 corresponding to Ery F, 720.45 to Ery C and 192.12 to DPEry192, a glycosidically bound sugar of the erythromycin structure (Figure 7). No degradation of the photoproduct could be observed for DPEry192 at pH 3 and 9 and for Ery F at pH 9. In these cases, the irradiation time was not sufficient long to observe total degradation of the intermediate product. Nevertheless, the formation rate constant could be determined by using equation 5, which corresponds to a final product.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57434/57434fig1.jpg" /> 
Figure 1. Comparison of the samples from the Rhine river after SPE (left) and ultrapure water(right) treatment. The green coloration is indicative for chlorophyll-containing substances. <a href="/files/ftp_upload/57434/57434fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57434/57434fig2.jpg" /> 
Figure 2. BPC of a water sample after SPE measured with HPLC-ESI-Q-TOF-MS. All chromatograms were normalized to the highest peak. Illustrative m/z-values as obtained from the corresponding MS spectrum are marked. <a href="/files/ftp_upload/57434/57434fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57434/57434fig3.jpg" /> 
Figure 3. Q-TOF-MS spectrum of erythromycin A (bottom) and the MS/MS spectrum of the ion m/z = 734.4689 (top). The spectra show the quasi-molecular ion of erythromycin A with its isotopic pattern and the fragments at an applied collision energy of 30 eV. <a href="/files/ftp_upload/57434/57434fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57434/57434fig4.jpg" /> 
Figure 4. Normalized EICs of (A) metoprolol, (B) carbamazepine, (C) erythromycin A and (D) anhydroerythromycin A in a Rhine river sample (blue) and in ultrapure water from reference compounds (red). The retention times of the reference compounds and the ones of the pharmaceuticals in the water sample are the same. The signal-to-noise ratios of metoprolol (A) and anhydroerythromycin (D) are higher than those of carbamazepine (B) and erythromycin (C), which indicates the latter were present only in traces. <a href="/files/ftp_upload/57434/57434fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57434/57434fig5.jpg" /> 
Figure 5. Normalized concentration-time curves of the photodegradation of erythromycin A at pH 3 (red), pH 7 (green) and pH 9 (blue). Solutions were irradiated for 10 min. At pH 7, erythromycin was completely removed from the sample. The concentration-time curves could be described using first-order kinetic equations. The kinetic rate constants were 0.10 (pH 3), 0.59 (pH 7) and =0.21 (pH 9). <a href="/files/ftp_upload/57434/57434fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57434/57434fig6.jpg" /> 
Figure 6. Comparison of the fits of the concentration-time curves of the photoprodegradates of erythromycin with m/z = 720 at pH 9 following equations 3 (A) and 4 (B). Goodness of the fit of the consecutive reaction (A): R2 = 0.9898, RMSE = 4.645E+04, and of the subsequent follow-up reaction (B): R2 = 09976, RMSE = 2.366E+04. <a href="/files/ftp_upload/57434/57434fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57434/57434fig7.jpg" /> 
Figure 7. Structure of erythromycin A, erythromycin B and anhydroerythromycin and their photdegradation products. This figure has been modified from Voigt et al.27. The products were formed after 10 min of UVC-irradiation and identified using HPLC-Q-TOF-MS and MS/MS. <a href="/files/ftp_upload/57434/57434fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Liquid Chromatography</td>
