Table of contents. N, O and B isotopes to evaluate nitrate pollution in water Analytical and practical manual

Transcription

1

2 Table of contents 1 Introduction ISONITRATE project Isotope method Sampling procedure Groundwater samples Surface water samples Required sample volumes and conservation of water samples Source material samples Monitoring strategy Analytical analysis methods Chemical monitoring Isotope monitoring Data analysis Chemical data analysis Isotopic data analysis Implementation of isotope analysis in nitrate monitoring Benefits of the multi-isotope approach Potential improvements for monitoring Potential improvements for management Cost/benefit economic evaluation Conceptual framework of valuing information Theoretical background: the Bayesian decision analysis References European stable isotope laboratories

3 1 Introduction The present document is a user-friendly manual dedicated to water managers, policy makers and administrations. It is an analytical and practical guide for introducing the multi-isotope approach in nitrate monitoring programs for achieving good water quality status according to the Water Framework Directive. The manual was developed in the framework of the EU-LIFE demonstration project ISONITRATE (Improved management of nitrate pollution in water using isotopic monitoring). The manual includes method descriptions and step-by-step guidelines to implement the methodology for different water bodies. This document also introduces the economical cost/benefit evaluation of the isotope approach. 1.1 ISONITRATE project The ISONITRATE project ( is an EU-LIFE sponsored project that aims at demonstrating the added value of isotope analysis for a more effective river basins management concerning nitrate pollution. The multi-isotope approach in the ISONITRATE demonstration project includes analysis of the isotopic composition of oxygen (δ 18 O) and nitrogen (δ 15 N) in nitrate and analysis of the isotopic composition of boron (δ 11 B). The demonstrated technology and methodology allows to - characterize water bodies; - trace and distinguish source(s) of dissolved nitrates in water; - analyse pressure and impact of nitrate pollution Background, problem and objectives Current approach to environmental management and control of water quality regarding nitrate is based on monitoring concentrations. Chemical data alone do not always permit to establish unambiguously the type, location and contribution of its different sources in a river basin. In particular, differentiating urban and agricultural origin is often impossible (even by increasing the number of monitoring stations or samples). Thus, design and application of specifically targeted management plans for nitrate control is improbable. Research showed the great added value of using isotopes to precisely distinguish nitrate sources, trace them in water and potentially quantify their respective contributions. This isotope approach inherently provides more information, but had to be fully demonstrated through a suitable integrated pilot project. The objective of ISONITRATE was to demonstrate, to policy makers, the technical/economical feasibility of integrating an isotope approach as part of characterising water bodies and analysing pressure and impact of nitrate pollution, for a more effective implementation of environmental management measures in river basins Description of technical/methodological solution The demonstration monitoring of the ISONITRATE project relied on a survey over 15 months on a pilot site (Alsace, Rhine river basin, France-Germany) where 4 studied cases were selected, trying to cover the different natural contexts encountered when dealing with nitrate pollution. 3

4 1) Natural, pristine water with a low NO 3 concentration (not impacted by any anthropogenic input and not resulting from denitrification); 2) Simple contamination (only one source of nitrate pollution involved); 3) Multiple-sources contamination (varying inputs from distinct nitrate sources) 4) Denitrification (natural attenuation of nitrate by biotic/abiotic conversion into N 2 ). Chemical and isotope analyses (δ 15 N, δ 18 O and δ 11 B) of both water (groundwater and surface water) and nitrate sources were carried out within partnership laboratory facilities. Acquired data were processed, evaluated for quality and interpreted according to appropriate statistical procedures Results and environmental benefits The results of the project generated the present user manual (as well as a separate guideline document which can be downloaded on the project website demonstrating the practical feasibility (technologically and economically) of implementing the isotope methodology for an improved monitoring of water quality with regards to nitrate pollution in groundwater and surface water (i.e. nitrate source apportionment via distinct isotopic fingerprints) and for a more efficient planning of induced environmental management measures. 1.2 Isotope method Isotopes to trace sources of dissolved nitrates in water The isotopic composition of the dissolved nitrogen species has been used extensively to better constrain the sources and fate of nitrogen in water (e.g. Kendall et al., 2007 and references included). Nitrogen cannot be considered conservative because it is biologically modified through e.g. nitrification and denitrification reactions causing isotopic fractionation that modifies the isotopic signatures of the dissolved nitrogen species. DNRA (dissimilatory nitrate reduction to ammonium) is another nitrate reducing pathway that occurs under reducing conditions when carbon is abundant and nitrate limited. Given the complexity of the biogeochemical nitrogen cycle, both the nitrogen isotope composition (δ 15 N) and oxygen isotope composition (δ 18 O) of the nitrate molecule are included to define the isotopic signature of nitrate. This isotope signature can be used to trace natural and anthropogenic nitrate pollution sources, to identify conversion processes (e.g. denitrification, nitrification and biological fixation) and to assess the nitrogen budget in water bodies. For various nitrate sources contributing to groundwater and surface water pollution, characterising the isotopic signature (δ 15 N and δ 18 O) of nitrate is required. Due to the wide variety of sources, nitrogen can enter the soil in several forms: nitrate (NO 3 - ), nitrite (NO 2 - ), ammonium (NH 4 + ), organic nitrogen compounds Moreover, nitrogen can be converted from one form into another through various reactions and processes. Since oxygen is usually plentifully available in unsaturated soils, most of the nitrogen that reaches the groundwater has undergone nitrification and is present in the oxidized form nitrate (NO 3 ). 4

