This project has received funding from the European Union’s Horizon 2020 research

and innovation programme under grant agreement No 731065

Project Title: AQUACOSM: Network of Leading European AQUAtic MesoCOSM Facilities Connecting Mountains to Oceans from the Arctic to the Mediterranean

Project number: 731065

Project Acronym: AQUACOSM

Proposal full title: Network of Leading European AQUAtic MesoCOSM Facilities Connecting Mountains to Oceans from the Arctic to the Mediterranean

Type: Research and innovation actions

Work program topics addressed:

H2020-INFRAIA-2016-2017: Integrating and opening research infrastructures of European interest

Deliverable No 4.1.5: Standard Operating Protocol (SOP) on High Frequency Measurements

Due date of deliverable:

Actual submission date:

Version: V1.0

Main Authors: Deniz Başoğlu, Meryem Beklioğlu, Robert Ptacnik, Behzaad Mostajir, Lisette de Senerpont Domis

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Project ref. number 731065

Project title

AQUACOSM: NETWORK OF LEADING EUROPEAN AQUATIC MESOCOSM FACILITIES CONNECTING MOUNTAINS TO OCEANS FROM THE ARCTIC TO THE MEDITERRANEAN

Deliverable title Standard Operating Protocol (SOP) on High Frequency Measurements

Deliverable number D4.1.5

Deliverable version V1.0

Contractual date of delivery

Actual date of delivery

Document status

Document version V1.0

Online access Yes

Diffusion Public

Nature of deliverable Report

Work package WP4.1

Partner responsible METU, WCL

Author(s) Deniz Başoğlu, Meryem Beklioğlu, Robert Ptacnik, Behzaad Mostajir, Lisette de Senerpont Domis

Editor Deniz Başoğlu, Meryem Beklioğlu, Robert Ptacnik

Approved by Jens Nejstgaard (IGB)

EC Project Officer Agnes Robin

Abstract This Standard Operating Procedure (SOP) describes methods for measuring temperature, dissolved oxygen concentration and saturation, conductivity, underwater light (PAR), pH, turbidity, depth,

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Chl-a, flow rate, light spectrum and nitrogen data with specific probes as well as with multi-parameter sensors in mesocosms (fresh and marine). It gathers best practice advice for field observation, cleaning, calibration, use and storage of probes as well as Quality Assurance/ Quality Control (QA/QC) measures for high frequency measurements and monitoring. In order to gather more information on the procedures for the computation of data and publication of final records, please refer to the Data-related QA/QC SOP. Use of this SOP will ensure consistency and compliance in collecting and processing high frequency (some min to h) data from mesocosm experiments across the AQUACOSM community, in Europe and beyond.

Keywords High frequency measurement

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Table of Contents 1. Executive summary 6

2. Definitions and terms 6

3. Cross References 8

4. Equipment and supplies 8

5. Health and safety indications 9

6. Field Parameters and Sensors 9

6.1. Temperature 9

6.2. pH 10

6.3. Dissolved Oxygen (concentration and saturation) 10

6.4. Conductivity 11

6.4.1. Salinity 12

6.5. Underwater Light (PAR) 13

6.6. Turbidity 15

6.7. Depth 17

6.8. Chl-a (including fluorescence per algal groups and total) 17

6.9. Nitrogen (Nitrate) 18

6.10. Multi-probes (Sensors, profilers) 18

7. Maintenance of the station and the equipment 20

7.1. Regular Sensor Inspection at the Field 20

7.2. Best Practice Advice on Sensor-specific Field Cleaning and Calibration 23

7.2.1. General Considerations 23

7.2.2. Temperature sensors 23

7.2.3. pH Sensor 24

7.2.4. Optical DO Sensors 25

7.2.5. Conductivity Sensor 26

7.2.6. Underwater Light (PAR) Sensor 26

7.2.7. Turbidimeter 27

7.2.8. Depth Sensor 28

7.2.9. Fluorometers (Chl-a) 29

7.2.10. ISE probes for measuring nitrogen 30

7.2.11. Multi Probes 31

8. Overcoming Drift 32

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8.1. Common Causes 32

8.2. Elimination strategies 32

9. Troubleshooting of sensors and record-keeping equipment 32

10. Quality Assurance and Quality Control (QA/QC) of high frequency measurement 34

11. Appendices 36

Appendix A: General Procedures for Calibration of Field Thermometers 36

Appendix B: General Procedures for Calibration of pH Sensors 38

Appendix C: General Procedures for Calibration of DO Sensors 39

Appendix D: General Procedures for Calibration of Conductivity Sensors 41

Appendix E: General Procedures for Calibration of in situ Fluorescent Sensors 43

Appendix F: General procedures for 3-points calibration of ammonium and nitrate sensors 44

12. References 45

13. Checklist for the next version 46

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1. Executive summary

This Standard Operating Procedure (SOP) describes methods for high frequency measurements of parameters accessible by specific non-invasive submersible probes as well as with multi-parameter sensors in mesocosms (fresh and marine). It gathers best practice advice for field observation, cleaning, calibration, use and storage of probes as well as Quality Assurance/ Quality Control (QA/QC) measures for high frequency measurements. To gather more information on the procedures for the computation of data and publication of final records, please refer to the QA/QC SOP and data management SOP. Use of this SOP will ensure consistency and compliance in collecting and processing high frequency data from mesocosm experiments across the AQUACOSM community, in Europe and beyond.

2. Definitions and terms

Calibration To standardize or correct sensors after determining, by measurement or comparison with a standard, the correct value [1].

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Drift The lack of repeatability caused by fouling of the sensor, shifts in the calibration of the system, or slowly failing sensors [2].

Deployment The way that the sensor comes into contact with the ambient water [3].

Multiprobe The combination of several sensors, electrodes, or probe assemblies into a complete, stand-alone piece of equipment which simultaneously measures several parameters for profiling, spot-checking, or logging readings and data.

Long-term drift The slope of the regression line derived from a series of differences between reference and measurement values obtained during field testing expressed as a percentage of the working range over a 24 h period [4].

Probe A small tube containing the sensing elements of electronic equipment. It is an essential part of the water quality monitoring system since it obtains measurements and data which can be stored, analyzed, and eventually transferred to a computer.

Profiling Lowering a probe through a water column to characterize the vertical distribution of parameters.

Sensor The fixed or detachable part of the instrument that measures a particular field parameter [5].

Short-term drift The slope of the regression line derived from a series of measurements carried out on the same calibration solution during laboratory testing and expressed as a percentage of the measurement range over a 24 h period [4].

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3. Cross References

The SOPs that will be provided by AQUACOSM will be listed here in the following versions when the different SOPs are completed. The SOPs that are provided by AQUACOSM by:

✓ Phytoplankton (Deliverable 4.1.1) ✓ Zooplankton (Deliverable 4.1.2) ✓ Periphyton (Phytobenthos) (Deliverable 4.1.3) ✓ Water Chemistry (Physical and Chemical Elements of Water) (Deliverable 4.1.4) ✓ High-Frequency Data Collection (this SOP) ✓ QA/QC (Deliverable 4.1.6) ✓ Data Management (Deliverable 4.1.7)

4. Equipment and supplies

Equipment used in automated sampling are (Adopted from (Canadian Council of Ministers of the Environment, 2011)):

1. the sensors (electrical, electrochemical, or optical) used to collect the data, which respond to changing water conditions with an output signal that is processed, displayed and recorded. The choice of the sensor depends on the parameters, the required specifications, the operating conditions, and required lifespan.

2. the accessory equipment:

✓ a data logger which may be contained within a (multi-) probe or connected externally. Data filtering and processing are completed within the data loggers. The time interval of the recorded samples is determined by the user. The duration of individual samples is a function of the sensors.

✓ power supply can be internal batteries (which are contained within the sensor), external batteries, which should be a good quality gel-cell type, or a deep discharge sealed lead-acid style; and solar panels (used for satellite transmission).

✓ Best Practice Advice: Residential (220V) and solar power sources can be used as auxiliary power to the primary battery for recharge purposes. Residential and solar power sources should not be directly connected to an instrument, as voltage spikes can occur and cause the entire system to fail. Use of a voltage regulator is recommended when connecting an auxiliary power source to the primary battery.

✓ a means of retrieving the data; Communication and data retrieval can be done on-site with a laptop or hand-held display. Data retrieval can also be achieved remotely in real-time using phone or satellite communication.

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3. the cables and adapters, (instrument and site-specific) to connect the external batteries to the sensor or the solar panel to the external battery.

5. Health and safety indications

✓ Please see the Water Chemistry SOP for working and personal protection equipment suggested for use in water sampling.

✓ Conductivity standard solutions, pH buffers, DO and pH reference solutions are nontoxic but can irritate eyes and other sensitive areas because of their high salt content.

✓ Rhodamine dye; used as a calibrant for chlorophyll-a measurements, is also nontoxic, but stains everything it contacts.

