Variable survival across low pH gradients in freshwater fish species



Variable survival across low pH gradients in freshwater fish species
Journal of Fish Biology (2014) 85, 1746–1752
doi:10.1111/jfb.12497, available online at
Variable survival across low pH gradients in freshwater fish
P. G. Jellyman* and J. S. Harding
School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch,
New Zealand
(Received 2 February 2014, Accepted 9 July 2014)
A series of 14 day experiments was conducted on five common New Zealand fish species (redfin bully
Gobiomorphus huttoni, inanga Galaxias maculatus, brown trout Salmo trutta, longfin eel Anguilla
dieffenbachii and koaro Galaxias brevipinnis) to assess the effect of pH on survival and changes in
body mass. No species survived in water of pH <4 although there was 100% survival of all adults at
pH 4⋅5, G. maculatus larvae were also tested and had high mortality at this pH. Results suggest that
adults are tolerant of low-pH waters; however, successful remediation of anthropogenically acidified
streams will require an understanding of the susceptibility to low pH on different life cycle stages.
© 2014 The Fisheries Society of the British Isles
Key words: experimental ecology; mortality; naturally acidic waters; New Zealand; tolerance.
Acidification of fresh waters is widespread owing to the natural leaching of soil acids,
pollution resulting in acid rain and human activities such as coal mining (Schindler,
1988; Hogsden & Harding, 2012). Understanding the effect these types of acidification have on freshwater communities can be complex because systems affected by
anthropogenic acidity often also have elevated concentrations of bioavailable toxic
metals (Petrin et al., 2008). Untangling the separate and interactive effects of stream
acidification (i.e. reduced pH) and metal toxicity is critical for the remediation of freshwater environments. Knowing which factors play the most important role in limiting
the recovery of riverine communities could greatly improve the chances of successful
For freshwater fish communities, pH can be a major determinant of which species
will be present because species vary widely in their tolerance of acidity (West et al.,
1997; Mills et al., 2000; Greig et al., 2010). One common remediation strategy has
been to treat water so as to improve quality to a level that is suitable for a sub-set of
representative species, with the expectation that this will render it acceptable for others
as well. Adopting such an approach requires species tolerance values to be measured for
*Author to whom correspondence should be addressed at present address: National Institute of Water and
Atmospheric Research Ltd., P. O. Box 8602, Christchurch, New Zealand. Tel.: +64 3 348 8987; email:
[email protected]
© 2014 The Fisheries Society of the British Isles
representative species and, whilst this can be done in the field, field tolerance values are
often confounded in acidified waters by the influence of dissolved metal concentrations
and other physico-chemical factors. Moreover, field data are rarely logged continuously so using pH from the day of sampling to determine fish tolerance limits may not
reflect the conditions during long-term exposure. Using one-off pH measures increases
the risk of over-estimating tolerance limits (or vice versa). For example, if transient
migratory species are captured during a field survey, they may skew results because
these fishes have not tolerated long-term exposure to water chemistry at a site. Thus,
determining accurate fish tolerances to a single factor such as pH is liable to be more
robust using an experimental approach. To determine the lower pH tolerance limits for
five common New Zealand freshwater fish species (from a region with streams affected
by acidified mine leachate), a series of 2 week laboratory experiments was conducted.
