PAPER
www.rsc.org/jem | Journal of Environmental Monitoring
A suite of microplate reader-based colorimetric methods to quantify ammonium, nitrate, orthophosphate and silicate concentrations for aquatic nutrient monitoring Stephanie Ringuet,a Lara Sassanoa and Zackary I. Johnson*ab Received 16th June 2010, Accepted 11th November 2010 DOI: 10.1039/c0em00290a A sensitive, accurate and rapid analysis of major nutrients in aquatic systems is essential for monitoring and maintaining healthy aquatic environments. In particular, monitoring ammonium (NH4+) concentrations is necessary for maintenance of many fish stocks, while accurate monitoring and regulation of ammonium, orthophosphate (PO43), silicate (Si(OH)4) and nitrate (NO3) concentrations are required for regulating algae production. Monitoring of wastewater streams is also required for many aquaculture, municipal and industrial wastewater facilities to comply with local, state or federal water quality effluent regulations. Traditional methods for quantifying these nutrient concentrations often require laborious techniques or expensive specialized equipment making these analyses difficult. Here we present four alternative microcolorimetric assays that are based on a standard 96-well microplate format and microplate reader that simplify the quantification of each of these nutrients. Each method uses small sample volumes (200 mL), has a detection limit #1 mM in freshwater and #2 mM in saltwater, precision of at least 8% and compares favorably with standard analytical procedures. Routine use of these techniques in the laboratory and at an aquaculture facility to monitor nutrient concentrations associated with microalgae growth demonstrates that they are rapid, accurate and highly reproducible among different users. These techniques offer an alternative to standard nutrient analyses and because they are based on the standard 96-well format, they significantly decrease the cost and time of processing while maintaining high precision and sensitivity.
1. Introduction A sensitive, accurate and rapid analysis of major nutrients in aquatic ecosystems is essential for monitoring and maintaining healthy aquatic environments. For example, monitoring ammonium (NH4+) concentrations is necessary for the health and maintenance of many aquaculture stocks such as salmon, mangrove snapper and shrimp.1–3 High concentrations of ammonium/ammonia can adversely affect growth and stocking density in a variety of species4 and strategies have been developed to remove this potentially harmful chemical.5 Alternatively, aquaculture based production of macroalgae or microalgae a Marine Laboratory, Nicholas School of the Environment, Duke University, 135 Duke Marine Lab Rd., Beaufort, North Carolina, 28516, USA. E-mail:
[email protected]; Tel: +1 252 504 7543 b Cellana BV, Carel van Bylandtlaan 30, 2596 HR The Hague, The Netherlands
requires precise monitoring and regulation of a suite of nutrients including ammonium, orthophosphate (PO43), silicate (Si(OH)4) and nitrate (NO3).6,7 In addition to carbon, these nutrients represent the major elements required by plants and regulating their concentration and elemental ratios is essential for commercial production. Understanding the concentration (and ratios) of these nutrients is necessary to understand the variability of the abundance and production of microalgae in natural environments as well.8,9 Finally, monitoring of wastewater streams from aquaculture, municipal or industrial sources is required for many facilities to comply with local, state or federal water quality effluent regulations.10 Thus, for both production and compliance, many aquaculture, municipal and industrial operations require precise and efficient monitoring of these major nutrient concentrations. Traditional methods for quantifying these nutrient concentrations typically use colorimetric based techniques in conjunction with
Environmental impact A sensitive, accurate and rapid analysis of major nutrients in aquatic systems is essential for monitoring and maintaining healthy aquatic environments. Traditional methods for quantifying these nutrient concentrations often require laborious techniques or expensive specialized equipment making these analyses difficult and time consuming. This report presents four alternative microcolorimetric assays that are based on a standard 96-well microplate format and microplate reader that simplify the quantification of each of these nutrients. Each assay has sufficient sensitivity for many common environmental monitoring applications such as aquaculture, industrial and municipal wastewater, non-point source discharge or surface run-off. These improved assays will facilitate more accurate and frequent environmental monitoring of aquatic environments. 370 | J. Environ. Monit., 2011, 13, 370–376
