Science of the Total Environment 669 (2019) 49–61

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Greenhouse gases, nutrients and the carbonate system in the Reloncaví

Fjord (Northern Chilean Patagonia): Implications on aquaculture of the
mussel, Mytilus chilensis, during an episodic volcanic eruption
Mariela A. Yevenes a,b, Nelson A. Lagos c,d, Laura Farías e,f, Cristian A. Vargas d,g,h,⁎
a
Departamento de Sistemas Acuáticos, Facultad de Ciencias Ambientales, Centro EULA, Universidad de Concepción, Chile
b
Centro de Recursos Hídricos para la Agricultura y la Minería (CRHIAM), Chile
c
Centro de Investigación e Innovación para el Cambio Climático (CiiCC), Facultad de Ciencias, Universidad Santo Tomas, Santiago, Chile
d
Center for the Study of Multiple-Drivers on Marine Socio-Ecological Systems (MUSELS), Universidad de Concepción, Concepción, Chile
e
Departamento de Oceanografía, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Chile
f
Centro de Ciencia del Clima y la Resiliencia (CR)2, Chile
g
Millennium Institute of Oceanography (IMO), Universidad de Concepción, Concepción, Chile
h
Aquatic Ecosystem Functioning Lab (LAFE), Department of Aquatic Systems, Faculty of Environmental Sciences & Environmental Sciences Center EULA Chile, Universidad de Concepción, Chile

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• A large bloom of phytoplankton was de-

tected in the surface waters of the
Reloncaví fjord following the Calbuco
volcano eruption.
Sal O2
• Subsequent to the eruption, higher N2O, Low due to low salinity/low
CH4 and SO42− concentrations were ob- alkalinity waters driven by
freshwater runoff
served in Fjord surface waters.
• Optimal juvenile mussel growth was Optimum for mussel
observed in refugee subsurface depths growth and calcification

coinciding with increased aragonite sat-

uration.
Low due to high pCO2
• The observed trends may be valuable for Low O2 waters by organic
developing effective management strat- matter remineralization

egies for mussel aquaculture in the

Fjord.

a r t i c l e i n f o a b s t r a c t

Article history: This study investigates the immediate and mid-term effects of the biogeochemical variables input into the
Received 19 November 2018 Reloncaví fjord (41°40′S; 72°23′O) as a result of the eruption of Calbuco volcano. Reloncaví is an estuarine system
Received in revised form 1 March 2019 supporting one of the largest mussels farming production within Northern Chilean-Patagonia. Field-surveys were
Accepted 3 March 2019
conducted immediately after the volcanic eruption (23–30 April 2015), one month (May 2015), and five months
Available online 7 March 2019
posterior to the event (September 2015). Water samples were collected from three stations along the fjord to de-
Editor: Daniel Wunderlin termine greenhouse gases [GHG: methane (CH4), nitrous oxide (N2O)], nutrients [NO3−, NO2−, PO43−, Si(OH)4, sul-
phate (SO42−)], and carbonate systems parameters [total pH (pHT), temperature, salinity, dissolved oxygen (O2),
Keywords: and total alkalinity (AT)]. Additionally, the impact of physicochemical changes in the water column on juveniles
Chilean Patagonian Fjord of the produced Chilean blue mussel, Mytilus chilensis, was also studied. Following the eruption, a large phyto-
Carbonates system plankton bloom led to an increase in pHT, due to the uptake of dissolved-inorganic carbon in photic waters, po-
Nutrients tentially associated with the runoff of continental soil covered in volcanic ash. Indeed, high surface SO42− and GHG
were observed to be associated with river discharges. No direct evidence of the eruption was observed within the

⁎ Corresponding author at: Aquatic Ecosystem Functioning Lab (LAFE), Department of Aquatic Systems, Faculty of Environmental Sciences & Environmental Sciences Center EULA Chile,
Universidad de Concepción, P.O. Box 160-C, Concepción, Chile.
E-mail address: crvargas@udec.cl (C.A. Vargas).

https://doi.org/10.1016/j.scitotenv.2019.03.037
0048-9697/© 2019 Elsevier B.V. All rights reserved.
50 M.A. Yevenes et al. / Science of the Total Environment 669 (2019) 49–61

