Name: a small-scale setup for algal toxicity testing of nanomaterials and other difficult substances
Uploaded: 2020-10-10T14:00:00.000+00:00
Duration: 8 min 18 s
Description: Watch this Scientific Journal Video about A Small-Scale Setup for Algal Toxicity Testing of Nanomaterials and Other Difficult Substances at JoVE.com

This research was funded by PATROLS &#8211; Advanced Tools for NanoSafety Testing, Grant agreement 760813 under Horizon 2020 research and innovation program.

1. Description of the LEVITATT setup
<ol>
	<li>Use 20 mL scintillation glass vials (Figure 1, insert 1) allowing light penetration. Alternatively, light penetrable plastic vials can be used. Quantify the light intensity using a photometer.</li>
	<li>Use at least a 4 mL test suspension at the beginning of the test to allow for quantification of biomass and for characterization/quantification of nanomaterials during and after incubation (Figure 1, insert 2).</li>
	<li>Fit the 20 mL scintillation vials with a cap (Figure 1, insert 3) where a small hole is drilled (approximately 1 mm in diameter) to allow for CO2 exchange with the atmosphere. This exchange is crucial to ensure stable pH and CO2 levels during testing.</li>
	<li>For volatile substances, use an air-tight Teflon coated cap to allow for CO2 enriching of the headspace using a syringe9 or completely closed flasks with no gas phase in which CO2 is maintained in solution by an enriched sodium bicarbonate (NaHCO3) buffer system10.</li>
	<li>Fasten the vials with clamps mounted on the exterior casing (Figure 1, insert 4).</li>
	<li>Use an LED light source located below the test vials (Figure 1, insert 5) providing a uniform fluorescent illumination of &#8220;cool-white&#8221; or &#8220;daylight&#8221; type and a light intensity in the range 60&#8211;120 &#181;E&#8729;m-2&#8729;s-1 measured in the photosynthetically effective wavelength range of 400 nm to 700 nm. The setup employs adjustable light intensity in the range 5&#8211;160 &#181;E&#8729;m-2&#8729;s-1 by fitting a light dimmer to the source. This allows for testing at higher and lower light intensities.</li>
	<li>Mount the setup on an orbital shaker to agitate samples throughout the duration of the test. This keeps the cells in free suspension and facilitates CO2 mass transfer from air to water (Figure 1, insert 6).</li>
	<li>Place the setup in a temperature-controlled room or a thermostatic cabinet to maintain stable temperatures throughout testing (Figure 1, insert 7).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61209/61209fig1v2.jpg" /> 
Figure 1: Picture of LED Vertical Illumination Table for Algal Toxicity Tests (LEVITATT).&#160;1) 20 mL glass scintillation vials for incubation, 2) 4 mL sample for analysis, 3) lid with drilled hole for CO2 exchange, 4) casing for defined light conditions, 5) LED light source located in the center of the casing, 6) orbital shaker for agitation during the experiment, and 7) a thermostatic cabinet. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61209/61209fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Preparation of algal growth medium
<ol>
	<li>The ISO 8692 algal growth medium consists of four different stock solutions. Weigh out the appropriate amount of salts and dilute in ultrapure water according to Table 1.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stock solutions</td>
			<td>Nutrient</td>
			<td>Concentration in stock solution</td>
			<td>Concentration in test solution</td>
		</tr>
		<tr>
