Ecuador protocol - 1

Contact in case of problems, questions, … Never hesitate to ask !

In case of problems with the material, lack of vials, filters, etc … or questions please
contact as soon as possible:
alberto.borges@ulg.ac.be

Before leaving to the field

- Check the status of the batteries of the probe

List of material needed for each sampling

Orange Peli box with Li-cor (switch on the Licor in the morning 1-2 h before first station)
Computer with Li-820 software
ProPlus probe
Field notebook
Pencils & permanent marking pens
Sampling bottle with silicone tubing
Micropipette (100 – 1000 µL, minimum delivery: 50 µL)
Box of blue tips
2 tweezers for handling filters
4 different graduated cylinders (100 ml, 250 ml, 500 ml, 1000 ml)
large syringes (60 ml) for filtrations
Encapsulated syringe filters (blue, 0.2 µm), see picture 10
1 filtration set for 47 mm filters + its bottle
1 filtration set for 25 mm filters + its bottle
HgCl2 flask
H3PO4 (50%) flask
Serum bottles (30 ml) for CH4/N2O concentrations; see picture 2
Grey butyl rubber stopper; see picture 2
Aluminum caps; see picture 2
Crimper to close the serum bottles see picture 2
Exetainers (12 ml) with grey stoppers for DIC; see picture 3
Bag with 5 syringes + four 3-way valves; see
Amber small glass vials with green cap (20 ml) for cDOM; see picture 11
Amber large glass vials with the green cap (40 ml) for DOC; see picture 15
Scintillation vials (20 ml) for major elements; see picture 13
Clear plastic vials with white lid for nutrient concentration; see picture 17
White plastic vials for total alkalinity measurement; see picture 10
Pre-weighted large GFF filters (47 mm) in petrislides
Small GFF filters (25 mm) in petrislides
4 large containers for the water samples (5 L)
 Ecuador protocol - 2

SAMPLING PROTOCOL
On the field :

I. Write in the notebook, the station name with sequential number for all stations (#1, #2,
etc…), the date ((DD/MM/YYYY), time when sampling started (HH:MM), name of the
river, any relevant information (fast/slow flow, steep, type of vegetation, etc…)
II. Look at geographical point (Lat/Long) on google map
III. Ask the name of the stream to locals
IV. If possible take a photograph of the sampling site.
V. Put the ProPlus probe in the water, wait 5 minutes at the surface, write down the value
in the notebook.
VI. Sample surface water with 4 syringes for pCO2 measurements (see (1) in page 3), and
do the sampling for pCO2.
VII. Sample for CH4/N2O with bottle device
VIII. Sample a large container for the other variables

-
 Ecuador protocol - 3

(1) pCO2 sampling with Li-Cor

Write the first value of the Li-cor that is stable, after injection. There is a very small leak in
the Li-cor so values will drift.

- Fill 4 syringes with surface water without making bubbles (plunge the syringes below
water)
- Expel water to adjust to 30 ml of water for the three syringes
- Add 30 ml of ambient air. When doing this keep the syringes away from your face or
something else’s face, and away for exhausts of boat engine or from cars. The idea is to
avoid contamination of CO2.
- Make sure the syringes are properly closed with the 3 way-valve (see picture 1)
- Shake the 4 syringes with water during 5 minutes (chronometer with a watch). Shake in
the shade to avoid warming of syringues.
- Inject 3 syringes of ambient air into the Li-cor
- Write down the value of the ambient air value (typically close to 400 ppm)
- Inject the headspace of gas from the first syringe, write down the value.
- Open the syringe and put the thermometer in the water of the syringe, and write down the
value.
- Do the same thing for the other 3 syringes.

Picture N°1

΃ϯ ϬŵůŽĨŐĂƐ
΃ϯ ϬŵůŽĨǁ ĂƚĞƌ

3-way valve closed 3-way valve open

 Ecuador protocol - 4

(2) CH4, N2O (2 serum bottles of 30 ml)

The quality of the CH4/N2O samples might be altered because of the biological activity, it is
therefore crucial to poison them with HgCl2.
Bubbles during the filling of the serum bottles and in the closed bottles will alter the quality
of the samples because the aim is to measure the dissolved gas concentration. Caution is
therefore needed during the sampling to avoid air bubbles in the bottles.

