Grant Agreement no : 614002 · Grant Agreement no : 614002 SCHeMA INTEGRATED IN SITU CHeMICAL MAPPING PROBE Collaborative project OCEAN.2013-2: Innovative multifunctional sensors

Grant Agreement no : 614002

SCHeMA

INTEGRATED IN SITU CHeMICAL MAPPING PROBE

Collaborative project

OCEAN.2013-2: Innovative multifunctional sensors for in situ monitoring of marine environment and related maritime activities

D 8.2 – Adaptation/improvement of laboratory based techniques

Due M24 (30th September 2015)

Actual submission date: 2.10.2015

Start date of project: October 1

st, 2013 Duration:48 months

Organisation name of lead contractor for this deliverable: UBX Revision 1.0

Project co-funded by the European Commission within the Seventh Framework Programme (2007-2013)

Dissemination Level

PU Public 10.03.2017

PP Restricted to other programme participants (including the Commission Services)

RE Restricted to a group specified by the consortium (including the Commission Services)

X

CO Confidential, only for members of the consortium (including the Commission Services)

Deliverable D8.2

Adaptation/improvement of

laboratory based techniques

RE: Restricted to the members of the SCHeMA consortium and the EU Commission Services

2.10.2015 RE: SCHeMA consortium members & EU Commission Services

SCHeMA – 614002_ D8.2 3

Project Number : 614002 Project Title : SCHeMA Deliverable Type : RE

Deliverable Number : D8.2 Title of Deliverable : Adaptation/improvement of laboratory based techniques Nature of Deliverable : Report Contractual Delivery Date : 30

September 2015

Actual Delivery Date : 2 October 2015 Contributing WPs : WP8, WP9 Author(s) : Jörg Schäfer – UBX Melina Abdou – UBX Emanuele Magi - UNIGE-IT Michela Castellano - UNIGE-IT Paolo Povero - UNIGE-IT

Abstract

SCHeMA’s overall aim is to develop, apply and field validate an autonomous system for

monitoring marine ecosystems and water quality. A range of pollutants will be detected by the

autonomous in situ system. The different partner laboratories are equipped with a wide range of

analytical techniques which will be used for laboratory inter-comparison and validation of the

analytical and technical developments. In fact parallel to in situ sensor-based monitoring, samples

will be collected and the same parameters as the SCHeMA system acquisition will be analysed with

established (standard) laboratory analytical methods. Therefore this deliverable consists in a review

of the adapted/improved established/alternative laboratory based techniques which will provide

validation and quality control of the sensor measurements. Laboratory-based validation techniques

are presented for the different sensor target pollutants: i) nutrients (spectrometry, ion

chromatography); ii) species relevant to the carbon cycle (commercial pCO2 probe and alkalinity

titration); iii) dissolved arsenic (Flow-Injection Analysis System for Absorption Spectrometry; FIAS-

AAS); iv) dissolved mercury (Gas Chromatography coupled to Inductively Coupled Plasma Mass

Spectrometer; GC-ICP-MS); v) dissolved trace metals (solid-liquid extraction followed by ICP-MS

detection); vi) biotoxins (High Performance Liquid Chromatography coupled to Fluorescence

Detection; HPLC-FLD); vii) algal species (inverted-microscope method); viii) volatile organic

carbon species (GC-MS). This deliverable aims at constituting a reference for laboratory protocols of

SCHeMA system parallel measurements in the field.

Keyword list

Laboratory-based techniques; ion chromatography; spectrometry; alkalinity titration; FIAS-AAS; GC-ICP-

MS; solid-liquid extraction; HPLC-ICP-MS; HPLC-FLD; inverted-microscope; GC-MS.


SCHeMA – 614002_ D8.2 4

Table of Content

1 Introduction 5 2 Laboratory-based validation methods for the different parameters measured by the

SCHeMA system 6 2.1 Nutrients: nitrite, nitrate, phosphate 6

2.1.1 Dishware preparation, sampling, sample handling and storage ......................................... 6 2.1.2 Laboratory protocol for measurement ............................................................................... 6 2.1.3. Performance check report ................................................................................................. 9

2.2 Species relevant to the carbon cycle (carbonate and carbon dioxide) 9 2.3 Dissolved arsenic (As(III), As(V) and As total) 12

2.3.1 Dishware preparation, sampling, sample handling and storage ....................................... 12 2.3.2 Laboratory protocol for measurement ............................................................................. 12 2.3.3 Performance check report ............................................................................................... 13

2.3.4 Improvement/adaptation of laboratory technique ............................................................... 14

2.4 Dissolved mercury (inorganic Hg(II) and organic CH3Hg(I)). 14 2.4.1 Dishware preparation, sampling, sample handling and storage ....................................... 14 2.4.2 Laboratory protocol for measurement ............................................................................. 14

2.4.3 Performance check report ................................................................................................ 17

2.5 Dissolved metals (Cd, Cu, Zn, Pb) 17 2.5.1 Dishware preparation, sampling, sample handling and storage ....................................... 17 2.5.2 Laboratory protocol for measurement ............................................................................. 17

2.5.3 Performance check report ................................................................................................ 21

2.6 Biotoxins (saxitoxin) 21 2.6.1 Dishware preparation, sampling, sample handling and storage ....................................... 22 2.6.2 Laboratory protocol for measurement ............................................................................. 22 2.6.3 Performance check report ................................................................................................ 22

2.7 Algal species detection 23 2.7.1 Dishware preparation, sampling, sample handling and storage ....................................... 23

2.7.2 Laboratory protocol for measurement ............................................................................. 23 2.7.3 Performance check report ................................................................................................ 26

2.8 Hydrocarbons and Volatile Organic Constituents (VOCs) 26 2.8.1 Dishware preparation, sampling, sample handling and storage ....................................... 27

2.8.2 Laboratory protocol for measurement ............................................................................. 27 2.8.3 Performance check report ................................................................................................ 28

3 Conclusion and Outlook 29

4 List of acronyms 30 5 References 33


SCHeMA – 614002_ D8.2 5

1 Introduction

Key targets of the SCHeMA system are chemicals that may adversely affect marine ecosystems,

living resources and ultimately human health. To achieve this, SCHeMA partners will develop a

range of chemical miniaturized sensors using innovative analytical procedures to insure reliable and

selective electrochemical and optical measurements of inorganic (micro-) nutrients/pollutants,

VOCs, biotoxins, species relevant to the carbon cycle. The WP8 consists in the prototype field

evaluation and application in coastal sites. One of the objectives is the validation of sensor

performance and ruggedness under contrasting real-life environmental conditions. The present

deliverable introduces the different laboratory based techniques that will be applied in order to insure

the validation of the SCHeMA probe measurements. For the different parameters, laboratory

protocols will be presented from the cleaning of the laboratory glassware to the chemical detection

by introducing as well the sample treatment from the field sampling. The performances of the

different laboratory techniques were checked. The review of the different laboratory techniques

presented in this deliverable is essential to ensure parallel measurements for the validation steps and

quality control of the SCHeMA sensors.


SCHeMA – 614002_ D8.2 6

2 Laboratory-based validation methods for the different parameters measured by the SCHeMA system

2.1 Nutrients: nitrite, nitrate, phosphate

In the frame of the WP2, the SCHeMA system will include potentiometric sensors based on

selective membranes for the detection of nutrients nitrite, nitrate and phosphate in seawater. This

highlights the need for a better understanding of nutrients processes in those ecosystems. This

section introduces the techniques used for the nutrients measurement in laboratory.

Spectrometry

2.1.1 Dishware preparation, sampling, sample handling and storage

In order to study nutrient content, seawater is sampled in the field by pumping through a

polypropylene pipe with a vacuum pump and collected into a polypropylene bottle. Seawater is

filtered through a 0.2 µm porosity polycarbonate filter (Minisart Syringe filters, hydrophilic,

Sartorius®) with a 10mL syringe (Terumo

®) and placed into polypropylene tubes of 10mL. This

sample should immediately be placed into the freezer in order to avoid any ammonium degradation.

2.1.2 Laboratory protocol for measurement

This technique consists in triggering the formation of a coloured complex (colorimetric method)

by adding one or several reagents to a sample (Figure 2). The colouration intensity is therefore

measured with a spectrophotometer set up with a specific wavelength. The sample is placed into a

spectrophotometric transparent cuvette and then inside the spectrophotometer (Figure 3) where a

light beam goes through: the more the sample is coloured, the less the light can go through the

sample (I0 > I1, Figure 1). This light intensity difference correspond to the sample absorbance.