6. Field Parameters and Sensors

Sensors can be in the form of individual instruments or as a single instrument including different sensors with many combinations of field parameters (multi probes) [5].

In this section, the information on the field parameters (included in this SOP) that can be measured with automated sensors will be explained in detail. In addition, best practice advice will be provided for measurements.

6.1. Temperature

Temperature is a critical environmental factor affecting physiological processes in organisms, the density of water, solubility of constituents (such as oxygen in water), pH, conductivity, and the rate of chemical reactions in the water [6], [7]. Monitoring of temperature thus is mandatory in any outdoor experiment. Depending on the fluctuations, and whether temperature itself is a treatment factor, the frequency of monitoring must match the experimental design. The monitoring of temperature along a vertical profile within a mesocosm provides information on the degree of stratification or homogenization of the water column [7].

Among many temperature sensors, thermistors are well adapted to measure the natural water temperature in mesocosms [7]. Thermistors that are made of a solid “semiconductor having resistance that changes with temperature” are incorporated into digital thermometers, other parameter instruments such as conductivity and pH meters and/or multi-parameter instruments [5]. The thermistor thermometer converts changes in resistance into temperature units. The resistance varies inversely with temperature, but, more importantly, their baseline resistance value and coefficient of variation are generally large. In some applications, an improvement can be reached with a software correction for minimization of linearization errors.

✓ The measurement range of temperature sensors are wide (-40 to +95°C, commonly). The measurement range of a thermistor used in a high-frequency monitoring station in a recent

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mesocosm study was from 0 to 40 °C [7]. ✓ Thermistors are one of the most accurate types of temperature sensors. The accuracy of

thermistors is from 0.01 to 0.02°C in a recent mesocosm study [7]. ✓ Thermistors require little maintenance and are relatively inexpensive. ✓ Diel fluctuations in water temperature tend to follow the fluctuations of atmospheric

temperature [7]. ✓ Many other parameters measured by automated probes (conductivity, DO, pH) are affected by

temperature. In a multiprobe, wrong calibration of the temperature probe affects multiple parameters.

6.2. pH

pH is the mathematical notation defined as “the negative base-ten logarithm of the hydrogen-ion activity, measured in moles per liter of a solution” [8, p. 3]. It is a primary factor governing the chemistry of natural water systems that affect physiological functions of plants and animals.

✓ The amount of dissolved gases, such as carbon dioxide, hydrogen sulfide, and ammonia affect pH level of water.

1) The instrument system that is commonly used to measure pH consists of a pH electrode; gel-filled or liquid-filled, which is a special type of ion-selective electrode (ISE), designed specifically for the measurement of the hydrogen ion (summarized from [8]). The pH electrodes have “a glass membrane, a reference and a measurement electrode, ionic (filling) solution, and a reference junction” [8, p. 6]. Sensors used in submersible monitors typically are combination

electrodes in which a proton (H+

)-selective glass-bulb reservoir is filled with an approximate

pH-7 buffer. ✓ A clean, undamaged glass membrane is necessary for performing an accurate

measurement of pH. ✓ Remember to check the concentration of the filling solution as any change in

concentration level will result in sensitivity loss. ✓ Remember to check that the junction on the pH electrode is not clogged; a clogged

electrode may not function properly.

6.3. Dissolved Oxygen (concentration and saturation)

Sensors of dissolved oxygen (DO) do not measure oxygen in milligrams per liter or parts per million but, the actual sensor measurement is proportional to the ambient partial pressure of oxygen; which can be displayed either as percent saturation or in milligrams per liter, depending on user input. The concentration of dissolved oxygen is an output of the calculations based on temperature and salinity of water [9]. The atmospheric air pressure is another factor in changing the concentration of DO of the water.

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✓ Since salinity and water temperature has an impact on the calculation of the dissolved O2 concentration, DO sensors should be associated with a temperature and a conductivity or salinity sensor via a data logger for necessary correction in real time [7].

✓ In surface waters, DO concentrations typically range from 2 to 10 milligrams per liter (mg/L). Luminescence-based optical sensors, which are based on dynamic fluorescence quenching, have a light-emitting diode (LED) to “illuminate a specially designed oxygen-sensitive substrate that, when excited,

emits a luminescent light with
a lifetime that is directly proportional to the ambient oxygen concentration” [5]. Most of the oxygen sensors used in marine mesocosm experiments are “based on oxygen dynamic luminescence quenching of a platinum porphyries complex”, or in short, luminescence-based optical sensors which can be used for long-term recording of dissolved O2 due to their strength, their immunity to biofouling and their easiness of cleaning [7].

✓ Remember that contact with organic solvents can compromise sensor integrity or performance.

● The maintenance routine and schedule for optical sensors is infrequent. ✓ Optical-sensor maintenance is recommended by manufacturer guidelines that are specific to

the type of sensor in use and the conditions to which the sensor has been subjected [9]. ✓ In a mesocosm experiment, calibration is recommended at the start of the experiment (plus

bi-monthly calibration in long-term experiments). In addition, sensors should be checked for potential drift at the end of the mesocosm experiments.

✓ The manufacturers generally recommend annual to biannual replacement of the luminophore-containing module as well as calibration check by them.

6.4. Conductivity

Conductivity is “a measure of the capacity of water to conduct an electrical current” and it is also a function of the types and quantities of dissolved substances in water [10, p. 3]. Specific conductance is the conductivity expressed in units of micro Siemens per centimeter at 25°C (μS/cm at 25°C).

✓ As concentrations of dissolved ions increase, the conductivity of the water increases. The water conductivity in mesocosms can be measured by two techniques: electrode cell method and electromagnetic induction method. The first method is commonly used for measuring low conductivities, especially in pristine environments. Use of the second method can be preferred for high conductivity measurements, especially in marine mesocosms [7]. Based on the methods, conductivity sensors generally are of two types, contact sensors with electrodes and sensors without electrodes. Specific conductance sensors with electrodes require the user to choose a cell constant (the distance between electrodes (in centimeters) divided by the effective cross-sectional area of the conducting path (in square centimeters) for the expected range of specific conductance. One shall choose a cell constant on the basis of expected conductivity based on Table 7-1.

✓ The greater the cell constant, the greater the conductivity that can be measured. ✓ Conductivity is temperature dependent, hence must be monitored in combination with

temperature. Wrong calibration of the temperature probe gives biased conductivity.

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Table 6-1. The cell constants for contacting-type sensors with electrodes and corresponding conductivity ranges (Adopted from [10])

Conductivity range, in microSiemens per centimeter

Cell constant, in 1/centimeter

0.005 – 20 0.01

1 – 200 0.1

10 – 2000 1.0

100 – 20000 10.0

1000 – 200000 50.0

Electrodeless-type sensors operate by inducing an alternating current in a closed loop of the solution, and they measure the magnitude of the current.

✓ Electrodeless sensors avoid errors caused by electrode polarization or electrode fouling as they are immune to biofouling [7].

6.4.1. Salinity

Salinity is the total quantity of dissolved salts in water. It can be calculated based on conductivity measurements, as conductivity is a tool to estimate the amount of chloride in water [5]. Salinity is most commonly reported using the Practical Salinity Unit (PSU), described by Lewis (1980) [11] which is, a scale developed relative to a standard potassium-chloride solution and based on conductivity, temperature, and barometric pressure measurements [5]. Currently the salinity expressed dimensionless.

Salinity may not be directly measured but is derived from conductivity – temperature – depth/pressure measurements. For conversion among related parameters (conductivity, salinity, temperature, and pressure) several packages are provided within the R statistical language (e.g., marelac; seacarb).

Recently, “a compact microstrip feed inset patch sensor” has been developed for measuring the salinity in seawater, which was claimed to bring better sensitivity to salinity changes (than commonly used sensors using conductivity change to measure the change in salinity) [12].

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6.5. Underwater Light (PAR)

Photosynthetically Active Radiation (PAR) is defined as radiation in the range from 400 to 700 nm, corresponding to the spectral part of daylight used by phototrophic organisms for photosynthesis. Measuring PAR can provide “an excellent gauge of how much light is available to photosynthetic organisms.

Upwelling and downwelling radiation are two aspects of underwater PAR. Upwelling radiation is radiation received from below the sensor due to reflectance off a lower surface of some type, while downwelling radiation is a measure of radiation from above the sensor, usually due to sunlight or other external light sources.

There are mainly two types of sensors used commonly to measure underwater PAR: planar and scalar sensors. Planar sensors have a flat light collecting surface that responds to light that impinges on their surface from downward directions. Planar sensors tend to underestimate PAR because the collecting surface does not absorb upwelling radiation or light that reflects off particles in the water and the sediment surface [13]. On the other hand, scalar PAR sensors have a hemispherical or spherical collecting surface that functions to absorb light from 2p to 4p steradians, (sr, i.e., Standard International (SI) unit of solid angular measure) respectively. They record more accurate measurements of total underwater PAR [13] as they absorb diffuse radiation from most directions [14].