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standard spectrophotometers, which although precise and reliable, often require laborious procedures or specialized equipment making these analyses difficult. For example, many of these classical manual techniques require large sample and reagent volumes and each sample must be processed separately.11,12 Alternatively, more modern techniques use flow injection or segmented flow autoanalyzers to automate the nutrient analysis process.13,14 But these instruments are expensive and require specialized training to successfully operate. Towards developing more streamlined alternatives, several researchers have advanced microplate-based nutrient analyses techniques.15–18 These techniques, in particular those currently available for ammonium and phosphate,16,19 are based on the standard 96-well microplate format that can greatly reduce sample volume, while at the same time speed up the processing time by taking advantage of the standard format and multichannel pipettes. Microplate reader methods for nutrient analyses have proven to be a quick, low-cost alternative to conventional auto-analyzer techniques because they speed up the analysis and do not require expensive specialized equipment. However, to date there is not a comprehensive suite of techniques that utilize a single, colorimetric based instrument to measure the four major nutrients (ammonium, nitrate, orthophosphate and silicate) that are found in abundance in natural and aquaculture systems. In addition to this goal, we also sought to eliminate the use of highly toxic cadmium powder, which has been used as a reducing agent by others for microplate-based assays of nitrate.15 Here we present four alternative assays that use improved, miniaturized colorimetric methods in a standard 96-well microplate format for the rapid determination of dissolved orthophosphate, ammonium, nitrate and silicate concentrations. Each method has better than 1 mM sensitivity in freshwater (2 mM in seawater) and favorably compares with the more laborious and expensive techniques. These techniques offer an alternative to standard nutrient analyses and because they are based on the standard 96-well plate format, they reduce the time of processing, while maintaining high precision and sensitivity necessary for environmental monitoring and aquaculture applications.
2. Experimental 2.1 General All labware and sample bottles were ‘acid cleaned’ by soaking overnight in a 10% HCl solution and rinsed at least three times with ultrapure deionized water (ddH2O, >18 mU cm1) from a Nanopure Diamond water system (Thermo # D11931). Except where noted, all reagents were prepared in ddH2O with analytical-grade chemicals (Sigma). To remove particulates, environment samples were passed through 0.22 mm syringe filters prior to analysis. Analyses in seawater were performed in 0.22 mm filtered surface seawater collected from 30 46.90 N, 72 45.50 W (in the Sargasso Sea) on May 2010 with a salinity of 35 where all major nutrient concentrations are 0.05) among slopes for increasing concentration ranges (0– 5 mM, 0–10 mM, and 0–25 mM). There is no statistical difference in the variability (standard deviation) among sample replicates between the developed microplate reader and autoanalyzer methods, thus the two methods are analytically equivalent. The concentration measured on certified reference material diluted in ddH2O to the target concentration of 10 mM was 10.4 0.2 mM and within the manufacturer’s acceptable tolerance range. 3.2 Ammonium
2.4 Standards for methods comparison A pre-determined set of mixed standards covering the analytical concentrations range for the microplate reader methods were prepared in ddH2O. Each standard (including blanks) was divided into four previously acid-cleaned PTFE bottles. All
The ammonium technique is the least sensitive of the four methods, but still has a low limit of detection, precision is comparable to standard assays (Table 1) and sensitivity curves (and related calibration curves) are highly linear (r ¼ 0.999) (Fig. 1B). However, decomposing the regression of absorption
Table 1 Major characteristics of dissolved nutrient analyses
Nutrient
Sample volume/mL
200 Orthophosphate (PO43) Ammonium (NH4+) 200 100 Nitrate (NO3) Silicate (Si(OH)4) 200
Range/ Detection limit/mM mM (ddH2O)
Precision at 10 mM/mM (ddH2O)
Detection limit/mM (seawater)
Precision at 10 mM/mM (seawater)
1–25
0.5
0.2
0.6
0.6
1–50 1–50 1–100
0.9 0.2 0.2
0.6 0.4 0.1
2.1 0.6 1.1
0.8 0.3 0.3
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Fig. 1 Representative sensitivity curves for each analyte as measured in ddH2O. When not visible, standard deviation bars (n ¼ 3) are contained within the symbol.