			<td rowspan="5">1: Macronutrients</td>
			<td>NH4Cl</td>
			<td>1.5 g/L</td>
			<td>15 mg/L (N: 3.9 mg/L)</td>
		</tr>
		<tr>
			<td>MgCl2&#8729;6H2O</td>
			<td>1.2 g/L</td>
			<td>12 mg/L (Mg: 2.9 mg/L)</td>
		</tr>
		<tr>
			<td>CaCl2&#8729;2H2O</td>
			<td>1.8 g/L</td>
			<td>18 mg/L (Ca: 4.9 mg/L)</td>
		</tr>
		<tr>
			<td>MgSO4&#8729;7H2O</td>
			<td>1.5 g/L</td>
			<td>15 mg/L (S: 1.95 mg/L)</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>0.16 g/L</td>
			<td>1.6 mg/L (P: 0,36 mg/L)</td>
		</tr>
		<tr>
			<td rowspan="2">2: Fe-EDTA</td>
			<td>FeCl3&#8729;6H2O</td>
			<td>64 mg/L</td>
			<td>64 &#181;g/L (Fe: 13 &#181;g/L)</td>
		</tr>
		<tr>
			<td>Na2EDTA&#8729;2H2O</td>
			<td>100 mg/L</td>
			<td>100 &#181;g/L</td>
		</tr>
		<tr>
			<td rowspan="6">3: Trace elements</td>
			<td>H3BO3a</td>
			<td>185 mg/L</td>
			<td>185 &#181;g/L (B: 32 &#181;g/L)</td>
		</tr>
		<tr>
			<td>MnCl2&#8729;4H2O</td>
			<td>415 mg/L</td>
			<td>415 &#181;g/L (Mn: 115 &#181;g/L)</td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>3 mg/L</td>
			<td>3 &#181;g/L (Zn: 1.4 &#181;g/L)</td>
		</tr>
		<tr>
			<td>CoCl2&#8729;6H2O</td>
			<td>1.5 mg/L</td>
			<td>1.5 &#181;g/L (Co: 0.37 &#181;g/L)</td>
		</tr>
		<tr>
			<td>CuCl2&#8729;2H2O</td>
			<td>0.01 mg/L</td>
			<td>0.01 &#181;g/L (Cu: 3.7 ng/L)</td>
		</tr>
		<tr>
			<td>Na2MoO4&#8729;2H2O</td>
			<td>7 mg/L</td>
			<td>7 &#181;g/L (Mo: 2.8 &#181;g/L)</td>
		</tr>
		<tr>
			<td>4: NaHCO3</td>
			<td>NaHCO3</td>
			<td>50 g/L</td>
			<td>50 mg/L (C: 7.14 mg/L)</td>
		</tr>
	</tbody>
</table>
Table 1: Concentrations of nutrients in stock solutions for algal growth medium
NOTE: H3BO3 can be dissolved by adding 0.1 M NaOH. EDTA should be removed when testing metals, to avoid complexation with metal ions. Sterilize the stock solutions by membrane filtration (mean pore diameter 0.2 &#181;m) or by autoclaving (120 &#176;C, 15 min). Do no autoclave stock solutions 2 and 4, but sterilize them by membrane filtration. Store the solutions in the dark at 4 &#176;C.
<ol start="2">
	<li>To produce 1 L of algal growth medium, transfer 500 mL sterilized ultrapure water into a 1 L sterilized volumetric flask and add 10 mL of stock solution 1: Macronutrients, 1 mL of stock solution 2: Fe-EDTA, 1 mL of stock solution 3: Trace elements, and 1 mL of stock solution 4: NaHCO3.</li>
	<li>Fill up to 1 L with sterilized ultrapure water, stopper the flask and shake thoroughly to homogenize the algal growth medium.</li>
	<li>Equilibrate the solution before use by leaving it overnight in contact with air or by bubbling with sterile, filtered air for 30 min. After equilibration, adjust the pH, if necessary, to pH 8.1 &#177; 0.2, with either 1 M HCl or 1 M NaOH.</li>
</ol>
3. Setting up the algal test
NOTE: A flow diagram of the algal test procedure is shown in Figure 2.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61209/61209fig2v6.jpg" /> 
Figure 2: Flow diagram of the algal test setup. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61209/61209fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Prepare a stock solution of the test compound at the desired highest test concentration in the algal growth medium prepared according to step 2. For preparation of stock solutions/suspensions, follow OECD 201 (for soluble compounds) or OECD 318 (for nanomaterials).</li>
	<li>Measure the pH in the stock solution. If it deviates more than one unit from the algal growth medium, adjust the pH to 8 with either 1 M HCl or 1 M NaOH.</li>