✓ According to Long and colleagues (2012), “cosine-corrected planar sensor”, which is another sensor for PAR measurements; will produce more accurate measurements of PAR than a planar sensor without cosine correction especially under light conditions which are not ideal for accurate results such as during sunrise and sunset [14, p. 417].

✓ The planar sensors are insufficient for studies, for instance, involving phytoplankton residing within the water column where diffuse radiation may be a significant form of available light [14]. Instead, spherical sensors should be used for quantification of light in the water column.

✓ Advantages and disadvantages of all three types of sensors (planar, cosine-corrected planar and scalar sensors) are summarized in Table 7-2 below (Adopted from [14]).

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Table 6-2. The advantages and disadvantages of the three different instruments that can be used in PAR measurements (Adopted from [14]). Note for quantification of light in the water column, ideally spherical sensors are used (planar sensors will only capture e.g. the incoming light, depending on their exposition in the water column).

Sensor type Advantages Disadvantages

Planar Sensor (ex. HOBO pendant logger)

✓ Inexpensive ✓ Simple field deployment ✓ Temperature sensor ✓ Use of multiple data loggers,

reduction in data loss ✓ Small, easy to handle and mount ✓ Can be used for microscale

measurements ✓ Average out variations with multiple

loggers

✗ Data requires heavy post-processing ✗ Limited data logging period ✗ Light intensity sensor, measuring in

the unit LUX, rather than PAR ✗ Records at user-specified intervals

(no integration) ✗ No stability or accuracy reported by

the manufacturer ✗ Housing scratches and degrades

easily, shading sensor

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Cosine-corrected planar sensor (ex. Odyssey Integrating PAR sensor

✓ Fairly inexpensive ✓ Simple field deployment ✓ User-specified integration periods ✓ Small, easy to handle and mount

✗ Difficult to download/open data if using often

✗ Needs to be calibrated ✗ Difficult battery replacement ✗ No stability/accuracy reported by

the manufacturer

Spherical Sensor (ex. LICOR LI-193SA)

✓ Guaranteed factory calibration (+-5%) ✓ User-specified integration periods ✓ Excellent angular response, stability,

sensitivity

✗ Expensive

6.6. Turbidity

Turbidity is “an expression of the optical properties of a liquid that causes light rays to be scattered and absorbed rather than transmitted in straight lines through a sample” [15]. Turbidity is caused by the presence of suspended and dissolved matter, such as clay, silt, finely divided organic matter, plankton, and other microscopic organisms, organic acids, and dyes [15]. Color, either of dissolved materials or of particles suspended in the water can also affect turbidity [5]. The most common unity of turbidity is Nephelometric Turbidity Units (NTU).

Turbidity indicates the PAR availability in the water column of the mesocosm. Turbidity is typically measured at a wavelength near 850-880 nm (documentation turner sensors).

✓ Note that some combined chl-a & turbidity sensors measure turbidity at 700 nm for correcting chl-a for background turbidity (Seabird documentation).

Numerous methods and instruments can be used to measure turbidity. Because different measurement technologies result in different sensor responses, the available turbidimeters are categorized according to the instrument design as follows:

1. Type of incident light source (incandescent, LED, laser) 2. The detection angle (90°, 180° (attenuated angle), 0 - 45° (backscatter angle)) 3. Number of the scattered/ attenuated light detectors used (multiple detection angles, dual

light source-detector) The nature of particles could affect turbidity readings with 0.5-1.0 NTU. In long-term studies, this can be up to 10 NTU [16]. In addition, Ruzycki and colleagues (2014) reminded that the readings could be affected due to probable discharge, color, particle size, sediment concentrations, mineral composition

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and organic matter [17].

Measurement and documentation for submersible turbidity sensors discussed thoroughly in Anderson (2005) [15].

It is important to note that, for a valid comparison of turbidity data over time, between sites, and among projects, it is recommended to use with identical optical and data-processing configurations. Because of the potential to generate data with a high degree of variability when different technologies are used, use of units in reporting is recommended to express the measure of turbidity accordingly. In other words, units indicate the type of technology used in measuring turbidity (See Table 6-3).

✓ Nephelometric (90°) near-infrared wavelength technology is used commonly, which report data in NTU.

Table 6-3: The reporting units employed when using given instrument design (Adopted from [18]).

Instrument Design Reporting Unit

Nephelometric non-ratio turbidimeters (NTU)

Ratio white light turbidimeters (NTRU)

Nephelometric, near-IR turbidimeters, non-ratiometric (FNU)

Nephelometric, near-IR turbidimeters, ratiometric (FNRU)

Surface scatter turbidimeters (SSU)

Formazin backscatter unit (FBU)

Backscatter unit (BU)

Formazin attenuation unit (FAU)

Light attenuation unit (AU)

Nephelometric turbidity multibeam unit (NTMU)

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6.7. Depth

Depth is routinely measured by many multi probes as to relate other parameters to a given depth. Depth is measured with the help of a vented or non-vented pressure sensor (ex. YSI EXO1 and EXO2). With a non-vented sensor, “a differential strain gauge transducer measures pressure with one side of the transducer exposed to the water and the other side exposed to a vacuum” [19, p. 17]. Then, depth is calculated from the pressure exerted by the water column minus atmospheric pressure.

✓ In non-vented sensor, depth measurement can be influenced by barometric pressure, water density, and temperature.

On the other hand, vented level sensors use “a differential transducer with one side exposed to the water” [19, p. 137] (YSI, 2018, p. 137). Unlike non-vented ones, the other side of the level transducer is vented to the atmosphere. Accordingly, the transducer will measure the water pressure exerted by the water column, with a particular sensor that is vented to the outer atmosphere as a tube that runs through the sonde and cable. According to the YSI handbook (2018), this tube must remain open and vented to the atmosphere, without any distraction of foreign objects, to function appropriately (YSI, 2018) [19].

6.8. Chl-a (including fluorescence per algal groups and total)

Chlorophyll-a absorbs light in the blue and red parts of the visible electromagnetic spectrum. Chlorophyll fluorescence is the red light re-emitted by chlorophyll molecules when excited by a light source [20]. Chlorophyll fluorescence is a non-invasive method for analyzing photosynthetic energy conversion of higher plants, algae, and bacteria [20]. As previously put forward by Mostajir and colleagues (2012), “the continuous measurement of chlorophyll a and phycoerythrin concentrations by fluorescence sensors (such as YSI EXO2 sensors) is used to monitor the presence and the temporal dynamic of the algal bloom (chlorophyll a measurement) or cyanobacteria (phycoerythrin measurement)” in a mesocosm experiment [7]. The measurements can also be used to determine “the diel variations of pigment concentration (particularly chlorophyll a), which is affected by change of phytoplankton biomass and photo acclimation processes” [7, p. 313]. In situ fluorometers with an excitation (light) source, such as a Xenon lamp, laser or LED, are regarded as highly sensitive tools for the quantification and analysis of phytoplankton, which offer continuous measurement of chlorophyll concentrations in the field. They neither require pre-treatment nor a large sample volume [20]. They can be used with a profiler and integrated CTD sensor to collect data at different depths of a mesocosm [21].

✓ It should be noted that the excitation (light) source can greatly affect the quality of fluorescence signal because of its importance as a prerequisite of light-induced fluorescence measurement. Different light sources have their own advantages and disadvantages. For a brief comparison of the light sources used, please read Zeng and Li (2015) [20].

✓ In addition, the type of detector, which is the receiver of the fluorescence, have an important impact on the detection limit and measurement frequency. Selecting a high-performance

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detector with low noise can improve both the detection limit and the measurement accuracy [20].

✓ In-situ measurement of chl-a by autofluorescence is dependent on the physiological state of the algal cells. In general, dark adaptation is recommended for comparable measurements. For probes that are permanently installed for in-situ measurements in mesocosms, dark adaptation is not an option. Here, nighttime readings may be more comparable than readings from daytime. However, as the most classical measurements (e.g. HPLC) realized during the day, the comparison of the results could not be possible.

6.9. Nitrogen (Nitrate)

The continuous nutrient concentration monitoring in real time by in situ nutrient probes allows determining sources, sinks, and dynamics of different nutrients in natural environments and can be adapted for the mesocosm experiment [7]. Most in situ nutrient (nitrate, nitrite, phosphate, ammonia, etc.) analyzers automatically measure dissolved nutrients by “using wet chemical techniques of in-flow analysis based on standard laboratory analytical methods (spectrophotometry and fluorometry)” [7, p. 311]. These analytical probes are equipped with one or multiple reagent-delivering modules and standards, and one or multiple electro-optical detectors, number of which depends on the measurement of one or several nutrients.

✓ The frequency of measurement is affected by the amount of reagent and standard loaded in the probe [7].

✓ To avoid the drifts and the degradation of optics, reagents, calibration standards, and also biofouling in the sensor sampling line, frequent maintenance is necessary, which includes reagents and standards change, complete clean up and in situ recalibration of the probe [7]. Please see the next section for basic calibration recommendations.

