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Gill damage and delayed mortality of Northern shrimp (Pandalus borealis) after short time exposure to anti-parasitic veterinary medicine containing hydrogen peroxide

  • NORCE Norwegian Research Centre
  • Norwegian Research Centre AS


Hydrogen peroxide (H 2 O 2 ) is used as anti-parasitic veterinary medicine in salmon farms worldwide. In the period from 2009 to 2018 a total of 135 million kg of H 2 O 2 was used in Norway, the world's largest producer of Atlantic salmon. Since the treatment water is discharged to the sea, concerns have been raised about effects of H 2 O 2 on the coastal ecosystem. In the present study, Northern shrimp (Pandalus borealis) have been exposed to short pulses of H 2 O 2 in the PARAMOVE ® formulation, followed by a recovery period in clean seawater. The exposure concentrations represented 100, 1000 and 10 000 times dilutions of the prescribed treatment concentration for salmon; 15 mg/L, 1.5 mg/L and 0.15 mg/L H 2 O 2 . Significantly increased mortality was observed after 2 h exposure to 15 mg/L H 2 O 2 (50%) and after 2 h exposure to 1.5 mg/L H 2 O 2 on 3 consecutive days (33%), but no mortality was observed after 2 h exposure to 0.15 mg/L. The mortality occurred 2–4 days after the first pulse of exposure. The patterns of acute effects (immobility and death) could be captured with a toxicokinetic-toxicodynamic model (GUTS), which allows extrapolations to LC50s for constant exposure, or thresholds for effects given untested exposure profiles. Effects of H 2 O 2 were also detected in shrimp that survived until the end of the recovery period. The feeding rate was 66% lower than in the control after 12 days of recovery for the three-pulse 1.5 mg/L exposure. Furthermore, dose dependent tissue damage was detected in the gills and evidence of lipid peroxidation in the hepatopancreas in shrimp exposed for 1 h to 1.5 mg/L and 15 mg/L and kept in recovery for 8 days. Fluorescence intensity in the hepatopancreas of treated shrimp increased 47% and 157% at 1.5 mg/L and 15 mg/L, respectively, compared to the control. Local hydrodynamic conditions will determine how fast the concentration of H 2 O 2 will be diluted and how far it will be transported horizontally and vertically. Results from dispersion modelling (literature data) together with the current experiments indicate that treatment water with toxic concentrations of H 2 O 2 (1.5 mg/L) could reach P. borealis living more than 1 km from a treated salmon farm.
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Ecotoxicology and Environmental Safety
journal homepage:
Gill damage and delayed mortality of Northern shrimp (Pandalus borealis)
after short time exposure to anti-parasitic veterinary medicine containing
hydrogen peroxide
Renée Katrin Bechmann
, Maj Arnberg
, Alessio Gomiero
, Stig Westerlund
, Emily Lyng
Mark Berry
, Thorleifur Agustsson
, Tjalling Jager
, Les E. Burridge
NORCE Norwegian Research Centre, Mekjarvik 12, 4072, Randaberg, Norway
DEBtox Research, NL-3731, DN De Bilt, Netherlands
Burridge Consulting Inc., 61 Emmalee Dr Stratford PE, Canada, C1B 0B5, Canada
Delayed eects
Hydrogen peroxide
Toxicokinetic-toxicodynamic (TKTD) model
General unied threshold model for survival
Hydrogen peroxide (H
) is used as anti-parasitic veterinary medicine in salmon farms worldwide. In the
period from 2009 to 2018 a total of 135 million kg of H
was used in Norway, the world's largest producer of
Atlantic salmon. Since the treatment water is discharged to the sea, concerns have been raised about eects of
on the coastal ecosystem. In the present study, Northern shrimp (Pandalus borealis) have been exposed to
short pulses of H
formulation, followed by a recovery period in clean seawater. The
exposure concentrations represented 100, 1000 and 10 000 times dilutions of the prescribed treatment con-
centration for salmon; 15 mg/L, 1.5 mg/L and 0.15 mg/L H
. Signicantly increased mortality was observed
after 2 h exposure to 15 mg/L H
(50%) and after 2 h exposure to 1.5 mg/L H
on 3 consecutive days (33%),
but no mortality was observed after 2 h exposure to 0.15mg/L. The mortality occurred 24 days after the rst
pulse of exposure. The patterns of acute eects (immobility and death) could be captured with a toxicokinetic-
toxicodynamic model (GUTS), which allows extrapolations to LC50s for constant exposure, or thresholds for
eects given untested exposure proles. Eects of H
were also detected in shrimp that survived until the end
of the recovery period. The feeding rate was 66% lower than in the control after 12 days of recovery for the
three-pulse 1.5 mg/L exposure. Furthermore, dose dependent tissue damage was detected in the gills and evi-
dence of lipid peroxidation in the hepatopancreas in shrimp exposed for 1 h to 1.5 mg/L and 15mg/L and kept in
recovery for 8 days. Fluorescence intensity in the hepatopancreas of treated shrimp increased 47% and 157% at
1.5 mg/L and 15 mg/L, respectively, compared to the control. Local hydrodynamic conditions will determine
how fast the concentration of H
will be diluted and how far it will be transported horizontally and vertically.
Results from dispersion modelling (literature data) together with the current experiments indicate that treatment
water with toxic concentrations of H
(1.5 mg/L) could reach P. borealis living more than 1 km from a treated
salmon farm.
1. Introduction
Norway is the world's leading producer of Atlantic salmon, and the
Norwegian Ministry of Trade, Industry and Fisheries aims to increase
the production of salmon in Norway (Nærings- og skeridepartementet,
2015). Continued growth of salmon aquaculture may lead to increased
discharge of anti-salmon lice chemicals. Despite the increasing use of
non-chemical methods to remove sea lice from farmed salmon, several
chemicals (bath treatment and medicated feed) are still used ex-
tensively (Aaen et al., 2015;Burridge et al., 2014;Lillicrap et al., 2015).
Received 6 February 2019; Received in revised form 10 May 2019; Accepted 13 May 2019
Corresponding author.
E-mail addresses: (R.K. Bechmann),, (M. Arnberg), (A. Gomiero), (S. Westerlund), (E. Lyng), (M. Berry), (T. Agustsson), (T. Jager), (L.E. Burridge).
Present address (from 1 June 2019): Akvaplan-niva, Postboks 1268, 7462 Trondheim, Norway.
Present address: Vålandsbakken 23, 4010 Stavanger, Norway.
Present address: Marine Management Organisation (MMO), Room 13, Ground Floor, Crosskill House, Mill Lane, Beverley, HU17 9JB, UK.
Ecotoxicology and Environmental Safety 180 (2019) 473–482
0147-6513/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
Updated information about the use of chemicals in Norwegian aqua-
culture is available on the web page of the Norwegian Institute of Public
Most sh farms depend on chemical treatments to keep the
parasite numbers below national maximum permitted levels (Helgesen
et al., 2015). The emerging occurrence of reduced sensitivity of salmon
lice towards other chemicals led to the re-introduction of hydrogen
peroxide (H
) formulations for salmon lice treatment in 2009. In the
period from 2009 to 2018 a total of 135 million kg of (100%) H
been used in Norwegian salmon farms, according to data available on
the web site of the Norwegian Institute of Public Health
. The use of
has, however, been reduced from 43 million kg in 2014 to 7
million kg in 2018. Chemicals used for bath treatments are either added
directly into the net pen surrounded by a tarpaulin or administered in
well-boats. After completed treatment, chemicals are released untreated
directly into the environment, either by release of the tarpaulin or from
the well-boat. During the production cycle, salmon can be treated
several times with chemicals, hence marine organisms, such as shrimp,
living in areas with aquaculture could be exposed repeatedly for a re-
latively short period each time. Limited data are available on pulsed
exposure and reports on repeated pulses or uctuating exposures are
even rarer (Dennis et al., 2012). Repeated short-term exposure studies
have, however, been performed with American lobsters (Homarus
americanus) (Burridge et al., 2000,2008) and crab larvae (Metacarcinus
edwardsii) exposed to anti-parasitic veterinary medicines (Gebauer
et al., 2017). Langford et al. concluded that repeated short-term ex-
posures of coastal ecosystems are likely to occur in Norway due to the
number and proximity of sh farms (Langford et al., 2014). Norwegian
shermen have observed that Northern shrimp (Pandalus borealis) have
disappeared from several shrimp elds in areas with salmon farms and
this observation was conrmed by the Institute of Marine Research for
the coast of Helgeland (Eraker, 2016;Steinhold and Thonhagen, 2017).
The shermen claim that shrimp disappear from areas where chemicals
are used to treat salmon against lice. Concern has also been raised about
potential negative eects on the kelp community, due to the sensitivity
of sugar kelp (Saccharina latissimi) to H
(Haugland et al., 2019).
Formulations with hydrogen peroxide as the active ingredient are
generally considered to be the bath treatment method with lowest en-
vironmental risk, because H
has a relatively short half-life in sea-
water and degrades to oxygen and water (Burridge et al., 2014;Haya
et al., 2005). The half-life of H
in seawater varies from 1 to 28 days,
depending on formulation, temperature, start concentration and or-
ganic content of the seawater (Bruno and Raynard, 1994;Fagereng,
2016;Haya et al., 2005;Lyons et al., 2014). Hydrogen peroxide is a
strong oxidizing agent, and the mechanism of action is lipid peroxida-
tion of cellular membranes and mechanical paralysis, in addition to
inactivation of enzymes and DNA replication (Cotran et al., 1989). In
sea lice, H
causes mechanical paralysis when air bubbles form in the
gut and hemolymph, causing the sea lice to detach and oat to the
surface (Bruno and Raynard, 1994). Gill damage and mortality have
been observed for Atlantic salmon exposed to 14502580 mg/L H
for 1020 min (Kiemer and Black, 1997). There is, however, limited
information published on the eects of the use of this chemical on non-
target marine crustaceans (Burridge et al., 2014;Urbina et al., 2019).