5 From the sole concentration measurements, it is generally hard to distinguish to what extent different sources are contributing to the observed nitrate level in water. It requires an extended dataset of spatially and temporally staggered concentration measurements as well as detailed information about potential nitrogen sources in the surrounding area, hydrology, soil characteristics Even if there is abundant data available, budgeting the different sources based on concentration measurements remains difficult and uncertain. Moreover, such a characterisation of the observed nitrate pollutions might become very expensive and time consuming in areas that are exposed to multiple possible nitrogen sources. These problems and shortcomings can be tackled by applying isotopic analyses in addition to the concentration measurements. Scientific research has shown that multi-isotope analyses can be used to distinguish sources of nitrate and to evaluate whether nitrate concentrations are changing due to mixing of different sources or due to conversion processes (or even both). In case of nitrogen pollution, the isotopic methodology encloses the measurement of nitrogen, oxygen and boron isotope ratios. The isotope composition is defined as a delta value which expresses parts per thousand deviations from an international standard. For nitrogen, oxygen and boron the respective delta notations are δ 15 N, δ 18 O and δ 11 B. The international standards are air for δ 15 N, the Standard Mean Ocean Water (SMOW) for δ 18 O, and a boric acid (NBS951) for δ 11 B: 15 δ N = 15 N ( 14 ) N 15 N ( 14 ) N sample AIR δ O = 18 O ( 16 ) O 18 O ( 16 ) O sample SMOW and 11 δ B = 11 B ( 10 ) B 11 B ( 10 ) sample B NBS Due to the complexity of the biogeochemical nitrogen cycle, the δ 15 N isotope composition is usually combined with the δ 18 O isotope composition of the NO 3 - molecule to trace nitrate sources and to identify the effect of potential conversion processes (e.g. natural attenuation) on the nitrogen budget. Mixing processes, which are generally controlling nitrate pollution, lead to a modification of the isotope composition of dissolved nitrate. This effect can be amplified by the superimposition of conversion reactions such as denitrification. Therefore, discriminating multiple NO 3 - sources based on the δ 15 N and δ 18 O isotope composition of nitrate alone becomes sometimes difficult, especially when conversion processes occur. Boron is a ubiquitous element, which happens to be a co-migrant of nitrate. The isotope composition of boron (δ 11 B) is not affected by nitrate conversion processes (e.g. denitrification). Therefore, boron isotopes can be used to improve the identification of nitrate pollution sources in case conversion processes (e.g. natural attenuation) are involved. The boron isotope composition can also provide very valuable additional information in case multiple sources and/or mixing processes affected the nitrate pollution. Several studies have shown that a multi-isotope analysis based on the use of δ 15 N and δ 18 O compositions in nitrate combined with δ 11 B signatures is more successful to trace the origin of nitrate contamination compared to nitrate concentration monitoring alone (e.g. Seiler, 2005; Widory et al., 2006) Isotope composition of various source materials The main potential pollution sources of nitrate contamination in water include mineral fertilizers, organic fertilizers (i.e. animal manure) and sewage effluents. In order to be able to distinguish 3 5

6 sources and to assess their contribution, it is recommended to collect samples in the investigated area to characterize the real local pressure and to determine the isotopic signature of potential pollution sources. Literature data about signature ranges can also be used, but it might increase uncertainty as the isotope compositions are known to depend on both the source type and the local conditions (temperature, humidity, wind ). The ranges of δ 15 N and δ 18 O values for dissolved nitrate originating from various sources are shown in Figure 1 and Figure 3. The ranges of δ 11 B signatures for various sources are shown in Figure 2. Figure 1: Box plots of δ 15 N and δ 18 O signatures of nitrate from various sources, sinks and processes. Data collected from literature. The box plots illustrate 25 th, 50 th and 75 th percentiles; the whiskers indicate the 10 th and 90 th percentiles; and the circles represent outliers. (Figure adapted from Xue et al., 2009) 6

7 Figure 2: Range of δ 11 B signature for various sources. Data collected from literature. (Figure adapted from Widory et al., 2004) Isotope fractionation due to conversion processes Several fractionation processes during the nitrification process (which occurs during nitrogen migration in the soil) lead to specific isotopic signatures for different nitrate vectors. Therefore, δ 15 N and δ 18 O values in nitrate can be used to identify the origin and the fate of nitrate in water. Isotope fractionation during conversion reactions results from the preference to convert the lighter isotopes (e.g. preference for 14 N over 15 N and preference for 16 O over 18 O). As a result, the products are selectively enriched in lighter isotopes (depleted δ-values) while the substrates become enriched in heavier isotopes (enriched δ-values). Fractionation mechanisms for nitrogen isotopes include volatilization of NH 3(g) after ammonification, conversion of ammonium to nitrate during nitrification, conversion of nitrite to N 2(g) during denitrification and nitrate assimilation towards organic nitrogen. Denitrification affects the signature of both the nitrogen and oxygen isotopes of nitrate. As denitrification occurs, the nitrogen and oxygen isotopes in nitrate are typically enriched in heavy isotopes as indicated in Figure 3 (this will be discussed later in the manual). 7

8 Figure 3: Summary of δ 15 N and δ 18 O signatures for various nitrate sources. (Figure adapted from Kendall et al., 2007 and Widory et al., 2004) Multi-isotope approach for nitrate monitoring and pollution issues Measuring the δ 15 N and δ 18 O isotope composition of nitrate and the δ 11 B composition of boron in groundwater and surface water is potentially useful as a monitoring and evaluation method. - Application of the multi-isotope approach to distinguish sources of nitrate pollution and to assess their respective contributions. - Application of the multi-isotope approach to monitor variations and trends in nitrate contamination. - Application of the multi-isotope approach to quantify the impact of denitrification and other conversion processes. - Application of the multi-isotope approach to define adequate measures to reduce nitrate pollution and to evaluate implemented measures. 8

