New Nitrate and Total Ammonia Testing Procedures in use at the Monterey Bay Aquarium

1. New Nitrate and Total Ammonia Testing Procedures in use at the Monterey Bay Aquarium Eric Kingsley, Nicholas Modisette, Roger Phillips Monterey Bay Aquarium 886 Cannery Row, Monterey, CA 93940 I want to talk to you today about New Nitrate and Total Ammonia Testing Procedures in use at the Monterey Bay Aquarium. First a bit of an introduction into what factors you should consider when developing a monitoring program.I want to talk to you today about New Nitrate and Total Ammonia Testing Procedures in use at the Monterey Bay Aquarium. First a bit of an introduction into what factors you should consider when developing a monitoring program.

2. Life Support System Water Quality Good water quality = well maintained LSS To assess water quality, monitoring various parameters is necessary Example of some of these parameters is the nitrogen cycle Total Ammonia (NH4+ + NH3) Nitrite (NO2-) Nitrate (NO3-) NH4+ + NH3 ? NO2- ? NO3- As we all know you achieve good water quality with a well maintained life support system. In order to asses water quality, monitoring of various parameters is necessary. An example of some of these parameters would be the nitrogen cycle. Total NH3 which comes primarily from animal waste. Nitrite and nitrate which are byproducts of the nitrification reactions that occur in an established bio-filter.As we all know you achieve good water quality with a well maintained life support system. In order to asses water quality, monitoring of various parameters is necessary. An example of some of these parameters would be the nitrogen cycle. Total NH3 which comes primarily from animal waste. Nitrite and nitrate which are byproducts of the nitrification reactions that occur in an established bio-filter.

3. Monitoring Program Considerations An monitoring program should Be flexible Adapt to changing testing requirements Updates to analytical equipment New testing methodologies Generate meaningful data Accuracy and precision Low Detection limits Meet budgetary and time constraints Analyzing a large number of samples each day by a highly accurate and precise time intensive method may not be practical Some of the factors to consider for a monitoring program include the following. It needs to be flexible so it can adapt to changing testing requirements, updates to analytical equipment, and new testing methodologies. It must generate meaningful data with good accuracy and precision, along with low detection limits. Given the environment we all operate under, a monitoring program also must meet budgetary and time constraints. Analyzing a large number of samples each day by a highly accurate and precise, time intensive method may not be practical.Some of the factors to consider for a monitoring program include the following. It needs to be flexible so it can adapt to changing testing requirements, updates to analytical equipment, and new testing methodologies. It must generate meaningful data with good accuracy and precision, along with low detection limits. Given the environment we all operate under, a monitoring program also must meet budgetary and time constraints. Analyzing a large number of samples each day by a highly accurate and precise, time intensive method may not be practical.

4. Monitoring Program Considerations An monitoring program should also Consider hazards of the reagents used for the analysis Consider how test results compare to other testing Especially important to prevent misleading information when there have been Changes to testing methodologies Changes to analytical equipment A monitoring program should also consider the hazards of the reagents used in the analysis. Probably one of the most important would be how the test results compare to other testing. That is especially important to prevent misleading information when there have been changes to either testing methodologies or to analytical equipment.A monitoring program should also consider the hazards of the reagents used in the analysis. Probably one of the most important would be how the test results compare to other testing. That is especially important to prevent misleading information when there have been changes to either testing methodologies or to analytical equipment.