The main objective in this study was to investigate the eects in P.
borealis of exposure to short (12 h) pulses of diluted H
in the
formulation. A recovery period in clean seawater was
included in the experiments to study potential delayed eects of ex-
posure to PARAMOVE
. Mortality, feeding, swimming, gill histo-
pathology and lipid peroxidation of the hepatopancreas were in-
vestigated in order to assess the eects of exposure. The toxicokinetic-
toxicodynamic (TKTD) model GUTS (Jager et al., 2011) was used to
explain the dynamic mortality pattern of shrimp exposed to various
pulse treatments.
2. Materials and methods
2.1. Collection of shrimp and holding conditions
The experimental plan was approved by the Norwegian Animal
Research Authority (FOTS). Female shrimp (P. borealis) were collected
from Hillefjord (North of Åmøy, Rogaland County, Norway; 59° 0400
N, 5° 4500E) using a shrimp bottom trawl on the 10th of January
2017. Trawling lasted 40 min at 100 m depth. The animals were sorted
by hand, transferred to 50 L tanks that were regularly oxygenated to
maintain > 80% oxygen saturation, and transported to the laboratory
within 2 h of capture. Upon arrival at NORCE marine research facility
(Randaberg, Norway), shrimp were randomly divided between eight
500 L tanks (approximately 100 shrimp per tank). Each tank had a
continuous ow of sand ltered seawater adjusted to 7 °C, pumped from
75 m depth in the fjord close to the laboratory facilities. Shrimp were
fed 3 mm pellets of sh feed (Spirit supreme, Skretting, Norway) ad
libitum and acclimatized in laboratory conditions (7 °C, 34) for two
weeks before the rst screening test was performed. All experiments
were performed in the period from 24th of January to 1st of March
2017. Female shrimp were used in all experiments, with a mean wet
weight of 8.3 g (SD 1.2 g), and mean carapace length 23 mm (SD 1 mm).
2.2. Experimental design, set-up and exposure concentrations
The experiments were performed in a continuous ow exposure
system with temperature regulated seawater. The mean temperature
was 6.81 ± 0.03 °C and the mean oxygen saturation in the tanks was
95.3 ± 0.8%. The mean salinity in the inlet water during the experi-
mental period was 34.3 ± 0.2. Female shrimp were exposed to one
or three pulses of diluted PARAMOVE
solution for either one or 2 h
followed by a recovery period in clean seawater to investigate delayed
eects of the short time exposure.
In Exp. 1 and Exp. 2. shrimp were exposed to short pulses of 15 mg/
L and 1.5 mg/L H
, respectively, to investigate eects on mortality,
immobilization, swimming activity and feeding rate (details below). Six
replicate tanks with 10 shrimp, were used for each treatment in both
experiments. The shrimp were exposed for 2 h to H
for three con-
secutive days, followed by two days of recovery in Exp. 1 and 12 days of
recovery in Exp. 2. In Exp. 3 shrimp were exposed to one pulse of
0.15 mg/L, 1.5 mg/L and 15 mg/L H
lasting either 1 h or 2 h fol-
lowed by 8 days of recovery. Eects of H
on gill histopathology, and
lipid peroxidation of the hepatopancreas were investigated in addition
to mortality, immobilization, swimming activity and feeding rate (de-
tails below). Four replicate tanks with 6 shrimp, were used for each
treatment. An overview of Exp. 13 is presented in Supplementary
Table 1 and the experimental set-up is illustrated in Supplementary
Figure 1.
The concentration of H
; 49.5% w/w H
) used
to treat infestations of sea lice on Atlantic salmon is 1500 mg H
for 20 min (Felleskatalogen, 2017). All experiments were conducted
using the PARAMOVE
formulation. During the experiments shrimp
were exposed to 0.1515 mg/L H
, representing 10 000100 times
dilution of the recommended treatment concentration for salmon. A
stock solution of PARAMOVE
in distilled water was prepared for each
test concentration. The density of PARAMOVE
was regularly measured
to ensure that PARAMOVE
still contained approximately 50% H
(density 1.2 g/cm
). To create the stock solutions for the 0.15 mg/L,
1.5 mg/L and 15 mg/L H
treatments, 0.255, 2.55 and 25.5 mL
was added to 4 L distilled water in 5 L Schott bottles. The
bottles were agitated and placed on magnetic stirrers. A small amount
(3 mL/min) of stock solution was pumped by a peristaltic pump (model
520, Watson and Marlow, Cornwall, UK, Marprene pump tubing) into
the seawater inlets of each exposure tank (volume 46 L) to achieve the
R.K. Bechmann, et al. Ecotoxicology and Environmental Safety 180 (2019) 473–482
desired exposure concentrations of H
. The mean ow of seawater
into each exposure tank was 753 mL/min (Supplementary Figure 1).
The ow of stock solution and seawater into the tanks was monitored
during the experiments (Supplementary Figure 1). To verify the H
concentration in the exposure tanks and in diluted samples from the
header tanks a kit for semi-quantitative detection of H
was used
(Quantox1100 mg/L, Macherey-Nagel Germany supplied by Sigma
Aldrich). This semi-quantitative method could not be used in the
0.15 mg/L exposure which was below the detection limit for the
2.3. Mortality, immobilization and swimming activity
The number of standing shrimp (normal control behaviour), swim-
ming shrimp (indication of stress), and immobilized shrimp in each
tank were recorded before and after each exposure pulse and once a day
in the recovery period in all three experiments. In Exp. 3, more frequent
observations of the shrimp were made during and right after the ex-
posure (0, 15, 30, 45, 60, 75, 90, 105, 120 and 240 min after start of the
experiment). Dead shrimp were recorded and removed daily.
2.4. Feeding rate
Feeding was determined as follows: new pellets of sh feed (one
pellet per shrimp) were added to the tanks and number of remaining
pellets were counted the following day. This gave information about
feed consumption which was expressed as number of pellets consumed
per shrimp per day. In Exp. 1 the feeding rate was determined once in
the exposure period (between rst and second pulse) and the rst day of
the recovery period. In Exp. 2 the feeding rate was determined once in
the exposure period (between second and third pulse), and every
second day of the recovery period. In Exp. 3 the feeding rate was de-
termined every second day of the experiment.
2.5. Gill histopathology
At the end of Exp. 3, gills from control shrimp and shrimp exposed
to 1.5 mg/L and 15 mg/L H
, were sampled for gill histopathology
(n = 15 samples per treatment). Gill samples were dissected out, xed
in Davidson's xative for 48 h and transferred to 10% buered formalin
solution. Later, the samples were processed by serial alcohol dehydra-
tion, embedded in paran wax and cut into 6 μm transsections before
being stained by Mayer's hematoxylin and Eosin for observation by the
light microscope according to Boudet et al. (2015). The degree of his-
tological damage was scored in four elds on each of the 15 slides from
each treatment. Scores were based on the number of elds in which
histological changes were observed with (class 0) no histopathology in
any eld, (class 1) = mild histopathology present in < 25% of the
elds, (class 2) = moderate histopathology present in 25%75% of the
elds, and (class 3) = severe histopathology present in > 75% of the
eld, following the scale suggested by Zodrow et al. (2004). Parasites
were scored as 0 = absent or 1 = present.
2.6. Lipid peroxidation in hepatopancreas
Hydrogen peroxide is a well-known oxidative stress inducer in
marine organisms. Among all toxicity mechanisms, one of the most
documented is Reactive Oxygen Species (ROS) induction in cells. ROS
are a major initiator of lipid peroxidation, and membrane bound
polyunsaturated fatty acids are their major targets. Linoleic acid (LA) is
the most abundant polyunsaturated fatty acid found in marine organ-
isms, including shrimp (Olsen et al., 2012;Ouraji et al., 2011). Fol-
lowing oxidative stress, lipids are oxidized by H
to organic per-
oxides (hydroperoxides), which further decompose to multiple α,β-
unsaturated aldehydes. The end-products of the peroxidation process
readily modify proteins at nucleophilic side chain sites. These are
insoluble and persistent alkyne-containing proteins accumulated and
stored in the lysosomal compartment. They can be selectively stained
and detected using 5-Carboxamido-(6-Azidohexanyl), Bis (Triethy-
lammonium Salt) 5-isomer based uorescent probes in xed cells and
tissues, using the Click-iT Lipid peroxidation detection kit (Life Tech-
nologies, Carsbad, USA).
At the end of Exp. 3, the hepatopancreas from control shrimp and
shrimp exposed to 1.5 mg/L and 15 mg/L H
, were sampled for his-
tochemical examination (n = 15 samples per treatment).