9 2 Sampling procedure 2.1 Groundwater samples Sampling is done from observation wells already characterised. Normally a peristaltic pump is used for the sampling unless the depth of the water level is lower than 6 to 7 meters below the surface. The following items have to be considered when sampling groundwater: measurement of piezometric level; purging of the well; on-site measurements; filtration. Piezometric level Before the sampling starts, the groundwater level and the depth of the well have to be measured. Measurements have to be carried out at the same reference point (e.g. side of the pipe). Well purging Prior to groundwater sampling, purging of a volume equal to five times the water content of the well is required. Purging has to be carried out taking into account the characteristics of the well (depth, water level ). Complete emptying of the well should be avoided (the filter element should not be exposed to air). On site measurements Prior and during the sampling, it is necessary to record the following field parameters: temperature (T ), ph, dissolved oxygen level (O 2 ), electrical conductivity (E c ) and redox potential (E h ). These measurements are carried out in a closed system. The sampling can only be performed when the field parameters are stable (not changing). Filtration of groundwater samples In case metals are analysed on the samples, filtration of a sub sample is required. The sub sample (250 ml) has to be separated and filtered immediately after sample collection. Preferably a Teflon filter (0.45 µm) is used. Avoid contact between the sample and the air as much as possible. The same protocol is applied for collection of samples dedicated to boron isotope analysis. Depending on the laboratory requirements, filtered sub-samples can also be prepared on-site for other parameters. 9

10 Required sample volumes The required sample volumes are summarized in Table 1 (see further). 2.2 Surface water samples Sampling is done from rivers or other types of surface water. A grab sample is taken. The following items have to be considered when sampling surface water: on-site measurements; filtration. On site measurements The location of the sampling site is recorded. Prior to and during sampling, it is necessary to record the following field parameters: temperature (T ), ph, dissolved oxygen level (O 2 ), electrical conductivity (E c ) and redox potential (E h ) Filtration of surface water In case metals are analysed on the samples, filtration of a sub sample is required. The sub sample (250 ml) has to be separated a filtered immediately after sample collection. Preferably a Teflon filter (0.45 µm) is used. Avoid contact between the sample and the air as much as possible. The same protocol is applied for collection of samples dedicated to boron isotope analysis. Depending on the laboratory requirements, filtered sub samples can also be prepared on-site for other parameters. Required sample volumes Summarized in Table Required sample volumes and conservation of water samples Classical chemical analyses A summary of typical sample volumes required for classical chemical analysis and the conservation requirements for different non-isotopic parameters is given in Table 1. The table includes all parameters that have been analysed during the monitoring campaigns of the ISONITRATE project. The listed parameters are considered to be relevant for nitrate pollution 10

11 monitoring. However, the list in Table 1 is not compulsory and for specific case other parameters might be considered Isotope analyses (δ 15 N & δ 18 O of NO 3 and δ 11 B) For δ 15 N & δ 18 O analysis of NO 3, the sampling volume depends on the nitrate concentration. It is therefore necessary to have at least an approximate value of the nitrate concentration present in the water to be sampled (this can be done on-site by using a nitrate electrode or by collecting data previously measured at the considered site). The ion-exchange resin technique (AgNO 3 method; see further) for isotope analyses requires ~150 µmol NO 3 - to be passed and adsorbed onto the anion exchange resin columns, while the denitrification method (see further) requires much lower amounts (~100 nmol NO 3 - ). This means that the volume of sampled water has to be adjusted on-site, taking into account the nitrate concentration of the sample, in order to reach the required amount of nitrate. It is highly recommended to collect, when possible, even a larger volume of water to have some reserve in case unpredictable analytical problems occur. For low nitrate concentrations several litres of water might be required. Recipients of appropriate volume should be available. For boron isotope analysis, to prevent any sorption/desorption processes (causing great isotope shifts), water is filtered (PVFD; 0.45 µm) upon sampling. A 1 litre bottle, rinsed several times with the sample water, is filled leaving no air space in the neck. Borosilicate bottles are prohibited as they could alter the measured δ 11 B by isotope exchange with the dissolved boron. 2.4 Source material samples Potential source materials present in the study area have to be collected for chemical and isotopic characterisation. It is recommended to take at least one representative sample for each type of pollution source. Collection can be eased by getting assistance from local authorities and stakeholders. Sample size: About 500 g Recipient: Plastic bottle (± 1 litre) Pre-treatment Leach 100 g of the dried and homogenized solid sample with 1 L of Milli-Q water. The complete analysis of the leached parameters can be carried out according to Table 1. All analysis should be performed on leachates. 11

12 Table 1: Sample volume and conservation requirements for different non-isotopic parameters. Parameter Typical sample volume (ml) Type of container Preservation technique ISO :2003 Maximum recommended preservation time Nitrate NO 3-50 Plastics Cool to between 1 C and 5 C 7 days Total N 50 Plastics Acidify to between ph1 to 2 with H 2 SO 4 1 month Ammonium NH Plastics Cool to between 1 C and 5 C 21 days Boron B DOC 100 Plastics Fill container completely to exclude air 1 month 50 Plastics Cool to between 1 C and 5 C 7 days DIC (alkalinity) 50 Plastics Cool to between 1 C and 5 C 7 days o-phosphate 100 Plastics Cool 1-5 C 1 month Calcium Ca Plastics Acidify to between ph1 to 2 with HNO 3 1 month Sodium Na + 20 Plastics Acidify to between ph1 to 2 with HNO 3 1 month Potassium K + 20 Plastics Acidify to between ph1 to 2 with HNO 3 1 month Magnesium Mg Plastics Acidify to between ph1 to 2 with HNO 3 1 month Sulphate SO 4 10 Plastics Cool to between 1 C and 5 C 1 month Chlorine Cl - 10 Plastics Cool to between 1 C and 5 C 1 month Zinc Zn Total S Total P Bromide Br Fluoride F Dry residue 20 Plastics Acidify to between ph1 to 2 with HNO 3 1 month 20 Plastics Acidify to between ph1 to 2 with HNO 3 1 month 10 Plastics Cool to between 1 C and 5 C 1 month 50 plastics Cool to between 1 C and 5 C 1 month 100 Plastics - Ash content - Plastics - 12