			<td colspan="6"></td>
		</tr>
		<tr>
			<td>Column:</td>
			<td colspan="6">reversed-phase C-18</td>
		</tr>
		<tr>
			<td>Column:</td>
			<td colspan="6">CoreShell column;</td>
		</tr>
		<tr>
			<td>Column:</td>
			<td colspan="6">50 mm x 2.1 mm dimensions, 2.6 &#956;m particle size</td>
		</tr>
		<tr>
			<td>Column temperature</td>
			<td colspan="6">40 &#176;C</td>
		</tr>
		<tr>
			<td>Injection volume:</td>
			<td colspan="6">5 &#181;L</td>
		</tr>
		<tr>
			<td>Flow:</td>
			<td colspan="6">0.3 mL/min</td>
		</tr>
		<tr>
			<td>Mobile phase:</td>
			<td colspan="6">Solvent A: water containing 0.1% formic acid</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="6">Solvent B: methanol containing 0.1% formic acid</td>
		</tr>
		<tr>
			<td>Gradient program:</td>
			<td colspan="6"></td>
		</tr>
		<tr>
			<td>Time /min</td>
			<td>0</td>
			<td>1</td>
			<td>10</td>
			<td>11.1</td>
			<td>11.2</td>
			<td>12</td>
		</tr>
		<tr>
			<td>A:B solvent ratio</td>
			<td>99:1</td>
			<td>70:30</td>
			<td>25:75</td>
			<td>1:99</td>
			<td>1:99</td>
			<td>99:1</td>
		</tr>
		<tr>
			<td>Mass Spectrometry</td>
			<td colspan="6"></td>
		</tr>
		<tr>
			<td>Source:</td>
			<td colspan="6">Dual AJS ESI (positive mode)</td>
		</tr>
		<tr>
			<td>Gas and source</td>
			<td colspan="6"></td>
		</tr>
		<tr>
			<td>Gas Temperature:</td>
			<td colspan="6">300 &#176;C</td>
		</tr>
		<tr>
			<td>Drying Gas:</td>
			<td colspan="6">8.0 L/min</td>
		</tr>
		<tr>
			<td>Nebulizer:</td>
			<td colspan="6">14 psig</td>
		</tr>
		<tr>
			<td>Sheath Gas Temperature:</td>
			<td colspan="6">300 &#176;C</td>
		</tr>
		<tr>
			<td>Sheath Gas Flow:</td>
			<td colspan="6">8 L/min</td>
		</tr>
		<tr>
			<td>Mass Range:</td>
			<td colspan="6">100 - 1000 m/z</td>
		</tr>
		<tr>
			<td>Acquisition Rate:</td>
			<td colspan="6">1 spectrum/s</td>
		</tr>
		<tr>
			<td>Acquisition Time:</td>
			<td colspan="6">1000 ms/spectrum</td>
		</tr>
		<tr>
			<td>Transient/ spectrum</td>
			<td colspan="6">10014</td>
		</tr>
		<tr>
			<td>For targeted MS method</td>
			<td colspan="6"></td>
		</tr>
		<tr>
			<td>Collision energy (CE):</td>
			<td colspan="6">0 eV</td>
		</tr>
		<tr>
			<td>Preferred Mass - Table</td>
			<td colspan="6">734.4685</td>
		</tr>
		<tr>
			<td>For MS/MS (typically auto MS/MS mode)</td>
			<td colspan="6"></td>
		</tr>
		<tr>
			<td>Collision energy (CE):</td>
			<td colspan="6">30 eV</td>
		</tr>
		<tr>
			<td>Absolute threshold</td>
			<td colspan="6">3000 counts</td>
		</tr>
		<tr>
			<td>Relative threshold</td>
			<td colspan="6">0.01 %</td>
		</tr>
		<tr>
			<td>Mass Range:</td>
			<td colspan="6">100 - 100 m/z</td>
		</tr>
		<tr>
			<td>Acquisition Rate:</td>
			<td colspan="6">1 spectrum/s</td>
		</tr>
		<tr>
			<td>Acquisition Time:</td>
			<td colspan="6">1000 ms/spectrum</td>
		</tr>
		<tr>
			<td>Transient/ spectrum</td>
			<td colspan="6">9964</td>
		</tr>
		<tr>
			<td>For targeted MS/MS method</td>
			<td colspan="6"></td>
		</tr>
		<tr>
			<td>Preferred Mass - Table</td>
			<td colspan="6">734.4685</td>
		</tr>
	</tbody>
</table>