Hepatopancreas of each individual was quickly dissected out, snap-
frozen in liquid nitrogen and stored at 80 °C. The quantication of
lipid peroxidation was conducted using a modied protocol of Click-iT
Lipid Peroxidation Detection (catalog no. 10446, Life Technologies
Corporation) using the shrimp's natural linoleic acid levels as indicator
of no treatment control. The histochemical procedures were applied on
frozen tissue sections of hepatopancreas. Serial transsections of the
tissue (10 μm) were placed on glass slides, positioned in Hellendal's jars
and xed in Baker's solution for 15 min at 4 °C. Sections were rinsed
twice with Hanks' saline solution to remove the xative and incubated
with 0.5% TritonX-100 in 0.01M phosphate buered saline (PBS), pH
7.4 at room temperature (RT). Cell membrane permeability reaction
was blocked by replacing the solution with 1% bovine serum albumin
(BSA) in PBS, pH 7.4 and incubating the sections at RT for 30 min. The
blocking solution was removed by washing tissue sections twice with
PBS before the uorescent probe incubation, and then the slides were
placed in humidity chambers. Click-iT
reaction buer, buer additive
and CuSO
reaction promoter solution were prepared and mixed ac-
cording to the manufacturer's instructions. 200 μL of the reaction so-
lution was added to each slide, then left to react at RT in dark and
humid conditions for 30 min. The reaction solution was removed by
washing incubated tissue sections twice with 1% BSA in PBS and twice
with PBS. Sections were mounted in ProLongGold Antifade Mountant
(P36930, Invitrogen Corporation) and analysed in a Zeiss AxioVert 100
Inverted Fluorescence Phase Microscope with a FITC lter and coupled
to an Axiocam MRc5. Digital image analysis was carried out by ImageJ
1.51k software (NHI, USA). Each hepatopancreas section was divided
into quarters for statistical interpretation and scored for maximal
staining. The total area of digestive cells scanned in each measurement
was approximately 2000 μm
and ve measurements were made per
section. Mean optical density was calculated for each sample and results
are reported as percentage of variation respect to control organisms
(Gomiero and Viarengo, 2014).
2.7. Toxicokinetic-toxicodynamic modelling
The complete data set for survival over time (Exp. 13) is analysed
with a toxicokinetic-toxicodynamic (TKTD) model, namely a special
case of the General Unied Threshold model for Survival (GUTS) (Jager
et al., 2011). As TKTD models explicitly deal with the mechanisms
underlying the eects over time, they oer the possibility to analyse the
survival data for the various treatments (dierent pulse length, dif-
ferent pulse intensity, and dierent number of pulses) in a single con-
sistent analysis. Thus, a single set of model parameters should be able to
capture the results from all of the treatments. For this model analysis,
we used the data for immobile (lying down) and dead animals com-
bined, as only two animals recovered from the immobile state, and in
the eld, immobility likely is equivalent to death.
Details of the model and the modelling procedure are provided in
the supporting information. In short, the reduced GUTS model for
stochastic death was used, extended with a module to account for sa-
turation of the eect. This extension was needed as it was found that
increasing the exposure concentration (or the number of consecutive
pulses) led to less than the expected increase in the toxic eect. All
calculations were performed with the BYOM platform in Matlab (see, maximising the likelihood func-
tion based on the multinomial distribution, corrected for animals
R.K. Bechmann, et al. Ecotoxicology and Environmental Safety 180 (2019) 473–482
sampled for histological analysis in the three-pulse experiment.
Condence intervals were calculated by likelihood proling.
2.8. Statistical analysis
All data analyses were performed in v 23 SPSS
(IBM, Chicago,
USA). The Kolmogorov-Smirnov test and Levens test was used to test for
normal distribution and variance. For normally distributed variables,
the General Linear Model (GLM) tests, two-way ANOVA was used to
test if signicant the eect of Paramove
on biological parameters could
be found. The criterion for signicance was set at p < 0.05. The
Wilcoxon Rank sum test was used to analyse the biological parameters
when normality or homogeneity criteria were not met. The criterion for
signicance was set at p < 0.05.
3. Results
3.1. Chemical analyses
The semi-quantitative method showed that the concentration of
in the header tanks was stable for several days. The nominal
exposure concentration of H
was reached 30 min after starting the
pump delivering H
into the exposure tank. When the pump was
stopped 1 or 2 h later, the concentration was rapidly reduced
(Supplementary Figure 2).
3.2. Mortality, immobilization and swimming activity
There was no mortality of control shrimp in any of the experiments.
Mortality was signicantly increased for shrimp exposed to 1.5 mg/L
and 15 mg/L H
(Wilcoxon, p < 0.05, signicant dierence from
control indicated by * in Fig. 1). In the three-pulse experiments, there
was no immediate eect on mortality after the rst and second pulse of
exposure to 1.5 mg/L or 15 mg/L H
(Fig. 1, Exp. 1 and 2). However,
after the third pulse of exposure to 15 mg/L, 46% mortality was ob-
served, and two days later the mortality increased to 80%. Im-
mobilization of shrimp was observed two days earlier than mortality in
the three-pulse exposure to 15 mg/L (Supplementary Figure 3). Fur-
thermore, after the third pulse of exposure to 1.5 mg/L mortality started
to increase and mortality reached 33% after one day of recovery.
Delayed eect of PARAMOVE
was also observed in the one-pulse ex-
periment (Fig. 1, Exp. 3). There was no mortality in any treatment the
rst two days after the pulse, but on the third day there was 38%
mortality in the 2-h 15 mg/L exposure, increasing to 50% later in the
recovery period (Fig. 1, Exp. 3). Although there was a tendency to a
dose- and time dependent increase in mortality also for the other
treatments in the one-pulse experiment, the dierence from the control
was not statistically signicant. Exposure to three pulses of 1.5 mg/L or
15 mg/L H
caused higher mortality than exposure to one pulse of
the same concentration (Wilcoxon, p < 0.05, Fig. 1). All the shrimp
exposed for 1 h or 2 h to 0.15 mg/L H
survived to the end of the
recovery period.
Swimming activity (i.e. percentage of shrimp that were swimming
at each observation point) was signicantly increased during the 1 h
and 2 h exposure to 15 mg/L (signicant dierence from control
Fig. 1. Mortality of shrimp exposed to H
formulation (mean + SD). In Exp. 1 and 2 shrimp received 2 h exposure for 3 consecutive days. In
Exp. 3 shrimp were exposed once for 1 or 2 h. No mortality was observed in any of the controls in Exp. 13, nor in the 1 or 2 h exposure to 0.15 mg/L in Exp. 3.
Signicant dierence from control indicated by * (Wilcoxon, p < 0.05).
Fig. 2. Percent swimming P. borealis in 15 mg/L H
treatment compared to in
the control (white bars) before, during and after the 1 h (grey bars) or 2 h (black
bars) exposure in Exp. 3 (mean + SD). Signicant dierence from control in-
dicated by * (Wilcoxon, p < 0.05).
R.K. Bechmann, et al. Ecotoxicology and Environmental Safety 180 (2019) 473–482
indicated by * in Fig. 2, Wilcoxon, p < 0.05). The mean percentage of
swimming shrimp was 48% in the control compared to 2938% during
exposure to 15 mg/L. The increased swimming activity lasted
throughout the pulse but went down to control level once the pulse had
stopped (Fig. 2). In the three-pulse experiments, no observations of
swimming activity were done during the pulse, and no signicant dif-
ference in swimming activity was observed after each pulse
= 3.35, p = 0.09, ANOVA).
3.3. Feeding rate
The feed intake of shrimp was signicantly reduced after exposure
to 1.5 and 15 mg/L in Exp. 1 and Exp. 2 and remained so during the
recovery period (signicant dierence from control indicated by * in
Fig. 3, Wilcoxon, p < 0.05). After 12 days of recovery the feeding rate
was 66% lower for shrimp in the three-pulse 1.5 mg/L exposure than in
the control. Feed intake was documented for all 6 replicate control
tanks at all sampling days. At both sampling days in Exp. 1 and at 6 of
the 7 sampling days in Exp. 2 no feed intake was observed in ve of the
six replicate tanks with shrimp exposed to H
. The feeding rate was
not consistently reduced in the recovery period after the one pulse
exposure in Exp. 3 (Supplementary Table 2).
3.4. Histopathology
Minor changes were observed in the gills of control shrimp (Figs. 4a
and 5a). Analysed individuals showed uniform arrangement of lamellae
with normal hemocoel and limited fusion of the lamellae (Fig. 4a).
Signicant and progressive changes in the histoarchitecture of the gills
were observed in shrimp exposed to both 1.5 mg/L and 15 mg/L H
for 1 h followed by 8 days recovery in clean seawater (Wilcoxon, p-
values in Fig. 5). Swelling of gills, as well as the accumulation of he-
mocytes in the hemocoelic space of the gill lamellae, was the most re-
current observation in the 1.5 mg/L treatment (Figs. 4b and 5b). A si-
milar eect, but with more severe impairment was observed in the
15 mg/L treatment (Figs. 4c and 5c). Swelling and haemocyte inltra-
tion phenomena induced the formation of diuse disorganized tissues
masses in disrupted gills together with severe gill fusion and necrosis
phenomena, lifting of lamellar epithelium and hyperplasia in most in-
dividuals. The occurrence of parasites was generally low, and there was
no dierence between treatments. Parasites were detected in two of the
15 shrimps from each treatment.
3.5. Histochemistry, lipid peroxidation in hepatopancreas
The 1 h H
exposure to both 1.5 mg/L and 15 mg/L induced
signicant increase of the uorescence intensity in the hepatopancreas
of treated shrimp compared to the control (+47% increment compared
to the control at 1.5 mg/L and +157% increment compared to the
control organisms at 15 mg/L, KruskalWallis test, p < 0.05, Fig. 6).
Supplementary Figure 4 shows an example of the image analysis of
tissue damage in the hepatopancreas of shrimp.
3.6. Toxicokinetic-toxicodynamic modelling
The GUTS model t is shown in Fig. 7, and the model parameters
are given in Supplementary Table 3. The model is able to explain all
data simultaneously with ve parameters. In fact, one parameter
(background hazard rate) could have been excluded as there were no
deaths in the control over the entire duration of the tests. Note that for
this analysis, we treated the immobile animals (lying down) as dead.
The model cannot capture the slow onset of eects over the rst days,
but overall provides a reasonable explanation for the overall pattern
across all treatments.