	<li>Calculate the inoculum volume needed to reach a final cell concentration of 1 x 104 cells/mL in a 25 mL test solution. 
	NOTE: The inoculum should come from a culture of uncontaminated exponentially growing Raphidocelis subcapitata grown using the LEVITATT setup.</li>
	<li>Calculate the amount of stock solution to add to each 25 mL volumetric flask to obtain the desired test concentrations. The factor between each concentration should not exceed 3.2.</li>
	<li>Mark one 25 mL volumetric flask for each chosen concentration and an additional 25 mL volumetric flask marked control.</li>
	<li>Add the amount of stock solution of the test compound needed to reach the desired concentrations to the 25 mL volumetric flask. Do not add stock solution to the control.</li>
	<li>Add the medium to each 25 mL volumetric flask to reach a volume of approximately 20 mL.</li>
	<li>Add the volume of inoculum calculated in step 3.3 to each 25 mL volumetric flask. Add the medium to each 25 mL volumetric flask to a final total volume of 25 mL.</li>
	<li>Stopper the flasks and mix thoroughly by turning the flasks two times vertically.</li>
	<li>Transfer 0.4 mL from each flask into individual screw cap vials and add 1.6 mL of acetone (saturated with MgCO3): one sample for each test concentration and the control. Close the lids tightly and store in the dark at room temperature until fluorescence measurements (section 4).</li>
	<li>Pipet 4 mL of each test solution into 20 mL scintillation vials (3 replicates per concentration and 5 replicates for the control). Screw lids on the scintillation vials. Remember that the lids must have a drilled hole (approximately 1 mm in diameter) to allow for CO2 exchange.</li>
	<li>After 24 h, 48 h, and 72 h, pipet 0.4 mL from each vial into screw cap vials and add 1.6 mL of acetone (saturated with MgCO3). Close the lids tightly and store in the dark at room temperature until fluorescence measurements (section 4).</li>
	<li>After the last sample is taken at 72 h, gently pool the three replicates for a given concentration in one vial and measure the pH. Repeat for all concentrations and the control. The pH should not deviate more than 1.5 units from the initial pH for any of the samples measured.</li>
	<li>Discharge the remaining liquids into a waste container following your institutional rules and regulations.</li>
</ol>
4. Analyzing algal test samples
<ol>
	<li>Use a fluorescence spectrophotometer to measure the algal biomass (here expressed as chlorophyll A). The peak emission for chlorophyll A is 420 nm for the excitation wavelength and 671 nm for the emission wavelength.</li>
	<li>Measure the fluorescence of each individual sample three times and calculate the average value for each sample.</li>
	<li>Use equation 1 to calculate the growth rate. The measured fluorescence (relative units) can be used directly as the biomass parameter in equation 1. 
	Equation 1: &#181; = (ln Nt &#8211; ln N0) / t 
	where &#181; is the growth rate (d-1), N0 is the initial biomass, Nt is the biomass at time t, and t is the length of the test period (d). Note, N0 and Nt should be expressed in the same unit.</li>
	<li>Use a statistical software to fit a non-linear regression curve (e.g., a log-logistic or Weibull function) to the growth rate data to obtain effective concentration values at 10%, 20%, and 50% inhibition. In the supplementary information an example of code for fitting in the statistical software R using the DRC package11 is given.</li>
</ol>