Table 1. Conditions and parameters used for HPLC-ESI-Q-TOF-MS analysis of pharmaceuticals in water matrices. It is advisable to introduce a rinsing step between the chromatographic runs through running a sample of pure ultrapure water between two analyses or through extending the run time of the chromatographic method in order to elute all substances.
<img alt="Table 2" src="/files/ftp_upload/57434/57434table2.jpg" /> 
Table 2. Pharmaceuticals found in the Rhine river sample with their retention time, theoretical and observed [M+H]+ and their structure. The ESI mode was set to positive, so that [M+H]+-ions were detected. The retention time may vary minimally for usual experimental known reasons.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>pH 3</td>
			<td>pH 3</td>
			<td>pH 7</td>
			<td>pH 7</td>
			<td>pH 7</td>
			<td>pH 7</td>
			<td>pH 7</td>
			<td>pH 7</td>
			<td>pH 9</td>
			<td>pH 9</td>
			<td>pH 9</td>
			<td>pH 9</td>
		</tr>
		<tr>
			<td>Product</td>
			<td>k1 [min-1]</td>
			<td>t1/2 [min] (k1)</td>
			<td>k1 [min-1]</td>
			<td>k2 [min-1]</td>
			<td>k3 [min-1]</td>
			<td>t1/2 [min] (k1)</td>
			<td>t1/2 [min] (k2)</td>
			<td>t1/2 [min] (k3)</td>
			<td>k1 [min-1]</td>
			<td>k2 [min-1]</td>
			<td>t1/2 [min] (k1)</td>
			<td>t1/2 [min] (k2)</td>
		</tr>
		<tr>
			<td>Ery A&#160;</td>
			<td>0.1</td>
			<td>6.81</td>
			<td>0.59</td>
			<td>-</td>
			<td>-</td>
			<td>1.18</td>
			<td>-</td>
			<td>-</td>
			<td>0.21</td>
			<td>-</td>
			<td>3.37</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ery B&#160;</td>
			<td>0.05</td>
			<td>14.23</td>
			<td>0.66</td>
			<td>-</td>
			<td>-</td>
			<td>1.04</td>
			<td>-</td>
			<td>-</td>
			<td>0.22</td>
			<td>-</td>
			<td>3.21</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ery A &#8211; H2Oa&#160;</td>
			<td>0.11</td>
			<td>6.53</td>
			<td>0.59</td>
			<td>-</td>
			<td>-</td>
			<td>1.17</td>
			<td>-</td>
			<td>-</td>
			<td>0.19</td>
			<td>-</td>
			<td>3.72</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ery A &#8211; H2Ob</td>
			<td>0.15</td>
			<td>4.76</td>
			<td>1.11</td>
			<td>-</td>
			<td>-</td>
			<td>0.63</td>
			<td>-</td>
			<td>-</td>
			<td>0.21</td>
			<td>-</td>
			<td>3.35</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ery F&#160;</td>
			<td>not observed</td>
			<td>-</td>
			<td>0.89</td>
			<td>0.35</td>
			<td>-</td>
			<td>0.78</td>
			<td>1.98</td>
			<td>-</td>
			<td>1.09*</td>
			<td>-</td>
			<td>0.64</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ery C</td>
			<td>not determined</td>
			<td>-</td>
			<td>0.74</td>
			<td>5.27</td>
			<td>0.78</td>
			<td>0.94</td>
			<td>0.13</td>
			<td>0.89</td>
			<td>0.17</td>
			<td>0.18</td>
			<td>4.04</td>
			<td>3.92</td>
		</tr>
		<tr>
			<td>DPEry192</td>
			<td>0.35*</td>
			<td>1.97</td>
			<td>not observed</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>0.30*</td>
			<td>-</td>
			<td>2.34</td>
			<td>-</td>
		</tr>
		<tr>
			<td colspan="13">* No further degradation observed</td>
		</tr>
	</tbody>
</table>
Table 3. Kinetic rate constants and corresponding half-lives of the degradation of erythromycin and its photodegradates adapted from Voigt et al.27. Erythromycin consists of erythromycin A, erythromycin B and two forms of anhydroerythromycin. Three photodegradates were observed. There are referred to as Ery F, Ery C and DEry192.