13 2.5 Monitoring strategy In order to design a monitoring strategy, it is advised to take into account all available knowledge and expertise concerning both the characteristics of the area and the potential sources of nitrate. Hydrogeology Connection between groundwater and surface water Expected levels of pollution Vicinity and type of pollution sources Spatial heterogeneity Land use and land use history Expected temporal variability Analysis of field measurements and non-isotopic data from earlier monitoring programs can be very helpful to optimize monitoring strategies and to determine whether and where an isotopic approach might allow for considerable improvements (see 4.2. and Figure 8) The purpose of the monitoring campaign also affects the design of the network. One should keep in mind both the expectations and the interests. Concerning the sampling frequency, for example, one should consider whether temporal dynamics are of any interest, as well as and whether there are any proxy of potential variations on the sampling time scale (such information can be obtained from previous measurements, field data, expert knowledge, ). Note that variations in concentration are not necessarily linked to variations in isotopic composition as concentrations are related to quantity while isotopic signatures are related to fate and origin. As shown in the ISONITRATE project, both non-isotopic measurements and isotopic methods can be applied for nitrate pollution monitoring. The extent of a measurement program depends on the complexity of the contamination, the required information, the knowledge that needs to be obtained and a cost-benefit analysis. Often, there is no need to determine all parameters, nonisotopic and isotopic, at every sampling location and for every sampling campaign. Easier/cheaper measurements (i.e. non-isotopic) can be used as a preliminary screening method to characterise potential contamination problems and to distinguish sites or periods that require further investigation. Depending on the requirements more detailed monitoring can involve either including more water quality parameters or increasing sampling intensity (in time and/or space) or including isotopic measurements. Table 2 provides an overview of the information obtained from different types of measurements. 13

14 Table 2: Monitoring methods for nitrate pollution and information obtained from the data. Non-isotopic Isotopic Measured parameters NO 3 -, NH 4 +, Kjeldahl N, total N ph, conductivity, ion (balance) O 2, COD, BOD, TOC, TIC, DOC, DIC O 2, NO - 3, NH + 4, Kjeldahl N, total N PO 3-4, total P B -, Cl - in addition to (1), (2), (3) and/or (4) Heavy metals in addition to (1), (2), (3) and/or (4) δ 15 N and δ 18 O composition of NO 3 - in addition to (1) δ 11 B composition in addition to (7) and (5) Water Quality Pollution status (1) - Nitrogen pollution level (2) (3) (4) - Water type - General water quality status - Organic pollution level - General water quality status - Eutrophication status - Nitrogen pollution level - Phosphorus pollution level (5) - Anthropogenic pollution Obtained information Conversion processes Identification of pollution sources - Single / multiple sources - Anthropogenic sources (6) - Anthropogenic pollution - Anthropogenic sources (7) - Denitrification - Mixing/Dilution - (Nitrification) (8) - Mixing/Dilution - Precipitation - Mineral fertilizers - Organic fertilizers - Soil nitrogen - Sewage - Organic fertilizers - (Mineral fertilizers) 3 Analytical analysis methods 3.1 Chemical monitoring For the non-isotopic monitoring and characterization of the ground and surface waters, European standardized methods are available. Generally, standardized methods play an important role in the execution of EU directives. When using standardized methods, it is easier to obtain reliable and comparable results, which facilitates national and international co-operation in both private and public sectors. In the Water Framework Directive, CEN (European Commission of Standardization) and ISO (International Organization for Standardization) standards are specified for classification and monitoring of several elements. CEN TC 230 is the Technical Committee responsible for standardization of biological, chemical and microbiological methods in water. 14

15 Many of the standards produced until now have been developed collaboratively between ISO TC 147 and CEN TC Data quality control It is advised to include data quality control in the monitoring program. The potential influence of losses or conversions of nitrite, ammonia and ortho-phosphate during sampling and transport can be checked using a field spectrophotometer with test-kits (cf ). The quality of obtained measurement data can be further evaluated based on ion balances, piper diagrams and statistical comparison of parameters measured on the same sample using different protocols Ion balances In normal situations a 10% difference in the ion balance is acceptable. Larger deviations might indicate that errors have occurred during sampling or analysis or the presence of not quantified organic acids. Deviations in the ion balance can also be related to carbonate composition and/or calculation errors. Piper diagrams Piper diagrams allow visualizing the composition of waters (there are other graphical methods available). This can be used to classify water types and to distinguish differences in the water composition. However, it only gives indications in terms of ion proportions. In case the ionic composition of the water is stable, sample data should cluster on the piper diagram. Shifts in the piper diagram over time indicate changes in the water composition which might be related to multiple pollution sources. Statistical comparison The statistical comparison includes comparing parameters that have been measured on the same sample using different analytical methods, and/or comparing parameters measured both in the laboratory and on-site (i.e. during sampling) Analysis of groundwater and surface water Several chemical parameters are available for groundwater and surface water characterisation. The following parameters have been measured during the ISONITRATE monitoring campaigns. The listed parameters are considered to be relevant for nitrate pollution monitoring. However, the list is not compulsory and for specific case other parameters might be considered. Field parameters: temperature (T ), ph, dissolved oxygen level (O 2 ), electrical conductivity (E c ), redox potential (E h ) Parameters directly related to the investigated isotopes: NO 3 -, NH 4 + and B concentrations Parameters of interest for a better understanding of the site, the water quality and the pollution status: PO 4 3-, SO 4 2-, carbon speciation (TOC, TIC, DOC, DIC, ), Ca, Na, Mg, K, Cl, Br, Zn and alkalinity 15