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The authors have nothing to disclose.

Phytoplankton converts solar energy and carbon dioxide to organic matter and thus holds a pivotal role in the aquatic ecosystem. For this reason, algal growth rate inhibition tests are included as one of three mandatory aquatic toxicity tests required for regulatory risk assessment of chemicals. The ability to perform a reliable and reproducible algal toxicity test is key in this regard. Test setups using Erlenmeyer flasks introduces a range of variabilities and inconveniences as described in the introduction. To circumvent this issue, microtiter plates have been proposed3. While the microtiter plates minimize the volume and space required for testing, concerns have been raised in literature with regard to the compliance of such setups with the test validity criteria of test guidelines6. For example, significant loss of semivolatile chemicals as well as cross-over to other wells in microtiter plates at 37 &#176;C in cellular growth media (DMEM GluteMAX, Opti-MEM, and Hams F12 GlutaMAX) was recently demonstrated by Birch et al.12. Testing of volatile substances may be successfully conducted with the LEVITATT setup using 1) closed vials with CO2 enriched headspace9, 2) directly dosing the volatile compound through the headspace13 or in a filled test vial10. In terms of space requirements, LEVITATT provides a test setup that fills the gap between microtiter plates and Erlenmeyer flask setups while still providing benefits from both setups, e.g., maintaining a concise and compact test environment and sufficient test volume for destructive sampling (e.g., for characterization of nanomaterials during the test).
The inter-sample variability for the LEVITATT test system ranged from 3.4% (3,5-Dichlorophenol and ZnO NP) to 5.6% (BaSO4 NP). This is within the requirement of coefficient of variation of average growth in replicate control cultures of 15% indicated by the OECD 201 guideline7 (for the LEVITATT inter-sample variability all exposures were also included).
The reproducibility with regards to EC50-values of the test setup for the reference substances showed a coefficient of variance of 13% for K2Cr2O7 (n = 4) and 21% for 3,5-Dichlorophenol (n = 3). This is comparable to tests carried out with conventional 250 mL Erlenmeyer flask bioassays for the reference substance K2Cr2O7 showing 16.8%14 and 25.4%15 coefficient of variation for EC50-values. Microtiter plates have shown lower variation coefficient for some reference substances (e.g., K2Cr2O7 (9%14,15), while higher coefficients of variation have been observed for phenol (34.9%16) and Dichlorophenol (38%17). Limited information is available with regard to reproducibility of studies with nanomaterials. However, comparing the inter-sample coefficient of variation for ZnO NPs using the LEVITATT system (3.4%) with microtiter plates (67%18) or Erlenmeyer flasks (13%19 and 35%20) it has less variation. To further test the robustness of the LEVITATT system, a round-robin study of two test materials (one reference substance and one difficult to test substance) have been initiated.
Maintaining an uncontaminated algal culture can be difficult if not handled in sterile conditions. Exponential algal growth is a key prerequisite for performing guideline algal testing. Measuring the growth at multiple time points (e.g., 24 h, 48 h, 72 h) can identify whether exponential growth is occurring throughout the test period. Fluctuations in temperature and pH can influence the algal growth; thus, these parameters must be stable throughout the test period. In small volumes of test sample, fluctuations in temperature and pH occur more rapidly compared to larger volumes and the practical issues of measuring these parameters are increasingly difficult with decreasing test volume. Parameters such as pH and temperature using 4 mL test volume was stable throughout a 72 h testing period in the LEVITATT both in a temperature-controlled room at 20 &#176;C &#177; 2 &#176;C and in an incubator at similar conditions.
Analytical methods for quantification of algal growth are many: cell counting in a haemocytometer, coulter counter, or fluorescence of pigment extracts. For difficult substances, considerations should be made with regard to the most suitable method for biomass quantification. For metal oxide nanoparticles, fluorescence of pigment extracts has been found to perform best due to interference of agglomerates when counting algae by haemocytometer or coulter counter21. In contrast, Farkas and Booth22 found that quantification of biomass by fluorescence was not a suitable method for ecotoxicity testing of carbon-based nanomaterials due to autofluorescence and absorbance of pigments to the nanomaterials. For colored substances, there may also be interference of the color with the fluorescence emission signal, thus, requiring additional controls or dilution to a level where this interference is negligible.
In the guidance document for aquatic toxicity testing of difficult substances2, one of the recommendations for testing colored materials is to increase the light intensity. Similarly, increased light intensity has been mentioned to circumvent shading issues when testing nanomaterials23,24. However, such modifications are often associated with increased temperatures, thus, requiring additional cooling or ventilation of the samples. In the LEVITATT setup, this is solved using LED light that produces little heat compared to conventional light bulbs or fluorescent tubes. Additionally, choosing an LED with a sufficiently high light intensity output and the installation of a dimmer allow for increasing the light intensity to test colored substances or nanomaterials and regular chemicals without altering the overall setup between tests. Furthermore, the placement of the light source below the samples and in separate casings allows for a consistent and homogenous light field.
In conclusion, the LEVITATT provides a compact platform for algal toxicity testing of regular chemicals compliant with international standardized guidelines. Furthermore, the setup provides a robust platform for testing of difficult substances that interfere with the passage of light toward the algal, e.g., nanomaterials.