The estimated threshold for eects on mortality, as estimated using
the GUTS model, is extremely low at just 4.6 μg/L. This model para-
meter should be interpreted as the model prediction for the water
concentration that can be tolerated by the shrimp for an arbitrary
amount of time without any eect on survival. Clearly, for short ex-
posure pulses, higher concentrations can be tolerated, and the model
can be used to predict a threshold value for specic exposure scenarios.
Furthermore, the calibrated model can be used to predict an LC50 for
constant exposure concentrations, which facilitates comparison to more
standard laboratory tests. The LC50 depends on exposure time, and the
model predicts a 1-day LC50 of 2.7 (1.54.2) mg/L, a 2-day LC50 of
0.53 (0.320.88) mg/L and a 4-day LC50 of 0.14 (0.0920.24) mg/L.
The threshold m
equals the incipient LC50.
4. Discussion
4.1. Mortality, immobilization, swimming activity and feeding rate
The eects documented in P. borealis exposed to one or three pulses
of 1.5 mg/L and 15 mg/L hydrogen peroxide in the present study de-
monstrate that P. borealis is more sensitive to H
formulation than the crustaceans tested by (Burridge et al., 2014) (see
Supplementary Table 4 for an overview of the main eects in P.
Fig. 3. Feeding rate for P. borealis exposed to 15 mg/L and 1.5mg/L H
. The shrimp were exposed for 2 h on day 1, 2 and 3 of each experiment. Signicant
dierence from control indicated by * (Wilcoxon, p < 0.05).
R.K. Bechmann, et al. Ecotoxicology and Environmental Safety 180 (2019) 473–482
borealis). These authors exposed three species of crustaceans to H
1 h followed by 95h recovery in clean seawater. The LC
was 973 mg/
L for Mysid sp., 3182 mg/L for Crangon septemspinosa and 1673 mg/L for
larvae of Homarus americanus. Crab larvae (Metacarcinus edwardsii) and
the shrimp Pandalus montagui were more sensitive to H
2016;Gebauer et al., 2017). High mortality of crab larvae was docu-
mented after multiple pulses (20 min per day for 7 days) of 188 mg/L
in the Hyperox
formulation (Gebauer et al., 2017), and the 6 h
LC50 for P. montagui was 170 mg/L H
(Fagereng, 2016). The co-
pepod Calanus nmarchicus also appeared to be more sensitive to H
than many other crustaceans (Escobar Lux, 2016;Hansen et al., 2017).
The 24 h LC
was 6 mg/L (Hansen et al., 2017) and increased mortality
of C. nmarchicus was observed after 1 h exposure 17170 mg/L H
The 24 h LC
for P. borealis was recently determined to be 37 mg/L
)(Refseth et al., 2016), but there are no literature
data on the eects of shorter H
exposure than 24 h for P. borealis. In
the present experiments, mortality did not occur during or right after
the rst pulse of exposure, but 24 days later. However, in the three-
pulse experiment a higher percentage of immobilized shrimp were
observed early in the experiment, and increased mortality later in the
experiment. Immobilization of copepods has also been observed during
and after 1 h exposure to 10 mg/L H
Paramove50) (Van
Geest et al., 2014). Furthermore, reduced swimming activity of P.
montagui was observed after exposure to H
, and the 6 h EC
estimated to be 10 mg/L H
(Fagereng, 2016). Other studies may
have underestimated the eects of exposure to H
because delayed
eects were not investigated, i.e. the duration of these experiments was
too short (24 h or shorter) (e.g. (Brokke, 2015;Fagereng, 2016;Refseth
et al., 2016;Van Geest et al., 2014). The results show that standard
toxicity tests with constant exposure concentration and xed (short)
duration may underestimate the risk to aquatic organisms as delayed
eects and carry-over toxicity due to multiple exposure pulses cannot
be detected (Ashauer et al., 2010).
During the one-pulse exposure to 15 mg/L H
there was a tran-
sient increase in swimming activity. This may indicate an escape re-
sponse or a stress response (Shumway et al., 1985), as the shrimp in
these treatments also showed increased mortality. The feeding rate of
shrimp exposed to three pulses of 1.5 mg/L was still signicantly lower
than in the control 12 days after the last pulse. This indicates that the
shrimp were not able to recover from the exposure. Feeding inhibition
has also been documented for copepods after exposure for 1 h to
2.610 mg/L H
Paramove50) (Van Geest et al., 2014).
4.2. Gill damage
Ultra-structural changes in target organs of shrimps have been
shown to be useful tools to characterize both the health status of or-
ganisms in led and laboratory exposure (Boudet et al., 2015;Sousa
et al., 2005). In this study, control shrimp showed a well-organized gill
structure, like that described for other species of shrimp (Bhavan and
Geraldine, 2000;Li et al., 2007;Wu et al., 2008), indicating good nu-
tritional status of individuals and normal function of the gill system.
Shrimp exposed to 1.5 mg/L of H
for 1 h showed clear evidence of
gill damage. Many adverse eects can be reversed by the immune
system reaction (hemocytes inltration), tissue restructuration and
swelling of the gills. The exposure to a well-known oxidative stress
promoter such as hydrogen peroxide is expected to induce biochemical,
cellular and subcellular alterations in crustaceans, as already docu-
mented in other marine organisms (Kiemer and Black, 1997). Elevated
hydrogen peroxide concentrations increased the adverse eects, with
even more evident alterations in the gill tissue of shrimp exposed to
15 mg/L of H
for 1 h, indicating a dose-response relationship. Due to
the severe tissue destruction at this concentration, it is not likely that
the shrimp will recover, it is also likely that this is the cause of the
Fig. 4. Gills from control shrimp P. borealis
(a), shrimp exposed for 1 h to 1.5 mg/L (b)
or 15 mg/L H
(c) and sampled after 8
days recovery in clean seawater. Signicant
and progressive structural alterations were
observed in the gill lamellae of shrimp ex-
posed to 1.5 mg/L and 15 mg/L. The accu-
mulation of hemocytes (HeM) in the he-
mocoelic space, swelling (SV) and lifting of
lamellar epithelium (Li), and marked hy-
pertrophy and hyperplasia in the gill epi-
thelium. Furthermore, necrotic (N), mod-
erate fusion of lamellae (FuS), clavate-
globate lamella and formation of a dis-
organized mass (DM) of disrupted gill la-
mellae are symptoms of necrosis and tissue
de-organisation phenomena.
R.K. Bechmann, et al. Ecotoxicology and Environmental Safety 180 (2019) 473–482
mortality in this treatment. For the 1 h 1.5 mg/L exposure to hydrogen
peroxide, the degree (and the pattern of alteration) indicates that very
little recovery and tissue restructuring is expected. This trend is further
supported by the results of the GUTS model pointing out an estimated
low damage-repair rate. However, in this context the ability of shrimp
to recover needs to be investigated in an experiment with even longer
recovery time than in the current experiment (i.e. more than 8 days).
Similar gill damage was observed for P. montagui exposed for 6 h to
3.5 mg/L H
(Fagereng, 2016).
4.3. Lipid peroxidation in hepatopancreas
Evidence of lipid peroxidation was observed in the hepatopancreas
already after 1 h exposure to 1.5 mg/L H
, and more evident in the
15 mg/L treatment. The analysis of the cryosections shows the in-
corporation of the peroxidation end-product in the lysosomes: this is a
well-known cell strategy to store unknown or potentially harmful
substances and make them less available in the cytosol. The analysis of
uorescent marked peroxidation end-products (lipofuscins, carbony-
lated proteins etc.) shows a distribution indicating that lysosomes are
no longer able to store the large quantity of these oxidative stress de-
rived molecules in the study, and therefore they are more widespread in
the cytosol. A certain level of peroxidation is a naturally occurring ef-
fect of the biological ageing systems, and the end-products of such
Fig. 5. Gill histopathology in control shrimp (a) and shrimp exposed 1 h to 1.5 mg/L (b) or 15 mg/L H
(c) and sampled after 8 days recovery in clean seawater.
Score 0: no histopathology in any eld, score 1: mild histopathology present in < 25% of the elds, class 2: moderate histopathology present in 25%75% of the
elds, and score 3: severe histopathology present in > 75% of the eld.
Fig. 6. Observed increment of membrane peroxidation in hepatopancreatic
cells of P. borealis exposed to 1.5 mg/L and 15 mg/L H
for 1 h followed by 8
days recovery in clean seawater. Average values as percentage increase with
respect to the control.
R.K. Bechmann, et al. Ecotoxicology and Environmental Safety 180 (2019) 473–482
process are accumulated over the lifespan of the organisms. As no ef-
cient homeostatic mechanisms are present in the cells of organisms the
continuous accumulation of peroxided material is considered a per-
manent eect.
4.4. Toxicokinetic-toxicodynamic modelling
The GUTS model provided a very reasonable explanation for the
patterns of eects across all treatments but could not capture the slow
onset of eects over the rst days. It is conceivable that the mechanism
of damage accrual and repair is more complex than simple one-com-
partment rst-order kinetics. The estimated threshold for eects on
mortality (m
) is very low (4.6 μg/L), and within the range of reported
background concentrations for H
in sea water. This indicates that
even very low exposure concentrations (over a long period of time) may
have some additional eect on mortality. However, this prediction rests
on the assumption that the model is true and cannot be veried as we
have not tested the consequences of very low constant exposure con-
centrations. Mortality among the individuals in the tested cohorts
continues for a long time after the last exposure event, which is re-
ected in the rather low estimate for the dominant rate constant (k
This rate constant is the one-compartment approximation of a more
complex chain of damage processes, likely reecting the slowest pro-
cess in the chain. This rate constant indicates that it takes 17 days for
the survivors of a pulse event to recover to 5% of the maximum damage
level. In other words, only if the interval between two pulses is at least
17 days, we could consider the two events to be toxicologically in-
dependent. For shorter intervals, carry-over toxicity (Ashauer et al.,
2010) would occur, which is reected in the much stronger eects in
Exp. 12 with three pulses (separated by 1 day), compared to the eects
observed due to a single pulse. Interestingly, in a similar analysis by
Smit et al. (2008) for other crustaceans, a mist of the initial response
was not observed, and there was also no indication of saturating eects.