Mussel farming carbonate system. Notwithstanding, a vertical pattern was observed, with an undersaturation of aragonite (ΩAr
Volcanic event b 1) both in brackish surface (b3 m) and deep waters (N10 m), and saturated values in subsurface waters (3 to
7 m). Simultaneously, juvenile mussel shells showed maximized length and weight at 4 m depth. Results suggest
a localized impact of the volcanic eruption on surface GHG, nutrients and short-term effects on the carbonate sys-
tem. Optimal conditions for mussel calcification were identified within a subsurface refuge in the fjord. These
specific attributes can be integrated into adaptation strategies by the mussel aquaculture industry to confront
ocean acidification and changing runoff conditions.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction
Natural disasters such as earthquakes and volcanic eruptions pro-
vide excellent opportunities to evaluate effects of mesoscale and large
disturbances on ecosystems (Wang et al., 2012). These disturbances
can leave ephemeral or long lasting ecological effects on spatial and
temporal patterns of marine communities (e.g. Jaramillo et al., 2017).
Thus, disasters within ecosystem have a fundamental effect on material
flux (minerals) across the land-ocean interface. Fjords are good exam-
ples of these events; they cover approximately 7 to 10% of the global
ocean (Sauer et al., 2016) and represent an important interface and reg-
ulating area between freshwater systems (i.e., lakes and rivers), glaci-
ated terrain, and the coastal ocean. Moreover, fjord ecosystems are
highly valuable in terms of different ecosystems services, such as nutri-
ent availability, carbon cycling, water quality for aquaculture produc-
tion, and pollution control (Iriarte et al., 2010). In consequence, many
studies highlight the threat of global changes on estuarine and fjord
coastal systems (Iriarte et al., 2010; Letelier et al., 2011; Silva and
Vargas, 2014; Vargas et al., 2017a).
The impacts of anthropogenic activities (such as land use changes,
nutrient loading, river damming, fishing, mussel farming) on biogeo-
chemical cycles have been well documented for fjord environments,
for example research by Filgueira et al. (2010) in Sweden and Norway.
Recently, similar impacts have also been documented in fjord ecosys-
tems of Chilean Patagonia (Silva and Vargas, 2014; Mayr et al., 2014;
Iriarte et al., 2017; Farías et al., 2017; Vargas et al., 2017b; Yevenes
et al., 2017). Aquaculture production in fjord ecosystems largely de-
pends on the integrity of different ecological, biophysical, and biogeo-
chemical processes, which are subjected to multiple sources of
variability (Barria et al., 2012; Lara et al., 2016). Fjords are sensitive to
local acidification processes, mostly due to the influence of riverine wa-
ters with a low total alkalinity (AT), which in turns reduces the buffering
capacity of receiving waters (Chierici and Fransson, 2009). Thus, an in-
crease in freshwater fluxes into fjord ecosystems has the potential not
only to decrease seawater pH and reduce the saturation state of calcium
carbonate (Ω), but also induce a shift in the NH3–NH4+ equilibrium to-
wards NH4+ (Freing et al., 2012), affecting nitrification and associated
N2O generation. Moreover, fjords store large amounts of organic carbon,
with burial rates exceeding double the average rates in global oceans
(Smith et al., 2015; Mohr et al., 2017). However, the influence of epi-
sodic disturbances, such as volcanic eruptions, in mobilizing nitrogen,
carbon and greenhouse gases in fjord environments remains poorly
studied (Mohr et al., 2017).
The Reloncaví Fjord area has been affected numerous times by the
eruption of the Calbuco Volcano (41°20′S, southern Chile), with erup-
tions registered in 1961, 1972, and more recently during April 22th
2015. During the last eruption, Calbuco Volcano ejected a significant
ash plume that required the evacuation of nearby areas, disrupted aerial
traffic, and damaged buildings in Chile (Van Eaton et al., 2016). The vol-
canic eruption produced a stratospheric column (N15 km height;
0,27 km3) of porphyritic basaltic andesite (SiO2). The tephra fall oc-
curred over the northeast area of the volcano, while the finest ash was
transported and deposited over northern Patagonia in Chile and
Argentina (Romero et al., 2016). It is widely acknowledged that an
increase in volcanic ash covering soils may result in high levels of dis-
solved silica in rivers, thus increasing the flux into fjord environments.
As a result, phytoplankton productivity and community structure may
be affected (Balseiro et al., 2014; Vandekerkhove et al., 2016). More-
over, ash fallout can also induce acidification of soils (Borie et al.,
2002), thus leading to low pH events in adjacent fjord areas (Santana-
Casiano et al., 2013). It is well known that low pH/high pCO2 conditions
can have a negative impact on marine calcifiers, such as corals, sea ur-
chins, gastropods, and mussels (Kroeker et al., 2010). Specifically, the
endemic Chilean mussel, Mytilus chilensis, is present in the intertidal
zone down to 25 m depth throughout the Chilean coastline. This specie
can withstand high salinity and brackish water, for example in river
mouths or close to glaciers, subsisting through the filtration of
microalgae (Molinet-Flores et al., 2015; Ríos et al., 2018). Mytilus
chilensis is the main farmed shellfish, and represents a considerable frac-
tion of aquaculture production in Chile of up to approximately 238,088
tons annually, representing the 96,6% shellfish production (Sauer et al.,
2016; Molinet-Flores et al., 2015; Castillo et al., 2016). Relative to this,
the Reloncaví Fjord is one of the most important areas for mussel seed
production (with 99% of production concentrated within these waters),
predominantly made up by the Chilean mussel specie, Mytilus chilensis.
Therefore, the treat of volcanic eruptions also has major implications
on the socio-ecological systems of mussel farming in the region. Re-
cently, Salas-Yanquin et al. (2018) identified the physiological impact
of the presence of volcanic ash from Calbuco Volcano on the diet of
Mytilus chilensis and their ability to select particles during feeding.
This study aims to investigate the posteriori potential effect of a high
magnitude volcanic eruption through the study of greenhouse gases
(such as CH4 and N2O), nutrients, and the carbonate system in the
Reloncaví Fjord during autumn (April), winter (May) and spring (Sep-