If the colouration intensity is proportional to the X element concentration, we can determine it

with the Beer-Lambert rule:

A = ɛ x L x C

A: the sample absorbance without unit

ɛ: the molar extinction coefficient in L.mol-1

.cm-1

L: the cuvette length crossed by the light in cm

C: the molar concentration in mol.L-1

Figure 1: Spectrophotometric cuvette crossed by the light beam


SCHeMA – 614002_ D8.2 7

Figure 2: Sample preparation

Figure 3: Cuvette placed in the spectrophotometer (visible-

UV type, Thermo®, Genesys 20)

The sample concentrations are determined with a standard curve obtained with the absorbance

values of the standards.

2.1.2.1. Phosphate measurements

Principle: In acidic media and in presence of antimony, phosphate ions react with the

ammonium molybdate to create a complex turning into blue through contact with ascorbic acid. The

blue intensity, proportional to the phosphate concentration in the solution, is measured with the

spectrophotometer.

Reagents:

(1) Ammonium molybdate solution:

Above the hot plate (50°C) dissolve 15g of ammonium molybdate in 500mL of MilliQ water (18.2

MΏ , Millipore®). Keep it away from the light.

(2) 2.5M sulfuric acid solution

Pour 140mL of sulfuric acid in 900mL of MilliQ water.

(3) Potassium oxytartre and antimony solution

Dissolve 0.68g of K(SbO)C4H4O6 in 500mL of MilliQ water.

(4) Ascorbic acid solution

Dissolve 1.08g ascorbic acid in 10mL of MilliQ water.

The final reagent is a mix between the fourth solutions, in the following proportions:

2/10 of (1) + 5/10 of (2) + 1/10 of (3) + 2/10 of (4)

MSA solution

The MSA is a stable solution that can be kept in the fridge for several months. However the

solution (4) is very unstable and should be prepared just before the measurement.


SCHeMA – 614002_ D8.2 8

Detection: The different standards should be prepared beforehand. Those standards

correspond to solutions with known phosphate concentrations. They are realised by dilution of a

stock solution very concentrated in phosphate (5mM). The standard concentrations range from 0µM

(blank) to 5µM.

The mix standard/sample – reagent is done in a spectrophotometric cuvette as following: 1/11th

reagent + 10/11th

standard/sample.

After 5min the absorbance is read with a wavelength of 885nm.

2.1.2.2. Nitrite measurements

Principle: In acidic media (pH<2), the nitrite ions create nitrous acid HNO2 that reacts with

the sulphanilamide to create a diazoic complex. With N-Naphtyl-1-ethylenediamine this complex

turns into a pink colouration. The colouration intensity is proportional to the nitrite concentration.

Reagents:

(1) Sulphanilamide solution

Dissolve 2.5g of sulphanilamide with 13mL of concentrated HCl and 250mL of MilliQ water.

(2) N-Naphtyl-1-ethylenediamine solution

Dissolve X mg of N-Naphtyl-1-ethylenediamine in X mL of MilliQ water. This solution is unstable

and must therefore be prepared the same day as the sample detection.

The final reagent is a mix 50:50 of solutions (1) and (2).

Detection: The different standards should be prepared beforehand. Those standards

correspond to solutions with known nitrite concentrations. They are prepared using a stock solution

1mM of NaNO2. The standard concentrations ranged from 0µM (blank) to 10µM.

The mix standard/sample – reagent is done in a spectrophotometric cuvette as following:

- 40µL of final reagent

- 1mL of standard/sample

After 30min the absorbance is read with a wavelength of 543nm.

2.1.2.3. Nitrate measurements (nitrate + nitrite)

Principle: The method corresponds to the reduction of nitrate into nitrite by the vanadium

chloride. Nitrites are then measured as previously described. The sum of the nitrites initially present

in the sample and the nitrites created by the reduction of the nitrates is therefore measured.

Reagents:

(1) Sulphanilamide solution

Dissolve 2.5g of sulphanilamide with 13mL of concentrated HCl with 250mL of MilliQ water.


SCHeMA – 614002_ D8.2 9

(2) N-Naphtyl-1-ethylenediamine solution

Dissolve X mg of N-Naphtyl-1-ethylenediamine in X mL of MilliQ water. This solution is unstable

and must therefore be prepared the same day as the sample detection.

(3) Vanadium chloride solution

Dissolve 1.6g of VCl3 in 200mL of MilliQ water and add 16.8mg of concentrated HCl (this solution

is stable for at least a month).

The final reagent can be used for at least a month and corresponds to the following mix:

1/7 of (1) + 1/7 of (2) + 5/7 of (3)

Detection: The different standards should be prepared beforehand. Those standards correspond

to solutions with known nitrate concentrations. They are prepared using a stock solution 5mM of

KNO3. The standard concentrations ranged from 0µM (blank) to 100µM.

The mix standard/sample – reagent is done in a spectrophotometric cuvette as following:

- 250µL of final reagent

- 300µL of standard/sample

After a night in the dark the absorbance is read with a wavelength of 543nm.

2.1.3. Performance check report

For phosphate, nitrite as well as for nitrate measurements the error percentage is less than 5%.

This protocol for nutrient analyses is based on Poirier and Charbonnier, 2015.

Ion Chromatrography

The developed electrodes were validated using ion chromatography (IC). Note that the sample

used was desalinated seawater in developed and reference technique. A Metrohm 881 compact IC

pro chromatograph with anion exchanging column (6.1006.520 Metrosep A Supp 5) was used as

reference method. The eluent was a solution composed of 1 mM NaHCO3 + 3.2 mM Na2CO3, along

with 50 mM H2SO4 for regeneration of the anion suppressor (0.8 mL min-1).

2.2 Species relevant to the carbon cycle (carbonate and carbon dioxide)

The development of new electrochemical systems for the determination of species relevant to

the carbon cycle, i.e. carbon dioxide (CO2), carbonate (CO32-

), and total alkalinity detection, is also

involved in the general aim of SCHeMA project. Validation of the prepared sensors consists in the

measurement of CO2 and pH using a commercial probe

(http://www.idronaut.it/cms/view/products/sensors/pcosub2sub/s333) and a pH electrode. With these

two measurements, carbonate levels can also be calculated. On the other hand alkalinity is obtained

also by using alkalinity titration.


SCHeMA – 614002_ D8.2 10

Detection of CO2

Principle: The pCO2 sensor has been developed by our partner Idronaut. The direct

measurement of pCO2 using the pCO2 electrode is an adaptation of a pH measurement. A

combination of pH measuring and reference electrodes makes contact with a solution behind a gas

permeable membrane. Carbon dioxide diffuses across the membrane in either direction in response to

a partial pressure difference, equilibrating the inner electrolyte with the external gas pressure.

Hydration of pCO2 in the electrolyte water produces carbonic acid causing a change in hydrogen ion

activity as shown in Figure 4.

CO2 + H2O

H2CO3

H+ + HCO3

The pH electrode senses the change in pCO2 concentration as a change in pH of the electrolyte

and develops a voltage exponentially related to pCO2. Thus a ten-fold increase in pCO2 is nearly

equivalent to a decrease of one pH unit (Idronaut website).

Measurement of total alkalinity via titration

Principle: Titration techniques belong to the classical methods in the analytical chemistry

laboratory. Recall that carbonate obeys the equilibrium reactions as mentioned above (Figure 4), the

proportion of these species varying with the pH. Monitoring the pH variation as a function of the

volume of acid added to the sample allows for the determination of total alkalinity as well as

carbonate speciation. Titration can be performed manually using pH indicators or automatically

using an automatic titrator (Metrohm 765 Dosimat) and a pH electrode.

The titration instrument consists of a single burette, a voltmeter, and a computer. For simplicity, a

combination pH electrode is used in a Metrohm titration vessel of 130 mL and a magnetic stirrer.

Protocol:

Manual titration

- Prepare 0.1M HCl and 0.1 M NaOH solution

- Pour the 0.1M HCl into a 50 mL burette

- Add 10 mL of sample, 10 mL of 0.1M NaOH and 30 mL of MilliQ water to the titration

beaker

- Place the beaker on a magnetic stirrer and add a clean stirring bar

- Add few drops of phenolphthalein indicator to the solution; the solution should turn purple

red.

Figure 4: Carbon species equilibrium reactions


SCHeMA – 614002_ D8.2 11

- Titrate the solution with acid to the first equivalence point, where the solution becomes

colorless. Note the volume of 0.1M HCl added.