5. Outline of Paper Introduction Testing Procedures Sources MBA Water Quality Lab Overview Development of Multi-Wavelength UV Absorption NO3 Method Development of Modified Salicylate Total NH3 Method OK, I’ve given you the Introduction. Here’s an outline of what’s still to come in this talk. Next I’m going to go over some possible sources to look at when developing a testing procedure. Then I’m going to take brief step back and give you an overview of the Monterey Bay Aquarium and our water quality lab for anyone not familiar with our facility. Following that will be the meat of this talk, starting with the steps we went thru to develop the multi-wavelength UV absorption NO3 method we use today. Following that with the development of the modified saliyclate Total NH3 method we use at MBA.OK, I’ve given you the Introduction. Here’s an outline of what’s still to come in this talk. Next I’m going to go over some possible sources to look at when developing a testing procedure. Then I’m going to take brief step back and give you an overview of the Monterey Bay Aquarium and our water quality lab for anyone not familiar with our facility. Following that will be the meat of this talk, starting with the steps we went thru to develop the multi-wavelength UV absorption NO3 method we use today. Following that with the development of the modified saliyclate Total NH3 method we use at MBA.

6. Testing Procedures Sources An example of some of the sources you can turn to when developing testing procedures at your facilities include: For general water quality methods Standard Methods (top left) is a good place to start. For seawater specific testing procedures I’d recommend Grassholf, Kremling, Ehrhardt or Parsons, Lalli, and Maita (bottom center). Commercial references such as Hach’s Water Analysis Handbook (top center) General aquarium seawater references such as Spotte’s Captive Seawater Fishes (top right) Published scientific literature (bottom left or right) Colleagues in the industry There is no one stop reference for testing procedures all of the above are good sources of information. You should keep the monitoring program considerations we just went thru and your own facilities requirements in mind when picking a method to examine further.An example of some of the sources you can turn to when developing testing procedures at your facilities include: For general water quality methods Standard Methods (top left) is a good place to start. For seawater specific testing procedures I’d recommend Grassholf, Kremling, Ehrhardt or Parsons, Lalli, and Maita (bottom center). Commercial references such as Hach’s Water Analysis Handbook (top center) General aquarium seawater references such as Spotte’s Captive Seawater Fishes (top right) Published scientific literature (bottom left or right) Colleagues in the industry There is no one stop reference for testing procedures all of the above are good sources of information. You should keep the monitoring program considerations we just went thru and your own facilities requirements in mind when picking a method to examine further.

7. MBA Water Quality Lab Overview OK, lets take a step back and give you an overview of our facilities for those not familiar with us. The Monterey Bay Aquarium is located in the city of Monterey, CA right next to the ocean. We draw in natural seawater from the bay, circulate it through our exhibits, and after treatment as necessary discharge it back to the bay. In 2010 we opened an offsite holding facility called the Animal Research and Care Center, or ARCC for short, in the city of Marina, CA about 14 miles north of the main campus. Seawater is trucked from MBA to the ARCC where it is kept in holding towers for use in periodic water changes. Water that is changed out is brought back to the main campus where after treatment it is discharge back to the bay. Our main water quality lab is located at MBA on top of the Kelp Forest Exhibit. We also have a small satellite water quality lab located at the ARCC.OK, lets take a step back and give you an overview of our facilities for those not familiar with us. The Monterey Bay Aquarium is located in the city of Monterey, CA right next to the ocean. We draw in natural seawater from the bay, circulate it through our exhibits, and after treatment as necessary discharge it back to the bay. In 2010 we opened an offsite holding facility called the Animal Research and Care Center, or ARCC for short, in the city of Marina, CA about 14 miles north of the main campus. Seawater is trucked from MBA to the ARCC where it is kept in holding towers for use in periodic water changes. Water that is changed out is brought back to the main campus where after treatment it is discharge back to the bay. Our main water quality lab is located at MBA on top of the Kelp Forest Exhibit. We also have a small satellite water quality lab located at the ARCC.