16 An overview of the International (ISO) or European (EN) standardized analytical methods applied in the ISONITRATE project is given in Table 3. For the preservation of the samples, the following standard was used: EN ISO :2003 Water quality - Sampling - Part 3: Guidance on the preservation and handling of water samples (ISO :2003). Table 3: Analytical methods for analysis of groundwater, surface water and source material. Parameter Standardized method Applied for Ca, Na, Mg, K, Zn, B Cl, Br, SO 4 2- TOC, TIC DOC, DIC NO 3 -, NO 2 - PO 4 3- NH 4 + EN ISO 11885:1997 Water quality - Determination of 33 elements by inductively coupled plasma atomic emission spectroscopy (ISO 11885:1996) EN ISO :2009 Water quality - Determination of dissolved anions by liquid chromatography of ions - Part 1: Determination of bromide, chloride, fluoride, nitrate, nitrite, phosphate and sulphate (ISO :2007) EN 1484:1997 Water analysis - Guidelines for the determination of total organic carbon (TOC) and dissolved organic carbon (DOC) EN ISO 13395:1996 Water quality - Determination of nitrite nitrogen and nitrate nitrogen and the sum of both by flow analysis (CFA and FIA) and spectrometric detection (ISO 13395:1996) EN ISO :2004 Water quality - Determination of orthophosphate and total phosphorus contents by flow analysis (FIA and CFA) - Part 2: Method by continuous flow analysis (CFA) (ISO :2003) ISO 11732:1997 Water quality - Determination of ammonium nitrogen - Method by flow analysis (CFA and FIA) and spectrometric detection Total N EN ISO : 1998 Water quality -- Determination of nitrogen -- Part 1: Method using oxidative digestion with peroxodisulfate Total S, total P EN ISO 11885:1997 Water quality Determination of 33 elements by inductively coupled plasma atomic emission spectroscopy (ISO 11885:1996) F Determination of Fluoride Method by flow analysis (CFA) and spectrometric detection Groundwater Surface water Source material extract Groundwater Surface water Source material extract Groundwater Surface water Source material extract Groundwater Surface water Source material extract Groundwater Surface water Source material extract Groundwater Surface water Source material extract Groundwater Surface water Source material extract Source material extract Source material extract 16

17 3.1.3 Analysis of source material The source material was extracted with Milli-Q water (Liquid/Solid ratio = 10). The extraction solution was filtered and subsequently the chemical parameters (identical to those determined for the water samples) were determined. Additionally, the following parameters might also be analyzed in the extraction solution of the source materials: total S, total P, and F -. The corresponding International (ISO) or European (EN) standardized methods are given in Table 3. The nitrate, nitrite, ammonia and total N were measured amongst other parameters on the extracts. The organically bound nitrogen concentration was calculated as follows: [Organically bound nitrogen] = [total N] [NO 3 - ] [NO 2 - ] [NH 4 + ] Preservation of critical parameters It is known that for some parameters (e.g. nitrite) analysis needs to be performed in the shortest time possible. In order to evaluate possible losses or conversions of some parameters during the sampling campaign and subsequent transport to the laboratory, a field spectrophotometer (testkits) can be used for the on-site determination of nitrite, nitrate, ortho-phosphate and ammonium. Upon arrival in the laboratory the samples should be re-analysed with the same test-kits and standardized method EN ISO 13395:1996. For example, the following test-kits can be used for on-site analysis with a Hach-Lange DR 2800 spectrophotometer: Parameter - NO 3 3- PO 4 + NH 4 - NO 2 Test-kit LCK 339 ( mg N L -1 ) based on ISO LCK 349 ( mg P L -1 ) based on ISO 6878 LCK 304 ( mg N L -1 ) based on ISO LCK 341 ( mg N L -1 ) Figure 4 : Example of spectrophotometer (mounted in a car trunk). 17

18 3.2 Isotope monitoring In this paragraph a brief overview is given of the analytical methods used for the isotopic analysis of δ 15 N and δ 18 O of nitrate and δ 11 B. Processed and validated procedures are available for the measurements of nitrate and boron isotopes. Two distinct and comparable methods coexist for the measurement of δ 15 N and δ 18 O of nitrate: the AgNO 3 conversion method (described in ) and the bacterial denitrification method (described in ). For isotope monitoring, quantitative determinations of nitrate and boron concentrations are needed, not only as a prerequisite for the correct sample intake of the isotopic analysis but also for the evaluation of mixing or denitrification patterns and for tracking sources. In the frame of the ISONITRATE project some additional analytical research and evaluation was performed. Comparison between field analysis and EN/ISO standardized methods for quantitative nitrate determination. Comparison between the bacterial denitrification method and the AgNO 3 method for determining the isotopic composition of nitrate. Comparison between ICP-AES and ICP-MS for quantitative boron determination. Comparison between TIMS and SF-ICP-MS for δ 11 B analysis Quantitative determination of nitrate and boron For the quantitative determination of nitrate and boron European standardized methods exist and can be used as such for the purpose of isotope monitoring. The field method for nitrate is recommended because (i) it is accurate when compared to laboratory methods and (ii) on-site knowledge of the nitrate concentration can be used to adjust sample volume intake needed for the δ 15 N and δ 18 O analysis of nitrate. This aspect is of economical relevance due to the fact that sample size and transport can be organized efficiently and that insufficient sample volume for isotope analysis (leading to possible need for re-sampling) can be avoided. Quantitative determination of nitrate For quantitative determination of nitrate in water samples the following methods can be used: Parameter - NO 3 - NO 3 - NO 3 Standardized method EN ISO :2009 Water quality - Determination of dissolved anions by liquid chromatography of ions - Part 1: Determination of bromide, chloride, fluoride, nitrate, nitrite, phosphate and sulphate (ISO :2007) EN ISO 13395:1996 Water quality - Determination of nitrite nitrogen and nitrate nitrogen and the sum of both by flow analysis (CFA and FIA) and spectrometric detection (ISO 13395:1996) Test kit- LCK 339 ( mg N L -1 ) based on ISO and field spectrophotometer (Hach-Lange DR 2800) 18