- Add few drops of bromocresol green indicator to the solution; the solution should turn blue

green.

- Continue the titration with the acid to the next equivalent point, where the solution turns

yellow. Note the volume of acid added.

The values of the volume of acid needed to reach both equivalence points allow for the calculation of

the two major carbonate species (CO32-

and HCO3-) concentrations in the sample as indicated in the

data analysis section below.

Automatic titration

The titration instrument consists of an automatic burette, a voltmeter, a Metrohm combined pH-

reference electrode and a computer.

- Prepare 0.1M HCl and 0.1 M NaOH solution

- Pour the 0.1M HCl into the automatic burette

- Add 10 mL of sample, 10 mL of 0.1M NaOH and 30 mL of MilliQ water to the titration

beaker

- Place the beaker on a magnetic stirrer and add a clean stirring bar

- Install the combined pH electrode previously calibrated using two pH buffer solutions

(buffers pH 4 and 7)

- Start the stirrer

- Start the automatic titration in the software. The complete titration experiment lasts about 30

minutes.

Data analysis: The pH can be plotted as a function of volume of acid added. By doing first order

differentiation for this curve, the equivalence points can be obtained by the maxima. The estimates of

error in the volumes can be obtained by locating the neighbouring points in the graphs or by using

the Lorenzian function near the equivalence point. From the two volumes needed to reach the first

and second equivalence points and the respective concentrations, one can calculate the number of

moles for these equivalence point, which will be denoted as (1)

HCln and (2)

HCln , respectively. Since we

know that the principal species are OH-, CO3

2- and HCO3

-, the following relationship holds:

𝑛𝐻𝐶𝑙(2)

− 𝑛𝐻𝐶𝑙(1)

= 𝑛𝐻𝐶𝑂3−

Where nHCO3− is the number of moles of total carbonate species in the titration vessel initially, which

also corresponds to the number of moles of total carbonate species in the sample added into the

titration vessel. From the pH value of the sample and the relationship between carbonate speciation

and pH, we can then get the concentration for every carbonate species in the sample.


SCHeMA – 614002_ D8.2 12

Figure 5: Atomic Absorption

Spectrophotometer (Perkin Elmer®

AA300)

2.3 Dissolved arsenic (As(III), As(V) and As total)

In the frame of the SCHeMA project, the WP3 aims at designing interdigitated electrodes for

the detection of As(III) and As(V) in seawater. Validation of those measurements in the laboratory

consists in As detection by Flow Injection Analysis System -Atomic Absorption Spectrophotometer

(FIAS-AAS).


Prior sample collection polypropylene vials should be acid-washed. Cleaning procedure

consists in filling the vials with 10% hydrochloric acid (made from14mol.L-1

HCl Baker Analyzed)

for 3 days, then cleaning them 3 times with deionised water and finally 3 times with MilliQ water. In

order to study As speciation, seawater is sampled in the field by pumping through a polypropylene

pipe with a vacuum pump and collected into a polypropylene bottle. Seawater is filtered through a

0.2 µm porosity polycarbonate filter (Minisart Syringe filters, hydrophilic, Sartorius®) with a 10mL

syringe (Terumo®) and placed into a 60mL polypropylene vial filled up to the very top (to avoid any

change in the speciation by oxidation of the sample). A replicate of each sample is realised. Samples

should be stored in the dark at 4°C until analysis.


Principle: Hydride Generation Flow Injection Analysis System -Atomic Absorption

Spectrophotometer (FIAS-AAS) is used for As species detection (Perkin Elmer® AA300, Figure 5)

as described by Yamamoto et al. (1985). Hydride generation is a very effective analytical technique

developed to separate hydride forming metals from a range of matrices. Because only gaseous

arsines are introduced to the AAS detector and the sample matrix is removed, spectral interference

encountered in the detection system is essentially eliminated (Gong et al., 2002). Only the As(III)

species is detected by the spectrophotometer. Therefore in order to detect the part of As(III) present

in the system, the sample is run directly. However to obtain the part of As(V), the sample is pre-

reduced so that all the As(V) already present in the sample turns into As(III) which will be detected

by the apparatus. This give the total As concentration, by subtraction of the As(III) concentration

already measured we can obtain the concentration of As(V).


SCHeMA – 614002_ D8.2 13

Solution preparation:

(1) Carrier 1.2M HCl

Pour 15% HCl 37% (Normapur) in MilliQ water qs 1L.

(2) Reductant

Dissolve 0.5g of NaOH and 5g of NaBH4 in MilliQ water qs 1L.

This solution is unstable and should be done the same day of the analysis.

(3) Pre-reductant

Dissolve 5g of potassium iodide (KI) and 5g of ascorbic acid in MilliQ water qs 100mL.

This solution should be kept in the dark until it turns into a yellow colouration.

Detection: Atomic absorption spectrometer was used with a Electrodeless Discharge Lamps

(EDL; Perkin Elmer®) as light sources (set up at 380mA) and an electrically heated quartz cell unit,

for atomization. Peak height measurements were done from signals recorded on a strip chart

recorder.

Different standard solutions should be prepared. Those standards correspond to solutions with

known arsenic concentrations. They are prepared using a stock solution 100µg.L-1

of As. The

standard concentrations range from 0µg.L-1

(blank) to 10µg.L-1

. Those solutions are pre-reduced in

order to have all the As in the As(III) form in the solution and therefore detect the total As.

For the analysis of As(III), the samples are prepared as followed:

- 8mL of sample

- 2mL of HCl 37%.

For the analysis of total As, the samples are prepared as followed:

- 5mL of sample

- 2mL of HCl 37%

- 2mL of pre-reductant KI

After 1h – 1mL of MilliQ water.

For As(III) concentration measurements, samples must be prepared the same day of analysis in

order to avoid any transformation of the species. After the opening of the sample tube, tests have

shown that after a day in an openned tube, samples have lost all the As(III) originally present. The

same sample analysed after a day in a closed tube have lost about 30% of the original As(III).

Furthermore, even when the sample tube remains closed, As(III) analysis should be quick in order to

preserve the As(III) originally present (within few days, weeks, Gong et al., 2002). Even total As

analysis should be done in the hour following the pre-reduction of As(V) in order to avoid As(III)

oxidation (no more As(III) left after 2hours). Therefore for a big set of samples, samples should be

prepared by sub-set of 10 for pre-reduction and As detection.

2.3.3 Performance check report

This analytical method is checked against international certified reference material TMRAIN-04.

Accuracy was about 80% and precision (%RSD; n=6) generally better than 10% for concentrations at

least 5 times higher than detection limit (3 times the blank standard deviation; 0.1µg.L-1

).


SCHeMA – 614002_ D8.2 14

Furthermore this technique has been applied successfully to seawater samples from the Arcachon

Bay confirming the possible application of this technique to field seawater samples.

2.3.4 Improvement/adaptation of laboratory technique

Another laboratory-based technique the Hydride Generation coupled to Inductively Coupled

Plasma Mass Spectrometry (HG-ICP-MS) could be developed in order to validate the sensor

measurements. In fact matrix elimination due to hydride generation will allow the introduction of

seawater samples in the ICP-MS without matrix problems and mass interferences while giving

higher detection limits.

2.4 Dissolved mercury (inorganic Hg(II) and organic CH3Hg(I)).

Another objective of the WP3 is to optimise functionalized polymer brushes sensors and

analytical methodology for Hg(II) and CH3Hg(I) measurements. The following section describes

laboratory-based validation technique that will be applied in order to check the sensor data.


In order to study mercury species content, seawater is sampled in the field with an ultra-cleaned

NOEX bottle rinsed off thoroughly with seawater from the site and collected into a 10% HNO3-

cleaned Teflon bottle which has also been rinsed with water sample and avoiding the introduction of

air which can degas the volatile Hg species. Seawater is immediately filtered using Teflon syringe

filters (0.45mm porosity; Millex, Millipore®) and HCl-cleaned polypropylene syringes (10mL,

Norma-Ject®) and placed in 30mL HCl-cleaned Teflon bottles and acidified (Ultrapur HCl; ~ 0.5%

v/v, J.T. Baker). A replicate of each sample is realised. Samples should be stored in the dark at 4°C

until analysis (Schäfer et al., 2010).