8. MBA Water Quality Lab Overview In our main water quality lab we were lucky enough to get capital funding to purchase an Agilent HP8453 UV/Vis Spectrophotometer with an XY Autosampler as our main analytical instrument (top right) a number of years ago. However when the ARCC was being built funding was tighter and we couldn’t afford the same instrument so we ended up getting a Hach DR5000 UV/Vis Spectrophotometer with a pour-thru cell (bottom right). This presented an additional challenge for us in developing the methods that are presented in this talk since the instruments are different at each of our facilities.In our main water quality lab we were lucky enough to get capital funding to purchase an Agilent HP8453 UV/Vis Spectrophotometer with an XY Autosampler as our main analytical instrument (top right) a number of years ago. However when the ARCC was being built funding was tighter and we couldn’t afford the same instrument so we ended up getting a Hach DR5000 UV/Vis Spectrophotometer with a pour-thru cell (bottom right). This presented an additional challenge for us in developing the methods that are presented in this talk since the instruments are different at each of our facilities.

9. Development of Multi-Wavelength UV Absorption NO3- Method Previous testing 1995-1998 UV Spectrophotometric Screening Method (4500-NO3- in Eaton et al.) Absorbance at 220nm gives NO3- concentration when compared to standard curve High detection limit as organic matter also absorbs in UV Cadmium Reduction Method (Grasshoff et. al 1999 and Parsons, Maita, and Lalli 1984) Cu coated Cd metal reduces NO3- to NO2- Time consuming, many reagents to prepare Can buy commercially prepared reagents (i.e. Method #8192 Hach 2008) All use cadmium metal which is hazardous to staff running the test and must be disposed of as toxic waste. First the development of our multi-wavelength UV absorption NO3 method. In 1995 the water quality group was split off into its own department. At that time we used the UV Spectrophotometric Screening Method that is listed in Standard Methods for our NO3 testing, which works by measuring the absorbance of your sample at 220nm and comparing it to a standard curve. It has a fairly high detection limit as organic matter also absorbs in the UV, even when applying the correction factor listed in the procedure. Since the Monterey Bay is a clean water body we also needed a method with a lower detection limit. So we ran the cadmium reduction method of Grasshoff et al. at the same time and in duplicate for several months. This method works by reducing NO3 to NO2 in a Cd metal column that is coated with copper. It is very time consuming as there are many reagents to prepare. Time can be saved if you are willing to buy commercial reagents such as those for Hach’s Method #8192. However all use Cd metal which is hazardous to staff running the test and must be disposed of as toxic waste.First the development of our multi-wavelength UV absorption NO3 method. In 1995 the water quality group was split off into its own department. At that time we used the UV Spectrophotometric Screening Method that is listed in Standard Methods for our NO3 testing, which works by measuring the absorbance of your sample at 220nm and comparing it to a standard curve. It has a fairly high detection limit as organic matter also absorbs in the UV, even when applying the correction factor listed in the procedure. Since the Monterey Bay is a clean water body we also needed a method with a lower detection limit. So we ran the cadmium reduction method of Grasshoff et al. at the same time and in duplicate for several months. This method works by reducing NO3 to NO2 in a Cd metal column that is coated with copper. It is very time consuming as there are many reagents to prepare. Time can be saved if you are willing to buy commercial reagents such as those for Hach’s Method #8192. However all use Cd metal which is hazardous to staff running the test and must be disposed of as toxic waste.

10. Development of Multi-Wavelength UV Absorption NO3- Method New NO3- Method Needed – 1998 Fairly low detection limits Minimal amount of preparation time required Did not contain hazardous materials Checked literature & talked with contacts At the time a Moss Landing Marine Laboratories student was doing research into a multi-wavelength UV absorption procedure for measuring among other things NO3- in seawater (Guenther 1999) In 1998 we started looking around for a new NO3 method that had fairly low detection limits, a minimal amount of preparation time, and did not contain hazardous materials. After checking the literature and talking with contacts I discovered a student at Moss Landing Marine Laboratories was doing her thesis research on a multi-wavelength UV absorption procedure for measuring among other things NO3 in seawater.In 1998 we started looking around for a new NO3 method that had fairly low detection limits, a minimal amount of preparation time, and did not contain hazardous materials. After checking the literature and talking with contacts I discovered a student at Moss Landing Marine Laboratories was doing her thesis research on a multi-wavelength UV absorption procedure for measuring among other things NO3 in seawater.