19 During the ISONITRATE monitoring campaigns, a good correlation was observed between the testkit measurements and the laboratory measurements for nitrate. The field spectrophotometer testkit method, which gives a result of the nitrate concentration on site within 5 minutes, has the advantage of determining the correct sample volume needed for the isotopic measurement ( ml depending on the nitrate concentration). Quantitative determination of boron For quantitative determination of boron two analytical techniques, which are widespread within commercial environmental laboratories, can be used: ICP-AES (inductively coupled plasma atomic emission spectrometry) and SF-ICP-MS (inductively coupled plasma mass spectrometry). For both techniques European standardized methods are available: Parameter B B Standardized method EN ISO 11885:1997 Water quality Determination of 33 elements by inductively coupled plasma atomic emission spectroscopy (ISO 11885:1996) EN ISO :2004 Water quality - Application of inductively coupled plasma mass spectrometry (ICP-MS) - Part 2: Determination of 62 elements (ISO :2003) There is a good correlation between both analytical methods. The ICP-MS technique is considered to be a factor of at least 10 more sensitive then ICP-AES (detection limit ~1 µg B L -1 ) for the determination of boron. However for groundwater and surface water analysis in isotopic monitoring both methods fit for the purpose. When using SF-ICP-MS an advantage, compared to TIMS, is that in one single analysis both boron concentrations and its isotope composition can be obtained simultaneously Determination of δ 15 N and δ 18 O of dissolved nitrate AgNO 3 method for δ 15 N and δ 18 O isotope analysis The isotopic composition of oxygen (δ 18 O) and nitrogen (δ 15 N) in nitrate are simultaneously determined after total chemical conversion of dissolved NO 3 to AgNO 3 by Continuous Flow Isotope Ratio Mass Spectrometry (CF-IRMS). The mass spectrometer is coupled to a High Temperature Conversion/Elemental Analyzer (TC/EA) peripheral. The analytical precision is of 0.4 vs. air for δ 15 N in nitrate and 0.6 vs. VSMOW for δ 18 O in nitrate. 19

20 Figure 5: Schematic summary of (left) the Ion Exchange Resin Technique for concentration and purification of nitrate samples and (right) the AgNO 3 method for δ 15 N and δ 18 O isotope analysis. Nitrate is collected by passing the water-sample through pre-filled, disposable, anion exchanging resin columns. Nitrate is then eluted from the anion exchange columns with 15 ml of 3 M HCl. Because HNO 3 is volatile, it must be neutralized before freeze drying. Thus, the eluded nitrate is converted to AgNO 3 by neutralization using silver oxide (Ag 2 O) in agreement with the following reaction: HCl + HNO 3 +Ag 2 O=> AgCl (s) + Ag NO 3 +H 2 O 20

21 Ion Exchange Resin Technique for concentration and purification of nitrate samples A. Introduction This method is used for the concentration and purification of surface water nitrate samples for simultaneous δ 15 N and δ 18 O determination using TC/EA-IRMS. Cation exchange resin columns are used to sorb cationic DOC and protonate anionic DOC (to reduce interference of DOC on the anion exchange columns) while anion exchange resin columns are used to concentrate NO 3 - (150 µmole NO 3 - ) for TC/EA-IRMS analysis. This method has been modified from Chang et al., 1999; Silva et al., 2000 and personal communication with Dr. Frederik Accoe from EU-JRC-IRMM. B. Equipment and Supplies Equipment and supplies have to be located in a constant temperature room. Ion Exchange Column Preparation anion exchange resin (Bio-Rad AG1X-X8; mesh; Cl - form; # ) cation exchange resin (Bio-Rad AG50 W-X8; mesh; H + form; # ) Poly-Prep chromatography columns (Bio-Rad 10ml columns; # ) 1.25 M CaCl 2 demineralised water (Milli-Q) disposable pipettes vacuum pressure bulb Cleaning Ag 2 O Ag 2 O (VWR; # ) 2-3 L Erlenmeyer flasks demineralised water (Milli-Q) orbital shaker Whatman #1 filters (VWR; 47 mm; # ) Pall glass filtration unit (VWR; 47 mm base, 300 ml funnel; #PALL4011) Sample Collection and Nitrate Determination 1 L Nalgene bottles (VWR; HDPE; # ) polycarbonate or nylon filters (VWR; 0.45µm, 47 mm; # ) Whatman GF/F filters, pre-filter for slow samples (VWR; 0.7 µm, 47 mm; # ) HACH nitrate test kits and/or Auto Analyzer for NO 3 - determinations. Loading Samples onto Ion Exchange Columns 2 ml anion and 2 ml cation exchange columns Poly-Prep column stack cap (Bio-Rad; # ) 21

22 2-way stopcocks (Bio-Rad; # ) separatory funnels with Teflon stopcocks (Nalgene; 1 litre) or Econo-Column funnels (Bio-Rad; 250 ml; # ) ring stands with ring and clamp rubber stoppers 1 L filter flasks with rubber stoppers vacuum pump with flow regulator and tubing Stripping the Nitrate from Anion Exchange Columns 3 M HCl to strip nitrate from anion exchange columns disposable pipettes 100 ml glass Duran or Pyrex beakers specimen cups with ice vacuum pressure bulb Neutralizing and Filtering HNO 3 Eluant clean (nitrate-free) Ag 2 O specimen cups with ice glass stirring rods with flattened bottom ph paper / ph meter glass filtration unit Whatman #1 filters 50 ml plastic centrifuge tubes with conical bottoms Precipitation and Filtration of Non-Nitrate O-Bearing Compounds 1 M BaCl 2 for removal of non-nitrate O-bearing compounds disposable pipettes nylon filters (VWR; 0.2 µm, 47 mm; # ) glass filtration unit 50 ml plastic centrifuge tubes with conical bottoms Removal of Excess Cations in Sample 4 ml cation exchange columns Econo Column funnels 100 ml glass beakers DI water (Milli-Q) vacuum pressure bulb Neutralization and Filtration clean (nitrate-free) Ag 2 O glass stirring rods 22