The nutrient (ammonium, nitrate, and chloride) can also be measured by ion-selective electrodes (ISEs), which are commonly used in in situ water quality monitoring as they can be used “directly in the medium to be tested, have a compact size, and are inexpensive” [22].

6.10. Multi-probes (Sensors, profilers)

Multi-probes are the sensors that allow for the continuous monitoring of several common field parameters altogether. These sensors are available individually or bundled together into a multi-parameter sensor with several sensors attached to a single unit [23].

✓ Note that the accuracy of the temperature probe affects multiple other parameters in multi-probes (e.g., Oxygen, pH, conductivity).

The types and number of sensors that can be bundled in each sensor depend on the instrument model and manufacturer. CTD sensors (Conductivity, Temperature, and Depth) are a good example of a basic multiparameter sensor. CTDs measure pressure (rather than depth) based on the relationship between

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pressure and depth, which also involves water density and compressibility as well as the strength of the local gravity field. The output data of CTDs can be used to calculate salinity, density, and sound velocity. Aside from temperature, conductivity and depth, dissolved oxygen, pH, PAR and turbidity sensors are commonly bundled together into a multiprobe in mesocosm studies. YSI EXO Series, Hydrolab DS5X and Aanderaa Seaguard series are the commonly used multiparameter sensors throughout the Aquacosm community. YSI EXO series (ex. EXO2) allow the user to collect data from up to six user-replaceable sensors and an integral pressure transducer (YSI) for 17 parameters (temperature, conductivity, salinity, depth, pressure, pH, Oxygen Reduction Potential (ORP), dissolved oxygen, Turbidity, Chlorophyll a, Blue-Green Algae (Phycocyanin and Phycoerythrin), fDOM (CDOM), Ammonium, Nitrate, Chloride, Total Dissolved Gas (TDG) and Total Suspended Solids (TSS)). In addition, optionally, they have a bulkhead (made from titanium) port for a central wiper (or an additional sensor) and an auxiliary port on top of the sensor. Hydrolab Datasonde series (ex. DS5X) allow the user to collect data for 17 different parameters (temperature, conductivity, depth, pH, Oxygen Reduction Potential (ORP), dissolved oxygen (LDO and Clark Cell), Turbidity, Chlorophyll a, Blue-Green Algae, Rhodamine WT, Ammonium, Nitrate, Chloride, Total Dissolved Gas (TDG) and Ambient Light (PAR)) with up to seven sensor ports. It has a central cleaning system to minimize (bio)fouling of the sensors; DO, pH, ion-selective electrodes, chlorophyll, blue-green algae, rhodamine, and turbidity (See Figure 6-1). Aanderaa SeaGuard Series (ex. SeaGuard String) are commonly used in marine mesocosms. The SeaGuard String is designed to be connected to the SeaGuard String logger; which can be connected with up to 25 sensor nodes. In addition, up to 6 sensors can be mounted onto the Top-end Plate of the multiparameter instrument.

Figure 6-1. Hydrolab DS5X instrument design, a type of multiparameter sensor, commonly used in mesocosm studies Some researchers prefer to use the single temperature sensors together with temperature sensor bundled into a multiparameter (ex. Testo AG; Testo 108, Campbell Scientific; Temperature probes). Other than that, Underwater PAR sensors (LICOR; LI-192 and 193), Chlorophyll-a sensors (Sea Bird Scientific-Wetlabs; ECO Series) and Light spectrum sensors (Ocean Optics; USB Series Spectrometers) are commonly selected to be single parameter probes.

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7. Maintenance of the station and the equipment

To obtain the most accurate and most complete records possible; periodic verification of sensor calibration, maintenance of the equipment and the monitoring station, troubleshooting of sensors and data loggers (if applicable) should be performed regularly. Maintenance frequency Maintenance frequency generally is governed by the study objectives as well as the biofouling rate of the sensors, which vary by sensor type, hydrologic and environmental conditions [5].

✓ According to Wagner and colleagues (2006), the performance of temperature and specific conductance sensors tends to be less affected by fouling than DO, pH, and turbidity sensors [5].

For a high degree of accuracy, maintenance should be weekly or more often. In nutrient-enriched mesocosms and moderate to high temperatures, maintenance as frequently as every third day is recommended [5]. In addition, monitoring disruptions as a result of recording equipment malfunction, sedimentation, electrical disruption, debris, ice, pump failure, or vandalism also may require additional site visits.

● Satellite telemetry is recommended for sensors where lost records will critically affect research objectives. Satellite telemetry (if applicable) can be used to verify proper equipment operation on a daily basis and can help in recognizing and correcting problems quickly [5].

Maintenance actions (adopted from [5]): Daily maintenance acts (for sites equipped with telemetry)

✓ Daily review of sensor function and data download ✓ Battery (or power) check ✓ Deletion of spurious data, if necessary

Maintenance acts during weekly field visits ✓ Inspection of the site for signs of physical disruption ✓ Inspection and cleaning of the sensor(s) for fouling, corrosion, or damage ✓ Inspection and cleaning of the deployment tube ✓ Battery (or power) check ✓ Time check ✓ Regular sensor inspection at the field (Calibration check, explained in details in Section 8.1) ✓ Calibration of the field meter(s) ✓ Downloading of data

7.1. Regular Sensor Inspection at the Field

Sensor inspections are required to verify that a sensor is working properly. Field trips for sensor inspection will provide “an ending point for the interval of water-quality record” since the last visit and “a beginning point for the next interval of water-quality record” [5].

1. Record the initial sensor readings (1) in the mesocosm: The initial sensor readings (1) of the equipment are compared to readings from a calibrated field meter, which can provide a reasonable comparison basis and an indication of potential electronic calibration drift and

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fouling errors, ideally located at the same measuring point” in the mesocosm. The initial sensor reading will be the last data recorded since the previous inspection visit. ✓ Readings from a calibrated field meter should not be used in computations (exceptional:

the temperature sensors), they are only used to assess “cross-section variability and environmental changes that may occur while the monitor is being serviced”, as well as to detect the fouling and probable calibration drift (in case the environmental conditions are stable or slowly changing) [5].

2. Clean the sensors and recording equipment (if required)

✓ The sensor should be removed from water for servicing while the field meter remains in its place. First, the removed sensor should be inspected for any sign of fouling; such as chemical precipitates, stains, siltation, or biological growths. If any are observed, it should be recorded in the field notes (see Field Form) before cleaning.

✓ For all the sensors, during the cleaning process, care should be given to ensure that the electrical connectors are kept clean and dry.

✓ The sensors (and the recording equipment) should be cleaned according to the specifications provided by the manufacturer.

✓ Best practice advice on cleaning for each parameter is provided in Section 8 of this SOP.

3. Record the cleaned-sensor readings (2) in the mesocosm: The cleaned sensor is returned to the water for the cleaned-sensor readings (2). The cleaned-sensor readings, field meter readings and reading times should be recorded in the field notes. ✓ The difference between the initial sensor reading (1) and the cleaned-sensor reading (2)

is the sensor drift caused by fouling (including chemical precipitates, stains, siltation, or biological growth).

✓ One should note that difference might not be always representative due to change in growth or loss of organisms in time. Those should also be considered in calculations.

4. Do the calibration checks of sensors by using appropriate calibration standards (solutions):

The sensors should be removed from water for calibration checks when all readings are recorded. Calibration checks of the sensor are performed in calibration standard solutions. The cleaned-sensor readings (2a) in the calibration standard solutions are recorded in the field form. ✓ During field visits, calibration of all sensors should be checked with (two) standard

solutions that bracket the range of expected environmental conditions. A third standard solution can be used additionally near the ambient environmental conditions, before any adjustments are made to the monitor calibration.

✓ The difference between the cleaned-sensor readings in calibration standard solutions (2a) and the expected reading ((2) or the calibration criteria) in these solutions is the sensor (calibration) drift error.

✓ Best practice advice on calibration for each parameter is provided in Section 8 of this SOP.

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5. Re-calibrate the sensors if the readings are beyond the accuracy ranges: Field re-calibration

should be performed if the cleaned-sensor readings obtained during the calibration check differ by more than the calibration criteria (See Table 8-1). ✓ Spare monitoring sensors might be needed to replace the ones that fail calibration after

re-calibration, proper maintenance by the user and troubleshooting steps have been applied.

✓ Best practice advice on troubleshooting are provided in Section 10 of this SOP.

6. Record the final sensor readings (3) in the mesocosm: A set of initial readings (3) should be taken as the start of the new record.

✓ Under rapidly changing conditions (i.e., change in the parameter that exceeds the calibration

criteria within 5 minutes) or when measurements are fluctuating, the site inspection acts should be modified accordingly. For more details on sensor inspection at rapidly changing conditions, please refer to (Wagner and colleagues (2006) [5].