11. Development of Multi-Wavelength UV Absorption NO3- Method http://www.satlantic.com/details.asp?ID=11&CategoryID=2&SubCategoryID=0 This research was continued by folks at our sister institution, the Monterey Bay Aquarium Research Institute. It eventually led to the production of an in situ sensor called ISUS marketed by the company Sat Atlantic (left) to generate continuous long term NO3 data (right). An example of such data is in the picture on the right which is some recent data I downloaded from MBARI’s Chem Sensor web page for a unit they have deployed on a buoy out in the bay.This research was continued by folks at our sister institution, the Monterey Bay Aquarium Research Institute. It eventually led to the production of an in situ sensor called ISUS marketed by the company Sat Atlantic (left) to generate continuous long term NO3 data (right). An example of such data is in the picture on the right which is some recent data I downloaded from MBARI’s Chem Sensor web page for a unit they have deployed on a buoy out in the bay.

12. Development of Multi-Wavelength UV Absorption NO3- Method Johnson and Coletti 2002 Procedure Allows for the determination of NO3-, Br-, HS- in seawater Low nutrient seawater used for calibrations Low detection limit due to the approximation of the contribution from colored dissolved organic matter by a quadratic function of wavelength (Equation 3) The original ISUS data was published by Johnson and Coletti in 2002 in the journal Deep-Sea Research. Their procedure allows for the determination of NO3, Br- and HS- in seawater. It uses low nutrient seawater for calibrations. It has a low detection limit due to the approximation of the contribution of colored dissolved organic matter by a quadratic function of wavelength.The original ISUS data was published by Johnson and Coletti in 2002 in the journal Deep-Sea Research. Their procedure allows for the determination of NO3, Br- and HS- in seawater. It uses low nutrient seawater for calibrations. It has a low detection limit due to the approximation of the contribution of colored dissolved organic matter by a quadratic function of wavelength.

13. Development of Multi-Wavelength UV Absorption NO3- Method Adapting Johnson and Coletti (2002) procedure to our lab Access to low nutrient seawater & storing it correctly were problematic for us Alternative calibration solution needed UV spectrum in seawater dominated by Br-, NO3-, and to a lesser degree organic matter So preparing NO3- standards in a NaBr solution at the seawater Br- concentration was a possible alternative calibration solution for seawater Freshwater uses NO3- standards prepared in laboratory water So could we adapt their procedure for use in our lab. The biggest problem was that regular access to a source low nutrient seawater and storing it correctly would be problematic for us at the aquarium. An alternative calibration solution that we could make up in the lab was needed. In their paper Johnson and Coletti point out that the UV spectrum in seawater is dominated by Br-, NO3, and to a lesser degree organic matter. Organic matter is already taken into account by the quadratic equation they use to approximate it. So that gave me the idea that NO3 standards prepared in a NaBr solution as seawater Br- concentration might be a possibility. Since freshwater usually doesn’t have a lot of Br- in it NO3 standards prepared in laboratory grade water should work there. So we decided to test this out if our HP8453 spec would generate sufficient data.So could we adapt their procedure for use in our lab. The biggest problem was that regular access to a source low nutrient seawater and storing it correctly would be problematic for us at the aquarium. An alternative calibration solution that we could make up in the lab was needed. In their paper Johnson and Coletti point out that the UV spectrum in seawater is dominated by Br-, NO3, and to a lesser degree organic matter. Organic matter is already taken into account by the quadratic equation they use to approximate it. So that gave me the idea that NO3 standards prepared in a NaBr solution as seawater Br- concentration might be a possibility. Since freshwater usually doesn’t have a lot of Br- in it NO3 standards prepared in laboratory grade water should work there. So we decided to test this out if our HP8453 spec would generate sufficient data.