Principle: This laboratory techniques is based on the Hg species separation by gaseous

chromatography (GC) followed by analyte ionisation in an Ar plasma and a detection of the Hg

content by inductively coupled mass spectrometry (ICP-MS; Leermakers et al., 2005). The ICP

coupled with GC allows a rapid, highly sensitive, quantitative analysis (in the pg.L-1

range). For this

purpose, the University of Bordeaux had acquired a new GC instrumentation (Trace1310; Thermo-

Fischer Scientific®) as well as new autosampler (TriPlus, Thermo-Fischer Scientific®).

The compound separation by GC requires that those compounds have volatile properties as well

as an affinity for organic phases. For this prior to analysis we realise a derivatisation of the chemical

forms in order to obtain only alkyl forms of Hg, more stable at high temperature and more volatile.

We realise this derivatisation by propylation of the compounds i.e. the anionic radicals of the

compounds (Cl-, OH-, Br-…) are replaced by sodium tetrapropylborate (NaBPr4). The derivatised

compounds are then extracted from the aqueous solution in isooctane by liquid-liquid extraction

(Castelle, 2008). External calibration is not recommended for this extraction-derivatisation technique

because the extraction recovery is much lower for a sample than for a standard solution, this protocol


SCHeMA – 614002_ D8.2 15

being very dependent from matrix effect. In order to avoid this, a calibration is done by isotopic

dilution. This method is based on an isotopically enriched solution spike of the sample with a known

amount of the studied species. The mono-isotopic species added represents therefore an ideal internal

standard because after the equilibration, it will be exposed to the same physico-chemical

transformations, due to extraction-derivatisation protocol, than the natural species in the sample

(Castelle, 2008).

Protocol: Prior sample preparation glass vials should be acid-washed. Cleaning procedure

consists in soaking the glass vials with RBS detergent for 1h, then in 10% aqua regia for 3 days, and

finally in 10% HCl for 3 days. Glass vials are then rinsed 3 times with deionised water and finally 3

times with MilliQ water.

Sample preparation for dissolved mono-methyl mercury (MeHgD) and divalent mercury (Hg(II)D)

analysis requires 2 days of sample preparation.

Day 1

1. Aliquots of 10mL of sample are diluted with 10% HCl and placed in 50mL pre-washed glass vials

with Teflon-lined-caps

2. Prepare isotope dilutions, for e.g. 201

Hg(II) and 202

CH3Hg(I) and add them to the sample for the

isotopic dilution

3. Mix slowly and keep the samples at 4°C for 2h to allow the equilibrium to take place with the

isotopes

Prepare in the meantime 100mL of acetate buffer (8.2g of acetate + 15mL of acetic acid + 76.8mL of

MilliQ water)

4. Take back the samples and add:

- 3mL of acetate buffer

- 100µL of ammonium hydroxide NH4OH and check that the pH corresponds to 3.9 (add more

NH4OH if needed)

- 500µL of isooctane

- 500µL of sodium tetra(n-propyl)borate NaBPr4

5. Cap immediately the vials and shake them manually during 5min

6. Keep the samples at 4°C overnight.

Day 2

7. Take back the samples (do not shake the vials)

8. Extract the isooctane (organic) phase above the water phase with a pipette and place it into a 2mL

glass vial with Teflon-lined-caps

9. Analysis to GC-ICP-MS or preservation in the freezer at -80°C

This method is following the protocol of Schäfer et al., 2010.


SCHeMA – 614002_ D8.2 16

Figure 6: Isotopic dilution principle (modified after Castelle, 2008)

Detection:

Analysis of the Hg species in the organic extract are performed by Gas Chromatography

coupled to ICP-MS (Thermo-Fischer Scientific; X7). Calibration is performed by species-specific

isotope-dilution by adding known amounts of 202

Hg-enriched MeHg and 201

Hg-enriched Hg(II)

solutions directly to water aliquots of each sample before the extraction procedure. The figure 6

presents the principle of the isotopic dilution in the case of 202

MeHg isotope addition (X isotope) and

the 200

MeHg reference isotope (Y isotope).

The GC-ICP-MS analysis allows the determination of the R ratio corresponding to the peak

area of the X isotope over the peak area of the Y isotope (Figure 6). The determination of this ratio

allows for the solution of the following equation which will give the MeHg concentration of the

sample:

c = [c’ w’ Ar ( R Y’ – X’)] / [(w Ar’ (X – R Y) ]

c : MeHg sample concentration (e.g. ng.L-1

)

c’ : MeHg concentration of added mono-isotopic solution

w: volume of sample (L)

w’: volume of mono-isotopic addition

Ar: atomic mass of natural Hg (200.6 g.mol-1

)


SCHeMA – 614002_ D8.2 17

Ar’: atomic mass of Hg in the mono-isotopic solution (201.8 g.mol-1

)

X: X isotope natural abundance in the sample (%)

X’: X isotope abundance in the mono-isotopic solution (%)

Y: Y isotope natural abundance in the sample (%)

Y’: Y isotope abundance in the mono-isotopic solution (%)

R: X/Y ratio measured by GC-ICP-MS in the mixed solution

(According to Castelle, 2008)

In order to obtain a consistent analysis of the isotope content one should consider the mass-bias

that is related to the inherent functioning of the mass spectrometer. For this an isotopically certified

thallium solution (SRM 997) is analysed continuously in order to correct for this bias.


The detection limits of the method (3 times the standard deviation of the blank values) is

4pg.L-1

and 0.5pg.L-1

for MeHg ad Hg(II) respectively. The analytical results are quality checked by

analysing international certified reference material IAEA 405. Accuracy as well as precision based

on replicate analysis (%RSD; n=33) are better than 10% (Castelle, 2008).

2.5 Dissolved metals (Cd, Cu, Zn, Pb)

The SCHeMA project will associate new functionalized GIME sensors for As and Hg target

analytes to the GIME sensor developed previously for Cd, Pb, Cu, and Zn. Laboratory-based

validation techniques of trace metals Cd, Pb, Zn and Cu are therefore needed. Those will consist in

ICP-MS analyses which are described in the following section.


Prior sample collection polypropylene vials should be acid-washed. Cleaning procedure consists

in filling the vials with 10% nitric acid (made from14mol.L-1

HNO3 Baker Analysed) for 3 days, then

cleaning them 3 times with deionised water and finally 3 times with MilliQ water. In order to study

dissolved metal concentrations, seawater is sampled in the field by pumping through a polypropylene

pipe with a vacuum pump and collected into a polypropylene bottle. Seawater is filtered through a

0.2 µm porosity polycarbonate filter (Minisart Syringe filters, hydrophilic, Sartorius®) with a 10mL

syringe (Terumo®) and placed into polypropylene a 60mL vial. The sample is acidified (HNO3,

0.1%, Ultrex). A replicate of each sample is realised. Samples should be stored at 4°C until analysis.


Analysis of trace elements in seawater is challenging due to extremely low concentrations of

most trace elements in seawater and the considerable influence of matrix elements such as Na, Mg,

Ca, K and Cl. This salty matrix triggers spectral (due to polyatomic species that interfere on the

analyte masses such as 35

Cl16

O+ on

51V

+ and

40Ar

23Na

+ on

63Cu

+) and non-spectral (due to the

influence of easily ionized matrix elements on the plasma, Na and K in particular, as well as signal

drift caused by accumulation of salts on the cones and lenses of the ICP-MS) interferences


SCHeMA – 614002_ D8.2 18

(Søndergaard et al., 2015). Simple dilution of the sample can be foreseen but can lead to sensitivity

problem. Therefore, a common way of solving the analytical problem of seawater analysis is to pre-

concentrate the trace elements on a chelating resin followed by a rinse of matrix elements from the

resin, an elution of the trace elements and detection using ICP-MS (Søndergaard et al., 2015). The

sample preparation consists therefore in a solid-liquid extraction with the resin. Different types of

resins are commercially sold. The first attempt was done with DigiSep type resin while the second is

using Chelex type resin. Results of certified material analysis are presented in the following section.

2.5.2.1. DigiSep type resin

Protocol:

Resins:

- Chelex: 50-100 mesh particle size

- DigiSep: DigiSEP Bleu® (SCP Science)

Reagents:

- 1M Ammonium acetate:

Dissolve 38g of ammonium acetate in 500mL MilliQ water. Adjust the pH to 5.5 by adding acetic

acid (Suprapur). If it is too acidic add NH4OH (Suprapur). Make a slurry of Chelex resin and shake it

for 20min.

- 0.1M Ammonium acetate:

Dissolve 7.7g of ammonium acetate in 500mL MilliQ water. Adjust the pH to 5.5 by adding acetic

acid (Suprapur). If it is too acidic add NH4OH (Suprapur). Make a slurry of Chelex resin and shake it

for 20min.