versus concentration, there are statistical differences among the slopes for different portions (concentration regions) of the curve. For example, for the sample calibration curve presented (Fig. 1B), the slope for 0–5 mM region is 6.4 0.2 103 OD mM1, for 0–10 mM region is 6.7 0.1 103 OD mM1, for 0–25 mM region is 5.7 0.2 103 OD mM1, and for 0–50 mM region is 5.3 0.1 103 OD mM1. These differences in slopes are not the result of absorption saturation and deviation from the Beer– Lambert law and repeated generation of the standard curves shows that these differences in slopes are not consistently observed. At present we do not know the mechanism responsible for these deviations from linearity, but they may be due to differences in the timing of reagent additions, errors in pipetting volumes, contamination during handling or the age of the reagents. To minimize this effect for high precision work, the calibration curve can be subdivided into different regions (e.g. low versus high concentrations) and concentrations calculated based on these regions. Nevertheless, using a single calibration curve with this technique, the 2-way regression25 between the microplate reader and autoanalyzer methods yields a strong correlation (r ¼ 0.996). Thus, for most applications a single regression should be adequate, but if high precision results are necessary, care should be taken to ensure that the entire calibration curve is linear and if not, it should be decomposed into regions of interest. The concentration measured on certified reference material diluted in ddH2O to the target concentration This journal is ª The Royal Society of Chemistry 2011
of 10 mM was 10.8 0.8 mM and within the manufacturer’s acceptable tolerance range. 3.3 Nitrate The nitrate technique is the most sensitive of the four methods and has a high precision, low detection limit and sensitivity curves (and related calibration curves) that are highly linear (r ¼ 0.999) (Fig. 1C), and all of these properties compare favorably to standard assays.11 Sensitivity curves are linear throughout the range and the slope does not significantly differ among different regions. There are no differences in the variability between the microplate- and autoanalyzer-based methods (Table 2). Although this technique is highly sensitive and reproducible and eliminates the need to use harmful cadmium common to most other nitrate analyses,15,26 care must be taken to properly mix the samples. In particular we found that not all microplate vortexers adequately homogenize the mixtures, thus if other vortex models are used their efficacy should be verified. Further, because the reduction step used here is enzymatically based, it is essential that standard matrix be matched with the samples (see below). Similarly, different concentrations of NADH can affect the sensitivity and detection range achieved with this enzymatic reaction27 and reaction time can be significantly reduced (to 20 min) if only freshwater samples are analyzed. The protocol reported here represents a compromise between achieving a high J. Environ. Monit., 2011, 13, 370–376 | 373
Table 2 The mean standard deviation for microplate- and autoanalyzerbased assays for all concentrations compared using ddH2O as the matrix. None of the microplate standard deviations are significantly different (p > 0.05) from the autoanalyzer standard deviations
Nutrient
Microplate s/mM
Autoanalyzer s/mM
Orthophosphate (PO43) Ammonium (NH4+) Nitrate (NO3) Silicate (Si(OH)4)
0.1 0.5 0.2 0.3
0.0 0.2 0.1 0.8
(better than 1 mM, Table 1) sensitivity and an extended range of concentrations expected for many natural and aquaculture systems. The concentration measured on certified reference material diluted in ddH2O to the target concentration of 10 mM was 9.5 0.1 mM and within the manufacturer’s acceptable tolerance range. Nitrate (NO3) is the most abundant form of nitrogen in the oceans that is readily biologically available, with nitrite (NO2) typically found only in low concentrations in localized areas.28 Nitrate is also a dominant form of nitrogen added to aquaculture systems7 and is a major accumulation waste product of many aquaculture fisheries.2 However, if significant levels of nitrite are expected and it is necessary to distinguish between nitrite and nitrate, this method can easily be modified to measure nitrite concentrations by excluding the reduction (i.e. nitrate reductase + NADH) steps. Thus, as with other approaches, the method as described measures nitrite + nitrate, but if significant levels of nitrite are expected, these components can be separately quantified by performing an additional analysis similar to steps taken for other standard methods.12 3.4 Silicate The silicate technique has a high precision, low detection limit, sensitivity curves and related calibration curves that are highly linear (r ¼ 0.999) through concentration ranges to 100 mM (Fig. 1D), and all of these properties compare favorably to standard assays.11 There is no statistical difference in the variability (standard deviation) among sample replicates between the developed microplate reader and autoanalyzer methods and the 2-way regression between the methods yields a slope that is not statistically different from unity (r ¼ 0.999). Sufficient mixing is critical to achieving the levels of precision reported. The concentration measured on certified reference material diluted in ddH2O to the target concentration of 10 mM was 10.2 0.1 mM and statistically identical to the assay concentration provided by the manufacturer. Similar to the other analyses, appropriate matching of the sample and standard matrix is required (see below), but for silicate there may be additional considerations. In particular, for some saline samples a precipitate developed that interfered with precise quantification of absorption. This precipitate was not observed in freshwater samples and was not present in standards made in artificial seawater29 without nutrients, but was found using ultraclean, oligotrophic water collected 100 miles north of Oahu, Hawaii and in coastal brackish water from the 374 | J. Environ. Monit., 2011, 13, 370–376