14. Development of Multi-Wavelength UV Absorption NO3- Method HP8453 allows for absorbance to be measured at multiple wavelengths 1nm apart Wavelengths used for calculations must be adjusted An Excel spreadsheet was designed to do calculations Method comparison was conducted running both Multi-Wavelength and UV Screening Method for 1 year Only portion of comparison data is shown here Our HP8453 spec does allows you to record absorbance's from multiple wavelengths 1nm apart in the UV region as shown in this screen capture of the Chem Station software (left). After some initial testing we found that we had to adjust the analytical wavelengths used for the calculations slightly to take in account the alternative calibration solution we were using. An Excel spreadsheet was designed to do the calculations. For the 1st year we ran both the multi-wavelength and UV screening methods side by side for comparison. If you plot the results of samples ran using one method against samples ran using the other method, the slope of a regression line should be close to 1 if they are comparable. I’ve plotted up a subset of some of this comparison data to show you how the methods compared (right).Our HP8453 spec does allows you to record absorbance's from multiple wavelengths 1nm apart in the UV region as shown in this screen capture of the Chem Station software (left). After some initial testing we found that we had to adjust the analytical wavelengths used for the calculations slightly to take in account the alternative calibration solution we were using. An Excel spreadsheet was designed to do the calculations. For the 1st year we ran both the multi-wavelength and UV screening methods side by side for comparison. If you plot the results of samples ran using one method against samples ran using the other method, the slope of a regression line should be close to 1 if they are comparable. I’ve plotted up a subset of some of this comparison data to show you how the methods compared (right).

15. Development of Multi-Wavelength UV Absorption NO3- Method DR5000 Spectrophotometer at ARCC No method can be built to measure enough wavelengths for calculations 2nm bandwidth Has a wavelength scan option Absorbance at individual wavelengths can be saved to individual Excel files from Hach’s DataTrans software Excel file had to be modified As I already mentioned though we ended up having to buy a different spec for the ARCC, a Hach DR5000. Looking into the design of the DR5000 it was found that we couldn’t build a user designed method capable of measuring enough wavelengths for the calculations. Plus it also had a slightly bigger bandwidth of 2nm. However the DR5000 also has a wavelength scan option (top) that allows the absorbance of multi wavelengths to be saved into individual Excel files from Hach’s Data Trans software (bottom). So with a modification of our Excel spreadsheet program it looked like we could run the multi-wavelength UV absorption NO3 method on the DR5000 too.As I already mentioned though we ended up having to buy a different spec for the ARCC, a Hach DR5000. Looking into the design of the DR5000 it was found that we couldn’t build a user designed method capable of measuring enough wavelengths for the calculations. Plus it also had a slightly bigger bandwidth of 2nm. However the DR5000 also has a wavelength scan option (top) that allows the absorbance of multi wavelengths to be saved into individual Excel files from Hach’s Data Trans software (bottom). So with a modification of our Excel spreadsheet program it looked like we could run the multi-wavelength UV absorption NO3 method on the DR5000 too.

16. Development of Multi-Wavelength UV Absorption NO3- Method Comparison study of DR5000 vs. HP8453 Multi-Wavelength NO3 Results To make sure we could get the same result on different specs we did another inter-comparison study looking at the DR5000 vs. HP8453 NO3 results of the same samples ran on each instrument. While the slope is not exactly 1, it is pretty close so we now use the multi-wavelength UV absorption method at both of our water quality labs today.To make sure we could get the same result on different specs we did another inter-comparison study looking at the DR5000 vs. HP8453 NO3 results of the same samples ran on each instrument. While the slope is not exactly 1, it is pretty close so we now use the multi-wavelength UV absorption method at both of our water quality labs today.