tember) 2015. Furthermore, this study aims to determine the impact
of potential changes in the carbonate system on the condition of mussel
shells in a nearby farming area.
2. Materials and methods
2.1. Study area
This study was carried out in the Reloncaví Fjord (41° 40′S–72° 32′W
′) located in northern Chilean Patagonia (Fig. 1). From the head to the
mouth, the fjord has a total length of 55 km and a width of almost
3 km in its widest section, and is characterized to have a glacial origin
(Valle-Levinson et al., 2007; Araya-Vergara, 2008). The main source of
freshwater comes from the Puelo River, with an annual average of
650 m3 s−1 (León-Muñoz et al., 2013). Winds have a marked seasonal
variability, during winter predominant winds are from the north
while during summer winds from the south and southeast are preva-
lent, with magnitudes that are generally between 9 m s−1 to 15 m s−1
(Saavedra et al., 2010; Castillo et al., 2012). The fjord area presents a
marked hydrographical variability, related to regimes of high rainfall
and ice melt (Castillo and Pizarro, 2009). High levels of freshwater
input from tributary rivers generate considerable salinity variations
that characterize the distinct water masses present in this region,
made up of channels and fjords (León-Muñoz et al., 2013). As a result
 M.A. Yevenes et al. / Science of the Total Environment 669 (2019) 49–61
Fig. 1. Study area and stations located in Reloncaví Fjord, Northern Chilean Patagonia.
a strong spatial salinity gradient exists in the surface fraction (González
et al., 2010), with a stratified water column characterized by a marked
pycnocline between the upper layer (0 to 5 m depth) and the subsurface
layer below 5 m depth (Valle-Levinson et al., 2007). Below this narrow
layer, there is a relatively homogenous layer of marine water predomi-
nantly from sub-Antarctic origin, described as modified sub-Antarctic
water or MSAAW (Sievers and Silva, 2006).
2.2. Water sampling
Seasonal water sampling was made during autumn (30th April),
winter (29th May 2015) and spring (25th September 2015). Water
samples from three stations (st.1: −41° 43′ 57′ S–72° 32′ 50.8″W′;
st.2: −41° 41′ 40.4′ S–72° 26′ 24.6″W′; st.3: −41° 37′ 50.3′ S–72° 20′
55.6″W′) were collected along the Reloncaví estuary over five depths
(1, 5, 10, 25, 50 m depth) (Fig. 1). Our study was focused on the
short-term response during autumn, winter and the long-term con-
sequences on early spring conditions. The longitudinal distribution
of sampling stations was based on the location of one of the largest
mussel-farming sites near the fjord mouth (ORIZON Company near
Chaparano Bay, Stn. 1), and the location of one of the main river dis-
charges in the area (Puelo River in the mid- portion of the fjord at Stn
3). Discrete water samples were collected in triplicate using Niskin
bottles (General Oceanic®, GO-FLO Niskin bottles sampling), for
SO42−, Chlorophyll a (Chl-a), Total pH (pHT), Total Alkalinity (AT),
N2O, CH4 and nutrients (i.e., NO3−, NO2−, PO43−, Si(OH)4). Continuous
profiles of temperature, salinity, and dissolved oxygen were ob-
tained using a Conductivity-Temperature-Dissolved Oxygen Sonde
51
(CTDO) (SeaBird 25) to 50 m depth. Chlorophyll measurements
(Chl-a) were conducted with a Fluoroprobe sensor (bbe-Moldaenke,
Kiel, Germany) (Beutler et al., 2002); a highly sensitive measuring
instrument for the analysis of chlorophyll, including the determina-
tion of algal class. Chlorophyll measurements were calibrated by
contrasting with fluorometry analysis (Turner Design TD-700),
using acetone (90% v/v) for the pigment extraction of discrete sam-
ples according to standard procedures (Parsons et al., 1984). Sub-
samples for pH measurements were stored in borosilicate BOD
bottles with ground-glass stoppers without air bubbles and
transported to the field lab where pH was measured within 2 h of
collection. Water samples for AT were poisoned with 50 μL of satu-
rated HgCl2 solution and stored in 500 mL borosilicate BOD bottles
with ground-glass stoppers, lightly coated with Apiezon L® grease,
and kept in darkness at room temperature (Dickson et al., 2007).
For logistic support, samples for N2O and CH4 analysis were only col-
lected in April and May, in 20 mL glass vials (by triplicate), treated
with 50 μL of saturated HgCl2 solution (6 g L−1) and sealed with her-
metic stoppers and aluminium caps (Wilson et al., 2018). These sam-
ples were stored at ambient temperature for posterior analysis in the
laboratory. Additionally, water samples for nutrient analysis were
filtered through a 0.45 μm Cellulose acetate (CA) membrane and col-
lected in 15 mL falcon tubes, and then frozen for later analysis in the
laboratory. Samples for SO42− concentrations were collected in falcon
conical tubes of 50 mL and analysed with ion chromatographic
method with chemistry suppression, based on standard methods
4110 B, using Dionex ICS2100 equipment, through an anionic AS11
High Capacity, with concentrations above 0.1 mg L−1.
52 M.A. Yevenes et al. / Science of the Total Environment 669 (2019) 49–61
2.3. Carbonate system parameters
Samples for pH measurement were collected and directly
transported inland so that pH was measured within a 2 h timeframe
after collection (Vargas et al., 2016). pH samples were collected in
50 mL syringes and immediately transferred to a 25 mL thermo stated
cell at 25.0 ± 0.1 °C for standardization, with a pH meter Metrohm®
using a glass combined double junction Ag/AgCl electrode (Metrohm
model 6.0258.600) calibrated with 8.089 Tris buffer at 25.0 °C (DOE,
1994), and therefore registered on the total hydrogen ion scale (pHT).
AT was determined using the open cell titration method (Dickson
et al., 2007), using an automatic Alkalinity Tritrator Model AS-ALK2
Apollo SciTech. The AS-ALK2 system is equipped with a combination
pH electrode (8102BNUWP, Thermo Scientific, USA) and temperature
probe for temperature control (Star ATC probe, Thermo Scientific,
USA) connected to a pH meter (Orion Star A211 pH meter, Thermo Sci-
entific, USA). All samples were analysed at 25 °C (±0.1 °C) with temper-
ature regulation using a water bath (Lab Companion CW-05G). The
accuracy was controlled against a certified reference material (CRM,
supplied by Andrew Dickson, Scripps Institution of Oceanography, San
Diego, USA) and the AT repeatability averaged 2–3 μM kg−1.
pHT and AT data were applied to the program CO2SYS (Pierrot et al.,
2006) to calculate aragonite saturation state (ΩAr), and other carbonate
parameters (e.g. pCO2). The dissociation constants for carbonic acid (K1