- Label 2 serum bottles (30 ml) with the station name, river name, and the date. The
date should always be written following this pattern : dd/mm/yyyy
- Fill directly from the tube of the sampling bottle the 2 serum bottles. Make sure to let
the water flow gently from the tube. It is important to make sure that the bottles are
flushed with at least 2x the volume of the bottles before creating a meniscus at the top
of the bottles, and removing the bottle from the water flow. (avoid bubbles during the
filling of the bottles !)
- Add in the 2 bottles 200 µl of the HgCl 2 solution (dip the blue tip of the micropipette
a few millimeters in the filled bottle. Avoid air bubbles ! : so stop the injection at the
first notch of the micropipette).
- Put a butyl rubber stopper and an aluminium cap, then seal the bottle with the
crimper.
- Keep and store the bottles in a dark and dry place, and at a relatively constant
temperature. (NEVER STORE THEM IN A FRIDGE OR FREEZER)
-
Picture N°2

2 serum bottles, butyl rubber HgCl2 flask, micropipette and blue tips
stopper, aluminium caps, crimper
 Ecuador protocol - 5

(3) δ13C-DIC (1 Exetainer)

The quality of the δ13C-DIC (in Exetainer vial) samples might be altered because of the
biological activity, it is therefore crucial to poison them with HgCl2.
Bubbles during the filling of the Exetainer vials and in the closed vials will alter the quality
of the samples because the aim is to measure the dissolved species stable isotope
composition. Caution is therefore needed during the sampling to avoid air bubbles in the
vials.

- Label 1 Exetainer vial (12 ml with grey stopper) with the station name, monitoring
cruise number, and the date. The date should always be written following this
pattern : dd/mm/yy
- Fill directly from the silicone tube of the Niskin bottle 1 Exetainer vial. Make sure to
let the water gently flow from the silicone tube, without bubbles. Create meniscus at
the top of the vial and remove the vial from the water flow. ( avoid bubbles during the
filling of the vial !)
- Add 50 µl of the HgCl2 solution in the vial (dip the blue tip of the micropipette a few
millimeters in the filled bottle. Avoid air bubbles ! : so stop the injection at the first
notch of the micropipette).
- Close the vial with its white lid.
- Keep and store the vial in a dark and dry place, and at a relatively constant
temperature. (NEVER STORE THE EXETAINER IN A FRIDGE OR FREEZER)

Picture N°3

1 Exetainer HgCl2 flask, micropipette and blue tips (right)

vial
 Ecuador protocol - 6

(4) Sample 2-5 L of water in the large container for subsequent sample processing
onshore

- Make sure to fill the right container (labelled with the station name and depth) at the
right station and depth.
- Rince THREE TIMES the container with a small amount of water from the Niskin
bottle
- Fill the container with 5 L of the water from the Niskin bottle
- Store the filled container in the shade
 Ecuador protocol - 7

Subsequent sample processing on shore

For every sampling point, the following samples should be processed :

- (9) TSM concentration (filtration)
- (10) POC concentration (filtration)
- (11) POP concentration (filtration)
- (12) Total alkalinity
- (13) cDOM composition
- (14) Major elements composition
- (16) DOC concentration and isotopic composition
- (17) Nutrients concentration

Before starting the processing of the samples, setup the lab with all the material needed :
- Notebook, pencil and permanent marking pen
- Large Filtration set 47 mm + white plastic bottle
- Small filtration set 25 mm + white plastic bottle
- Manual Vacuum pump
- Tweezers for handling the filters
- Graduated cylinders of 1000, 500, 250, 100 ml
- 1 clear plastic vial (50 ml) with white lid
- 1 scintillation vial of 20 ml
- 1 small amber glass bottle with white cap (20 ml)
- 1 large amber glass bottle with green cap (40 ml)
- 1 petrislide with a pre-weighted 47 mm GFF filters (TSM)
- 2 petrislides with 25 mm GFF filters
- Encapsulated syringe filter
- Large syringe (60 ml)
- H3PO4 flask
- Micropipette (100-1000 µl)
- Box of blue tips
 Ecuador protocol - 8