The algal toxicity test is one of only three mandatory tests used to generate the ecotoxicity data required for pre- and post-market registration of chemicals by European and international regulations (e.g., REACH1 and TSCA (USA)). For this purpose, standardized algal test guidelines have been developed by international organizations (e.g., ISO and OECD). These testing standards and guidelines prescribe ideal test conditions in terms of pH, temperature, carbon dioxide levels and light intensity. However, maintaining stable test conditions during algal testing is in practice difficult and the results suffer from problems with reproducibility and reliability for a range of chemical substances and nanomaterials (often referred to as &#8220;difficult substances&#8221;)2. Most of the existing algal toxicity testing setups operate with relatively large volumes (100&#8211;250 mL) situated on an orbital shaker inside an incubator. Such a setup limits the number of test concentrations and replicates achievable and high volumes of algal culture and test material. Additionally, these setups rarely have a uniform light field and reliable lighting conditions are furthermore difficult to obtain in large flasks, partly as light intensity decreases exponentially the further the light travels and partly due to the flask geometry. Alternative setups comprise plastic microtiter3 plates containing small sample volumes that do not allow for adequate sampling volumes to measure pH, additional biomass measurements, pigment extraction or other analyses requiring destructive sampling. One particular challenge using existing setups for algal toxicity testing of nanomaterials and substances forming colored suspensions is the interference or blocking of the light available to the algal cells, often referred to as &#8220;shading&#8221;4,5. Shading may occur within vials by the test material and/or interactions between the test material and the algal cells, or shading can occur between vials, due to their positioning relative to each other and the light source.
The method is based on the small-scale algal toxicity test setup introduced by Arensberg et al.6 that allows for testing in compliance with standards such as OECD 2017, and ISO 86928. The method is further optimized to address the limitations stated above by: 1) utilizing the LED light technology to ensure uniform light conditions with minimal heat generation, 2) providing adequate sample volume for chemical/biological analysis while maintaining constant pH, CO2 levels, and 3) enabling the use of versatile test container material for testing of volatile substances or substances with a high sorption potential.

<table><tbody><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>V179124</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma-Aldrich</td><td>254134</td><td></td></tr><tr><td>BlueCap bottles (1L)</td><td>Buch &amp;amp; Holm A/S</td><td>&amp;nbsp;9072335</td><td></td></tr><tr><td>Boric acid</td><td>Sigma-Aldrich</td><td>B0394</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>208290</td><td></td></tr><tr><td>Clear acrylic sheet (40x40 cm)</td><td></td><td></td><td></td></tr><tr><td>Cobalt(II) chloride hexahydrate</td><td>Sigma-Aldrich</td><td>255599</td><td></td></tr><tr><td>Copper(II) chloride dihydrate</td><td>Sigma-Aldrich</td><td>307483</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt dihydrate</td><td>Sigma-Aldrich</td><td>&amp;nbsp;E5134</td><td></td></tr><tr><td>Fluorescence Spectrophotometer F-7000</td><td>Hitachi</td><td></td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>258148</td><td></td></tr><tr><td>Iron(III) chloride hexahydrate</td><td>Sigma-Aldrich</td><td>236489</td><td></td></tr><tr><td>LED light source</td><td>Helmholt Elektronik A/S</td><td>H35161</td><td>Neutral White, 6500K</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma-Aldrich</td><td>M9272</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma-Aldrich</td><td>230391</td><td></td></tr><tr><td>Manganese(II) chloride tetrahydrate</td><td>Sigma-Aldrich</td><td>221279</td><td></td></tr><tr><td>Orbital shaker</td><td>IKA</td><td>2980200</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P0662</td><td></td></tr><tr><td>Raphidocelis subcapitata</td><td>NORCCA</td><td></td><td>NIVA-CHL1 strain</td></tr><tr><td>Scintillation vials (20 mL)</td><td>Fisherscientific</td><td>11526325</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>415413</td><td></td></tr><tr><td>Sodium molybdate dihydrate</td><td>Sigma-Aldrich</td><td>331058&amp;nbsp;</td><td></td></tr><tr><td>Spring clamp</td><td>Frederiksen Scientific A/S</td><td>472002</td><td></td></tr><tr><td>Thermostatic cabinet</td><td>VWR</td><td>WTWA208450</td><td>Alternative: temperature controlled room</td></tr><tr><td>Ventilation pipe (&amp;Oslash;125 mm)</td><td>Silvan</td><td>22605630165</td><td></td></tr><tr><td>Volumetric flasks (25 mL)</td><td>DWK Life Sciences</td><td>246781455</td><td></td></tr><tr><td>Zinc chloride</td><td>Sigma-Aldrich</td><td>208086</td><td></td></tr></tbody></table>