and K2) were used by Dickson et al. (2007) for salinities between N30
PSU, Millero (2010) for estuarine waters between 1 and 30 PSU, and
for freshwater samples in the mid- and upper river sampling stations.
KHSO4 was determined for both freshwater and seawater samples
(Dickson et al., 2007).
2.4. Determination of nutrients, nitrous oxide and methane
N2O and CH4 detection was made via the generation of a 5 mL ultra-
pure helium headspace into the vial, by a gas-tight syringe, and main-
taining the gas and liquid phases in equilibrium at 40 °C within the
vial, allowing for the equilibrium of the headspace in the vial (Farías
et al., 2015). N2O dissolved in the seawater was measured through
gas–liquid equilibration in the vial at 40 °C for 15 min under agitation,
using a headspace autosampler device (HP Agilent) followed by quanti-
fication via gas chromatography (GC). N2O was analysed in a
SHIMADZU-17A gas chromatograph equipped at 350 °C, using a capil-
lary column and injector operated at 60 °C and 300 °C, respectively,
and connected to the autosampler (Farías et al., 2015). Ar/CH4 gas mix
was used as a carrier gas with a flux of 6.5 mL min−1.
CH4 was analysed in a Shimadzu 17A gas chromatograph equipped
by a flame ionization detector (FID) at 250 °C, through and injector
and a capillary column GS-Q at operated to 180° and 30 °C, respectively,
using N2 as carrier gas with a flux of 3 mL min−1, and GC connected to
the mentioned autosampler (Farías et al., 2015).
A calibration curve was made using several concentrations for N2O
(0.10 air, 0.5 and 1 ppmv) and CH4 (1, air, 5 and 10 ppmv) using
Matheson gas standards whose nominal concentrations were checked
using set of high-pressure primary gas standards, prepared for the
SCOR Working Group by John Bullister and David Wisegarver at NOAA
Pacific Marine and Environmental Laboratory (PMEL). One batch, re-
ferred to as the air ratio standard (ARS), had N2O and CH4 mole fractions
similar to modern air, and the other batch, referred to as the water ratio
standard (WRS), had higher had N2O and CH4 mole fractions for the cal-
ibration of high-concentration water samples (Wilson et al., 2018).
Both, the ECD and FID detectors responded linearly to these concentra-
tion ranges. The analytical error of the N2O and CH4 analyses was b3%
and 5%, respectively. The uncertainty of the measurements was calcu-
lated from the standard deviation of the triplicate measurements by
depth. Measurements with a variation coefficient N10% were not in-
volved in the gas record (Farías et al., 2015).
The nutrient analyses (NO3−, PO43− and Si(OH)4) were carried out
using colorimetric techniques (Grasshoff et al., 1983), with an
autoanalyzer SEAL analytical (AA3). Calibration curves were performed
previous to each set of measurements, using primary standard. The pre-
cision and detection limit of the method was, ±50 nM and 20 nM for
NO3−, ±30 nM and 110 nM for PO4−3− and ±70 nM and 0.030 μM for
Si(OH)4, respectively.
2.5. Biological sampling
During May 2015, juvenile individuals of M. chilensis (b2 cms, mus-
sels' seeds) from the same cohort were randomly collected from pegged
ropes (6 m in length) at three different depths (1, 3 and 4 m). At each
depth, all mussels that were attached along ca. 10 cm length of the set-
tlement ropes were collected. Among 90–105 mussel seeds were col-
lected from each depth level, transported (ca. 4 °C using ice-pack
lasting for ca. 6 h), to the laboratory under chilled conditions, and
then frozen (−20 °C) for posterior processing in the laboratory, which
was performed during Sept. 2015. Taxonomic identification based on
shell shape and lack of external ribs was used to minimize potential
bias in including other species mussels in the processing. Several mor-
phometric variables were recorded for each mussel, including total
wet weight, dry shell weight, and soft tissue weight (after 6 h at 60 °C,
Memmert®) using an analytical balance (Metler Toledo); maximum
shell length, shell height, and shell thickness (measured using a digital
caliper, Mitutoyo®). Shell thickness corresponds to the average of 4 sep-
arate measurements taken from newly forming shell growth along the
edge of the mussel valves (e.g., Lagos et al., 2016). Finally, the relative
shell condition index is estimated through calculating the total dry
shell weight as a percentage of the total weight (i.e., [Dry shell weight
/ Total weight] × 100).
2.6. Statistical analysis
The Pearson correlation coefficient and lineal regression were used
to define relationships among variables, including GHG, nutrient con-
centrations and the carbonate system as parameters (Munro, 2005).
Mussel measurements were subjected to descriptive statistical analysis
to define trends in the frequency distribution of variables over depth.
The Kolmogorov-Smirnov test for normality was applied, and several
distribution moments were also calculated (e.g., mean, standard devia-
tion, median and its 95% confidence interval), due to the fact distribu-
tions presented significant deviations from the normality (see
Supplementary material S1). The Kruskal-Wallis test was applied to
compare the medians of each variable between mussels collected over
different depths, and results were described using median (±IQR,
Inter Quartile Range). A Principal Component Analysis was also esti-
mated to describe the variation in the set of physical-chemical and bio-
logical variables.
3. Results and discussion
The eruption of Calbuco volcano released high levels of particles and
gases throughout its area of influence, including the estuarine waters of
Reloncaví fjord. Throughout the three periods of field sampling, the
fjord presented a vertical salinity distribution that clearly divided the
water column into two layers, separated by a marked halocline. In the
upper 5 m of the surface water salinity was approximately 8.84 to
15.2, and in the subsurface layer below the 5 m from 28.2 to 32.87
(Fig. 2a–i). Several studies have identified that the water in the
Reloncaví fjord are seasonally dominated by estuarine waters (EW) in
the upper layer to 5 m depth, and in the deeper layer there is more
cold and dense modified subantarctic water (MSAAW) (González
et al., 2010; Castillo et al., 2016). In this study surface temperature
fluctuated between 10.4 °C and 13.3 °C, with the lowest values recorded
in Stn. 1 during May, and the highest at Stn. 1 in April. In terms of
 M.A. Yevenes et al. / Science of the Total Environment 669 (2019) 49–61 53