● Validation/ cross comparison: As a best practice advice, use your maintenance visits to collect other data to support the monitoring effort: e.g. Secchi disk and water temperature readings, water samples for chl-a extraction, nutrients, DOC, etc. For meteorological data, make an independent measurement of air temperature and a visual check on wind direction. While these measurements may seem unnecessary at the time they can easily be incorporated into your maintenance visits, and they are invaluable for confirming and strengthening patterns shown in the sensor data [24].

Table 7-1. Calibration criteria for continuous measurements (variation outside the value shown requires re-calibration)

Measurement Calibration Criteria Reference Respectively

Temperature ± 0.2 ºC ± 0.3 ºC

[5], [1] [25]

(specific) conductance ± 5 μS/cm or ±3 % of the measured value, whichever is greater ± 10% of reading

[5] [25] & [1]

Dissolved Oxygen ± 0.3 mg/L [5], [1] & [25]

Dissolved Oxygen (% saturation)

± 5% saturation [1]

pH ± 0.2 pH unit ± 0.5 pH unit ± 0.3 pH unit

[5] [25] [1]

Turbidity ± 0.5 turbidity unit or ± 5% of the measured value, [5]

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whichever is greater ± 10% of range

[25]

Underwater Light (PAR) ± 3 - 5% (for quantum sensors)

Depth

Chl-a Exceeding the accuracy of the sensor (the accuracy of the sensor is provided in the handbook provided by the manufacturer) ± 3% for signal level equivalents of 1 ppb rhodamine WT dye or higher using a rhodamine sensor

[25]

Light spectrum Each degree of separation from the NIST-calibrated light source introduces some uncertainty, yielding a total estimated uncertainty of within 10% for most Ocean Optics calibration light sources (a value that is typical for the industry).

[26]

Nitrogen Ammonium( NH4): ±10% of reading or ±2 mg/ L-N, whichever is greater Nitrate( NO3): ±10% of reading or ±2 mg/ L-N, whichever is greater

[19]

(±, plus or minus value shown; °C, degree Celsius; μS/cm, microsiemens per centimeter at 25 °C; %, percent; mg/L, milligram per liter; pH unit, standard pH unit; turbidity unit is dependent on the type of meter used)

7.2. Best Practice Advice on Sensor-specific Field Cleaning and Calibration

7.2.1. General Considerations

✓ The manual of the manufacturer should be the main source of information for calibration. For regular calibration frequency at the field site, manufacturer’s advice should be considered. Besides, calibrating individual sensors or multi-parameter sensors at least once a month is a common good practice advice. If recommended differently than this SOP, the manufacturer’s guidelines must be followed for both single and multi-parameter sensors.

7.2.2. Temperature sensors

Best Practice Advice on Cleaning: Commonly used temperature sensors (thermistors) can be cleaned with “a detergent solution and a soft-bristle brush” [5]. If recommended differently, the manufacturer’s guidelines on cleaning procedures must be followed for both single and multi-probes. Best Practice Advice on Calibration: Several choices of calibrations can be explored depending on availability, facility and time. The temperature sensors are quite durable and accurate and show low-drift characteristics. Temperature accuracy is especially important because of the effect of temperature on the performance of other

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sensors. Calibrating temperature sensors, a calibration thermometer (a non-mercury calibration thermometer can be used in the field to check or monitor temperature readings) and a temperature-controlled water bath should be used [5].

✓ The calibration thermometers should only be used for calibration rather than as field thermometers [6].

An annual five-point calibration methodology is recommended over the temperature range of 0 to 40°C [5]. In addition, two-point calibration checks over the maximum and minimum expected annual temperature range should be carried out at least every four months (three or more times per year) for thermistors [5].

✓ Quarterly or possibly monthly calibration can be required if the thermometer is in heavy use; exposed to thermal shock, an extended period of direct sunlight, aggressive chemical solutions, or extreme shifts in temperature.

A sample methodology for calibrating field thermometers with and without a commercial refrigerated water bath is provided in Appendix A.

7.2.3. pH Sensor

Best Practice Advice on Cleaning: In general, the only routine cleaning needed for (the body of) the pH electrode is rinsing thoroughly with deionized water. However, in cases of extreme fouling (or contamination), the manufacturer’s instructions on cleaning must be followed [5].

Best Practice Advice on Calibration: According to Wagner and colleagues (2006), “two standard buffer solutions bracketing the expected range of environmental values are used to calibrate a pH electrode, and a third is used as a check for calibration range and linearity of electrode response” [5]. Ionic buffer solutions (for pH 4, 7, and 10) are commonly used in calibration of the pH instrument system. Buffers can resist changes to the specific pH value. For that reason, the accuracy of the buffer solutions will have an impact on the accuracy of pH measurements.

✓ Use of buffer solutions with lower-than-standard molarity, in combination with the pH electrode with a low ionic-strength, are recommended for dilute water with conductivity lower than 100 µS/cm [8].

✓ Use of buffer solutions with a higher-than-standard molarity is recommended for pH measurements in high ionic-strength waters having conductivity greater than 20,000 µS/cm [8].

As a best practice advice, buffer bottles must be capped firmly after use to prevent evaporation and contamination from atmospheric CO2.

✓ Sensitivity of standard buffers to CO2 contamination: pH 10 buffer > pH 7 buffer > pH 4 buffer. ✓ Variation of buffer pH with change in temperature: pH 10 buffer > pH 7 buffer > pH 4 buffer.

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Prior to calibration, the temperature of the buffer solutions should be as close as possible to the mesocosm. In order to ensure that, upon arrival at the field site, tightly capped buffer solutions should be immersed in the mesocosms to allow time for temperature equilibration, usually 15 to 30 minutes.

✓ Waters with specific conductance values less than 100 µS/cm (low-ionic strength water) and greater than 20,000 µS/cm (high-ionic strength water) require special buffers and pH sensors. The extra preparations, precautions, and troubleshooting steps necessary to use these buffers and sensors to measure low- or high-ionic strength waters are described in Ritz & Collins (2008) [8].

A sample calibration process for pH meters, including a wide range of available equipment, is compiled from Wagner et.al. (2006) [5] and Ritz and Collins (2008) [8], which is provided in Appendix B of this SOP.

7.2.4. Optical DO Sensors

DO sensors are accessible from various manufacturers. As there are varieties DO sensors among manufacturers, which are different in the instrument design and instructions for use, calibration and maintenance, it is essential that one shall read the manual provided by the manufacturer thoroughly in addition to the guidance provided in this SOP. Manufacturer’s guidelines must be followed, if these recommendations deviate from the SOP. Best Practice Advice on Cleaning: Silt, outside the sensor, can be removed with a soft-bristle brush and the membrane should be wiped with a lint-free cotton swab. DO sensors, then, should be rinsed with de-ionized water [5]. Best Practice Advice on Calibration:

✓ The DO sensors should be temperature compensated. Because of the potential influence of altitude and temperature, the DO probe(s) should be calibrated at the field.

✓ It should be noted that luminescent (optical) DO sensors are mostly calibrated by the manufacturer, and the manuals indicate that calibration may not be required for up to a year. When calibrated, the user should follow the manufacturer’s guidance. Regardless of the manufacturer’s claims, the user must verify the correct operation of the sensor in the local measurement environment. The standard protocol for servicing should be used for luminescent-based DO sensors to quantify the effects of fouling and calibration drift. Rounds and colleagues advise the users to make frequent calibration checks and to recalibrate as frequently as required to meet the specific data-quality objectives [9]. Recalibration should not be necessary if calibration checks show the sensor to be in agreement with the calibration criteria [5].

DO sensors must be calibrated to 100-percent DO saturation and checked with a zero DO solution to provide “an indication of sensor-response linearity” [5].

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Three steps to calibration, as compiled from Wagner and colleagues (2006) [5] and Rounds and colleagues (2013) [9], are provided in Appendix C of this SOP.

7.2.5. Conductivity Sensor

Best Practice Advice on Cleaning: First, the manufacturer’s recommendations must be checked before using acid solution or solvents on sensors. Radtke and others (2004) recommend cleaning specific conductance sensors thoroughly with deionized water [10]. A detergent solution can be used to remove oily or chemical residues. In addition, a solvent or hydrochloric acid (5%) can also be used for dipping the sensor to be cleaned.

✓ The sensors can be soaked in detergent solution for hours without damage, however, care should be taken when cleaning with an acid solution. The sensor must never be in contact with acid solution for more than a few minutes.

✓ Sensors made of carbon and stainless-steel can be cleaned with a soft brush, but platinum-coated sensors must never be cleaned with a brush.

Best Practice Advice on Calibration: Calibration and operating procedures differ, depending on the instrument. The manufacturer’s instructions should be checked before starting calibration. The general procedures, that apply to most of the instruments used in conductivity measurements, described in Radtke and colleagues (2004) [10] and [5] are summarized in Appendix D of this SOP. ✓ For a cup-type cell, calibration and measurement procedures described for the dip-type cell apply;

the only difference is that standards are poured directly into the cup-type cell. ✓ “When using a dip-type cell, do not let the cell rest on the bottom or sides of the measuring

container” [10, p. 7].