23 ph paper / ph meter 0.2 µm nylon filter glass filtration unit 50 ml plastic centrifuge tubes with conical bottoms DOC Removal and Filtration Darco G-60 activated carbon (Sigma Aldrich; 100 mesh; # G) specimen cups orbital shaker 0.2 µm nylon filter glass filtration unit 50 ml plastic centrifuge tubes with conical bottoms Preparation for Freeze-Drying samples in 50 ml centrifuge tubes or 100 ml glass beakers covered with ParaFilm liquid nitrogen or overnight in freezer vessel for liquid nitrogen freeze-dryer to form solid AgNO 3 end-product aluminium block for silver capsules (optional) silver capsules for TC/EA-IRMS analysis (Sercon; 5 x 9mm) C. Procedures and Methods Ion Exchange Column Preparation Rinse the resin with demineralised water. Place anion exchange resin in a beaker and add ~10 times the volume Milli-Q water. Allow resin to sink to bottom of beaker, discard water and repeat 2-3 times. Place the empty columns in a rack, remove the lock, and use a disposable pipette to fill the columns with the according resin slurry. Do this for many columns at once since the packed columns can be stored. Continue to fill columns until you reach the 2 ml mark on the column after all of the DI has drained and been blown dry with the vacuum pressure bulb. As an optional step, you can drip 2 ml of 1.25 M CaCl 2 through the anion exchange columns to ensure that the exchange sites are fully occupied by Cl - ions. This is only a precautionary step and may not be necessary. If done, follow this by 5x 2mL rinses with Milli-Q water to ensure all excess chloride is removed. Be sure to store the columns with Milli-Q water filled to the 6 ml mark. The columns must be capped on both ends and stored upright at room temperature. Repeat the above steps for packing the 2 ml and the 4 ml (later cation removal step) cation exchange columns. The cation exchange resin does not need to be charged so just needs to be rinsed with Milli-Q water, packed to necessary volume, and stored with Milli- Q to 6 ml mark. 23

24 Cleaning Ag 2 O Silver oxide is used as a neutralizing agent for the acidic HNO 3 column eluant. This commercially available reagent has been found to contain trace levels of nitrate and therefore must be rinsed several times to remove any external nitrate sources that could introduce contamination. To remove contaminant nitrate, 2 L of Milli-Q water is added to 500 g of Ag 2 O in a 3-4 L Erlenmeyer flask. This is then placed on an orbital shaker for ~1 hour. The silver oxide in DI is then vacuum filtered using the glass filtration unit and Whatman #1 filters. The idea is the more nitrate-bearing water that is initially removed from the silver oxide, the fewer total number of rinses that are necessary to get your rinse water 0.01 mg N L -1. After filtration, Milli-Q rinse all of the Ag 2 O back into the Milli-Q rinsing flask and repeat the above rinsing process until the rinse water nitrate is low enough (may take anywhere from hour rinses so be patient and continue to check the nitrate - concentration of your rinse water using HACH low level NO 3 test kits or the Auto Analyzer). Before the used rinse water can be poured down the drain, it must be treated with an excess amount of NaCl to precipitate out dissolved Ag as AgCl. The treated rinse water is then vacuum filtered through a 0.45 µm nylon filter (this can be re-used) to remove AgCl before disposing of the water down the drain. Be sure to properly dispose of the AgCl on the filters, store as waste in a large container along with all used Ag 2 O for possible future regeneration. The rinsed, nitrate-free Ag 2 O is then oven dried at 50 o C and stored in the refrigerator in a dark container since Ag 2 O is photo reactive and can degrade when exposed to light. Sample Collection and Nitrate Determination Surface water samples are collected in the field in 1-2 L HDPE sample bottles (depending on NO 3 - concentrations). Since we are only determining NO 3 - concentrations back in the lab, we collect larger volumes of sample to ensure we have enough sample to concentrate the required 150 µmole NO 3 - onto the anion exchange resins (which have an exchange capacity of 1.2 meq ml -1 ). Samples are filtered immediately upon return to the lab to remove any particles that could clog the resins (0.45 µm polycarbonate or nylon filters (GF/F as a pre-filter if needed) with the glass filtration unit). After filtration, NO 3 - concentrations are measured either using the HACH nitrate test kits or an auto-analyzer. This is necessary to ensure we pass an adequate volume of sample through the ion exchange resins to achieve a mass of ~150 µmole NO 3 - onto the anion exchange resins. Loading Samples onto Ion Exchange Columns Samples are loaded onto the anion exchange columns using the general set-up portrayed in the photo below. 1 L separatory funnels with stopcocks (which contain your surface water samples) are joined by straight connectors and rubber stoppers to a 2 ml cation exchange column. The cation exchange columns are connected by stopcocks and stack 24

25 caps to the anion exchange columns which are inserted into larger rubber stoppers that sit on the 1L filter flasks under vacuum. Figure 6: Detail of the laboratory setup for concentrating and purifying nitrate samples. Samples must be passed through this set-up at a flow rate of ml/hour to ensure that DOC interference is minimized by the cation columns and that ~100 µmole NO 3 - is loaded onto the anion exchange columns. Flow can be regulated by the two stopcocks to be sure that sample does not pass too quickly through the columns. It is highly important to be sure not to overwhelm the exchange capacity of the 2 ml anion columns (1.2 meq ml -1 ) since this can cause fractionation of your sample. This can be checked by testing the NO 3 - concentration of the sample that is left in the vacuum flasks (it should be 0 or below detection limit). Be sure the sample passing through the columns remains below the rubber stoppers and stopcocks to minimize chance of contamination and/or overflow. Once you are certain that the sample nitrate has been completely loaded onto the anion exchange resins, the columns are filled with Milli-Q water, capped on top and bottom, labelled and stored upright in the refrigerator. This is the best way to store your samples if there is an unforeseen delay in further processing. Organic carbon in the sample may cause difficulties when analysing δ 15 N and δ 18 O. So if detected during chemical characterisation of the samples, cation exchange resin columns are used to remove cationic DOC and protonate anionic DOC (to reduce interference of DOC on the anion exchange columns). Stripping the Nitrate from Anion Exchange Columns The anion exchange columns are shaken and mounted on test tube racks to allow the simultaneous desorption of nitrate from multiple columns. 25