Fig. 2. Physical-chemical distribution for the three stations along the Reloncaví Fjord, including Temperature (°C) (A, B, C), Salinity (D, E, F), and Dissolved Oxygen (mL L−1) (G, H, I), during
field visits in April (A, D, G), May (B, E, H) and September (C, F, I).

dissolved oxygen, well-oxygenated waters of approximately 8 mL L−1
were observed at the surface of brackish waters, but surface O2 falls
below 4 mL L−1 during the second sampling period, especially at Stn.
1, which is the station closest to the mouth of the Reloncaví fjord
(Fig. 2a–i). Castillo et al. (2016) has described the oceanographic
conditions of Reloncaví fjord to have a well-stratified water column, in-
cluding a thin surface layer of brackish water with mean salinities be-
tween 10.4 ± 1.4 (spring) and 13.2 ± 2.5 (autumn).
Additionally, Chilean Patagonia is characterized by continual humid
conditions throughout the year, subjected to abundant precipitation
(around 3000 mm year−1) during winter (June to August) and glacial
meltwater during spring/summer (September–January) (Garreaud,
2009; León-Muñoz et al., 2013). Throughout the study period the pre-
cipitation was recorded at the Puelo meteorological station (41° 39′
3.96″S 72° 18′42.12″O); 353,4 mm recorded during April; 834 mm dur-
ing May, and 2430 mm during September 2015 (available at http://
explorador.cr2.cl). This precipitation will eventually contribute to river
runoff. Thus, river discharges of glacial origin (Petrohué, Puelo and
Cochamo Rivers) towards the Reloncaví fjord can reach a maximum
flow from July to September, with flow intensities reaching up to
~1500 m3 s−1 (Castillo et al., 2016), in turn this is an important driver
of biogeochemical activity in the case of a volcanic eruption.
3.1. Biogeochemistry of estuarine waters
The occurrence of volcanic eruptions in Patagonia has led high con-
centrations of SO42− in surrounding areas (Bia et al., 2015). As a conse-
quence high levels of SO42− have been observed in surface waters
(0–5 m depth) of the fjord, after April relatively high levels were re-
corded (Table 1), between b500 to 2300 mg L−1 that decreased over
time due to dilution from precipitation and snowmelt. During May,
the lowest surface SO42− concentrations were recorded in the surface
layer (b500 mg L−1). The fact that field sampling was carried out imme-
diately after the eruption, and in conditions of zero rainfall during the
eruption event may explain the SO42− increase in surface waters.
Flaathen and Gislason (2007) suggest that the effect of H2SO4 coming
from volcanic eruptions is both temporally and spatially dependent;
for example the location of Calbuco volcano is high latitude and is ex-
posed to high solar radiation during daytime (during autumn), thus
leading to an intense local H2SO4 contamination due to the high oxida-
tion rate of SO2 into SO42−. However, in general, studies in Patagonian
surface waters have indicated that SO42− concentrations do no exceed
the detection limits of the instrumental methods (Beamud et al., 2013).
Volcanic fallout into aqueous environments leads to the dissolution
of adsorbed metal salts and aerosols, increasing the bioavailability of