(5) TSM (“Total Suspended Matter”)

- Make sure to homogenize (shake) the large (5 L) plastic container filled with the
lake water just before collecting it for the filtration
- Collect from the large container a precise volume of water (for example, 500 ml), and
filter it through 1 preweighted filter (47 mm). The initial weight of the filter is already
written on the white label on the petrislide. The volume that can/should be filtered
depends on the turbidity/biomass of the sampling site, and can be modified if needed–
just make sure to note down the exact volume.
- After the filtration, take the filter with the tweezer and put it back in its petrislide. Make
sure to write the exact volume of water filtered in the notebook.
- Write on the label on the petrislide the station name, depth, the monitoring campaign
number, the date (dd/mm/yy), and the exact volume of water filtered.
- Let the filter dry 24h in a clean and dry place (avoid dust deposit on the filter). In case of
sunny weather, let the filter dry under the sun (in a clean place, without dust, sheltered
from the wind).
-
Picture N°7

Preweighted filter (47 mm) in its 47 mm filtration set

petrislide
 Ecuador protocol - 9

(6) POC (« Particulate Organic Carbon »)

- Make sure to homogenize (shake) the large (5 L) plastic container filled with the
lake water just before starting the filtration
- Use the small filtration set (25 mm). Make sure to not use the small filtration set
dedicated to the filtration of primary production measurement samples
- Collect from the large container a precise volume of water (for example, 100 ml), and
filter it through 1 small GFF filter (25mm).
- After the filtration, put the filter back in its petrislide and write the exact volume of water
filtrated in the notebook.
- Write on the label on the petrislide « POC », the station name, depth, monitoring cruise
number, date (dd/mm/yy) and exact volume of water filtrated.
- Let the filter dry 24h in a clean and dry place (avoid dust deposit on the filter). In case of
sunny weather, let the filter dry under the sun (in a clean place, without dust, sheltered
from the wind).
-
Picture N°8

Filters for POC and POP (25mm) Small filtration set (25 mm)
 Ecuador protocol - 10

(7) POP (« Particulate Organic Phosphorus »)

- Make sure to homogenize (shake) the large (5 L) plastic container filled with the
lake water just before starting the filtration
- Use the small filtration set (25 mm). Make sure to not use the small filtration set
dedicated to the filtration of primary production measurement samples
- Collect from the large container a precise volume of water (100 ml), and filtrate it
through 1 small GFF filter (25mm).
- After the filtration, put the filter back in its petrislide and write the exact volume of water
filtrated in the notebook.
- Write on the label on the petrislide « POP », the station name, depth, monitoring cruise
number, date (dd/mm/yy) and exact volume of water filtrated.
- Let the filter dry 24h in a clean and dry place (avoid dust deposit on the filter). In case of
sunny day, let the filter dry under the sun (in a clean place, without dust).
-
Picture N°9

Filters for POC and POP (25mm) Small filtration set (25 mm)
 Ecuador protocol - 11

(8) Total alkalinity

These samples are not altered by the biological activity. It is therefore unnecessary to poison
them. BUT they imperatively should be filtered with the encapsulated syringe filter (blue,
0.22 µm)

- Label 1 white plastic vial (50 ml) for total alkalinity (station name, number of the
monitoring cruise, depth, date (dd/mm/yy)
- Sample ~50 ml of water of the TSM filtration with the a large syringe (60 ml)
- Screw at the bottom of the syringe an encapsulated syringe filter (blue, 0.22 µm)
- Fill the white plastic vial with filtered water (pushing the piston).
- Repeat the previous step untill the vial is completely filled
- Never add poison/acids in these vials
- Store and keep the samples in the dark and room temperature (DON’T FREEZE THEM)

Picture N°10

White plastic vial The vial was filled with water filtered with the encapsulated
(50 ml) for total syringe filter, screwed in the syringe (right). NO POISON,
alkalinity sample NO ACID, NO FREEZING for these samples
with the lid and
the cap screwed
 Ecuador protocol - 12

(9) CDOM (« Coloured Dissolved organic Matter »)