However, those experiments were done with constant exposure. Smit
et al. report 4-day LC50s of 46 mg/L for Corophium volutator and
168 mg/L for Artemia salina, which are very high compared to the value
of 0.14 mg/L we could estimate from the GUTS parameters. Since Smit
and co-workers essentially applied the same model (apart from the
saturation constant C
), we can compare the model parameters between
the dierent species (Supplementary Table 3). The most striking dif-
ference is that the shrimp have, compared to the other crustaceans
tested, a much lower threshold and a much higher killing rate. This is a
clear indication that the shrimp are intrinsically more sensitive to H
formulation than some other crustaceans. Com-
paring our LC
-values to those reported for the copepod Calanus n-
marchicus (Hansen et al., 2017), the 1-day LC50 for the shrimp is
roughly half that for the copepod. However, the 4-day LC50 is almost a
factor of 20 less. This indicates that the shrimp are more sensitive than
the copepods, but also that the dynamics of the response may be quite
dierent for both species. At the moment, we have no explanation for
these inter-species dierences in response to H
; since species seem
to dier in more than one parameter of the TKTD model
(Supplementary Table 3), our analysis does not suggest a simple me-
chanistic hypothesis. However, it must be stressed that the LC50 only
considers the mortality at the end of the exposure period, and not the
delayed mortality that is observed after exposure has ended. The data in
Fig. 7 clearly show that one should not assess the eect of a 2 h pulse of
using a 2 h LC
value. The parameterised model can be used to
make predictions (with condence interval) for untested exposure
proles, or derive scenario-specic no-eect thresholds, which are
more meaningful exercises for risk-assessment purposes than focussing
on LC
4.5. Possible consequences in the eld
To estimate the potential consequences of discharging treatment
water containing H
on populations of P. borealis living in areas with
salmon farms, the dispersion and degradation of H
need to be
considered. Although H
degrades relatively rapidly to water and
oxygen, the half-life of H
has been reported to range from 1 day up
to 28 days (Bruno and Raynard, 1994;Fagereng, 2016;Haya et al.,
2005;Lyons et al., 2014) considerably longer than the time (12h)
needed to cause mortality and gill damage in P. borealis. All results
reported here are for exposures to the commercial formulation and,
while results are reported on the basis of hydrogen peroxide exposure
and concentration, the potential for formulation ingredients to play a
role cannot be discounted.
It is the transport and dilution of the treatment water in the hours
after the discharge that are the most relevant factors for estimating the
risk for P. borealis and other non-target crustaceans. There are few eld
studies available where the concentration of H
has been measured
in surrounding water after bath treatment of salmonids. In a recent
study, more than 600 water samples taken at 560 m depth 025 min
after treatment with H
were analysed (Andersen and Hagen, 2016;
Fagereng, 2016). The raw data from this eld study shows that the
concentration of H
was above 1.5 mg/L in 20% of the samples, and
that concentrations above 1.5 mg/L were detected in some samples
from all depths (560 m). High measured concentrations of H
(> 700 mg/L) in some samples from 60 m were most likely caused by
plume sinkingthat can happen when the water column is well mixed
in the period from October to May (Refseth et al., 2016). Most of the
measured values were, however, below 1.5 mg/L indicating that po-
tential exposure of P. borealis will depend on the transport and dilution
of water containing H
after the rst 25 min, as studied by Fagereng
Fig. 7. Observed survival probability over time as a result of various pulse treatments (animals lying down were considered to be dead). Solid lines are the
simultaneous model t on all data, and dotted lines connect symbols to the corresponding model curve. The right panel has been truncated for readability; ob-
servations in the control and the 3 × 2 h 1.5 mg/L continue till day 19 with only one additional death in the treatment.
R.K. Bechmann, et al. Ecotoxicology and Environmental Safety 180 (2019) 473–482
Hydrodynamic dispersion modelling showed that release of chemi-
cals in an open location could lead to a dispersion of several kilometres
(Brokke, 2015). Release in a sheltered location usually led to a lower
dilution and a shorter dispersion distance. Some areas may contain
concentrations of 13% of the initial dosages used 24 h after release
(Brokke, 2015). Page et al. (2014) added a uorescein dye into the
treatment volume along with the chemical to track the transport and
dispersal of the released chemicals following tarped net-pen or well-
boat application. Data from the dye experiment was combined with
modelling to estimate how far the chemicals were transported and the
dilution with time after release. Page et al. (2014) showed that releases
from tarp treatments are diluted by several orders of magnitude as they
are advected from their point of release. The dilution, however, may
take several hours to occur, thus increasing the opportunity for ex-
posure of non-target organisms (Page et al., 2014). More specically,
the estimated dilution factor was 1001000 after 3 h (i.e. 151.5 mg/L
) and the discharge could be transported several kilometres away,
depending on the water current speed (see (Page et al., 2014) for more
details). Dispersion modelling for an area on the west coast of Norway
estimated that concentrations as high as 10 mg/L H
could occur
several kilometres away from the treated farm up to 3 h after the dis-
charge (Refseth et al., 2016). Local hydrodynamic conditions will de-
termine how fast the concentration of H
will be diluted and how far
it will be transported horizontally and vertically. Results from disper-
sion modelling (Brokke, 2015;Page et al., 2014;Refseth et al., 2016)
and the current experiments indicate that treatment water with toxic
concentrations of H
(1.5 mg/L) could reach P. borealis living more
than 1 km from a treated salmon farm.
Pandalus borealis are found throughout the North Atlantic and
Pacic. They have a circumpolar distribution and are commercially
shed, not only in Norway but in the cold waters of the North Atlantic,
including the Bay of Fundy and Newfoundland in Canada (Bergström,
2000). Shrimp, in Norway, are often found in waters close to aqua-
culture activity (Olsen et al., 2012). If multiple farms are using the
product in the same area, or even in multiple cages at a
single site, there is a potential for negative consequences for shrimp
populations in the eld.
RKB designed the study, participated in the practical work in the lab
and is the main writer of this manuscript with the help of MA who
participated in the design, practical work, analyses of the data and
writing. SW was responsible for the exposure system including the
chemical measurements and input to writing. AG was responsible for
performing and writing about the histology. TJ was responsible for
GUTS modelling work including writing of the modelling sections. EL
and MB contributed to the daily practical work with the lab experi-
ments. TA and LB contributed to reading and commenting on the
manuscript. TA is WP leader in the ECOAST project #258833 COFASP
that co funded the experiments, LB is scientic advisor in the
PestPuls#267746/E40 project funded by the Research Council of
Norway, and RKB is project leader for PestPuls.
Declarations of interest
We thank Frederike Keitel-Gröner for technical assistance in the lab.
Financial support was received from EU through the ECOAST project
#258833 COFASP, the Research Council of Norway through the
PestPuls #267746/E40 project, and from Solvay Interox. Ltd.
Appendix A. Supplementary data
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... Although H 2 O 2 is an effective antiparasitic agent (Bruno and Raynard, 1994), it is also toxic for several non-target taxa (Munn et al., 2003;Urbina et al., 2019), especially crustaceanssuch as European lobster (Homarus gammarus; median lethal concentration after 1-h exposure (1-h LC 50 ) = 177-737 mg l − 1 ) , Northern krill (Meganyctiphanes norvegica; 1-h LC 50 = 35.2 mg l − 1 ) (Escobar-Lux and Samuelsen, 2020), Northern shrimp (Pandalus borealis; 24-h LC 50 = 2.7 mg l − 1 ) (Bechmann et al., 2019), copepods (Van Geest et al. (2014), Calanus spp.; 1-h LC 50 = 214.1 mg l − 1 , Escobar-Lux et al. (2019) -molluscs (Potamopyrgus antipodarum; 24-h LC 50 = 37.5 mg l − 1 ) (Oplinger and Wagner, 2015) and polychaetes (Capitella sp; 1-h LC 50 = 1227 mg l − 1 , and Ophryotrocha spp.; 1-h LC 50 = 296 mg l − 1 ) (Fang et al., 2018). In comparison, macroalgae exhibit a high interspecific variability in their response to H 2 O 2 (Dummermuth et al., 2003). ...
... Within salmon fish farms, delousing operations generally involve simultaneous and sequential applications of pesticides in many cages and non-target species are likely to face multiple exposure to H 2 O 2 over several days (Grefsrud et al., 2019). Multiple exposure may lead to cumulative impacts on non-target species with even more pronounced effects on their physiology than a single exposure (Bechmann et al., 2019). Exposure of thalli to oxidative stress over repeated and longer periods may drastically increase damages on PSII, potentially inhibiting repair systems through irreversible damages on cells. ...
... In addition to the impact on coralline algae, H 2 O 2 treatment in the vicinity of maerl beds may have severe impacts on associated faunal and floral species. For example, crustaceans represent one of the most abundant groups from maerl beds (De Grave, 1999;Barbera et al., 2003;Teichert, 2015) and are reported to be particularly sensitive to H 2 O 2 (Van Geest et al., 2014;Escobar-Lux et al., 2019Bechmann et al., 2019;Urbina et al., 2019;. The industry also relies on the use of other chemotherapeutants to regulate sea lice infestations, such as emamectin-benzoate, azamethiphos and deltamethrin, which affect directly or indirectly the nerve functions of many non-target species (Walsh et al. 2007;Overton et al. 2019). ...