Table 1
Average of environmental variables (mean ± standard deviation) values for the three stations along the Reloncaví fjord at different depth.

Depth N2O (nM) N2O (nM) AOU (μM) AOU (μM) % saturation % saturation CH4 (nM) CH4 (nM) % saturation % saturation SO2−
4 SO24-
April May April May N2 O N2O April May CH4 CH4 (mg L−1) (mg L−1)
April May April May April May

1 22.9 ± 3.75 9.7 ± 0.34 120 ± 8.0 95 ± 25.2 219 ± 33.2 84.3 ± 4.1 15.8 ± 13.1 36.3 ± 10.5 553 ± 456 1169 ± 318 1991 ± 379 257 ± 144
5 30.7 ± 1.52 11.9 ± 1.23 143 ± 16.5 125 ± 12.7 316 ± 14.9 119 ± 12.9 6.30 ± 5.19 6.00 ± 3.93 243 ± 200 227 ± 149 1292 ± 128 –
10 29.8 ± 4.52 12.9 ± 1.44 145 ± 8.2 136 ± 6.70 304 ± 46.0 131 ± 14.4 2.44 ± 0.78 2.18 ± 1.01 94.3 ± 30.4 83.6 ± 38.7 – –
25 31.7 ± 4.94 12.9 ± 0.34 38.8 ± 7.0 32.6 ± 14.6 319 ± 48.3 130 ± 3.40 2.93 ± 1.16 3.32 ± 2.65 112 ± 43.6 191 ± 15.4 – –
50 34.0 ± 1.50 13.5 ± 0.18 62.6 ± 18.1 56.8 ± 83.9 342 ± 15.8 137 ± 2.00 1.63 ± 0.67 8.74 ± 9.10 62.2 ± 25.6 335 ± 349 – 1794 ± 347
54 M.A. Yevenes et al. / Science of the Total Environment 669 (2019) 49–61
Fig. 3. Nutrient distribution for the three stations along the Reloncaví Fjord: NO−
3 (μM) (A, B, C), PO4 (μM) (D, E, F), Si(OH)4 (μM) (G, H, I) and N:P (J, K, L).
key nutrients (Jones and Gislason, 2008). However, low NO3− and PO43−
values (b5 μM) were found in the Reloncaví Fjord. NO3− concentrations
in the entire water column varied between 0.77 μM and 23.3 μM, with
the lowest concentrations in the surface waters up to 5 m depth within
the estuary (Stn. 1). These low concentrations are mainly due to the di-
lution effect of freshwater discharges from the Puelo River, due to melt-
waters and rainfall (León-Muñoz et al., 2013). In contrast, the highest
NO3− concentrations in subsurface waters (Stn. 3) were observed near
to the Puelo River in April during the first sampling period (Fig. 3a–c).
NO3− concentrations increased in subsurface waters as salinity in-
creased, this is a characteristic of sub-Antarctic waters entering the es-
tuary (Fig. 3a–c). The nutrient results are consistent with the previous
results reported by several authors, indicating that freshwater from riv-
ers inputs silicic acid into the surface waters, due to meltwater and rain-
fall (León-Muñoz et al., 2013; Farías et al., 2017). Instead, the overlaying
marine subsurface water is nutrient rich in both nitrogen and phospho-
rus (Yevenes et al., 2017; Farías et al., 2017; Iriarte et al., 2017). It is
likely that the highest Si(OH)4 levels recorded post eruption led to
high biogenic productivity in the area and contributed to the atypical
phytoplankton bloom (Fig. 3j–l). One important characteristic of north-
ern Chilean Patagonia is the presence of andosol-type soil, that brings
about abundant dissolved Si(OH)4 concentrations in the surrounding
aquatic system as a product of river runoff (Vandekerkhove et al.,
2016). High salinity and nutrient-rich subsurface waters were present
under 10 m depth (Fig. 3a–c), which is typically associated with the
intrusion of deep water masses, which is modified sub-Antarctic
water entering through the mouth of Reloncaví estuary (Castillo et al.,
2016). Nevertheless, estuarine stratification could limit mixing and
3−
entrainment of nutrients between layers, from subsurface waters to sur-
face waters, as observed in other estuarine systems (Silva and Vargas,
2014).
The region of Patagonia is understood to undergo a potential light
limitation during autumn (Iriarte et al., 2017), resulting in low chloro-
phyll and phytoplankton biomass in surface waters. However, this
study indicated that a maximum Chl-a of 20–25 mg L−1 was observed
during the April sampling period, in surface waters, at approximately
4 m depth (Fig. 4a). Thus indicating that the phytoplankton bloom is po-
tentially associated with the utilization of available nutrients. However,
during May, Chl-a concentration decreased and was homogeneous
throughout the entire water column (b2 μg L−1, Fig. 4b). Another phy-
toplankton bloom, but deeper and less intense, was observed in the sub-
surface layer during September, reaching a Chl-a maximum of 15 μg L−1
between 10 and 15 m depth (Fig. 4c). This has been previously reported
during spring (Iriarte et al., 2007).
3.2. Distribution of the carbonate system and greenhouse gases
Volcano eruptions can disturb the carbonate system, inducing
abrupt acidification in the top layer of the ocean (Flaathen and
Gislason, 2007). These results indicate that pHT and AT values ranged
from 7.67 up to 8.06 and 144 to 2136 in the upper 10 m depth of the
brackish layer, respectively (Fig. 4g–i). Relative to these results, no
clear effect of the volcanic eruption on local acidification events were
observed in the surface waters of the fjord during April and the subse-
quent months. It is likely that surface pHT decreased due to the CO2 up-
take associated with the high Chl-a concentration during these periods
 M.A. Yevenes et al. / Science of the Total Environment 669 (2019) 49–61
Fig. 4. Carbonate system distribution for the three stations along the Reloncaví Fjord: Chla (A, B, C), pHT (D, E, F), AT (G, H, I), pCO2 (J, K, L), ΩAr Ñ during field visits in April (A, D, G, J, M), May
(B, E, H, K, N) and September (C, F, I, L, Ñ) during 2015.
(Fig. 4a, c), mainly due to the development of a large bloom of phyto-
plankton during the days following the eruption. Vergara et al. (2016)
continuously studied the carbonate system within the top surface
layer of the fjord continually for one month after the eruption, using
high temporal resolution data (every 1 h) and with pH values as low
as 7.6 (≈0.4 pH units) during June. This value was closely related to
the minimum pHT values (7.2). It apparently took a few weeks after
the precipitation events to flush out the readily soluble constituents
near to the headwaters of the Reloncaví Fjord. pHT also decreased
below 7.7 in subsurface waters, similar to the situation observed for
O2 concentration. A good correlation between pHT and the Apparent
Oxygen Utilization (AOU) implied the influence of organic matter respi-
ration on the pHT levels (Fig. 5c), however; this was not the case for the
pCO2 and/or ΩAr (Fig. 5b, d). Instead, AT was highly correlated with sa-
linity due to the influence of low AT in freshwater, especially during
April (r2 = 0.81, r = p b 0.05), and very highly correlated during Sep-
tember (r2 = 0.96, r = p b 0.05) (Fig. 5a). Certainly, surface waters
were undersaturated in CO2, with pCO2 b 400 μatm mainly during
April and September, however levels rapidly increased with depth,
from subsurface waters up to N1000 μatm. The low pCO2 conditions in
55
brackish waters are associated with low-salinity conditions in these
permanently stratified waters. In turn, it is likely that corrosive waters
for CaCO3 in the surface layer (i.e. upper 4 m depth) are a frequent oc-
currence (Torres et al., 2011; Silva et al., 2011; Alarcón et al., 2015).
Low pHT, high pCO2, and CaCO3 sub-saturated waters (ΩAr) were ob-
served in subsurface waters below 10 m depth, mainly during April
and May field sampling periods, mostly associated with respiration of
both autochthonous and allochthonous organic matter (Waldbusser
and Salisbury, 2014). Zhai et al. (2015) discovered that bivalve species
were affected by the presence of short-term aragonite corrosive waters
(ΩAr) in the very shallow waters of the Northern Yellow Sea below 15 m
depth. This raised the concern that a greater effort is required by the lo-
cally cultured bivalve species in order to calcify.
Unexpectedly high N2O concentrations (22.9 ± 3.75 nM) were re-
corded in the surface layer of the Reloncaví Fjord, predominantly during
April. In comparison, previous studies report that the maximum average
reached 12 ± 2 nM (Yevenes et al., 2017). Higher concentrations were
observed in Stn. 3, which is closer to Puelo River discharge. However,
no significant correlations were found between N2O and salinity in the
surface water layer, implying a continental origin (Fig. 7a, b). Similarly,
56 M.A. Yevenes et al. / Science of the Total Environment 669 (2019) 49–61
Fig. 5. Relationship between A) Salinity and Alkalinity (AT), B) AOU and pCO2, C) AOU and pHT, D) AOU and ΩAr. Each figure includes data from the three field visits, April (Green), May
(blue), September (red). (*) Indicates a significant relationship between variables.
riverine waters within the surface layer showed N2O concentrations
that were twice as high as recorded by Yevenes et al. (2017), with satu-
ration levels reaching as high as 342% in April. This coincided with min-
imum nitrate (NO3−) levels during the same field-sampling period. The
observed N2O saturation is apparently the result of overall denitrifica-
tion in brackish water, since denitrification should not occur in well-
oxygenated waters (Yevenes et al., 2017). Excess N2O (ΔN2O) is one
of the most relevant biogeochemical factors in estimating N2O produc-
tion. If AOU and NO3− are associated with ΔN2O, it may indicate that
N2O is of biogeochemical origin (Yoshinari, 1976). This is supported
by the significant linear relationship between ΔN2O and AOU, and
ΔN2O and NO3−, during April and May, respectively (Fig. 8a, c). Apparent
N2O production can also be estimated through the consumption of O2
(AOU), based on the remineralization of particulate organic matter as-
sociated with nitrification (Nevison et al., 2003). Oxygen consumption
due to the oxidation of organic matter suggests that NO3− is formed dur-
ing nitrification, and as a result N2O is also produced (Farías et al., 2015).
CH4 concentrations varied between 0.27 and 44.4 nM throughout
the entire water column during April and May, with a maximum CH4
concentration of 44.4 nM, equivalent to 1411% saturation, observed at
1 m depth (Stn. 3) in May (Fig. 6c, d) near to the mouth of Puelo
River. The observed concentrations of CH4 in surface waters were within
the same range reported by previous studies between 16.97 and 151 nM
(Farías et al., 2017). Towards Stn. 3, surface CH4 concentrations doubled
throughout both campaigns. Moreover, a strong correlation between
surface CH4 concentrations with the pCO2 and Si(OH)4 confirm the
input into the fjord of continental CH4 sources from soils. Surface waters
were over-saturated with CH4, mainly during May. It is well known that
CH4 in fjords originates from anoxic sediments (Borges and Abril, 2011;
Borges et al., 2016). However, Farías et al. (2017) identified that the bot-
tom waters of the Reloncaví Fjord did not suggest consistently higher
CH4 concentrations compared to surface layers (Fig. 6c, d).
3.3. Implications for shellfish farming industry
Calcifying organisms, such as marine molluscs, are exceptionally vul-
nerable to fluctuations in carbon chemistry (Byrne, 2011; Gazeau et al.,
2013). Figs. 9 and S1 as supplementary material show the morphomet-
ric measurements recorded from juvenile's edible mussels, Mytilus
chilensis, collected from settlement ropes within the Reloncaví Fjord.
With the exception of shell thickness, that was homogeneous across
depth [Median ± IQR; Depth 1: 0.10 mm ± 0.05; Depth 3 m:
0.09 mm ± 0.03; Depth 4 m: 0.10 mm ± 0.04; Kruskal Wallis, H =
3.62; p = 0.164], the rest of variables showed significantly low values
in the upper 3 m of the water column, however maximum values are
consistently attained at 4 m depth (p b 0.05). In fact, we found increased
shell thickness in the less favorable conditions in the upper depth layer,
which may result as compensation in order to maintain shell strength
and functionality. Previous studies also evidenced that under favorable
environmental conditions for shell precipitation (NΩAr), mussel seeds
adapt to elongate a thin shell with a thicker periostracum, however, in
less favorable conditions the strategy is to increase the shell thickness
(Osores et al., 2017). Moreover, shell weight showed a significant in-
crease at 4 m depth (0,10 mg ± 0,04;) respect to more surface levels
(Depth 1 and 3 m: 0.05 mg ± 0.08) (H = 10.55; p = 0.005). Similar pat-
tern was found in the soft tissue weight of the mussel seeds (Depth 1:
 M.A. Yevenes et al. / Science of the Total Environment 669 (2019) 49–61 57