mesotrophic Albemarle-Pamlico estuary in North Carolina. This precipitate was also found when using the standard silicate protocol11 on these waters and is therefore not unique to this miniaturized microplate-based method. When reactions were done in microtubes, then centrifuged at 21 000 g for 5 min before transferring the supernatant to the microplate for quantification, the presence of a precipitate did not interfere with the stoichiometric development of color in the reaction. Thus, if a precipitate is encountered during pilot analyses of the matrix, it can easily be removed without any negative effects on the results. However, this additional step does increase the processing time of the method and requires additional sample volume. 3.5 Matrix/salinity Each of the described methods is based on colorimetric techniques with the concentration of analytes quantified using the stoichiometric development of attenuation (absorption) from a colored product. Appropriate matching of the standard matrix to the sample matrix is essential for high precision and sensitivity work because of potential interference of the matrix with attenuation and hence quantification. These effects are mainly the result of differences in the index of refraction (mostly salts) and interference or interaction with non-target constituents and reactants. For example, differences in salinity between samples and standards can significantly affect the results and some methods have been developed to minimize the contribution of these potential artifacts.30–32 In addition to salt effects, other media additives or components of the water or seawater can alter the reactivity of these techniques. In particular, the enzymaticbased nitrate assay requires proper matrix matching between the samples and standards.24 Matching the matrix of the standards to the sample matrix is straightforward and accounts for these potential artifacts. The salinity of the sample also affects the detection limit and precision of each of the techniques (Table 1). Each of the techniques is equally precise whether in freshwater or saltwater, but there is a reduction in the sensitivity for ammonium and silicate in saltwater. In particular, there is a significant reduction in the detection limit for ammonium. However, the detection limit of 2 mM in seawater is sufficient for monitoring aquaculture systems1–3 and other water quality guidelines for ammonium in seawater (e.g. EPA 440/5-88-004). 3.6 Example implementation To demonstrate the utility of this suite of analyses, we utilized the microplate reader based methods to measure silicate, ammonium and orthophosphate concentrations over the course of a growout experiment of marine microalgae (Fig. 2). Marine microalgae were grown exponentially with serial transfers over >10 generations,33 then transferred to replete media and the concentrations of ammonium, orthophosphate and silicate measured over time. (Nitrate was not added to this media and therefore it was not measured.) Because of known ammonium toxicity of some marine microalgae above 150 mM,34 ammonium was added two additional times over the course of the experiment (Fig. 2). Decreases in nutrient concentrations (i.e. uptake by the marine microalgae) are consistent with expected nutrient drawdown ratios35 and known variability among different microalgae This journal is ª The Royal Society of Chemistry 2011
chemical constituents of potential interest to aquaculture facilities or agriculture run-off including urea,17 but they require special heating of samples and additional equipment. Further, still other approaches have been optimized for specific environmental conditions (e.g. pore water of freshwater sediments) and the analytes that may be present.16 Beyond these other methods, the techniques refined here present a suite of simple and robust approaches for monitoring many major nutrients found in natural aquatic ecosystems and aquaculture facilities and can be used to maintain healthy and productive aquatic environments.
Acknowledgements Fig. 2 Example of the application of microplate based nutrient analyses—nutrient drawdown study of marine microalgae. When not visible, standard deviation bars (n ¼ 3) are contained within the symbol. Ammonium (NH4+) was added two times (day 179.8 and day 180.6) during the experiment. Nitrate was not used in this experiment (and therefore not measured), but shows similar reproducibility for other experiments (not shown). Concentrations that exceeded the recommended range (Table 1) were diluted with nutrient-free media and reanalyzed.