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The proliferation of sea lice (Lepeophtheirus salmonis) represents a major challenge for the salmonid aquaculture industry in Norway. Hydrogen peroxide (H2O2) is a chemotherapeutant frequently used on Norwegian farms, however, its toxicity to non-target benthic species and habitats remains poorly understood. Maerl beds are constructed by the accumulation of non-geniculate coralline algae and provide important ecological functions. Due to the rapid expansion of aquaculture in Norway and the continued use of H2O2 as an anti-sea lice treatment, it is crucial to understand the impact of H2O2 on the physiology of maerl-forming species. The effects of a 1 h exposure to H2O2 on the photophysiology and bleaching of the coralline alga Lithothamnion soriferum were examined here through a controlled time-course experiment. PAM fluorimetry measurements showed that H2O2 concentrations ≥ 200 mg l⁻¹ negatively affected photosystem II (PSII) in thalli immediately after exposure, which was observed through a significant decline in maximum photochemical efficiency (Fv/Fm) and relative electron transport rate (rETR). The negative effects on PSII induced by oxidative stress, however, appear to be reversible, and full recovery of photosynthetic characteristics was observed 48 h to 28 days after exposure to 200 mg H2O2 l⁻¹ and 2000 mg H2O2 l⁻¹, respectively. At 28 days after exposure, there was evidence of two- to four-times more bleaching in thalli treated with concentrations ≥ 200 mg H2O2 l⁻¹ compared to those in the control. This indicates that despite the recovery of PSII, persistent damages can occur on the structural integrity of thalli, which may considerably increase the vulnerability of coralline algae to further exposure to H2O2 and other chemical effluents from salmonid farms.
... Especially important is the assessment of delayed sub-lethal effects of bath treatment plumes on these non-target species, resulting from short time exposures. Shorter exposure times i.e. 1h followed by a 24h post-exposure time can provide a more realistic assessment of the impacts of bath treatment plumes on nontarget species (Medina et al., 2004;Van Geest et al., 2014;Bechmann et al., 2019;Frantzen et al., 2020). behavior reduces the encounter probabilities with predators, the escape responses minimize the successful attacks, and the defense responses decreases the probability of ingestion by a predator. ...
... The chemotherapeutants combating sea lice also affect non-target species following release into the sea. In the past years, several studies have tested the toxicity of H2O2 in 12 different non-target crustacean species (Smit et al., 2008;Burridge et al., 2014;Van Geest et al., 2014;Brokke, 2015;Gebauer et al., 2017;Hansen et al., 2017;Bechmann et al., 2019;Frantzen et al., 2020) (Fig. 2). In addition, there is available literature for the toxicity of H2O2 on the polychaetes Capitella sp. and Ophryotrocha spp., and sugar kelp (Saccharina latissima) Mitchell and Collins, 1997;Rach et al., 1997;Fang et al., 2018;Haugland et al., 2019). ...
... Another study focused on the effects of H2O2 on the deep-water shrimp P. borealis, and investigated immobilization, swimming activity and feeding rates after exposure (Bechmann et al., 2019). Even at low concentrations, reported effects were, inability to swim or increased swimming activity (depending of the chemical), inability to capture food and delayed molting Bechmann et al., 2019;Frantzen et al., 2020). ...
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I present new knowledge on the toxicity of three major bath treatment chemotherapeutants used in Norway. Previously, regarding the toxicity studies of hydrogen peroxide (H2O2) alone, a total of twelve non-target crustaceans have been examined across the globe, but only five species were relevant for the Norwegian marine ecosystem. The present study applied laboratory experiments to assess the toxicity of this chemotherapeutant to three non-target crustacean species that play a crucial role in the Norwegian marine ecosystem, bringing a better understanding of the risk posed by H2O2. Hydrogen peroxide has long been labeled as the most environmentally friendly bath treatment in use for the salmonid industry. It has also been considered that it poses little to no threat in terms of lethality to non-target crustaceans such as lobster, shrimps or crabs (Burridge et al., 2014; Gebauer et al., 2017). However, papers I, II and III show that the recommended H2O2 concentrations used by the salmonid industry across the globe are lethal to non-target crustaceans. Through the creation of species sensitivity distribution curves (SSD), this thesis identified the Northern krill (Meganyctiphanes norvegica) as the crustacean species that is most sensitive to H2O2 of those that have been tested so far. By including the sensitivity of six phyla other than the arthropods, this thesis takes a broader perspective on the impact of H2O2 on the marine environment. The hazardous concentration of H2O2 for 5% of the species (HC5) derived from the available toxicity data for marine species is 5.11 (1.52 – 16.15) mg/L. As SSD curves are a central tool for ecological risk assessments, showing the different sensitivities and variations between species, it is crucial that this tool continues to be used for the risk assessment of the other chemotherapeutants. Deltamethrin and azamethiphos have a detrimental effect on European lobster larvae (Homarus gammarus) in laboratory experiments (Paper IV). One-hour exposure to deltamethrin proved to be more toxic than H2O2 and azamethiphos to both stage I and stage II H. gammarus larvae. By examining the toxicity of all three chemotherapeutants to a single species this thesis, in combination with the results from previous studies, proposes a ranking of the toxicity of deltamethrin, H2O2 and azamethiphos based on the difference between the median lethal concentrations LC50 (Papers II & IV). With the available data from other studies, the toxicity ranking for Norwegian relevant species is: deltamethrin > H2O2 > azamethiphos. This thesis has also shown the importance of coupling sub-lethal studies with more conventional toxicity studies (Papers I & II). It was shown that behavior parameters linked with the predator avoidance and escape response of the European lobster juveniles and the copepod Calanus spp. were affected following short-term (1 h) exposures at concentrations ≤85 mg/L H2O2 (i.e. 5% of the recommended treatment). All three chemotherapeutants induced immobility at concentrations considerably lower than the reported lethal values. Furthermore, in paper IV the calculated effective median concentration EC50 values for both deltamethrin and azamethiphos were considerably lower than the reported LC50 values based on mortality. The results from the hydrodynamic model presented in paper IV plus the lethality findings from papers I, II and III coupled with both field studies and models should be considered by regulatory authorities in Norway and can be an important tool for other salmonid producer nations when carrying out future environmental risk assessments of H2O2, deltamethrin and azamethiphos. These results should thus be used to evaluate the potential risks associated with the expansion of salmonid aquaculture into new locations. To have a better understanding of the risks of these chemotherapeutants in the Norwegian marine environment, further studies should evaluate their broader impact by assessing chronic or pulse-like exposures that are certainly closer to real life delousing scenarios where multiple pens are treated over a cumulative period of time. Likewise, data from the flushing of well-boats should also be included in new hydrodynamic models, as this bath treatment method dilutes the effluent of waste treatments and thus reduces its environmental impact (Ernst et al., 2014). Overall, this study has shown that the recommended H2O2, deltamethrin and azamethiphos concentrations used by the salmonid industry have a detrimental effect in the survival of the non-target crustaceans Calanus spp., H. gammarus and M. norvegica.
... Unlike the other compounds considered in this report, hydrogen peroxide should not at all accumulate in the environment as it splits quickly into water and oxygen, and is therefore considered environmentally benign 37 . However, recent findings document potential short-term toxicity on non-target species present around farms [38][39][40] . We can note that overall chemical input in the environment was reduced in 2019 (not for hydrogen peroxide) and that this trend has not been consistent within the 4 years. ...
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We used 4 years of publicly available data (2016-2019) on chemical usage at salmon sites with information on production, stocking, locations and environmental conditions to explore patterns of anti-sea lice treatments in a Canadian province. Results show that sequential chemical treatments are prevalent, emamectin benzoate (EMB) with azamethiphos being the most used combination with a decrease in ivermectin usage. Relatively high rates of usage of EMB per fish biomass may point to potential lice resistance patterns with information needed on mechanisms and local populations. Lower or no chemical usage at some sites indicate less sea lice infestations likely influenced by localized site conditions (coves), and a lessened need for medication due to the usage of cleaner fish and possibly other non-chemical methods (not documented in this report). The year/climate influenced chemical input only in sites with higher treatment levels likely due to effects on sea lice growth and reproduction. Observed differences between years are warmer surface temperature in the fall, a higher freshwater input in spring, and stronger wind conditions for 2017 and 2018 with more medication usage for these two years. The lack of significant effect of site distances calculated in zones of influence based on 24 h potential connectivity patterns highlight the need to refine the resolution of hydrodynamic processes.
... In practice, cages are often treated successively, with pulses of chemicals released repeatedly into the ocean. Multiple exposures have the potential of being harmful to sensitive organisms even if the chemical is highly diluted (Bechmann et al., 2019). ...
Tarpaulin bath treatments are used in open net-pen finfish aquaculture to combat parasitic infections, in particular sea lice. After treatment, the toxic wastewater is released directly into the ocean, potentially harming non-target species in the vicinity. We model the dispersion of wastewater chemicals using a high-resolution numerical ocean model. The results are used to estimate the impact area, impact range, dissolution time, and exposure probability for chemicals of arbitrary toxicity. The study area is a fish-farming intensive region on the Norwegian western coast. Simulations are performed at 61 different release dates, each on 16 locations. In our base case where the chemical is toxic at 1% of the treatment concentration, the release of a 16000 m³ wastewater plume traverses a median distance of 1.9 km before being completely dissolved. The median impacted area is 0.9 km² and the median dissolution time is 6.8 hours. These figures increase to 5.9 km, 7.0 km², and 21 hours, respectively, if the chemical is toxic at 0.1 % of the treatment concentration. Locations within fjords have slower dissolution rates and larger impact zones compared to exposed locations off the coast, especially during summer.