7.2.6. Underwater Light (PAR) Sensor

Best Practice Advice on Cleaning: Underwater light sensors are prone to biofouling. Accordingly, they should be cleaned frequently for accurate measurements. The user should check the manufacturer’s guidelines for the best cleaning material. As a best practice advice, debris, dust and other organic deposits on the PAR sensor lens should be removed with water or window cleaner. One must not use an abrasive cleaner on the lens. The salt accumulated on the sensor lens (especially in marine mesocosm) should be dissolved with vinegar and removed from the lens with the help of a soft cloth or cotton swabs.

● Recording of pre- and post-cleaning results will help define the rate of sensor fouling, and the optimal time between cleanings” [24].

Best Practice Advice on Calibration:

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Most of the PAR sensors are already calibrated prior to deployment at the factory and most of them are recalibrated by the manufacturers when needed. For calibration, please refer to the manufacturer’s instructions or contact with the manufacturer. Quantum sensors (ie. LI-COR; LI-192 Underwater quantum sensor), are usually calibrated using a standard light source (i.e. working standard quartz halogen lamps), which have been calibrated against reference standard lamps. It is advised that every two years, the sensors should be calibrated by the manufacturer.

● The absolute calibration specification for quantum sensors is ± 5% (typically ± 3%). As a best practice advice, the calibration can be verified using the Clear Sky Calculator at www.clearskycalculator.com [27], which is an online calculator that reports theoretical photosynthetic photon flux (PPF) at any time of day at any location in the world on a cloudless day (it is most accurate at noon in spring and summer with completely clear skies).

7.2.7. Turbidimeter

Best Practice Advice on Cleaning: Turbidity sensors are vulnerable to fouling especially in mesocosms high in sediment, algae accumulation, larvae growth or other biological and chemical elements. Errors or drifts might occur also due to outgassing air bubbles. The mechanical cleaning devices, wiper and shutters (most of the time, equipped with the sensor), can be used to remove or prevent any accumulation.

✓ The probable algae accumulation on the wiper pad could prevent complete cleaning. Again, accumulation of inorganic or organic debris in shutters could prevent operation of the sensor efficiently. For that reason, the wiper pad or other cleaning device also should be inspected for wear, and cleaned or replaced if necessary.

As a best practice advice, the optic lens of the turbidity meter should be cleaned carefully with alcohol using a soft cloth (or as recommended by the manufacturer), rinsed three times with turbidity-free water, and carefully dried.

✓ If the readings are unusually high or erratic during the sensor inspection, entrained air bubbles may be present on the optic lens and must be removed.

Best Practice Advice on Calibration: Before starting calibration, be certain that the probe is cleaned and free of debris. Solid particles left will contaminate the standards during the calibration process and cause either calibration errors and/or inaccurate field data. It should also be noted that turbidity meters should be calibrated directly rather than by comparison with another sensor. The verification of calibration should be done with the same or similar technology, at least in terms of the light source and the detection angle. Field inspection or calibration of the turbidity sensor is made by using approved calibration turbidity and calibration verification solutions (calibrants) and by following the manufacturer’s calibration instructions.

● The use of standards other than those mentioned in the manufacturer’s guidelines will

http://www.clearskycalculator.com/

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result in calibration errors and inaccurate field readings. Calibration of the turbidity sensor should be checked in three standard solutions (although some instruments may be limited to calibration with only one or two standards) before any adjustments are made.

✓ If instrument calibration allows only a two-step process, two primary standard solutions covering the expected range must be used for calibration and a third midpoint standard solution is used to check for linearity. Similarly, if the instrument calibration requires only turbidity-free water and one standard solution, another midpoint standard solution must be used to check for linearity.

Turbidity calibrants can be instrument specific. Be careful to check the manufacturer's instructions. Use of calibrants with instruments for which they are not designed can introduce significant errors. The three types of turbidity calibrants generally recommended are (as summarized in Wagner and colleagues (2006), [5]): (1) reference turbidity solutions, which are calibrants that are synthesized reproducibly from traceable raw materials by a skilled analyst. The reference standard is fresh user-prepared formazin. (2) calibration turbidity solutions, that are used in calibration must be traceable and equivalent to the reference turbidity calibrants. Acceptable calibration turbidity solutions include commercially prepared formazin, stabilized formazin, and styrene divinylbenzene (SDVB) polymer standards.

✓ Formazin-based calibrants can be diluted by using a dilution formula; however, errors may be introduced during the dilution process, thus reducing the accuracy of the standard solution.

✓ Formazin-based calibrants are temperature dependent, and accurate readings may be difficult to obtain during field conditions. Anderson (2005) [15] suggests that the effect of thermal fluctuations can be minimized by calibrating the instrument at room temperature in an office laboratory using a reference or calibration turbidity solution. Instrument calibration can then be checked at the field site by using a calibration verification calibrant.

(3) calibration verification solutions and solids may include, but are not limited to, calibration turbidity solutions; however, calibration verification calibrants that are sealed or solid materials must not be used to adjust instrument readings [15].

✓ Before placing the sensor in a calibration verification calibrant, the sensor must be cleaned, rinsed three times with turbidity-free water, and carefully dried. Turbidity-free water is prepared as described by Anderson (2005) [15] and Wagner and colleagues (2006) [5].

7.2.8. Depth Sensor

Best Practice Advice on Calibration: Calibration of the depth sensors should be done in the atmospheric zero pressure and with respect to the local barometric pressure. For the calibration of depth sensors in multi probes, the user manual of the multi probes as well as the software provided by the manufacturer should be visited. For instance, YSI EXO2 multi probes are

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equipped with a non-vented strain gauge for measuring the depth. In order to calibrate the depth, the calibration software provided with the multi probe should be used.

✓ It is important to note that, for the calibration of depth sensors for both vented or non-vented, the sensor should be in air and not immersed in any solution.

7.2.9. Fluorometers (Chl-a)

Best Practice Advice on Cleaning: Chlorophyll fluorescence is vulnerable to variations due to the environment changes, biofouling and instrument design [28]. In order to improve and evaluate the accuracy, reliability, and stability of a fluorometer, the optical surface should be inspected frequently. If needed, the optical surface should be cleaned with a non-abrasive, lint-free cloth. One should take care to prevent scratches and damage to the sensing window. Best Practice Advice on Calibration: Pure Chlorophyll, dyes, algae cultures, and field water samples can be used as the calibration standards. Pure Chl-a or liquid dyes such as Fluorescein Sodium Salt, Rhodamine WT Red, Rhodamine B, and Basic Blue 3 offered by various suppliers, can be used to calibrate fluorometers in the laboratory by standard methods. Solid fluorescent materials, as trialed by Earp and colleagues (2011) are recommended as reference standards for field calibration [28].

● Zeng and Li (2015) reminded that the dyes as well as pure or extracted Chl-a, have different fluorescence intensity from the in vivo chlorophyll cell in natural populations [20]. They recommended; in order to improve the accuracy among various species, pure phytoplankton cultures should be used as a calibration sample (as in [29] and in [30]). Lawrenz and Richardson (2011) also recommended the use of “natural communities collected from the site of interest” for calibration of in situ fluorometers [31].

1 or 2-point calibration is recommended by most of the manufacturers. The probe/sensor can be calibrated according to the measurement unit used in monitoring (μg/L or RFU; relative fluorescence unit from 0 - 100%). The RFU method is recommended if grab samples are also used to post-calibrate in vivo chlorophyll readings. For both measurement units, 2 reference standards should be used in calibration:

(1) clear (deionized or distilled) water (either 0 μg/L or 0%RFU) (2) the standard solution with a known chlorophyll content OR dye solutions (not recommended in

field use; See the note in Calibration standards) ✓ Follow the manufacturer’s manual for the recommended standard solution/s and their

predetermined chlorophyll content/ratio. The general procedures, that apply to most of the instruments used in situ chlorophyll measurements are summarized in Appendix E. However, Zeng and Li (2015) reminded that “the general and manufacturer calibration procedures are too simple to meet scientific requirements; furthermore,

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calibration must be verified regularly due to species and environment variation within space and time and lamp and sensor performance degradation over time” [20]. For that reason, fluorometers should be calibrated with multistep, pre- or post-calibration procedures, and through special methods according to different situations. For more information and steps to the recommended calibration procedure, please visit Zeng and Li (2015) [20].

7.2.10. ISE probes for measuring nitrogen

Best Practice Advice on Cleaning: ISE-probes are (commonly) equipped with an automatic cleaning system using pressurized air, which proved to work reliably [32]. ● It is recommended to carry out a visual check of the probe before any calibration is started – if

necessary the probe should be cleaned manually [32]. Best Practice Advice on Calibration: A grab sample or a standard of known concentration can be used in calibration. The calibration standards need to have sufficient ion activity; TISAB-solutions (Total Ionic Strength Adjustment Buffer) can be used to adjust the ionic strength of the calibration samples [32].