These samples are not altered by the biological activity. It is therefore unnecessary to poison
them. BUT they imperatively should be filtered with the encapsulated syringe filter (blue, 0.22
µm)

- Label 1 amber glass vial with green lid (station name, number of the monitoring cruise,
depth, date (dd/mm/yy)
- Sample ~50 ml of water of the TSM filtration with the a large syringe (60 ml)
- Screw at the bottom of the syringe the same encapsulated syringe filter than used for
alkalinity
- Fill the amber glass vial with the filtered water (pushing the piston).
- Never add poison/acids in these vials
- Store and keep the samples in the dark at 4°C (DON’T FREEZE THEM)

Picture N°11

Amber glass vial The vial was filled with water filtered with the encapsulated
with green lid for syringe filter, screwed in the syringe (right). NO POISON,
cDOM sample NO ACID, NO FREEZING for these samples

Picture N°12

CDOM vial is DOC vial is

→
smaller (20ml) → larger (40ml)
White label Red label

CDOM and DOC vials look very similar but are different size !
 Ecuador protocol - 13

(10) Major elements

- Label 1 scintillation vial (20 ml) with the station name, number of the monitoring cruise,
depth, date (dd/mm/yy). Write on the vial in itself, not the cap.
- Sample ~50 ml of water of the TSM filtration with the a large syringe (60 ml)
- Screw at the bottom of the syringe the same encapsulated syringe filter than used for cDOM
and alkalinity samples
- Fill the scintillation vial with 20 ml of filtered water (pushing the piston).
- Never add poison/acids in these vials
- Store and keep the samples in the dark and at room temperature (DON’T FREEZE
THEM)

Picture N°13

20-ml scintillation vial The vial was filled with water filtered with the encapsulated
for major elements syringe filter, screwed in the syringe (right). NO POISON,
sample NO FREEZING for these samples.
 Ecuador protocol - 14

(11) DOC (« Dissolved Organic Carbon »)

These samples are altered by the biological activity. They should be filtered with an
encapsulated syringe filter and acidified with H3PO4 in order to insure good preservation of the
sample.
- Label 1 amber glass vial with the green cap (40 ml) with the station name, number of the
monitoring campaign, depth and date (dd/mm/yy).
- Sample ~50 ml of water of the TSM filtration with the a large syringe (60 ml)
- Screw at the bottom of the syringe the same encapsulated syringe filter than used for
alkalinity and cDOM
- Fill the amber glass vial with green cap with filtered water (pushing the piston), leave a
few mL of air space, i.e. do not fill the vials completely.
- Add 50 µL of phosphoric acid (H3PO4) in the vial
- Store and keep the samples in the dark and at room temperature (DON’T FREEZE
THEM, DON’T ADD HgCl2 !)
- Every few months, start using a new flask of phosphoric acid (H3PO4) and discard the old
one. Also, when you suspect the acid might have become contaminated with organics (e.g.
visual presence of dust, particles, a pipette tip that dropped in), start using a new batch.

Picture n°15

Amber glass vial The vial was filled with water filtered with the encapsulated syringe
(40 ml) with filter, screwed in the syringe AND acidified with 50 µl of H3PO4, using
green cap the micropipette (right). NO FREEZING for these samples

Picture N°16

CDOM vial is DOC vial is

→
smaller (20ml) → larger (40ml)
White label Red label

CDOM and DOC vials look very similar but are different size !
 Ecuador protocol - 15

(12) Nutrients (NO3-, NO2-, NH4+, SRP, P total)

- Label 1 clear plastic vials with white lid with the station name, number of the monitoring
campaign, depth and date (dd/mm/yy)
- Sample ~50 ml of water of the TSM filtration with the a large syringe (60 ml)
- - Screw at the bottom of the syringe the same encapsulated syringe filter than previously
used during the processing of the samples of this sampling point and fill the vial
- Add 100 µl of HgCl2 in the vial, put in a freezer if possible

Picture N°17

Clear plastic vial with white lid (50 ml) The vialsis filled with water filtered with the
encapsulated syringe filter, screwed in the
syringe, and poisoned with 100 µl of H gCl2,
using the micropipette