- 2M Nitric acid:

Add 63mL HNO3 (Suprapur) in a Teflon vial in MilliQ water qs 500mL.

Sample preparation:

With the help of a peristaltic pump (flow rate 2mL/min), the DigiSEP resin is first

conditioned by different reagents. Once conditioned the sample is eluted and the trace metals are

retained by the resin. After a rinse of the resin, the retained trace metals are eluted with HNO3 and

the solution is collected. This solution will be dosed at the ICP-MS.

1) DigiSep resin conditioning

- 5mL 2M HNO3

- 5mL MilliQ water

- 5mL 0.1M ammonium acetate.

2) Sample “loading”

- Sample (55 to 60mL) with adjusted pH (5.5)

3) Rinsing of the resin


SCHeMA – 614002_ D8.2 19

Table 1: Accuracy, precision and blank analyses from the solid-liquid extraction of

Cu, Cd, Pb and Zn measured by ICP-MS.

- 5mL MilliQ water

- 5mL 0.1M ammonium acetate.

4) Elution

- 5mL 2M HNO3.

The solution is collected in 14mL pre-cleaned with HNO3 and pre-weighed tube (to know the exact

sample volume eluted).

Detection: The sample is dissolved in 9mL MilliQ water before the ICP measurement in

order to have the sample lower than 5% acidic. Concentrations of trace metals Cu, Cd, Pb, and Zn

are then measured in certified reference material CASS-5 by ICP-MS (Thermo X7) with external

calibration under standard conditions.

Performance check report:

µg.L-1

Cu Cd Pb Zn

CASS-5 Certified value 0.38 0.0215 0.011 0.719

Standard deviation 0.028 0.0018 0.002 0.068

Measured mean (n=4) 0.429 0.030 0.096 0.907


Recovery (accuracy) 87% 63% -672% 68%

Precision (%RSD) 3% 6% 89% 0%

Blanks Measured mean (n=4) 0.036 0.009 0.266 10.4


While the quality of the measurements from the extraction seems good for copper and

cadmium, lead and zinc present some anomalies (Table 1). In fact Zn and Pb cannot be measured

using this method. While Zn blank were not “clean” enough, Pb measurement precision is not

acceptable.

Considering those results, we have decided to develop a new method in the laboratory, based

on High Pressure Liquide Chromatography (HPLC) coupling with ICP-MS (Figure 7).

2.5.2.2. Chelex-100 type resin

On-line method with coupling to HPLC gives an elution profile (concentrations vs. time) of

the elements eluted and usually the average peak intensity or the maximum peak intensity is used as

proportional to the sample element concentration (Søndergaard et al., 2015) while reducing possible

contamination problems. We are following Søndergaard et al., 2015 protocol using commercially

available iminodiacetate chelating resin Chelex-100 which has an appropriate affinity for most trace

elements of interest.


SCHeMA – 614002_ D8.2 20

Table 2: Summary of the pre-concentration and elution program (modified after Søndergaard et al., 2015).

Figure 7: HPLC coupled to ICP-MS Figure 8: Sample injection into the HPLC column

Protocol: The laboratory protocol follows the one established by Søndergaard et al., 2015.

The laboratory-made Chelex-100 column is prepared by making a slurry of 1 g Chelex-100 resin

(Na-form; 100–200 mesh) in 1M NH4OH and loading it into the column using a syringe until

perfectly packed. Prior to this, the resin was cleaned by soaking it in 5M HCl overnight before it was

collected on a glass microfiber filter and further cleaned with 2M HNO3 and Milli-Q water.

Polyether ether ketone (PEEK) filters was placed in the ends of the column. Before the column was

used, it was conditioned with 1M ammonium acetate buffer solution (pH 7.0) in order to convert the

active sites of the resin into ammonium form and raise the pH in the column to above pH 5.

A 2M stock solution of ammonium acetate was then prepared by diluting ∼140g of 25–35%

(m/m) analytical grade ammonia hydroxide (Merck) and ∼121g of 100% Suprapure acetic acid

(Merck) in 1000mL of Milli-Q water. The pH was adjusted to 7.0 by adding either ammonia

hydroxide or acetic acid. Last, the ammonium acetate buffer solution was purified by passing it

through a 100 mm Chelex-100 filled glass column. From the purified stock buffer solution a 0.05 M

ammonium acetate buffer solution was prepared by diluting the stock solution 1:40 with Milli-Q

water.

For the quantitative analysis, several standard solutions were prepared using the certified

seawater reference material NASS-6 with known quantities of 1g.L-1

mono-elementary standard

stock solutions. The standards were diluted 1:1 with the purified 2M ammonium acetate (NH4Ac)

buffer solution (pH 7.0) resulting in a final pH of 6.1–6.3.

Detection:

Step Time (s) Flow (mL.min-1

) Comment

1 0-150 1 Matrix separation and analyte pre-concentration (sample loading

into the colum; Figure 8)

2 150-180 1 Column rinsing with 0.05M NH4Ac

3 180-505 0.8 Analyte elution using 5% HNO3 and measurement by ICP-MS

(sample injection with injection valve on “inject”; Figure 7)

4 505-625 1 Column rinsing with 5% HNO3

5 625-655 1 Column reconditioning with 0.05M NH4Ac rinse solution


SCHeMA – 614002_ D8.2 21


Standard additions of NASS-6 certified reference material have been realised and eluted through

the resin following the steps presented in Table 2. However recoveries are higher than expected

revealing a pollution problem that might arises from the buffer purity. Therefore efforts are directed

towards purification of the overall reagents and chemicals in order to reduce the trace metal

pollution. Furthermore we plan on working on each elution phase, time and solutions in order to

obtain the optimal conditions for sample pre-concentration and elution avoiding pressure problems of

the column and reducing pollution problems. Finally bigger sample loops (1mL and 2mL) have been

purchased and will be soon tested in order to increase the sample pre-concentration (we are currently

using a 100µL sample loop).

2.6 Biotoxins (saxitoxin)

The SCHeMA project aims also at developing a miniaturized, fully integrated, versatile

algae/biotoxin optochemical multichannel probe. This is more specifically one of the objectives of

the WP4 which plan on the detection of saxitoxin (STX) in seawater. The sensor for saxitoxin is still

in development and tests in seawater have not been conducted yet. Therefore the following section

describes the validation method that will be used for general measurement validation during sensor

development. This involves the participation of our external collaborator IFREMER Nantes that will

apply HPLC standard analytical methods for STX detection. Another method consisting in ELISA

test will be described for validation of the data in seawater context.

HPLC

This method follows the protocol established by Lawrence et al., 1995 and described in Cervantes

Cianca et al., 2007.

Principle: This method is based on the hydrogen peroxide oxidation of STX to allow its detection

by fluorescence using a pre-column oxidation HPLC method with fluorescence detection.

Protocol: This method was based on the hydrogen peroxide oxidation of STX described by

Lawrence et al. (1995) and modified as follows by Cervantes Cianca et al., 2007:

- 250µL of 1M NaOH and 25µL of 3% hydrogen peroxide is added to 300µL of the STX standard

solution (NRC-CRM diluted in HCl) or the sample obtained after extraction procedure

- The mixture is allowed to react for 2 min at room temperature

- About 25µL of 99.7% acetic acid is added to stop the reaction.

- 20µL of this solution is injected into the HPLC system.

Detection: The pretreated sample is injected into the HPLC system using an injection valve. The

separation of STX is achieved using a reversed-phase column. The column is eluted with a gradient

program of solvents consisting of 0–100% (v/v) acetonitrile in 0.1M ammonium format, adjusted to

pH 6 with 0.1M HCl, as follows: 0–5% during the first 5 min, then 5–70% during the next 4 min,


SCHeMA – 614002_ D8.2 22

then 70–100% during the next 9 min, and finally, 100–0% during the last 2 min. The flow rate is

maintained at 1.5 ml/min. STX is detected with a fluorescence detector with an excitation

wavelength of 330 nm and an emission wavelength of 390 nm as described in Cervantes Cianca et

al., (2007).

Performance: Detection limit are about 12 STX pg and quantification limit about 32 STX pg

(20µl injected respectively). Reproducibility studies are carried out by injecting a 16 ng.mL-1

STX

standard solution into the HPLC-FLD system and give a peak area of about 2 FLD u.a. (n = 10), each

sample is injected in triplicate (Cervantes Cianca et al., 2007).