... However, because of its fat solubility, PAA has far more potent antimicrobial properties than H 2 O 2 [15]. In addition, concerns have been raised regarding the excessive use of H 2 O 2 , as in Atlantic salmon (Salmo salar) farming, due to toxicity threats to other organisms, particularly shrimp [16]. PAA degrades relatively faster than H 2 O 2 , and the difference in the degradation kinetics is important for the PAA byproducts not to persist for an extended period in the environment and present risks to other organisms [10]. ...
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Although chemotherapeutics are used to treat infections in farmed fish, knowledge on how they alter host physiology is limited. Here, we elucidated the physiological consequences of repeated exposure to the potent oxidative chemotherapeutic peracetic acid (PAA) in Atlantic salmon (Salmo salar) smolts. Fish were exposed to the oxidant for 15 (short exposure) or 30 (long exposure) minutes every 15 days over 45 days. Unexposed fish served as the control. Thereafter, the ability of the remaining fish to handle a secondary stressor was investigated. Periodic chemotherapeutic exposure did not affect production performance, though survival was lower in the PAA-treated groups than in the control. Increased ventilation, erratic swimming, and a loss of balance were common behavioural manifestations during the oxidant exposure. The plasma reactive oxygen species levels increased in the PAA-treated groups, particularly after the third exposure, suggesting an alteration in the systemic oxidative stress status. Plasma indicators for internal organ health were affected to a certain degree, with the changes mainly observed after the second and third exposures. Metabolomics disclosed that the oxidant altered several circulating metabolites. Inosine and guanosine were the two metabolites significantly affected by the oxidative stressor, regardless of exposure time. A microarray analysis revealed that the gills and liver were more responsive to the oxidant than the skin, with the gills being the most sensitive. Moreover, the magnitude of the transcriptomic modifications depended on the exposure duration. A functional analysis showed that genes involved in immunity and ribosomal functions were significantly affected in the gills. In contrast, genes crucial for the oxidation-reduction process were mainly targeted in the liver. Skin mucus proteomics uncovered that the changes in the mucosal proteome were dependent on exposure duration and that the oxidant interfered with ribosome-related processes. Mucosal mapping revealed gill mucous cell hypertrophy after the second and third exposures, although the skin morphological parameters remained unaltered. Lastly, repeated oxidant exposures did not impede the ability of the fish to mount a response to a secondary stressor. This study provides insights into how a chemical oxidative stressor alters salmon physiology at both the systemic and mucosal levels. This knowledge will be pivotal in developing an evidence-driven approach to the use of oxidative therapeutics in fish, with some of the molecules and pathways identified as potential biomarkers and targets for assessing the physiological cost of these treatments.
... Before we focus on innovation in breeding strategies for lice resistance, we need to have a better idea of how the salmon sector is currently responding to the lice situation. The resistance of lice to medicinal treatments has increased more rapidly than the effectiveness of the treatments themselves, and negative environmental effect (on shellfish in particular) of medical treatments is increasingly being documented (Bechmann et al., 2019;Olaussen, 2018). ...
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Lice is a persistent and major problem in the salmon aquaculture sector with serious environmental impacts and reducing growth potential and income of the salmon industry. This article discusses whether there is an untapped potential in breeding for improved lice resistant of Atlantic salmon. To this end, three sets of factors that may impact the state of breeding for lice resistance are examined, using document analysis and key actor interviews. First, our data material indicates that market-based factors will hardly stimulate this type of breeding, as the benefits from breeding for lice resistance is predominantly a public good, and because the polygenic nature of lice resistance does not enable patenting as a powerful instrument to secure private goods or privatize the benefits of genetic improvement. Second, the regulation of gene editing technologies is in flux, and increase the risk of investments in technologies as selective breeding that could handle the polygenic challenges of lice resistance. Finally, policy instruments aimed at stimulating relevant innovation has been applied generously for other types of innovations to deal with the lice problem. For instance, new technologies for delousing have resulted in increased treatments and caused higher stress and mortality the recent years. However, none of these have targeted major root causes of the salmon lice problem (e.g. big monocultures or susceptible fish) or been exploited by breeding actors. Seen from a social and environmental point of view this could paradoxically lead to increased demand for fish that better endures harsh delousing treatments rather than demand for more lice resistant fish.
... Oxidants could remain at the end of the reaction (Liu et al., 2021a,b). Common oxidants such as hydrogen peroxide and peroxymonosulfate have effect on the microorganisms (Bechmann et al., 2019;Moreno-Andrés et al., 2020), which could affect the wastewater toxicity. García-Espinoza et al. (2018) pointed out that the increase of carbamazepine solution' toxicity after electrochemical oxidation was mainly due to the remnant active chlorine. ...
Advanced oxidation processes (AOPs) have been widely applied for the advanced treatment of recalcitrant wastewater. However, the toxicity changes during AOPs are still unclear because in some cases the intermediate products could exhibit equivalent or even higher toxicity compared with the original pollutants. Therefore it is necessary to evaluate the toxicity evolution during AOPs. This review mainly focused on the toxicity changes of wastewater during various AOPs treatment, including Fenton/Fenton-like oxidation, ozonation/catalytic ozonation, persulfate radical-based oxidation, photocatalytic oxidation, ionizing radiation, electro-catalytic oxidation, ultrasound oxidation and wet oxidation. Firstly, the toxicity assessment methods were summarized, including acute toxicity (such as luminescent bacteria assay, fish assay, water flea assay and algae assay), genetic toxicity (Ames assay, SOS/umu assay, comet assay and micronucleus assay), estrogenic activity, immunity toxicity, and endocrine disrupting effect (such as yeast two-hybrid assay and fish endocrine disrupting assay), the applications of these methods in determining the toxicity changes of wastewater during AOPs treatment were summarized and analyzed in detail. Secondly, the toxicity prediction by computational simulation was introduced, mainly focusing on two common software, including TEST and ECOSAR. Thirdly, the strategies for eliminating the toxicity of wastewater after AOPs treatment were proposed. Finally, the concluding remarks and perspectives were presented for further study. This review will deepen to understand the toxicity evolution of wastewater during AOPs.
Oil spill clean-up measures using in situ burning can potentially result in seafloor contamination affecting benthic organisms. To mimic realistic exposure and measure effects, ovigerous Northern shrimp were continuously exposed for two weeks to the water-soluble fraction of oil coated on gravel followed by two weeks in clean seawater. North Sea crude oil (NSC) and field generated in situ burn residue (ISBR) of NSC were used (Low: 3 g/kg gravel, Medium: 6 g/kg gravel and High: 12 g/kg gravel). The concentrations of polyaromatic hydrocarbons (PAHs) in the water resulting from NSC were higher compared to ISBR. No mortality was observed in any treatment and overall moderate sublethal effects were found, mostly after exposure to NSC. Feeding was temporarily reduced at higher concentrations of NSC. PAH levels in hepatopancreas tissue were significantly elevated following exposure and still significantly higher at the end of the experiment in NSCHigh and ISBRHigh compared to control. Mild inflammatory response reactions and tissue ultrastructural alterations in gill tissue were observed in both treatments. Signs of necrosis occurred in ISBRHigh. No change in shrimp locomotory activity was noted from NSC exposure. However, ISBR exposure increased activity temporarily. Larvae exposed as pleopod-attached embryos showed significant delay in development from stage I to stage II after exposure to NSCHigh. Based on this study, oil-contaminated seafloor resulting from in situ burning clean-up actions does not appear to cause serious effects on bottom-living shrimp.
The active ingredients from 5 chemotherapeutant formulations (Slice® [active ingredient [AI] emamectin benzoate), Salmosan® (AI azimethiphos), Alphamax® (AI deltamethrin), Excis® (AI cypermethrin), and Interox® Paramove 50 (AI hydrogen peroxide) that have been used or continue to be used to treat sea lice infestations in salmon aquaculture were examined to generate data on their environmental partitioning and persistence in water-sediment microcosms, as well as their acute and subchronic toxicity to representative classes of marine organisms. Emamectin benzoate, cypermethrin and deltamethrin partitioned mainly to the sediment phase; azimethiphos and hydrogen peroxide remained mainly in the water phase. The persistence of chemicals in water was: CP > DM > AZ > HP (half-lives: 19.8, 17.9, 12.7 d, and 8.9 h, respectively). In sediments, the following trend in calculated half-lives was observed: CP > EB > DM (half-lives: 557, 230 and 45 d, respectively). Toxicity test results with a wide variety of marine organisms (macroalgae, echinoderms, bivalves, crustaceans and fish) showed no susceptibility trend for any species, or inherent toxicity trend for any chemical, although DM tended to be the most toxic and HP the least toxic to the majority of species. This information is useful for identifying risks; specifically, toxicological parameters calculated for several of the non-target marine organisms examined, indicate that recommended treatment concentrations could result in non-target organism toxicity following release in the immediate vicinity of aquaculture sites before significant dilution. This study provides valuable data on the environmental fate and associated risks of chemotherapeutant use to non-target marine organisms whose habitat coincides with salmon aquaculture sites.