Acid tolerance experiments were conducted from December 2010 to April 2011. Five
fish species were exposed to a range of waters with different pH levels. These species
were redfin bully Gobiomorphus huttoni (Ogilby 1894), inanga Galaxias maculatus
(Jenyns 1842), brown trout Salmo trutta L. 1758, longfin eel Anguilla dieffenbachii
Gray 1842 and koaro Galaxias brevipinnis Günther 1866. Instead of assessing their survival over a four-day (96 h) period, as per standard ecotoxicology experiments, survival
was measured over 14 days. This longer time period was selected because chronic exposure to low pH is relatively common in anthropogenically affected systems and acute
96 h experiments may not accurately reflect whether a species can tolerate longer-term
exposure. The water used in each of the five experiments was naturally acidic, brown
stream water (pH 6⋅4–6⋅6, 31⋅2 μS cm−1 ) collected from O’Malley Creek (42∘ 09′
S; 171∘ 47′ E) on the West Coast, South Island, New Zealand. For each experiment,
1200 l of stream water was transported to a temperature and light cycle-controlled laboratory at the University of Canterbury. The species used were collected from several
naturally acidic, brown-water streams (pH range: 6⋅0–6⋅6) on the west coast because
naturally acidic waterways (e.g. as low as pH 4⋅5) are common in this region and these
fishes may be adapted to tolerate more acidic stream water. Fishes were captured by
either electrofishing or Gee-minnow trapping, depending on species. Only one fish
species was tested during each 14 day experiment. Fish size was controlled to ensure
that only juvenile and small adult fishes were used and also of a size that could be held
in re-circulating tanks for 2 weeks without ammonium concentrations reaching lethal
levels. The size ranges were: G. huttoni (63–74 mm total length, LT , 2⋅6–4⋅5 g body
mass, M B ), G. maculatus (82–91 mm LT , 3⋅3–4⋅9 g M B ), S. trutta (69–82 mm LF ,
3⋅7–6⋅3 g M B ), G. brevipinnis (77–105 mm LT , 3⋅0–7⋅9 g M B ) and A. dieffenbachii
(250–340 mm LT , 23–77 g M B ). Whilst A. dieffenbachii are an order of magnitude
heavier than the other fish species, A. dieffenbachii shift from living within substrata
to open water only at sizes around 300 mm (Jellyman et al., 2002), so it was important
to test eels of approximately this size.
Fishes were acclimated in 40 l holding tanks of unmanipulated stream water for 48
h prior to commencing the experiment. The experiment used 30 re-circulating tanks
filled with 35 l of stream water. The tanks were split into five pH treatments (pH 3,
3⋅5, 4, 4⋅5 and 5⋅5) and a control (pH c. 6⋅5), each with five replicates and one fish
per replicate. The desired pH level was achieved by adding hydrochloric acid to the
stream water; direct acid addition to manipulate pH is the appropriate method given
the research objective (Esbaugh et al., 2013). The water was constantly sampled and
adjusted during initial pH manipulation and throughout the trials using a laboratory pH
© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 85, 1746–1752
P. G . J E L LY M A N A N D J . S . H A R D I N G
meter (Metrohm; All tanks were sealed to prevent fishes escaping
and had a flow circulation pump with an external air line fitted that drew in air as the
water circulated. Fishes were kept in an 18∘ C temperature-controlled room with a
12L:12D photoperiod. Fishes were weighed at the start and end of the experiment to
determine changes in fish mass in waters of different pH levels. The one exception to
this was the A. dieffenbachii experiment where on day 14 (22/2/11) a large earthquake
hit Canterbury and the experimental facility could not be accessed for 4 days; all A.
dieffenbachii that were alive on the morning of day 14 were still alive after 4 days but
they had not been fed for 96 h prior to their final weighing.
Tanks were monitored daily to check fish status, food consumption and pH; all treatments became slightly less acidic over a 24 h period (e.g. pH 4 would shift to 4⋅05–4⋅13
after 24 h) so pH was sampled and adjusted each morning. In addition, temperature, dissolved oxygen, conductivity and ammonium concentration were also measured daily.
If ammonium concentration in the tanks was >1 mg l−1 , then 1–10 ml of Stress Zyme
( (i.e. a biological filter) was added which decreased ammonium
levels without altering pH. All fishes were fed 1⋅8 g of commercial frozen blood worms
Chironomus spp. each afternoon, with any uneaten bloodworms cleaned from the tank
the following morning to reduce waste product build-up.
As the susceptibility of larval fishes to pH may differ from that of juvenile or adult
fishes, larval fish pH tolerance was also tested. Owing to life-history timing issues and
difficulties in locating the fish eggs from which to hatch larvae, only G. maculatus
larvae could be tested. Galaxias maculatus were collected as eggs (from adult fish site)
and were hatched in the laboratory; larvae size was c. 7 mm. Forty-eight hours after
hatching, larvae were placed into aerated 2 l containers. As per the trial for juvenile
and adult fishes, 30 containers were used and the same pH treatments were applied.
The major differences were that this experiment lasted only 96 h (as larvae were not
fed) and that five larvae were placed in each container instead of only a single fish.