✓ Supplementary sensors like temperature or pH should be calibrated before the ISE is calibrated, so that any errors of the automatic temperature or pH-compensation are corrected before the ISE-calibration is started [32].

✓ Sample readings should be taken after sensors have fully stabilized. Calibrating in a continuously stirred solution from 1 to 5 minutes has shown to improve sensor performance (YSI EXO2, 2018).

✓ For best performance sensors should be calibrated as close to the expected field conditions as possible (YSI EXO2, 2018).

✓ In multiparameter sensors, remove the ISEs to avoid exposing them to conductivity standards, Zobell solution, pH buffer, or any solution with significant conductivity, as exposure to such solutions will reduce data quality and response of the sensors.

Ammonium or nitrate sensors can be calibrated to one, two or three points. If preferred, a single-point calibration procedure can be followed. However, the concentration of the measurement-ion at time of the calibration should be in the upper half of the concentration range at the measurement location. In case the maximum concentration of the measurement-ion is below 5 mg/l a two-points calibration should be carried out in order to consider non-linearities in the lower measurement range” [32]. The 3-points calibration method assures maximum accuracy and best performance of ISE sensors.

For the two- and the multiple-points calibration the probe has to be removed from the mesocosm and put into a bucket with a grab sample or with a standard of known concentration.

✓ Using a grab sample of the actual measurement location has the advantage that influences due to disturbance-ions at the time of sampling are compensated automatically. By using standards, reference measurements can be omitted. However, a single-point calibration in-situ has to follow any calibration in standards in order to consider influences from disturbance-ions.

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For the two-points calibration the calibration measurements shall be carried out at approximately 20% and 80% of the concentration range of the measurement-ion at the measurement location and an approximate concentration ratio of the calibration samples of 1:10. A multiple-points calibration can be applied in case the concentration range at the measurement location has a wide span. Especially in the lower concentration range of ammonium (

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8. Overcoming Drift

8.1. Common Causes

Drift can be a major issue for deteriorating measurement quality. If the sensor is used in longer-term deployments, drift is almost certain to occur. The extent of the drift will vary depending on the age of the probe, the flow rate at the site, and the quality of the water as well as the quality of the instrument. The ion-selective electrodes have the greatest tendency to exhibit calibration drift over time. Optical DO sensors are stable and robust in maintaining calibration over long-term deployment and over a wide range of environmental conditions. Accordingly, sensor (calibration) drift over time is minimal when the sensor is kept clean. Chlorophyll fluorescence (fluorometers) is also vulnerable to sensor drift and calibration rigor [28]. Errors or drifts might occur in turbidimeters due to outgassing air bubbles. Signal drift can be a major concern with on-line management systems because continuous immersion of the sensors (especially ISEs) in water causes “electrode degradation, affecting the stability, repeatability, and selectivity over time” [22].

✓ It should be noted that, some sensors may need regular manufacturer’s calibrations at a regular interval. Otherwise, a user calibration may highlight a drift issue, in which case, sensors can be sent back to the factory [24].

8.2. Elimination strategies

Sensor drift, in general, can be ameliorated by frequent calibration of the sensors manually. In addition, if required, the authorized person should get in touch with the manufacturer as soon as possible. To improve and evaluate the accuracy, reliability, and stability of a fluorometer and to eliminate the sensor drift, the optical surface should be inspected periodically. In turbidimeters, the mechanical cleaning devices; wiper and shutters (equipped with the sensor commonly) can be used to remove or prevent any accumulation of air bubbles. Sensor drift in the ion-selective electrodes can be ameliorated by using an ISE that has been conditioned in the sample matrix, typically overnight, to produce a stable, reproducible and fast responding ISE, also minimizing sample contact times and the concomitant ISE release of analyte into the sample” [33]. In addition, the nitrate electrode drift can be fixed by “automatic calibration” [4]. For all monitoring studies using ion-selective electrodes, the user should acquire a few grab samples during the deployment for analysis in the laboratory or with another sensor that has been recently calibrated [19].

✓ Collecting grab samples from the site is encouraged to correct for drift [19].

9. Troubleshooting of sensors and record-keeping equipment

When a field parameter cannot be calibrated with specified methods, one shall determine the problem source. If the problem is due to the sensor or the monitor, the necessary corrections should be made to

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ensure the monitor is operational. For instance, spare sensors should be carried at every field visit so that troubleshooting can be accomplished at the time of the visit. Accordingly, need for extra trips and record loss can be prevented [5]. Some of the common problems that can be encountered in the field (and solutions) are listed in table 10-1. Table 10-1. Common troubleshooting problems and likely solutions (adopted from [5])

Symptom Possible Problem Likely Solution

Temperature

Thermistor does not read accurately

Dirty sensor Clean sensor

Erratic monitor readings

Poor connections at monitor or sensor Tighten connections

Monitor slow to stabilize

Dirty sensor Clean sensor

Readings off scale Failure in electronics Replace sensor or monitor

Dissolved oxygen

Meter drift or excessive time for monitor to stabilize

- Fouled sensor

- Wait for temperature equilibration - Clean or recondition - Check for obstructions or replace

Erratic monitor readings

- Bad connection at monitor or sensor

- Fouled sensor

- Tighten connections - Clean or recondition

Monitor will not zero - Zero-DO solution contains oxygen - Zero-DO solution is old

- Add additional sodium sulfite to zero-DO solution - Mix a fresh solution

Conductivity

Will not calibrate - Standard solutions may be old or contaminated

- Electrodes dirty - Air trapped around sensor - Weak batteries

- Use fresh standard solutions - Clean with soap solution - Thrust sensors up and down and tap gently to expel air - Replace batteries

Erratic monitoring - Loose or defective connections - Tighten or replace connections

Monitor requires frequent calibration

- Broken cables - Replace cables - Replace monitors

pH

Meter will not calibrate

- Buffers may be contaminated - Replace buffers

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- Faulty sensor - Replace sensor

Slow response time - Dirty sensor bulb - Water is cold or of low ionic

strength

- Clean sensor - Be patient

Erratic readings - Loose or defective connections - Defective sensor

- Tighten connections - Replace sensor

Turbidity

Unusually high or erratic readings

- Entrained air bubbles on the optical sensor

- Damaged sensor - Dirty sensor - Water in connections

- Follow manufacturer’s directions - Replace sensor - Clean, following manufacturer’s directions - Dry connector and reinstall

10. Quality Assurance and Quality Control (QA/QC) of high frequency measurement

Quality assurance during high-frequency measurements can be ensured by the following activities:

1. Timely and accurate documentation of field information in electronic and paper records: It is recommended to keep a log book for each field instrument (if single probes are available) to record the instrument repair, maintenance, and calibration history. The log book is recommended to be made of a waterproof paper/material [34].

2. Use of manufacturer’s handbook, procedures and protocols to ensure sample integrity and data quality: The manufacturer of the instrument(s) used for high frequency measurements should be the primary source of information on the use, calibration, maintenance, troubleshooting and storage of sensors. To ensure quality assurance and control, one shall read the manufacturer’s guidelines thoroughly first. The recommendations provided in this section are general guidelines and best practice advice related, prepared to ensure the level of quality assurance within the AQUACOSM community.

3. Training of the personnel in charge in measurement techniques and the collection of quality-control samples.

4. Second- or third-party auditing of such records, such as inter-(laboratory) calibrations. Quality control during high-frequency measurements can be checked by the following activities:

1. Records of replicate measurements: In a QA/QC program for high frequency measurements, determination of the “true value” of the measurement is fundamental [25]. The true value can be defined by applying a second means of measuring with another instrument that is regularly serviced and calibrated and only used for quality control purposes.

✓ Calibration standards may also be used as a reference value [25].

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Other QA/QC applications at the field (adopted from [25]) “When real time or remote instruments are first placed into the field, accompanying measurements should be taken with a handheld water quality instrument or by some other means. These field measurements – which are sometimes paired samples taken for laboratory analyses – serve as QC points. These independent field measurements (intermediate checks) are extremely important as they are the only check for the accuracy and performance of the real time or remote water quality measurement. Each time an instrument is removed and/or replaced, another complete set of field measurement should be collected” [25, p. 3]. Intermediate checks for a deployed multi-probe (eg., Hydrolab) should include (adopted from [1]): 1. “Upon deployment, field staff should collect a Winkler sample and, for other parameters, a check measurement with another” instrument (if available, can be a hand-held instrument).

✓ “For DO, Winkler samples should be collected to bracket the expected high and low points for DO”.

2. For the other parameters, checks with another instrument” (if available, a hand-held meter) should be conducted over the expected range of the parameter being measured.

✓ “At a minimum, one intermediate check should be completed for a short deployment (one week or less)”.