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Hydrogen peroxide (H2O2), a pesticide used in salmonid aquaculture, is released directly into the environment where nontarget organisms are at risk of exposure. We determined threshold concentrations for mortality of Calanus spp., the dominant zooplankton species in the North Atlantic, and assessed sublethal effects, focusing on the escape response and oxygen consumption rates (OCRs) as behavioral and physiological assays. One-hour exposure to 170 mg·L−1 (i.e., 10% of the recommended H2O2 treatment) was lethal to copepodite stage V (92% mortality) and adult females (100% mortality). The acute median lethal concentration (1h-LC50) was 214.1 (150.67–277.4) and 48.6 (44.9–52.2) mg·L−1 for copepodite V and adults, respectively. The 25-h LC50 was 77.1 (57.9–96.2) and 30.63 (25.4–35.8) mg·L−1 for copepodite V and adults, respectively. At concentrations of 0.5% and 1% of the recommended treatment level, Calanus spp. showed a decrease in escape performance and lower OCRs with increased concentration. At H2O2 concentrations of 5% of the recommended treatment levels (85 mg·L−1), exposed copepods showed no escape reaction response. These results suggest that sublethal concentrations of H2O2 will increase the risk of predation for Calanus spp. Furthermore, this study provides supporting evidence that theoretical “safe” values, traditionally used for predicting toxicity thresholds, underestimate the impact of H2O2 on the physiological condition of nontarget crustaceans.
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Reduced sensitivity towards chemotherapeutants in the salmon louse Lepeophtheirus salmonis (Krøyer) is an increasing problem for the fish farming industry. Most fish farmers are dependent on chemical treatments in order to maintain salmon lice numbers below permitted levels. However parasites showing reduced sensitivity contribute to complicating this task. Hydrogen peroxide (H2O2) is used as a delousing agent in bath treatments and until recently treatment failures due to reduced H2O2-sensitivity have not been documented in Norway. The aim of the current study was to develop a bioassay protocol suitable for testing H2O2-sensitivity in L. salmonis. If failed treatments were found to be caused by parasite insensitivity to H2O2 the possibility of this reduced sensitivity being hereditary was looked into. The results show that bioassays permit differentiation between strains of salmon lice with regards to H2O2-sensitivity, coinciding with treatment efficacies. Up to ten times variance in sensitivity between two strains was recorded. The progeny of the least sensitive salmon lice also showed reduced sensitivity to H2O2 in a bioassay, which indicates that reduced sensitivity towards H2O2 is hereditary. The current study presents the first case report of reduced sensitivity towards H2O2 in salmon lice in Norway. This change in sensitivity imposes a threat to the Norwegian fish farming industry and should be monitored closely.
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Sea lice are copepod ectoparasites with vast reproductive potential and affect a wide variety of fish species. The number of parasites causing morbidity is proportional to fish size. Natural low host density restricts massive parasite dispersal. However, expanded salmon farming has shifted the conditions in favor of the parasite. Salmon farms are often situated near wild salmonid migrating routes, with smolts being particularly vulnerable to sea lice infestation. In order to protect both farmed and wild salmonids passing or residing in the proximity of the farms, several measures are taken. Medicinal treatment of farmed fish has been the most predictable and efficacious, leading to extensive use of the available compounds. This has resulted in drug-resistant parasites occurring on farmed and possibly wild salmonids. Copyright © 2014 Elsevier Ltd. All rights reserved.
ABSTRACT: Sugar kelp Saccharina latissima is one of the dominating kelp species on sheltered sublittoral sites along the Norwegian coastline, providing important habitats and ecosystem services. In finfish aquaculture in Norway, infections caused by salmon lice Lepeophtheirus salmonis are one of the biggest challenges the industry is currently facing, and presently the most-used therapeutant is hydrogen peroxide (H2O2). Commonly, this treatment involves bathing the fish in a solution of approximately 1700 mg H2O2 l −1 seawater before releasing the solution into the surrounding waters. The present study was conducted to increase the knowledge on how these H2O2 emissions from farm operations could impact juvenile S. latissima. This was achieved by determining the effects of a 1 h exposure to realistic H2O2 levels on S. latissima mortality and photosyn - thesis. Effects on photosynthesis were determined by incubating plants at 3 time intervals postexposure. Toxicity potentials including lethal concentration for 50% of the population (LC50) and effective concentration (EC50) for photosynthetic capacity (PMAX) and efficiency (α) were determined based on these data. Juvenile S. latissima was highly sensitive, having an LC50 of 80.7 mg H2O2 l −1, which is less than 5% of the dose commonly used at farms and emitted to the environment. A concentration of 85 mg l−1 caused an immediate 90% reduction in both PMAX and α. The EC50 was found to be 27.8 and 35.4 mg l−1 for PMAX and α, respectively. This may indicate that natural S. latissima populations in the vicinity of fish farms can be negatively affected by H2O2 bath treatments.
Aquaculture is currently one of the best prospects to help meet the growing need for protein in the human diet. However, aquaculture development and production result in consequences for the environment and also impact other productive activities. Salmon and trout cage culture has required the use of large quantities of pharmaceuticals in order to control outbreaks and the persistence of different pathogens, including sea lice (parasitic copepods), which cause economic losses of around 0.39 € Kg−1 of salmon produced. The pharmaceuticals currently used for the control of sea lice (cypermethrin, deltamethrin, azamethiphos, hydrogen peroxide) are applied by in situ immersion treatments, enclosing net pens using tarpaulin and then bathing fish with the pharmaceutical. After treatment the pharmaceuticals are released into the surrounding environment, exposing non-target species. Although the effects of such pharmaceutical exposure has been studied in some species, to date a systematic and exhaustive review of these potential effects has not yet been performed. In this study, an exhaustive review of the literature evaluating lethal and sub-lethal effects of anti-sea lice pharmaceuticals on non-target crustaceans and bivalves was performed, in order to assess the extent of the effects, toxicity, variables affecting such toxicity and identify potential synergistic effects previously unexplored. Our results show clear negative effects at concentrations lower than those used in treatments against sea lice in all of the species studied. Likewise, this study demonstrates knowledge gaps that need to be addressed in order to improve our understanding of the effects of these pharmaceuticals on non-target species, ecosystems in general and other productive activities.
Use of hydrogen peroxide (H2O2) for removal of salmon lice in the aquaculture industry has created concern that non-target organisms might be affected during treatment scenarios. The aim of the present study was to examine the potential for H2O2 to produce oxidative stress and reduce survival in one of the most abundant zooplankton species in Norwegian coastal areas, the copepod Calanus finmarchicus. Copepods were subjected to two 96-hr tests: (1) acute toxicity test where mortality was determined and (2) treated copepods were exposed to concentrations below the No Observed Effect Concentration (0.75 mg/L) H2O2 and analyzed for antioxidant enzyme activities, as well as levels of glutathione (GSH) and malondialdehyde (MDA). Compared to available and comparable LC50 values from the literature, our results suggest that C. finmarchicus is highly sensitive to H2O2. However, 96-hr exposure of C. finmarchicus to 0.75 mg H2O2/L did not significantly affect the antioxidant systems even though the concentration is just below the level where mortality is expected. Data suggest that aqueous H2O2 exposure did not cause cellular accumulation with associated oxidative stress, but rather produced acute effects on copepod surface (carapace). Further investigation is required to ensure that aqueous exposure during H2O2 treatment in salmon fish farms does not exert adverse effects on local non-target crustacean species and populations. In particular, studies on copepod developmental stages with a more permeable carapace are warranted.
The pesticides used by the salmon industry to treat sea lice, are applied in situ via a bath solution and are subsequently discharged into the surrounding medium. The effects of cypermethrin, deltamethrin, azamethiphos and hydrogen peroxide were assessed on the performance of Metacarcinus edwardsii larvae, an important crab for Chilean fishery. All larvae were dead or dying after 30 min of exposure to cypermethrin and after 40 min to deltamethrin at concentrations 100 and 20 times lower (0.15 and 0.1 μg L⁻¹, respectively) than the concentrations and exposure times recommended by the manufacturers (CRM) to treat sea lice. Azamethiphos affected all larvae at a concentration 10 times lower than CRM. Hydrogen peroxide had the lowest detrimental effects, but at the CRM, 100% of the larvae were affected. Sub-lethal effects, i.e prolonged developmental time, were observed at concentrations lower than CRM. Repeated exposure to azamethiphos (0.0625-0.5 μg L⁻¹) and hydrogen peroxide (188-1500 mg L⁻¹) had effects on survival. In conclusion, the pesticides used against parasitic copepod tested here, negatively affect non-target crustacean larvae. Due to the product´s characteristics, the lethal effects of the pyrethroids probably are restricted to the time and area of application, while the action of azamethiphos may extend to a wider area. Current data are insufficient to accurately dimension the effects of these compounds in the field. More research is required to evaluate the consequences of prolonged developmental times and/or reduction in appendage mobility, so as the effects of these compounds on the pelagic and benthic communities.
The tissue damage induced by various organic pollutants in aquatic animals is well-documented, but there is a dearth of information relating to the histological alterations induced by pesticides in freshwater prawns of the genus Macrobrachium. In the present study intermoult juveniles of the freshwater prawn Macrobrachium malcolmsonii (total length. 4.5-5.0 cm; weight, 1.0-1.25 g) were exposed to three sublethal concentrations of the organochlorine pesticide, endosulfan (10.6, 16.0 and 32.0 ng/l) for a period of 21 days. The hepatopancreas and gills of the prawns were then dissected out and processed for light microscopic studies. Exposure to endosulfan was found to result in several alterations in the histoarchitecture of the hepatopancreas and gills of M. malcolmsonii. The alterations included: hemocytic infiltration in the interstitial sinuses, thickening of basal laminae and necrosis of the tubules of the hepatopancreas: accumulation of hemocytes in the hemocoelic space. swelling and fusion of lamellae, abnormal gill tips, hyperplastic, necrotic, and clavate-globate lamellae of the gills. The results obtained suggest that the hepatopancreas and gills of prawns exposed to endosulfan were structurally altered. Such alterations-could affect vital physiological functions, such as absorption, storage and secretion of the hepatopancreas, and respiration, osmotic and ionic regulations of the gills, which in turn could ultimately affect the survival and growth of,21. malcolmsonii. Thus, all possible remedial measures should be adopted to prevent the occurrence of pesticide contamination in the aquatic environment.