Enzyme Linked Immunosorbent Assay (ELISA)

Little information is available regarding the levels of extracellular Paralytic Shellfish Toxins

(PSTs) that may leak or be released into seawater from toxic cells during blooms (Lefebvre et al.,

2008). Even though it is present in low quantity, measurable extracellular PSTs were detected in

seawater for the first time in Atlantic waters (Costa et al., 2010). The laboratory-based method

allowing this detection consists in ELISA tests.


Surface seawater samples are collected by a submersible pump or with a Niskin bottle. Water

samples are passed through a 200µm plankton net in order to exclude large plankton (Costa et al.,

2010). For the analysis 5mL of seawater is filtered under light vacuum pressure on 0.45µm pore size

HA membrane filters (Durapore Millipore Inc.) and frozen at -20°C (Lefebvre et al., 2008).


Principle: Enzyme-linked immunosorbent assays (ELISAs) are a type of biochemical assay using

antibodies raised to the analyte of interest, with detection typically manifested as a color change. A

range of assays have been developed for saxitoxin and several of its derivatives that utilize both

direct and indirect formats, with poly- and mono-clonal antibodies (Cusick and Sayler, 2010). The

high sensitivity for STX of ELISA makes it useful in scientific research particularly for detecting

low levels in seawater (Lefebvre et al., 2008).

Protocol: In order to quantify extracellular STX levels, frozen filtrates from seawater samples are

thawed and analysed via ELISA following the protocol provided by the ELISA manufacturer

(Abraxis LLC, USA) and after a 1:10 dilution (with dilution buffer provided in the kit) to eliminate

matrix effects (Costa et al., 2010).


The Abraxis ELISA has a detection limit of 0.05nM for STX and cross-reactivities with other

toxin compounds is of 100% for STX as is stated by the manufacturer (Costa et al., 2010).

Due to the selectivity of the ELISA, the toxin profile can profoundly affect extracellular STX levels

quantified by the assay by largely underestimating the STX levels. While this shows the limitation of


SCHeMA – 614002_ D8.2 23

the ELISA, its advantage is its sensitivity compared to other techniques such as HPLC and therefore

its ability of detecting STX at very low levels.

Clearly, when trying to characterize STX levels in the marine environment, it is important to

consider the best toxin detection method for the circumstances. The newly available ELISA kit,

although more sensitive, underestimates toxicity of samples due to its substantially reduced cross-

reactivity with common toxins other than STX. Other methods should be applied for STX detection

when trying to evaluate potential threats to exposed organisms (Lefebvre et al., 2008).

2.7 Algal species detection

In addition to STX detection, the SCHeMA project aims at developing a multi-wavelength

fluorescence/absorbance detection unit for relevant algae species which will in the end be included in

its sensor system as a miniaturized, fully integrated, versatile STX/algae detection module.

Validation method of the algae species detection module consists in the inverted-microscope method

and is described in the following section. Furthermore in order to have an estimation of the main

phytoplanktonic groups with a different method, HPLC techniques will be applied in collaboration

with experts also from other research institution (SZN and Federico II University, Naples) for the

microalgal species assessment to better compare the results of the SCHEMA sensor.


In order to collect phytoplankton pre-cleaned sampling bottles (plastic or glass, minimum

500ml) have to be prepared.

A series of surface samples (500mL each) have been collected, manually or through Niskin

bottle, in the previous months (starting from February) in two pristine sites in the Portofino Marine

Protected area and preserved in buffered formaldehyde (4% final concentration). These samples will

allow us following the seasonal development of phytoplankton succession, to better characterize the

area prior the in situ field tests. Moreover, since the choice of the adequate volume of the subsample

to analyse depends on the material (coastal waters with abundant detritus or off-shore and more

oligotrophic waters) and on the abundance of the phytoplanktonic cells, we need to test samples

representative of different periods of the annual cycle. The same samplings will be carried out in

harbour waters. Fewer samples of the pristine Portofino MPA and of the Genoa Harbour are

collected in order to be analysed by HPLC by external collaborators. Samples for pigment analysis

are taken at different depths, using Niskin bottle, and filtered onto Whatman GFF filters for analysis.


Principle: In order to validate the results obtained by TU Graz sensor, regarding algal species

determination, DISTAV (UNIGe-IT) equipped its laboratory with all the instrumentation needed to

determine the quali-quantitative analysis of phytoplankton in seawater, following the Utermöhl

Technique. This technique is characterized by the combined use of an inverted-microscope and a

special counting chamber, where phytoplankton has been gathered through a sedimentation cylinder.

This method does not imply any transfer or loss of material and allows identifying the main

phytoplanktonic taxa: coccolithophorids, dinoflagellates and diatoms.


SCHeMA – 614002_ D8.2 24

Protocol:

The inverted-microscope method.

This method is described in details in Hasle, 1978. Lately this method was adopted as

reference method for Italian monitoring studies (National Environmental Agency), as described in

Zingone et al., (2010).

Figure 9: Inverted microscope

An initial screening of each sample must be done in order to determine the final settled volume

needed for analyses unless historical data is available to show what volumes have traditionally been

used for samples for the same site. This is done by sedimenting 10mL of each sample and counting

the total number of phytosynthetic organisms, within a selected area of the slide (Figure 9). No

identifications are done at this time, but any irregularities such as excessive sediment in the sample

are noted.

The phytoplankton sample is homogenized by gently inverting the sample bottle for 60

seconds. The predetermined sample volume is loaded into a settling Utermöhl chamber (Figure 10)

of appropriate volume (10mL, 25mL, 50mL, 100mL). The chamber is topped with a glass cover

slips. Algae are allowed to settle onto the base of the settling chamber. The time recommended for a

complete sedimentation varies with the heights of the chamber. Approximate settling times necessary

are as follow: 100mL – 100 hours, 50mL – 50 hours, 25mL – 25 hours; 10mL – 10 hours.

Figure 10: Sedimentation Utermöhl chambers


SCHeMA – 614002_ D8.2 25

There are three alternative counting strategies when using sedimentation chambers. We chose

to follow the “transect” strategy which is the most use. It entails the identification of the cells along

the transects of which the length is the same as the diameter of the sedimentation chamber and the

width is the same as the diameter of the visual field. The operator should read at least two transects

placed perpendicular to each other. The number of cells was estimated according to the formula:

C = N * π * r * 1000

2 * h * v * n

where

C = cell abundance, expressed as cells/litre

N = total number of cells counted on all transects

r = radius (in mm) of the sedimentation chamber

h = height (in mm) of the transect (diameter of the field)

v = volume (in mL) of the sample

n = number of transects used for the cell counts.

The HPLC method:

High Performance Liquid Chromatography allows rapid separation and quantification of several

component pigments from algal species in natural waters. Once thawed, pigments were extracted and

analysed using a HPLC (Hewlett Packard HPLC mod. 1100) according to Mantoura and Llewellyn,

1983 as modified by Brunet et al., 1993, Vidussi et al. (1996) and Brunet and Mangoni, 2010.

The filters will extract 100% methanol and filter on GF/F Whatman (diameter 25 mm). One ml of

extract will be added to 500 µl of P solution (Ion-Pairing: 1M of Ammonium acetate) and left for 5

minutes. This mixing will be injected into the loop of the instrument.

For the determination of chlorophylls and carotenoids, a spectrophotometer with diodesarray

detector (DAD) was set at 440 nm. It was thus possible to determine the absorption spectrum in the

350–750 nm interval for each peak in order to check the purity of single pigments. Calibration of the

instruments was carried out using different pigments provided by the International Agency for 14 C

Determination, VKI Water Quality Institute. The pigments resolved from the chromatograms are

shown in Table 3.

Table 3: Pigments resolved from the HPLC chromatograms


SCHeMA – 614002_ D8.2 26

A review of taxonomic pigments can be found in Jeffrey (1997). Divinyl-chlorophyll a is the

typical marker of prochlorophytes whereas Chl a is the universal descriptor of other phytoplankton

taxa. Fucoxanthin (Fuco) characterizes diatoms and peridinin (peri) dinoflagellates. Nano- and pico-

flagellates containing chlorophyll c are characterized by 19'-hexanoyloxyfucoxanthin (19'HF,

prymnesiophytes) and by 19'-butanoyloxyfucoxanthin (19'BF, chrysophytes and pelagophytes).

Phytoplankton community composition was derived using marker pigments and conversion factors

as in the work of Casotti et al., 2000.


Counts and measurements will be done in duplicate. For the inverted-microscope method

counting at least 100 cells leads to an error of ±20% with a probability of 95 % (Lund et al. 1958).