a small-scale setup for algal toxicity testing of nanomaterials and other difficult substances

Ecotoxicity data is a requirement for pre- and post-market registration of chemicals by European and international regulations (e.g., REACH). The algal toxicity test is frequently used in regulatory risk assessment of chemicals. In order to achieve high reliability and reproducibility the development of standardized guidelines is vital. For algal toxicity testing, the guidelines require stable and uniform conditions of parameters such as pH, temperature, carbon dioxide levels and light intensity. Nanomaterials and other so-called difficult substances can interfere with light causing a large variation in results obtained hampering their regulatory acceptance. To address these challenges, we have developed LEVITATT (LED Vertical Illumination Table for Algal Toxicity Tests). The setup utilizes LED illumination from below allowing for a homogenous light distribution and temperature control while also minimizing intra-sample shading. The setup optimizes the sample volume for biomass quantification and does at the same time ensure a sufficient influx of CO2 to support exponential growth of the algae. Additionally, the material of the test containers can be tailored to minimize adsorption and volatilization. When testing colored substances or particle suspensions, the use of LED lights also allows for increasing the light intensity without additional heat generation. The compact design and minimal equipment requirements increase the possibilities for implementation of the LEVITATT in a wide range of laboratories. While compliant with standardized ISO and OECD guidelines for algal toxicity testing, LEVITATT also showed a lower inter-sample variability for two reference substances (3,5-Dicholorophenol and K2Cr2O7) and three nanomaterials (ZnO, CeO2, and BaSO4) compared to Erlenmeyer flasks and microtiter plates.