17. Development of Modified Salicylate Total NH3 Method Total NH3 Testing Most common method uses the Berthelot Reaction Phenol one of reagents is very toxic MBA used Oxidative Method of Parsons, Maita, and Lalli (1984) Total NH3 oxidized to NO2 with OCl- in alkali using excess KBr as a catalyst Very time consuming 7 different reagents to add Minimum of 45 min in water bath required Moving on to the Total NH3 testing, the most common method you will find in the literature is the Berthelot Reaction. However this reaction uses phenol a very toxic reagent so we have never used this at MBA. Instead we used the alternative Oxidative Method of Parsons, Maita, and Lalli for a number of years. The Oxidative Methods works by oxidizing total NH3 to NO2 with OCl- in alkali using an excess of KBr as a catalyst. From there it is the pink sulfanilamide NO2 determination (left). In order to avoid having to use the toxic phenol reagent we accepted the trade off of our method being very time consuming, with 7 different reagents that must be added to your sample along with a minimum of 45min in a water bath.Moving on to the Total NH3 testing, the most common method you will find in the literature is the Berthelot Reaction. However this reaction uses phenol a very toxic reagent so we have never used this at MBA. Instead we used the alternative Oxidative Method of Parsons, Maita, and Lalli for a number of years. The Oxidative Methods works by oxidizing total NH3 to NO2 with OCl- in alkali using an excess of KBr as a catalyst. From there it is the pink sulfanilamide NO2 determination (left). In order to avoid having to use the toxic phenol reagent we accepted the trade off of our method being very time consuming, with 7 different reagents that must be added to your sample along with a minimum of 45min in a water bath.

18. Development of Modified Salicylate Total NH3 Method Water Quality Lab at ARCC 1st one, now two staff work there Do both analyst and system operator duties Time available to run testing very limited Other Total NH3 testing available Hach Salicylate Method (Method #8155, Hach 2008) most common in industry. Reagents sold commercially Only 18 min incubation required after reagent addition Hazardous material concerns minimized Initial inter-comparison were terrible However, when we opened the water quality lab at the ARCC we were first given the OK to hire 1 staff person, Nick my co-author. We have since been given the OK to hire a second person out there, Billy. Nick and Billy function as both analysts and as system operators so their time available to run testing is very limited. Adapting the Oxidative Method to the DR5000 wasn’t going to be practical as they simply wouldn’t have the time so, we needed another testing procedure. The Hach Salicylate Method is the most common procedure used in the industry today. Reagents are sold commercially saving you a lot of preparation time. It only requires 18 min of incubation of after reagent addition, which is another time savings. Hazardous material concerns of the reagents are minimized. We decided therefore to test this out with an initial inter-comparison study shown here (right), the results of which were terrible, showing a very poor R^2.However, when we opened the water quality lab at the ARCC we were first given the OK to hire 1 staff person, Nick my co-author. We have since been given the OK to hire a second person out there, Billy. Nick and Billy function as both analysts and as system operators so their time available to run testing is very limited. Adapting the Oxidative Method to the DR5000 wasn’t going to be practical as they simply wouldn’t have the time so, we needed another testing procedure. The Hach Salicylate Method is the most common procedure used in the industry today. Reagents are sold commercially saving you a lot of preparation time. It only requires 18 min of incubation of after reagent addition, which is another time savings. Hazardous material concerns of the reagents are minimized. We decided therefore to test this out with an initial inter-comparison study shown here (right), the results of which were terrible, showing a very poor R^2.