Fig. 6. Greenhouse gas distribution for the three stations along the Reloncaví Fjord: (N2O and CH4) during April and May field visits, including N2O (nM) (A, B) and CH4 (nM)(C, D).
September was not recorded.

0.07 mg ± 0.05; Depth 3 m: 0.07 mm ± 0.07; Depth 4 m: 0.10 mm ±
0.11; H = 6.96; p = 0.031). Molinet-Flores et al. (2015) also observed
that in natural banks, the condition index recorded in mussels living
in the upper 7 m depth was significantly greater than in mussels col-
lected from deeper habitats. In addition, shell size measurements
showed the same significant pattern of vertical variability (e.g., Shell
Length: Depth 1: 13.3 mm ± 5.9; Depth 3 m: 12.8 mm ± 5.77; Depth
4 m: 15.2 mm ± 6.8; H = 6.56; p = 0.038). Finally, the proportional
increments in length and weight of the mussel seeds as increased the
deep levels, the shell condition index, an integrative measure, indicate
a progressive increment of the shell weight in relation to tissue weight
of the individuals raised at 4 m depth (Shell condition index: 41.1% ± 9;
Depth 3 m: 44% ± 8; Depth 4 m: 48% ± 6; H = 6.56; p = 0.038). Inter-
estingly, morphometric analyses of mussel seeds demonstrated that at
4 m depth (or the “optimum layer”) some attributes related with shell
length, weight, and biomass reached maximum levels (Fig. 10). For

Fig. 7. Relationship between surface salinity, and N2O and CH4 during two field visits (April and May); N2O (A, B) and CH4 (C, D). (*) Indicates a significant relationship between variables.
58 M.A. Yevenes et al. / Science of the Total Environment 669 (2019) 49–61

Fig. 8. Relationship between ΔN2O vs. AOU and ΔN2O vs. NO−
3 during two field visits in April (A, B) and May (C, D). (*) Indicates a significant relationship between variables.

the Reloncaví areas previous studies indicated that a high load of seston mussels, as it is necessary to spend extra energy to eliminate these par-
(Mohr et al., 2017) and an increase in more corrosive waters may have ticles to their bodies (Salas-Yanquin et al., 2018). Therefore, under vol-
an impact on the suspension feeding processes and shell thickness in canic effects this finding may have significant implications for mussel

0.25 H = 8.23 0.12 H = 10.55 0.12 H = 6.96 16 H = 6.56

DF = 2 DF = 2 DF = 2 DF = 2
P = 0.016 P = 0.005 P = 0.031 15.5 P = 0.038
0.1 0.1
0.20 N total = 306
Dry shell weight (mg)