Analogue estimates on the precision of the method and considerations on the number of cells to be

counted and recognized (often estimated around 200 cells each sample), and validation criteria have

been discussed in the European Standard Guides (EN 15204, 2006).

Concerning the HPLC method, Mantoura and Llewellyn, 1983 report that the adsorbed

pigments are recovered with acetone with a recovery greater than 90%. The overall reproducibility of

the combined operations of filtration, extraction and HPLC on triplicate samples is 4.8% and that of

replicate injection is about 1% (n=3).

2.8 Hydrocarbons and Volatile Organic Constituents (VOCs)

A miniaturised optochemical mid-infrared sensor will be developed as part of the SCHeMA

system for the selective detection of range of dissolved VOCs as well as dissolved aromatic and

halogenated hydrocarbons. Validation of the obtained results with standard laboratory techniques

will be applied with GC-MS.

The new GC-MS instrument acquired by UNIGe-IT (Trace GC Ultra with FID and ITQ 1100

from THERMO-FISHER SCIENTIFIC), has been installed and employed to develop a screening

method for the identification and quantification of seawater contaminants at the trace level.

In particular, the chromatographic conditions have been optimized for the separations of

hydrocarbons (both HCs and PAH); experiments have been carried out both in MS and MS/MS

mode. The optimized methods have been applied to the analysis of two different marine fuel types,

provided by an Italian oil refinery. The aim of this investigation is to identify specific HC

compounds to be used as a marker of oil contamination in seawater, together with VOC.

Quantitative analyses are carried out on the basis of a protocol previously developed in our

laboratory (Magi et al., 2002) and adapted to seawater according to Bizkarguenaga et al., (Journal of

Chromatography A, 2012, 1247. 104– 117) and the Environmental Protection Agency Method

8270D.


SCHeMA – 614002_ D8.2 27


Glass Borosilicate Graduated Beakers

Glass column

Pipet tip boxes (until 250 mL)

Milli-Q water

Pipettes

SPE OASIS-HLB cartridge from Waters

Ethylene chloride was purchased from Riedel-de Haёn,

N-hexane from Merck

Isooctane from Merck

Methanol from Carlo Erba

Acetonitrile from Carlo Erba

Standard solution of the analytes (100 µg/ml each in cyclohexane) from LabService

Analytica. This standard contains the following sixteen PAHs:

naphthalene (Nap), acenaphthylene (Acl), acenaphthene (Ace), fluorene (Flu),

phenanthrene (Phe), anthracene (Ant), fluoranthene (Flt), pyrene (Pyr),

benzo[a]anthracene (BA), chrysene (Chr), benzo[b]fluoranthene and

benzo[k]fluoranthene (BF), benzo[a]pyrene (BP), indeno[1,2,3-c,d]pyrene (IP),

dibenzo[a,h]anthracene (dBA), benzo[g,h,i]perylene (BPer). Working standards were

prepared by dilution with isooctane. Quantitative determinations were performed by

means of five perdeuterated

PAHs standards (1000 µg/mL each in toluene, purchased from LabService Analytica):

d8-naphthalene, d10-acenaphthene, d10-phenanthrene, d12-crysene and d12-perylene.

Seawater samples are collected in bottles of amber-glass (500mL), filtered (0.45µm) and placed in

a cooler to be maintained at 4ºC. Before analysis, each seawater sample (500 mL) is passed through a

200-mg OASIS-HLB cartridge, previously conditioned. Then, 5mL of MilliQ water and MeOH

mixture are added for cleaning and the cartridges are dried under vacuum. Finally, the analytes are

eluted in a vial using EtOAc.


The extracts were analyzed by both GC-MS instruments present in our laboratories: a 7890 series

gas chromatograph coupled to a 5975N MSD quadrupole mass spectrometer from Agilent and a

Trace GC Ultra with FID and ITQ 1100 from Thermo-Fisher Scientific, using the same analytical

method.

Both GCs are equipped with a Phenomenex ZB5 (30 m x 0.25 mm i.d. x 0.25 µm) column coated

with 5% phenylpolysiloxane. The oven temperature was programmed as follows:

55 °C for 1 min, from 55 °C to 133 °C at 30 °C/min, from 133 °C to 285 °C at 12 °C/min and then

held at 285 °C for 10 min. Helium was used as carrier gas, at a flow rate of 1.4 ml/min. Samples

were injected by means of a Multi-Purpose Sampler (MPS-2) from Gerstel GmbH (Mülheim an der

Ruhr, Germany) using a split/splitless injector; injection of 1 µL was performed in splitless mode.


SCHeMA – 614002_ D8.2 28

The analytes were identified by acquiring their spectra in scan mode from 45 to 300 m/z. The

analyses of real samples were performed by single ion monitoring (SIM), measuring the molecular

ion of each compound or by MS/MS. Quantitative determination was made by isotopic dilution

analysis. A fixed quantity (1 µg) of the five perdeuterated PAHs was mixed with variable quantities

of the analytes standard solution. The resulting mixtures were then analyzed and a calibration curve

was obtained by plotting the ratios of the measured areas against the ratios between concentrations.

Since the perdeuterated compounds are subjected to the same matrix interferences as the analytes,

the isotopic dilution method enables recovery problems to be overcome.


Concerning the GC/MS method, we have performed three determinations for each PAH, on a

blank sample and three determinations on a sample giving measurable responses. The LOD and the

LOQ have been calculated by using the following equations:

LOD = C(std) x (Area(b) + 3xSD(b) ) / Area(std)

LOQ = 3 x LOD

Where,

C(std) : The analyte concentration that gives a measurable response ;

Area(b): The mean response, in terms of area, that is produced by a blank sample ;

Area(std) : The mean response, in terms of area, that is produced by a sample giving measurable

responses ;

SD(b) : The Standard Deviation of the blank sample responses.

The whole methodology was verified on a Certified Reference Material, obtaining results in good

agreement with the certified values.


SCHeMA – 614002_ D8.2 29

3 Conclusion and Outlook

The deliverable D8.2 is a review of the different laboratory-based techniques available for newly

developed sensor in situ measurements. Following the Task 8.2: Adaptation/improvement of

laboratory based techniques, completion and adaptation of the existing laboratory techniques in order

to cover all the parameters measured by the SCHeMA system have been performed.

The following table summarises the current state of laboratory-based validation techniques

development (Table 4). Work towards improvement and adaptation of some techniques will be

achieved in the following time period.

Table 4: Validation techniques and their development progress

Target

parameter

Validation

technique Progress Partner involved Comment(s)

Nutrients Spectrometry Fully developed

Quality checked UBX

Species

relevant to the

carbon cycle

Commercial

pCO2 probe

Titration

Fully developed

Quality checked UNIGE

Dissolved As FIAS-AAS

Fully developed

Sensitivity can be

improved

UBX

Work on sensitivity

improvement (new

EDL lamp, HG-

ICP-MS)

Dissolved Hg GC-ICP-MS Fully developed

Quality checked UBX

Dissolved trace

metals

Solid-liquid

extraction – ICP-

MS

In progress UBX

Work on chemical

purity and pollution

problem

Biotoxins HPLC-FLD Fully developed

Quality checked

Ifremer external

collaboration

Validation method

needed for seawater

samples (ELISA not

specific enough for

STX)