strains.36 Variability among triplicate samples across the analytes averaged 6 to 7% CV, including values near the limit of detection for all three nutrients in this experiment. Values show a clear and expected pattern and can readily be used to calculate nutrient uptake fluxes for the various nutrients as well as ratios among the elements. In addition, the timing of drawdown to analytical zero (i.e. the onset of nutrient starvation),37 or the practical limit of detection (1 mM) is readily observed for each of the analytes further demonstrating the utility of these techniques. In addition to these experiments, we have successfully used these techniques for monitoring nutrient concentrations in a pilot aquaculture facility to assess nutrient concentrations for operational guidance and have mapped nutrient concentrations in a brackish estuary to validate an on-going environmental nutrient monitoring effort.38 Thus, this suite of methods has proven valuable in a variety of applications representative of potential aquaculture and environmental monitoring uses.
4. Conclusion The suite of microplate reader-based colorimetric methods for quantifying major plant nutrients present in aquaculture systems and other environmental aquatic systems presented here are designed to be simple, rapid and reduce the need for toxic metals (specifically cadmium). These methods can all be used on a single colorimetric microplate reader, thus eliminating the need for costly, specialized equipment. The miniaturized techniques have analytical precisions that are consistent with advanced segmented flow analyses (Tables 1 and 2) and have working concentration ranges that are suitable for many aquaculture and environmental monitoring applications. Other higher precision and low detection limit microplate based techniques are available (e.g. high sensitivity ammonium analysis18), but they require different instrumentation (fluorescence or dual-mode microplate reader). Also, there exist other techniques to measure other This journal is ª The Royal Society of Chemistry 2011
This work was supported by funds from Cellana BV and by the US National Science Foundation (OCE05-26462, OCE0550798). The authors acknowledge valuable contributions to design of this experiment and critical reading of a prior version of the manuscript by Mark Huntley (Cellana BV) and Ian Archibald (Cellana BV) and discussions with William Cochlan (Cellana BV). The authors are also grateful for the helpful comments from the three anonymous reviewers and the editor.
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Errata for Ringuet S, Sassano L, Johnson ZI (2011) A suite of microplate reader-based colorimetric methods to quantify ammonium, nitrate, orthophosphate and silicate concentrations for aquatic nutrient monitoring. Division of Marine Sciences and Conservation, Marine Laboratory, Duke University, Beaufort, North Carolina 28516 Due to a transcription error some of the reagent concentrations listed in Ringuet et al. (2011) are incorrect. A comprehensive list of all reagents and their concentrations used in the manuscript is listed below. All of the experiments and their results and interpretation listed in the manuscript are based on the concentrations listed below: Assay PO 4 PO 4 PO 4
Reagent Ammonium Molybdate Tetrahydrate L-Ascorbic acid Antimony Potassium Tartrate
Chemical Formula (NH 4 ) 6 Mo 7 O 24 4H 2 O C6H8O6 C 8 H 4 K 2 O 12 Sb 2
mM 32 100 4.5
NH 4 NH 4 NH 4 NH 4
Sodium hydroxide tri-sodium citrate dehydrate phenol Sodium nitroprusside dihydrate
NaOH HOC(COONa)(CH 2 COONa) 2 2H 2 O C 6 H 5 OH Na 2 [Fe(CN) 5 NO] · 2H 2 O
500 1632 404 1.3
NO 3 NO 3 NO 3 NO 3 NO 3
Ethylenediaminetetraacetic acid disodium salt dihydrate Potassium phosphate monobasic Potassium hydroxide Sulfanilamide N-(1-Naphthyl)ethylenediamine dihydrochloride
C 10 H 14 N 2 Na 2 O 8 · 2H 2 O KH 2 PO 4 KOH H 2 NC 6 H 4 SO 2 NH 2 C 10 H 7 NHCH 2 CH 2 NH 2 · 2HCl
25 28 25 58 3.9
(NH 4 ) 6 Mo 7 O 24 · 4H 2 O HO 2 CCO 2 H C6H8O6
8.1 555 159
SiOH 4 Ammonium Molybdate Tetrahydrate SiOH 4 Oxalic Acid SiOH 4 Ascorbic Acid
References: A suite of microplate reader-based colorimetric methods to quantify ammonium, nitrate, orthophosphate and silicate concentrations for aquatic nutrient monitoring Stephanie Ringuet, Lara Sassano and Zackary I. Johnson J. Environ. Monit., 2011, 13, 370-376 DOI: 10.1039/C0EM00290A.