An initial test with a reference substance is carried out to determine the sensitivity of the algal strain. Reference substances regularly used for R. subcapitata are potassium dichromate and 3,5-Dichlorphenol7,8. Figure 3 and Table 2 show a representative result of an algal test including curve fitting and statistical outputs when the DRC package in R is applied to the growth rates.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61209/61209fig03.jpg" /> 
Figure 3: Representative concentration-response curve for 72 h exposure of a chemical compound to algae (R. subcapitata).&#160;The solid line represents the log-logistic fit and the shaded area is the 95% confidence interval for the fit. The open circles represent the calculated growth rate for each replicate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61209/61209fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2"></td>
			<td>EC10</td>
			<td>EC20</td>
			<td>EC50</td>
		</tr>
		<tr>
			<td>[mg/L]</td>
			<td>[mg/L]</td>
			<td>[mg/L]</td>
		</tr>
		<tr>
			<td>Chemical compound</td>
			<td>4.6 [1.8-7.5]</td>
			<td>7.4 [4.1-11]</td>
			<td>16.9 [11-22]</td>
		</tr>
	</tbody>
</table>
Table 2: Representative effective concentrations for 10%, 20%, and 50% inhibition of growth rate for a chemical compound using algae (R. subcapitata). The value in brackets represents the 95% confidence interval of a log-logistic fitting.
A successful test will have growth rates above 0.9 d-1 to comply with the OECD guideline and 1.5 d-1 to comply with ISO 8692 guideline and should contain at least one concentration between 0% and 100% inhibition. Low growth rates may occur as a result of various issues such as bacterial contamination or inoculum not being in the exponential growth phase at the beginning of the test. Microscopic investigation of the replicates should only show uniform sickle-shaped green algae (R. subcapitata) with dimensions of approximately 2 &#181;m width and 8 &#181;m length. If a single replicate is contaminated, it can be omitted and the analysis can be carried out without this data. However, if multiple replicates are contaminated, repeat the experiment with uncontaminated exponentially growing inoculum. To evaluate whether the inoculum was in the exponential growth phase at the beginning of the test, calculate the growth rate of the control replicates at 24 h intervals and use only the time interval at which the algal growth of the control is exponential for statistical analysis.
To test the robustness of the setup, a toxicity test using two reference substances (K2Cr2O7 and 3,5-Dichlorophenol) and three nanomaterials was repeated three times (3,5-Dichlorophenol, BaSO4 nanoparticles (NM-220), and ZnO nanoparticles (NM-111)) and four times (K2Cr2O7 and CeO2 nanoparticles (NM-212)). The results showed a coefficient of variation of the EC50-values between 11% and 39%, with the lowest coefficient of variation observed for ZnO nanoparticles (NM-111) and the highest coefficient of variation for CeO2 nanoparticles (NM-212) (Table 3).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td># of experiments</td>
			<td>Inter-sample coefficient of variation for growth rate 
			%</td>
			<td>EC50 
			(average) 
			mg/L</td>
			<td>Coefficient of variation for EC50 values 
			%</td>
		</tr>
		<tr>
			<td>K2Cr2O7</td>
			<td>4</td>
			<td>4.5</td>
			<td>0.73</td>
			<td>13</td>
		</tr>
		<tr>
			<td>3,5-Dichlorophenol</td>
			<td>3</td>
			<td>3.4</td>
			<td>1.9</td>
			<td>21</td>
		</tr>
		<tr>
			<td>BaSO4 NP (NM-220)</td>
			<td>3</td>
			<td>5.6</td>
			<td>22</td>
			<td>20</td>
		</tr>
		<tr>
			<td>CeO2 NP (NM-212)</td>
			<td>4</td>
			<td>4.3</td>
			<td>13</td>
			<td>39</td>
		</tr>
		<tr>
			<td>ZnO NP (NM-111)</td>
			<td>3</td>
			<td>3.4</td>
			<td>0.13</td>
			<td>11</td>
		</tr>
	</tbody>
</table>
Table 3: Results of an internal laboratory toxicity test with R. subcapitata exposed for 72 h to two reference substances and three nanomaterials from the JRC repository

Watch this Scientific Journal Video about A Small-Scale Setup for Algal Toxicity Testing of Nanomaterials and Other Difficult Substances at JoVE.com

A Small-Scale Setup for Algal Toxicity Testing of Nanomaterials and Other Difficult Substances

We demonstrate algal toxicity testing for difficult substances (e.g., colored substances or nanomaterials) using a setup illuminated vertically with an LED.

Ecotoxicity data is a requirement for pre- and post-market registration of chemicals by European and international regulations (e.g., REACH). The algal ...

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Nanyang Technological University

Research

JoVE Journal

Environment

5.6K Views.  Technical University of Denmark. Wanneer een chemisch bedrijf een nieuw chemisch of ander materiaal op de markt wil brengen, moeten ze gegevens leveren uit een reeks ecotoxiciteitstests. De algentest is een van de drie verplichte tests die moeten worden uitgevoerd. De algentest lijdt onder het feit dat het een hele uitdaging is om uit te voeren.Daarom hebben we het LEVITATT testplatform ontwikkeld. De LEVITATT maakt gebruik van LED-licht om problemen met overmatige warmteopwekking te voorkomen. Bovendien zorgt het voo...