3. The number of intermediate check measurements should be defined prior to the deployment and due to the length of deployment [1]. 4. At the end of deployment period, a final Winkler sample for DO should be collected and other check measurements should be completed with an equivalent hand-held meter. 5. “This field check regime will provide a minimum of three checks per deployment and help identify if instrument drift occurs” [1].

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11. Appendices

Appendix A: General Procedures for Calibration of Field Thermometers

To calibrate field thermometers when a commercial refrigerated water bath is available (Paraphrased from [6]):

1. Precool the sensor of the thermometer/thermistor being checked (field thermometer) to 0°C by immersing it in a separate ice/water bath. 2. Immerse the field and calibration temperature sensors in the refrigerated bath with a water temperature of approximately 0°C. 3. Position the temperature sensor(s) so that they are properly immersed and the scales can be read. Stir the water bath and allow at least 2 minutes for the thermometer readings to stabilize. 4. Without removing the temperature sensor from the refrigerated water bath, read the field thermometer(s) to the nearest graduation (0.1 or 0.5°C) and the calibration thermometer to the nearest 0.1°C.

a. Take three readings within a 5-minute span for each field thermometer. b. Calculate the mean of the three temperature readings for each field thermometer and compare its mean value with the calibration thermometer. c. If a liquid-filled field thermometer is found to be within ±1 percent of full scale or ±0.5°C of the calibration thermometer, whichever is less, set it aside for calibration checks at higher temperatures. d. If a field thermistor is found to be within ±0.2°C of the calibration thermometer, set it aside for calibration checks at higher temperatures. Troubleshooting steps must be taken. If troubleshooting fails, the sensor should be returned to the manufacturer for proper calibration, repair, or replacement [5].

5. Repeat steps 1–4 in 25°C and 40°C water. Keep the bath temperature constant. Check the thermistors at two or more additional intermediate temperatures (for example, 15°C and 30°C). 6. Tag the acceptable and calibrated thermometers/thermistors with calibration date and certifier’s initials. To calibrate field thermometers when a commercial refrigerated water bath is not available (Paraphrased from [6]): For the 0°C calibration 1. Freeze several ice cube trays filled with deionized water. 2. Fill a 1,000-milliliter (mL) plastic beaker or Dewar flask three fourths (3/4) full of crushed, deionized ice. Add chilled, deionized water to the beaker. Place the beaker of ice/water mixture in a larger, insulated container or Dewar flask. Place the calibration thermometer into the ice/water mixture and make sure that the temperature is uniform at 0°C by stirring and checking at several locations within the bath. 3. Precool the sensor of the field thermometer(s) to 0°C by immersing in a separate ice/water bath. 4. Insert the field thermometer(s) into the ice/water mixture. Position the calibration and field thermometers so that they are properly immersed, and the scales can be read. Periodically stir the

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ice/water mixture and allow at least 2 minutes for the thermometer readings to stabilize. 5. After the readings stabilize, compare the temperature of one field thermometer at a time with that of the calibration thermometer. Without removing the temperature sensor(s) from the test bath, read the field thermometer(s) to the nearest graduation (0.1 or 0.5°C) and the calibration thermometer to the nearest 0.1°C.

a. Take three readings for each thermometer within a 5-minute span. b. Calculate the mean of the three temperature readings for each thermometer and compare its mean value with the calibration thermometer. c. If the field liquid-filled thermometer is found to be within ±1 percent of full scale or ±0.5°C of the calibration thermometer, whichever is less, set it aside for calibration checks at higher temperatures. d. If the field thermistor is found to be within ±0.2°C of the calibration thermometer, set it aside for calibration checks at higher temperatures.

For the “room temperature” calibration (25°C ) 1. Place a Dewar flask or container filled with about 3.8 liters (1 gallon) of water in a box filled with packing insulation. (A partially filled insulated ice chest can be used for multiparameter instruments.) Place the calibration container in an area of the room where the temperature is fairly constant (away from drafts, vents, windows, and harsh lights). 2. Properly immerse the calibration and field thermometer(s) in the water. Cover the container and allow the water bath and thermometers to equilibrate. 3. Stir the water and, using the calibration thermometer, check the bath for temperature uniformity. Repeat this every 2 hours. It may be necessary to let the bath equilibrate overnight. 4. Compare one field thermometer at a time against the calibration thermometer, following the procedures described above in step 5 for the 0°C calibration. For each temperature that is greater than 25°C 1. Warm a beaker of 1,000 mL or more of water to the desired temperature (for example, 40°C) and place it on a magnetic stirrer plate. 2. Follow the procedures described above in step A5 for the 0°C calibration.

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Appendix B: General Procedures for Calibration of pH Sensors

Summary of calibration process for pH meters, including a wide range of available equipment, is compiled from Wagner and colleagues (2006) [5] and Ritz and Collins (2008) [8] as follows:

1. Rinse the pH sensor, thermistor or thermometer, and calibration cup with pH-7 buffer solution. 2. Pour the fresh pH-7 buffer solution into the rinsed calibration cup. Immerse the sensor/probe

into the solution, making sure the sensor’s glass bulb is in solution by at least 1 cm. ● Allow the instruments to equilibrate for at least 1 minute for temperature equilibration

before proceeding. 3. Measure and record the temperature, pH, and associated millivolt reading (if available), along

with lot numbers and expiration dates of the pH buffers. This standardization process is repeated with fresh pH-7 buffer solution until two successive values of the temperature-adjusted pH-7 readings are obtained.

4. Rinse the pH sensor, thermistor or thermometer, and calibration cup with de-ionized water. Repeat the standardization process with a pH-4 or pH-10 buffer solution to establish the response slope of the pH sensor.

5. Use for instance, pH-4 or pH-10 buffer to establish the slope of the calibration line at the temperature of the solution.

✓ A buffer that brackets the expected range of pH values in the environment should be selected.

6. Record the second temperature-corrected pH value, temperature, millivolt readings, lot numbers, and expiration dates. Rinse the pH sensor, thermistor or thermometer, and calibration cup are with de-ionized water again.

7. The pH-7 buffer solution is then used to rinse, fill, and check the pH-7 calibration measurement. If the pH sensor reading is 7 ±0.1 pH units (as a correctly calibrated pH sensor can accurately measure pH to ±0.2 pH unit, See Table 8-1) the slope adjustment has not affected the calibration. If the accuracy standard is not met, the calibration and slope adjustment steps must be repeated.

✓ If re-calibration and troubleshooting steps fail, the pH sensor or monitoring probe must be replaced.

8. Once the slope-adjustment step is completed satisfactorily, the third buffer solution can be used as a check for calibration range and linearity of electrode response.

● The temperature and pH values are read and recorded along with the lot numbers and expiration dates of the pH buffers; however, the ±0.1 pH accuracy should not be expected to be achieved over the full range from pH-4 to pH-10 for a monitoring sensor. The third buffer should be within ±0.2 pH unit value [5].

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Appendix C: General Procedures for Calibration of DO Sensors

Three steps to calibration, as compiled from Wagner and colleagues (2006) [5] and Rounds and colleagues (2013) [9], are as follows:

Step 1. Calibration of a DO meter at 100-percent oxygen saturation is made by adjusting the meter reading for air saturated with water vapor to a value obtained from a DO solubility table. One or two-points calibration can be carried out to ensure 100% saturation oxygen environment, followed by calibration at zero DO.

✓ The two-points calibration adds complexity to the calibration process and is not recommended by all manufacturers of optical sensors. Be sure first to understand the instrument capabilities and then determine the best course of action for your field work.

Step 2. A zero-DO sensor-performance check should be carried out. For that reason, the zero-DO sodium sulfite solution1 should be prepared.

✓ Before immersing sensor in the zero-DO solution, the wiper (if applicable) should be removed from the unit to avoid saturating it with the sodium sulfite solution. Then, the sensor and the wiper should be rinsed thoroughly and then reinstalled.

✓ Multiple and thorough rinses with deionized water are necessary to restore the sensor to proper operating condition and prevent bias to subsequent measurements.

Step 3. Calibration of DO sensors: Air-calibration chamber in air, calibration with air-saturated water, air-calibration with a wet towel and air calibration chamber in water methodologies were explained in details in [9]. As mostly recommended by the manufacturers for optical sensor calibration, calibration with air-saturated water method is explained in details in this SOP.

Calibration with air-saturated water:

This method is considered to provide the best accuracy for calibration of optical sensors. The calibration should be done while the instrument is kept in water that is saturated with oxygen at a known temperature and ambient atmospheric pressure.

1. Required Equipment (as adapted from [9]): a. 5-gallon (around 19 liters) bucket, three-quarters full of tap water, or manufacturer-

provided aeration chamber b. 10-gallon (around 38 liters) aquarium air pump with two outlets c. 10-foot-length (around 300 cm) of aquarium pump tubing d. 2 X Gas-diffusion (air) stones

2. The methodology (as adapted from [9]): 1. Attach the pump tubing to the pump and then the two air stones to the ends of