Algal species

Inverted-

microscope

HPLC

Fully developed

Quality checked

UNIGE-IT

SZN and Federico

II Univ., Naples

VOCs GC-MS Fully developed

Quality checked UNIGE-IT


SCHeMA – 614002_ D8.2 30

4 List of acronyms

19'BF 19'-butanoyloxyfucoxanthin

19'HF 19'-hexanoyloxyfucoxanthin

A Absorbance

AAS Atomic Absorption Spectrophotometer

Ace Acenaphthene

Acl Acenaphthylene

Ar Argon

As Arsenic

BA Benzo[a]anthracene

BF Benzo[b]fluoranthene and

benzo[k]fluoranthene

BP Benzo[a]pyrene

BPer Benzo[g,h,i]perylene

Ca Calcium

Cd Cadmium

CH3Hg; MeHg Methyl-mercury

Chl Chlorophyll

Chr Chrysene

Cl Chloride

CO2 Carbon dioxide

CO32-

Carbonate

CRM Certified Reference Material

Cu Copper

dBA Dibenzo[a,h]anthracene

ɛ Molar extinction coefficient

EDL Electrodeless Discharge Lamps

ELISA Enzyme Linked Immunosorbent Assay

EtOAc Ethyl acetate

FIAS Flow Injection Analysis System

Flt Fluoranthene


SCHeMA – 614002_ D8.2 31

Flu Fluorene

HCl Hydrochloric acid

HCs Hydrocarbons

HG Hydride Generation

Hg Mercury

HNO3 Nitric acid

HPLC High Pressure (Performance) Liquid

Chromatography

IC Ion Chromatography

ICP Inductively Coupled Plasma

IP Indeno[1,2,3-c,d]pyrene

K Potassium

KI Potassium iodide

LOD Limit of Detection

LOQ Limit of Quantification

Mg Magnesium

MS Mass Spectrometry

Na Sodium

NaBPr4 Sodium tetrapropylborate

NaNO2 Sodium nitrite

NaOH Sodium hydroxide

Nap Naphthalene

NH4Ac Ammonium acetate

NH4OH Ammonium hydroxide

PAHs Polycyclic Aromatic Hydrocarbons

Pb Lead

pCO2 Partial pressure of carbon dioxide

PEEK Polyether ether ketone

Phe Phenanthrene

PSTs Paralytic Shellfish Toxins

Pyr Pyrene

RSD Relative Standard Deviation


SCHeMA – 614002_ D8.2 32

SBAE Strong base anion exchange

STX Saxitoxin

VCl3 Vanadium chloride

VOCs Volatile Organic Constituents

Zn Zinc


SCHeMA – 614002_ D8.2 33

5 References

Ben Issa, N., Marinkovic, A., D., Rajakovic, L., V. 2012. Separation and determination of

dimethylarsenate in natural waters. Journal of Serbian Chemical Society 77, (6) 775–788.

Bizkarguenaga E., Ros O., Iparraguirre A., Navarro P., Vallejo A., Usobiaga A., Zuloaga O. 2012.

Solid-phase extraction combined with large volume injection-programmable temperature

vaporization–gas chromatography–mass spectrometry for the multiresidue determination of priority

and emerging organic pollutants in wastewater, Journal of Chromatography A, 1247, 104– 117.

Brunet, C., Brylinski, J. M. and Lemoine, Y. 1993 In situ variations of the xanthophylls

diadinoxanthin and iatoxanthin: photoadaptation and relationships with an hydrodynamical system of

the Eastern English Channel.Mar. Ecol. Prog. Ser.,102, 69–77.

Brunet C., Mangoni O. 2010. Determinazione quali-quantitativa dei pigmenti fitoplanctonici medinte

HPLC. In Metodologie di studio del Plancton Marino. Manuali ISPRA (Istituto Superiore per la

Protezione e la ricerca Ambientale) pp. 379-387.

Buchwalder, K.L. 2005. Jonction Titrator. Mode d'emploi, University of Geneva.

Casotti, R., Brunet, C., Aronne, B., Ribera d’Alcala, M. 2000. Mesoscale features of phytoplankton

and planktonic bacteria in a coastal area as induced by external water masses, Marine Ecology

Program Series, 195, 15-27.

Castelle, S. Spéciation et réactivité du mercure dans le système fluvio‐estuarien girondin. Ph.D.

Thesis report, University of Bordeaux I, 2008, 201p.

Cervantes Cianca, R.C., Pallares, M.A., Durán Barbosa, R., Vidal Adan, L., Leão Martins, J.M.,

Gago-Martínez, A. 2007. Application of precolumn oxidation HPLC method with fluorescence

detection to evaluate saxitoxin levels in discrete brain regions of rats. Toxicon, 49, (1), 89-99.

Costa, P.R., Botelho, M.J., Lefebvre, K.A. 2010. Characterization of paralytic shellfish toxins in

seawater and sardines (Sardina pilchardus) during blooms of Gymnodinium catenatum.

Hydrobiologia, 655, 89-97.

Cusick, K.D., Sayler, G.S. 2013. An Overview on the Marine Neurotoxin, Saxitoxin: Genetics,

Molecular Targets, Methods of Detection and Ecological Functions. Marine Drugs, 11, (4), 991-

1018.


SCHeMA – 614002_ D8.2 34

Gong., Z., Lu, X., Ma, M., Watt, C., Le, X.C. 2002. Arsenic speciation analysis. Talanta, 58 (1), 77-

96.

EN 15204 (2006). Water quality. Guidance standard on the enumeration of phytoplankton using

inverted microscopy (Utermöhl technique). European Commettee for Standardization, Brussels.

Hasle, G.R. 1978. “The Inverted-microscope method” In Phytoplancton Manual, ed. by A. Sournia,

UNESCO pp.88-96.

Jeffrey, S.W., 1997. Application of pigment methods to oceanography. In: Jeffrey, S.W., Mantoura,

R.F.C., Wright, S.W. (Eds.), Phytoplankton pigments in oceanography. UNESCO,Paris, pp. 127-178

Idronaut website. http://www.idronaut.it/cms/view/products/sensors/pcosub2sub/s333, last accessed

September 2015.

Lawrence, J.F., Ménard, C., Cleroux C. 1995. Evaluation of prechromatographic oxidation for liquid

chromatographic determination of paralytic shellfish poisons in shellfish. Journal of AOAC

International, 78(2), 514-20.

Leermakers, M., Baeyens, W., Quevauviller, P., Horvat, M. 2005. Mercury in environmental

samples: Speciation, artifacts and validation. Trends in Analytical Chemistry, 24, (5), 383-393.

Lefebvre, K.A., Bill, B.D., Erickson , A., Baugh, K.A., O’Rourke , L., Costa, P.R., Nance, S.,

Trainer, V.L. 2008. Characterization of Intracellular and Extracellular Saxitoxin Levels in Both Field

and Cultured Alexandrium spp. Samples from Sequim Bay, Washington. Marine Drugs, 6, 103-116.

Lund J.W.G., Kilpling C., Le Cren E.D. (1958). The inverted microscope method of estimating algal

numbers, and the statistical basis of estimation by counting. Hydrobiologia, 11 82): 143-170.

Magi E., Bianco R., Ianni C., Di Carro M. 2002. Distribution of Polycyclic Aromatic Hydrocarbons

in the sediments of the Adriatic Sea. Environmental Pollution, 119, 91-98.

Mantoura, R. F. C., Llewellyn, C.A. 1983. The rapid determination of algal chlorophyll and

carotenoid pigments and their breakdown products in natural waters by reverse-phase high-

performance liquid chromatography, Analytica Chimica Acta, 151, 297- 314.

Poirier, D., Charbonnier, C. 2015. Internal document on colorymetry-based laboratory techniques.

11-16.

http://www.idronaut.it/cms/view/products/sensors/pcosub2sub/s333


SCHeMA – 614002_ D8.2 35

Schäfer, J., Castelle, S., Blanc, G., Dabrin, A., Masson, M., Lanceleur, L., Bossy, C. 2010. Mercury

methylation in the sediments of a macrotidal estuary (Gironde Estuary, south-west France).

Estuarine, Coastal and Shelf Science, 90, 80-92.

Skoog, D.A., West, D. M., Holler, F. J. 1997. Chimie analytique, Traduction de la 7ème édition

américaine, de Boeck.

Søndergaard, J., Asmund, G., Larsen, M.M. 2015. Trace elements determination in seawater by ICP-

MS with on-line pre-concentration on a Chelex-100 column using a ‘standard’ instrument setup.

MethodsX, 2, 323–330.

Yamamoto, M., Yasuda, M., Yamamoto, Y. 1985. Hydride-generation atomic absorption

spectrometry coupled with flow injection analysis. Analytical Chemistry, 57 (7), 1382–1385.

Vidussi, F., Claustre, H., Bustillos-Guzman, J., Cailliau, C., Marty, J.C., 1996. Determination of

chlorophylls and carotenoids of marine phytoplankton : separation of chlorophyll a from divinyl-

chlorophyll a and zeaxanthin from lutein. Journal of Plankton Research 18, 2377-2382

Zingone, C., Totti, D., Sarno, M., Cabrini, C., Caroppo, M.G., Giacoppo, A., Lugliè, C., Nuccio, G.,

Socal. 2010. Fitoplancton: Metodiche di analisi quali-quantitativa. In Metodologie di studio del

Plancton Marino. Manuali ISPRA (Istituto Superiore per la Protezione e la ricerca Ambientale) pp.

213-237.

Documents

Grant Agreement no : 614002 · Grant Agreement no : 614002 SCHeMA INTEGRATED IN SITU CHeMICAL MAPPING PROBE Collaborative project OCEAN.2013-2: Innovative multifunctional sensors