Video: A Small-Scale Setup for Algal Toxicity Testing of Nanomaterials and Other Difficult Substances

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1&#8211;7. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e., replicate samples from the same treatment flask8) is often employed rather than true replicates (i.e., sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling.
The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65 x 8.5 x 2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9&#8211;11. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

Developmental Biology

Comparison of Scale in a Photosynthetic Reactor System for Algal Remediation of Wastewater

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ammonia removal, nitrogen removal and algal growth are compared over an 8-week period in paired sets of small (100 L) and large (1,000 L) reactors designed for algal remediation of landfill wastewater. Contents of the small and large scale reactors were mixed before the beginning of each weekly testing interval to maintain equivalent initial conditions across the two scales. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted to better equalize conditions occurring at both scales. During the short 8-week representative time period, starting ammonia and total nitrogen concentrations ranged from 3.1-14 mg NH3-N/L, and 8.1-20.1 mg N/L, respectively. The performance of the treatment system was evaluated based on its ability to remove ammonia and total nitrogen and to produce algal biomass. Mean &#177; standard deviation of ammonia removal, total nitrogen removal and biomass growth rates were 0.95&#177;0.3 mg NH3-N/L/day, 0.89&#177;0.3 mg N/L/day, and 0.02&#177;0.03 g biomass/L/day, respectively. All vessels showed a positive relationship between the initial ammonia concentration and ammonia removal rate (R2=0.76). Comparison of process efficiencies and production values measured in reactors of different scale may be useful in determining if lab-scale experimental data is appropriate for prediction of commercial-scale production values.

An experimental methodology is presented to compare the performance of small (100 L) and large (1,000 L) scale reactors designed for algae remediation of landfill wastewater. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted based on application.

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ...

Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the treatment of wastes. As most new research efforts begin at laboratory scale, there is a need for cost-effective methods for culturing microalgae in a reproducible manner. Here, we communicate an effective approach to culture microalgae in laboratory-scale photobioreactors, and to measure the growth and neutral lipid content of that algae. Instructions are also included on how to set up the photobioreactor system. Although the example organisms are species of Chlorella and Auxenochlorella, this system can be adapted to cultivate a wide range of microalgae, including co-cultures of algae with non-algae species. Stock cultures are first grown in bottles to produce inoculum for the photobioreactor system. Algae inoculum is concentrated and transferred to photobioreactors for cultivation in batch mode. Samples are collected daily for the optical density readings. At the end of the batch culture, cells are harvested by centrifuge, washed, and freeze dried to obtain a final dry weight concentration. The final dry weight concentration is used to create a correlation between the optical density and the dry weight concentration. A modified Folch method is subsequently used to extract total lipids from the freeze-dried biomass and the extract is assayed for its neutral lipid content using a microplate assay. This assay has been published previously but protocol steps were included here to highlight critical steps in the procedure where errors frequently occur. The bioreactor system described here fills a niche between simple flask cultivation and fully-controlled commercial bioreactors. Even with only 3-4 biological replicates per treatment, our approach to culturing algae leads to tight standard deviations in the growth and lipid assays.

Here, we present a protocol to construct lab-scale bubble column photobioreactors and use them to culture microalgae. It also provides a method for the determination of the culture growth rate and neutral lipid content.

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the ...

Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas electricity generation. Microalgae can biofix CO2 10 to 15 times faster than plants and convert algal biomass to products of interest, such as biofuels. Thus, this study presents a protocol that demonstrates the potential synergies of microalgae cultivation with a natural gas power plant situated in the southwestern United States in a hot semi-arid climate. State-of-the-art technologies are used to enhance carbon capture and utilization via the green algal species Chlorella sorokiniana, which can be further processed into biofuel. We describe a protocol involving a semi-automated open raceway pond and discuss the results of its performance when it was tested at the Tucson Electric Power plant, in Tucson, Arizona. Flue gas was used as the main carbon source to control pH, and Chlorella sorokiniana was cultivated. An optimized medium was used to grow the algae. The amount of CO2 added to the system as a function of time was closely monitored. Additionally, other physicochemical factors affecting algal growth rate, biomass productivity, and carbon fixation were monitored, including optical density, dissolved oxygen (DO), electroconductivity (EC), and air and pond temperatures. The results indicate that a microalgae yield of up to 0.385 g/L ash-free dry weight is attainable, with a lipid content of 24%. Leveraging synergistic opportunities between CO2 emitters and algal farmers can provide the resources required to increase carbon capture while supporting the sustainable production of algal biofuels and bioproducts.

A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density.

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

Neuroscience

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Biology

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

A Small-Scale Setup for Algal Toxicity Testing of Nanomaterials and Other Difficult Substances

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