N total = 299 N total = 299 N total = 296

Total weight (mg)

Shell Length (mm)

15
0.08 0.08
Tissue (mg)

0.15 14.5
0.06 0.06 14
0.10 13.5
0.04 0.04
13
0.05
0.02 0.02
12.5
0 0 0 12
1 3 4 1 3 4 1 3 4 1 3 4

9 H = 7.08 0.105 H = 3.62 50 H = 23.35

DF = 2 DF = 2 DF = 2
8.5 P = 0.029 P = 0.164 48 P < 0.001
0.100
Shell Condition (%)

N total = 295 N total = 291 N total = 295

Shell thickness (mm)
Shell Height (mm)

46
8
0.095 44
7.5
0.090 42
7
40
0.085
6.5 38

6 0.080 36
1 3 4 1 3 4 1 3 4
Depth (meter)

Fig. 9. Median (±SE) in the morphometric measurements recorded in Mytilus chilensis seeds collected over three depths in a mussel farm located in the Reloncaví Fjord. Inset presents a
summary of the Kruskal-Wallis statistic (H) test, measuring the equality of medians of mussel characteristics over different depths. DF = degrees of freedom; N total is the overall number
of mussel seeds used in the test; Significant p values (b0.05) are showed in bold.
 M.A. Yevenes et al. / Science of the Total Environment 669 (2019) 49–61 59

Sal O2
Low due to low salinity/low
alkalinity waters driven by
freshwater runoff

Optimum for mussel

growth and calcification

Low due to high pCO2

Low O2 waters by organic
matter remineralization

Fig. 10. Conceptual model of mussel calcification with an optimal refugee at 4 m depth in the Reloncaví Fjord.

farmers in this fjord environment, as the installation of mussels ropes in
this layer. Supposedly present optimal conditions for mussel seed pro-
duction, could implement a management strategy to reduce the impacts
of changes during different climatic scenarios, such as carbon chemistry
in surface waters, both for ocean acidification processes and/or chang-
ing runoff conditions. Furthermore, this layer of aragonite-saturated
waters could be an additional factor explaining the spatial distribution
−2 −1 0 1
0.4
pattern of Mytilus chilensis beds in the Reloncaví Fjord. At this depth,
of the optimal range for mussel growth and production in this estuarine
environment was evidenced for a good correlation between variables.
For instance, the first PCA component explained 66% of the total vari-
ance, and showed a positive correlation with Temperature, Salinity, AT,
ΩAr, N2O, NO3−, PO43−, pCO2, and Shell condition (Fig. 11). This area con-
stitute one of the most important areas for larval mussel recruitment
(Molinet-Flores et al., 2015; Lara et al., 2016), and hosts important ben-
thic fisheries such as the king crab, gastropods, and sea urchins, all of
2
which are liable to impacts from decreasing pHT. Furthermore, reduc-
tion in ΩAr could be a significant challenge for other calcifying pelagic

Tissue
Total.weight
Shell.height
Shell.length
Dry.shell.weight and benthic organisms inhabiting similar fjord ecosystems in the South-
1.5

ern Patagonia, such as pelagic pteropods (Roberts et al., 2011), echino-

derms, and gastropods (Newcombe and Cárdenas, 2011).
Nitrous.oxide
1.0
0.2

Depth 4. Conclusion
0.5

Shell.thickness
Silicate
This study investigates the potential effect of a high magnitude vol-
Methane
PC2

0.0

0.0

canic eruption through the study of nutrients, greenhouse gases, and

Chl_a pCO2
Temperature
Salinity
Nitrate the carbonate system in the Reloncaví Fjord during autumn (April and
O2 Phosphate
Alcalinity
Aragonite May) and spring (September) 2015. Additionally, the impact of poten-
−0.5

tial changes was study in the carbonate system on the condition of mus-
sel shells in a nearby farming area. The outputs indicated a localized
−0.2

Shell.condition
−1.0

impact of the volcanic eruption on surface greenhouses gases mainly ni-

trous oxide, nutrients and short-term effects on the carbonate system.
pH
Despite our results indicate the presence of CaCO3 corrosive waters
−1.5

(ΩAr) in the Reloncaví Fjord, however no direct relation with the vol-
−0.4

cano eruption was found. Best conditions for mussel calcification were
recognized within a subsurface band, or refuge at 4 m depth, within
−0.4 −0.2 0.0 0.2 0.4 the estuarine environment, which appear to be resilient to catastrophic
PC1 events such as volcanic eruptions occurring in the Patagonian region.
These specific aspects can be integrated into adaptation strategies by
Fig. 11. Principal components analysis (PCA) of biological and physicochemical variables the mussel aquaculture industry to tackle ocean acidification and
from the Reloncaví fjord waters. changing runoff conditions.
60 M.A. Yevenes et al. / Science of the Total Environment 669 (2019) 49–61
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2019.03.037.
Acknowledgments
This work was supported by the Millennium Nucleus “Center for the
Study of Multiple-drivers on Marine Socio-Ecological Systems (MUSELS)”
funded by MINECON NC120086, and the Center for Climate and Resilience
Research Center (CR)2, CONICYT-Chile. Additional support from the Mil-
lennium Institute of Oceanography (IMO) funded by MINECON IC120019
is also acknowledged. The authors would like to thank to Pia Leon for lab
processing, the Mussel Farming Company ORIZON for logistical support
during our fieldwork, as well as the logistical support from the Techno-
logical Institute for the Mussel Farming Industry (INTEMIT) and the Fisher-
ies Development Institute (IFOP). CAV was supported by the CONICYT/
FONDECYT/1170065 during the manuscript preparation. MAY was sup-
ported by CONICYT/FONDAP/15130015 during the stage of the manu-
script writing. NAL also acknowledge support by PIA CONICYT
ANILLOS ACT 172037 during the last stages of the manuscript.
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