19. Development of Modified Salicylate Total NH3 Method Could we adapt Hach Salicylate Method so would work in natural seawater? Close examination of vials in initial study showed a cloudy white precipitate Mg and Ca can precipitate as hydroxides and carbonates in seawater with pH>9.6 (Grasshoff, Kremling, Ehrhardt 1996) Hach’s (2008) interference table says method can handle Ca<1000mg/L as CaCO3 (400 mg/L as Ca) Mg<6000 mg/L as CaCO3 (1458 mg/L as Mg Natural seawater has (Libes 2009) Ca = 420 mg/L as Ca Mg = 1313 mg/L as Mg The time savings of commercial reagents and only an 18min incubation time were highly attractive so we didn’t want to reject this method right away. A close examination of the seawater vials in the initial study showed they had a cloudy white precipitate floating around in them. In Grassholf et al. it mentions that Mg and Ca can precipitate as hydroxides and carbonates in seawater with a pH>9.6. In the interference table listed in the Hach reference it says it can handle Ca <1,000mg/L as CaCO3 (400 mg/L as Ca if you do the conversions) along with Mg<6,000mg/L as CaCO3 (1,458 mg/L as Mg). In any general chemical oceanography reference (i.e. Libes 2009) you will find that Ca and Mg are minor elements in natural seawater with fairly constant concentrations of around 420mg/L Ca and 1,313mg/L as Mg. Aha! So the white precipitate we were seeing might have been identified!The time savings of commercial reagents and only an 18min incubation time were highly attractive so we didn’t want to reject this method right away. A close examination of the seawater vials in the initial study showed they had a cloudy white precipitate floating around in them. In Grassholf et al. it mentions that Mg and Ca can precipitate as hydroxides and carbonates in seawater with a pH>9.6. In the interference table listed in the Hach reference it says it can handle Ca <1,000mg/L as CaCO3 (400 mg/L as Ca if you do the conversions) along with Mg<6,000mg/L as CaCO3 (1,458 mg/L as Mg). In any general chemical oceanography reference (i.e. Libes 2009) you will find that Ca and Mg are minor elements in natural seawater with fairly constant concentrations of around 420mg/L Ca and 1,313mg/L as Mg. Aha! So the white precipitate we were seeing might have been identified!

20. Development of Modified Salicylate Total NH3 Method Could we adapt Hach Salicylate Method so would work in natural seawater? Our volunteer suggested adding more citrate to samples. Hach reagents contain citrate so made sense Improved reaction but not good enough for seawater analysis Reviewed literature again Salicylate reaction most sensitive at pH ~13 (Pym and Millham 1976) Buffering capacity in seawater found to be a problem in review of questionnaire of 64 labs doing Bertholet reaction (Aminot, Kirkwood, and Kérouel 1997) ‘pH-shift’ caused by Mg-citrate complex in seawater exhibit some buffering characteristics and lowering reaction pH (Pai, Tsau, and Yang 2001) Pai, Tsau, and Yang (2001) recommends adding additional hydroxide to compensate for ‘pH-shift’ So what to do about this precipitate? We couldn’t dilute our samples, as was initially suggested, as we were looking for a low detection limit. We were all scratching our heads on this one. We are lucky enough to have a volunteer who works for out lab that has a PhD in chemistry. After reviewing our data she suggested adding more citrate to samples. This made some sense as the Hach reagents already included citrate as one of the ingredients. Plus literature for the Berthelot reaction also says citrate will complex Ca and Mg such that they don’t react in alkaline seawater. More citrate improved things but didn’t quite get us where we needed. So we went back to the literature again. Pym and Millham say the salicylate reaction is most sensitive at a pH ~13. Buffering capacity in seawater was found to be a problem in the review of labs running the Berthelot reaction by Aminot, Kirkwood, and Kerouel. Pai, Tsau, and Yang describe this ‘pH-shift’ as being caused by the Mg-citrate complex in seawater exhibiting some buffering characteristics and lowering the reaction pH. They recommended adding additional hydroxide to compensate.So what to do about this precipitate? We couldn’t dilute our samples, as was initially suggested, as we were looking for a low detection limit. We were all scratching our heads on this one. We are lucky enough to have a volunteer who works for out lab that has a PhD in chemistry. After reviewing our data she suggested adding more citrate to samples. This made some sense as the Hach reagents already included citrate as one of the ingredients. Plus literature for the Berthelot reaction also says citrate will complex Ca and Mg such that they don’t react in alkaline seawater. More citrate improved things but didn’t quite get us where we needed. So we went back to the literature again. Pym and Millham say the salicylate reaction is most sensitive at a pH ~13. Buffering capacity in seawater was found to be a problem in the review of labs running the Berthelot reaction by Aminot, Kirkwood, and Kerouel. Pai, Tsau, and Yang describe this ‘pH-shift’ as being caused by the Mg-citrate complex in seawater exhibiting some buffering characteristics and lowering the reaction pH. They recommended adding additional hydroxide to compensate.