Watch this Scientific Journal Video about Identification of Pharmaceuticals in The Aquatic Environment Using HPLC-ESI-Q-TOF-MS and Elimination of Erythromycin Through Photo-Induced Degradation at JoVE

Identification of Pharmaceuticals in The Aquatic Environment Using HPLC-ESI-Q-TOF-MS and Elimination of Erythromycin Through Photo-Induced Degradation

We present a protocol for non-targeted analysis using time of flight mass spectrometry as a perfect tool to identify pharmaceuticals in waters. We demonstrate the application of UV irradiation for their elimination. Analysis involving irradiation, compound isolation, identification and kinetic modelling of the degradation profiles is illustrated.

Monitoring pharmaceuticals throughout the water cycle is becoming increasingly important for the aquatic environment and eventually for human health. ...

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Environment

12.9K Views.  Niederrhein University of Applied Sciences. We present a protocol for non-targeted analysis using time of flight mass spectrometry as a perfect tool to identify pharmaceuticals in waters. We demonstrate the application of UV irradiation for their elimination. Analysis involving irradiation, compound isolation, identification and kinetic modelling of the degradation profiles is illustrated.

Video: Identification of Pharmaceuticals in The Aquatic Environment Using HPLC-ESI-Q-TOF-MS and Elimination of Erythromycin Through Photo-Induced Degradation

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

Alternative Method of Removing Otoliths from Sturgeon

Extracting the otoliths (ear bones) from fish that have very thick skulls can be difficult and very time consuming. The common practice of making a transverse vertical incision on the top of the skull with a hand or electrical saw may damage the otolith if not performed correctly. Sturgeons (Acipenseridae) are one family in particular that have a very large and thick skull. A new laboratory method entering the brain cavity from the ventral side of the fish to expose the otoliths was easier than other otolith extraction methods found in the literature. Methods reviewed in the literature are designed for the field and are more efficient at processing large quantities of fish quickly. However, this new technique was designed to be more suited for a laboratory setting when time is not pressing and successful extraction from each specimen is critical. The success of finding and removing otoliths using this technique is very high and does not compromise the structure in any manner. This alternative technique is applicable to other similar fish species for extracting the otoliths.

The goal of the protocol is to show an effective method to extract the otoliths from sturgeon carcasses.

Extracting the otoliths (ear bones) from fish that have very thick skulls can be difficult and very time consuming. The common practice of making a ...

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. Yet the in situ study of DOM is difficult as the molecular complexity of the DOM pool cannot be easily reproduced for experimental purposes. Nutrient additions to streams however, have been shown to repeatedly alter the in situ and ambient DOM pool. Here we demonstrate an easily replicable field-based method for manipulating the ambient pool of DOM at the ecosystem scale. During nutrient pulse experiments changes in the concentration of both dissolved organic carbon and dissolved organic nitrogen can be examined across a wide-range of nutrient concentrations. This method allows researchers to examine the controls on the DOM pool and make inferences regarding the role and function that certain fractions of the DOM pool play within ecosystems. We advocate the use of this method as a technique to help develop a deeper understanding of DOM biogeochemistry and how it interacts with nutrients. With further development this method may help elucidate the dynamics of DOM in other ecosystems.

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter provides an important source of energy and nutrients to stream ecosystems. Here we demonstrate a field-based method to manipulate the ambient pool of dissolved organic matter in situ through easily replicable nutrient pulses.

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to individually track cohoused animals. We tested the tracking routine with four cohoused Norway lobsters (Nephrops norvegicus) under light-darkness conditions for 5 days. The lobsters had been individually tagged. The experimental setup and the tracking techniques used are entirely based on the open source software. The comparison of the tracking output with a manual detection indicates that the lobsters were correctly detected 69% of the times. Among the correctly detected lobsters, their individual tags were correctly identified 89.5% of the times. Considering the frame rate used in the protocol and the movement rate of lobsters, the performance of the video tracking has a good quality, and the representative results support the validity of the protocol in producing valuable data for research needs (individual space occupancy or locomotor activity patterns). The protocol presented here can be easily customized and is, hence, transferable to other species where the individual tracking of specimens in a group can be valuable for answering research questions.

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster Nephrops norvegicus

Here we present a protocol to individually track animals over a long period of time. It uses computer vision methods to identify a set of manually constructed tags by using a group of lobsters as case study, simultaneously providing information on how to house, manipulate, and mark the lobsters.

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to ...

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous environments. It also plays a crucial role in symbiotic interactions, disease, and global processes, such as biogeochemical cycling. However, current techniques restrict chemotaxis research to the laboratory and are not easily applicable in the field. Presented here is a step-by-step protocol for the deployment of the in situ chemotaxis assay (ISCA), a device that enables robust interrogation of microbial chemotaxis directly in the natural environment. The ISCA is a microfluidic device consisting of a 20 well array, in which chemicals of interest can be loaded. Once deployed in aqueous environments, chemicals diffuse out of the wells, creating concentration gradients that microbes sense and respond to by swimming into the wells via chemotaxis. The well contents can then be sampled and used to (1) quantify strength of the chemotactic responses to specific compounds through flow cytometry, (2) isolate and culture responsive microorganisms, and (3) characterize the identity and genomic potential of the responding populations through molecular techniques. The ISCA is a flexible platform that can be deployed in any system with an aqueous phase, including marine, freshwater, and soil environments.

Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread ...

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...