26 Milli-Q water is first blown dry from all columns into a waste beaker using the vacuum pressure bulb. Labelled 100 ml glass beakers are placed under each column to catch the HNO 3 eluant. Since HNO 3 is volatile, the beakers are placed in ice baths (pre-frozen water in specimen cups works well) to minimize any chance of nitrate loss. 3 ml of 3 M HCl is applied to each column with a disposable pipette and allowed to sit for 30 seconds. Positive flow is applied to the columns using the vacuum pressure bulb to start the stripping of NO 3 - by the HCl. Allow the HCl to gravity drip through the columns; sometimes it is necessary to use the vacuum pressure bulb to blow the remaining acid from the columns. Repeat steps 4-6 until ml of 3 M HCl has been applied and passed through all the columns. Neutralizing and Filtering HNO 3 Eluant Since HNO 3 is volatile, the samples now require neutralization with Ag 2 O. Again, ice baths are recommended to dissipate the heat of reaction to eliminate the production of vapour. The reaction is as follows: HCl + HNO 3 + Ag 2 O AgCl + AgNO 3 + H 2 O 6-7 g of Ag 2 O is added to each beaker containing the HNO 3 eluant. This is done in 1g increments to minimize the heat of reaction. Make sure Ag 2 O is dry. Each 1g addition of Ag 2 O is crushed and stirred with a glass stirring rod to break the crust which encapsulates the unreacted silver oxide. The sample will begin to turn a cloudy white colour. Continue this process until you begin to see unreacted (black-coloured) Ag 2 O and/or your ph raises to ~5-7. The sample should turn from milky white to clear (but not always). The ph is checked with ph paper or with a ph meter. Next, remove the AgCl precipitate and any excess Ag 2 O by vacuum filtration through a prerinsed Whatman #1 filter. The eluant is most easily captured in stacked 50 ml centrifuged tubes placed inside the filter flask under the filter base stem. Additional Milli-Q water is used to rinse the sample nitrate through the filter bringing the sample volume to ~40 ml. Since AgNO 3 is light sensitive, care should be taken to minimize exposure to light in all remaining steps. Precipitation and Filtration of Non-Nitrate O-Bearing Compounds For accurate δ 18 O analyses of dissolved NO 3 -, all non-nitrate O-bearing compounds must be removed from the sample. This is achieved by the addition of BaCl 2. Add 2 ml of 1 M BaCl 2 to each sample contained in the 50 ml centrifuge tubes, cap and shake. The sample will immediately turn milky white. These are precipitates of BaSO 4, BaPO 4, BaCO 3 and also AgCl. Place the labelled centrifuge tubes in the refrigerator overnight to allow precipitates to settle from solution. 26

27 The following morning, pass the samples through pre-rinsed 0.2 µm nylon filters (into 50 ml centrifuge tubes) to remove the precipitates for further sample nitrate purification. Removal of Excess Cations in Sample The samples are now passed through packed 4 ml cation exchange columns (topped with Econo Column funnels) for the removal of excess cations in the solution (use vacuum pressure bulb to place positive flow as previously described for stripping the anion exchange column). Labelled 100 ml beakers are used to catch the column eluant. Excess Ba 2+ and remaining Ag + are exchanged for H + on the cation columns and your eluant is once again slightly acidic. After all the sample passes through the cation exchange columns, blow dry with vacuum pressure bulb, add 5 ml Milli-Q water to rinse the resins and blow dry again. Neutralization and Filtration The column eluant must once again be neutralized with Ag 2 O. Add ~1 g silver oxide to the sample beakers and crush/stir as previously done. This will also remove any excess Cl - in the sample. Sample will turn milky white but should clear. Check to see that the sample is ph 5-7. Pass the sample through a pre-rinsed 0.2 µm nylon filter (into stacked 50 ml centrifuge tubes) and be sure to dispose of the excess AgCl and Ag 2 O in the silver waste container. DOC Removal and Filtration Cation exchange columns were initially used for the removal of naturally occurring DOC in the raw surface water samples. We now use activated carbon to ensure removal of any DOC introduced from the method through sample contact with plastic wares and resins. Add 10 mg Darco G-60 activated carbon to labelled specimen cups and pour each sample (50 ml) into its respective cup. Place the samples on an orbital shaker at 180 rpm for 20 minutes. Be sure to keep the ratio of 10 mg over 50 ml and 20 minute shaking time exact as activated carbon can absorb nitrate. Pass the samples through pre-rinsed 0.2 µm nylon filters into stacked 50 ml centrifuge tubes. Preparation for Freeze-Drying Now that the samples are completely purified, it s time to reduce the liquid samples to solid AgNO 3. This is accomplished through freeze-drying to drive off the remaining H 2 O. Samples are then combusted from AgNO 3 to N 2 and CO at ~1400 C on a TC/EA for simultaneous δ 15 N and δ 18 O determinations. The samples must first be frozen before placement in the freeze-dryer to reduce spattering and any possibilities of contamination. This is done by placing the samples (in 100ml beakers or 50 ml plastic centrifuge tubes) either in the freezer overnight or in liquid nitrogen immediately. 27