21. Development of Modified Salicylate Total NH3 Method Combo reagent created after several experiments 110g/L sodium citrate + 40g/L sodium hydroxide Add 1mL to each 25mL sample before Hach Reagents After addtion of the Hach reagents precipitate gone After several experiments we settled on a combo reagent containing 110g/L sodium citrate + 40g/L sodium hydroxide for which 1mL was added to each 25mL sample before the Hach Reagents were added. After addition of the Hach reagents the precipitate is now gone as you see from these recent samples ran at the ARCC (left). The far left is a MQ blank, then the QC prepared in MQ, the rest are seawater samples showing no precipitate.After several experiments we settled on a combo reagent containing 110g/L sodium citrate + 40g/L sodium hydroxide for which 1mL was added to each 25mL sample before the Hach Reagents were added. After addition of the Hach reagents the precipitate is now gone as you see from these recent samples ran at the ARCC (left). The far left is a MQ blank, then the QC prepared in MQ, the rest are seawater samples showing no precipitate.

22. Development of Modified Salicylate Total NH3 Method Inter-comparison study of Modified Salicylate vs. Oxidative Method Total NH3 showed good results Linear range extended up to 1.5ppm Total NH3 Inter-comparison HP8453 vs. DR5000 also looked good An inter-comparison study of the modified salicylate method ran on the DR5000 vs. the Oxidative Method ran on the HP8453 showed very good results. We also found that the addition of the combo reagent extended the linear range up to 1.5ppm Total NH3. A later study of samples ran using this Modified Salicylate Method vs. the Oxidative Method ran on the HP8453, that I haven’t shown here, also looked good. We are now using this Modified Salicylate Total NH3 method in our water quality labs at both MBA and the ARCC.An inter-comparison study of the modified salicylate method ran on the DR5000 vs. the Oxidative Method ran on the HP8453 showed very good results. We also found that the addition of the combo reagent extended the linear range up to 1.5ppm Total NH3. A later study of samples ran using this Modified Salicylate Method vs. the Oxidative Method ran on the HP8453, that I haven’t shown here, also looked good. We are now using this Modified Salicylate Total NH3 method in our water quality labs at both MBA and the ARCC.

23. Conclusions New NO3 and Total NH3 procedures developed at MBA are: Less time consuming then previous methodology Involve less environmental hazards Offer comparable results To previous testing methods To testing ran on different models of spectrophotometers Copies of written protocols or Excel spreadsheets used in calculations are available from authors by request In conclusion we were able to develop new NO3 and Total NH3 testing procedures that are less time consuming then previous methodology, involve less environmental hazards, and offer comparable results to both previous testing methods and between different instruments. This talk has deliberately been kept as an overview of some of the factors to consider when developing or changing your monitoring procedures. If anyone is interested in copies of our written protocols or the Excel spreadsheets we use for the calculations see one of us afterwards. We can either give you an electronic copy here at the conference or can send them to you via email.In conclusion we were able to develop new NO3 and Total NH3 testing procedures that are less time consuming then previous methodology, involve less environmental hazards, and offer comparable results to both previous testing methods and between different instruments. This talk has deliberately been kept as an overview of some of the factors to consider when developing or changing your monitoring procedures. If anyone is interested in copies of our written protocols or the Excel spreadsheets we use for the calculations see one of us afterwards. We can either give you an electronic copy here at the conference or can send them to you via email.

24. Acknowledgements Staff in the Water Quality Lab who ran many additional samples for the inter-comparison studies presented in this paper Monterey Bay Aquarium for funding this research Dr. Cristina Amarillas, a volunteer in our department whose insight pointed us down the road to developing the Modified Salicylate Total NH3 Method we use today. See slide.See slide.

25. Thank You For Listening