To examine whether temperature, dissolved oxygen, conductivity or ammonium concentration differed over time and between trials, one and two-way analysis of variance
(ANOVA) was performed using the R statistical package 3.0.2 ( To
determine lethal concentration (LC50 ) and lethal time (LT50 ) estimates for each species,
fish mortality data were fitted using four parameter logistic curves in Sigmaplot (version 12.5; One-way ANOVA was conducted for each species
to determine whether the change in mass during the experiment differed significantly
between pH treatments. Post hoc Tukey tests were then conducted in the R statistical
package to assess which pH treatments had significantly different changes in mass. A
P-value of <0⋅05 was considered significant for all tests.
Mean temperature, dissolved oxygen, conductivity and ammonium concentration
did not differ significantly among experiments (ANOVA, P > 0⋅05; for all factors).
There were significant increases over time for both dissolved oxygen (time: ANOVA,
F 1,55 = 33⋅0, P < 0⋅001) and ammonium concentration (time: ANOVA, F 1,55 = 58⋅2, P
< 0⋅001) but there was no significant difference between species over time for either
variable (time × species interaction, ANOVA, F 4,55 < 1⋅0, P > 0⋅05; for both variables).
No fish species survived exposure at pH 3 or 3⋅5, but after 14 days all juvenile and
adult fishes had 100% survival at pH ≥4⋅5 (Fig. 1). Adult S. trutta, G. huttoni and G.
brevipinnis experienced some or complete mortality at pH 4, whereas all adult G. maculatus and A. dieffenbachii survived at this pH. The LC50 estimates ranged from 3⋅74
to 4⋅24 for these juvenile and adult fishes (Fig. 1). Estimates of LT50 varied markedly
© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 85, 1746–1752
Mortality (%) after 14 days
Mortality (%) after 14 days
Mortality (%) after 14 days
Fig. 1. Effect of water of different pH on the mortality of (a) Salmo trutta [lethal concentration (LC50 ) = 3⋅99],
(b) Gobiomorphus huttoni (LC50 = 4⋅24), (c) Galaxias brevipinnis (LC50 = 3⋅97), (d) Anguilla dieffenbachii
(LC50 = 3⋅74), (e) Galaxias maculatus (LC50 = 3⋅74) after 14 days. (f) G. maculatus larvae (LC50 = 4⋅72)
after 4 days. Individual data points represent mean per cent mortality (n = 5) but for G. maculatus larvae,
each replicate contained five larvae so error bars were calculated for this 96 h experiment.
for each fish species (Table I). Whilst there was 100% mortality at pH 3⋅5, G. huttoni
and S. trutta had an LT50 of c. 2⋅6 and 3⋅1 h (respectively), whereas G. maculatus and
G. brevipinnis had LT50 estimates of 11⋅9 and 13⋅0 h, respectively (Table I). In contrast
to adult G. maculatus, all G. maculatus larvae died at pH 4 and >75% of G. maculatus
larvae died at pH 4⋅5 resulting in a 96 h LC50 estimate of 4⋅72 (Fig. 1). Although high
mortality of G. maculatus larvae occurred at pH 4⋅5, the LT50 was 72 h and it took
2 days until the first larvae died indicating that they can withstand that pH for short
periods (Table I).
© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 85, 1746–1752
P. G . J E L LY M A N A N D J . S . H A R D I N G
Table I. Time estimates (h) for 50% fish mortality (LT50 ) in waters of varying pH. Data for pH
6⋅5 are not shown as they are identical to data for pH 5⋅5 for all species
Salmo trutta
Gobiomorphus huttoni
Galaxias brevipinnis
Anguilla dieffenbachii
Galaxias maculatus
Galaxias maculatus (larvae)
pH 3
pH 3⋅5
pH 4
pH 4⋅5
pH 5⋅5
*Probable significant over-estimation as larvae were observed after 1 and then after 12 h.
At the end of the 14 day experiments, the percentage change in mass observed for
G. maculatus, G. huttoni and A. dieffenbachii were not significantly different across
the various pH treatments (ANOVA, P > 0⋅05; for these species) (Table II). Although
G. maculatus and G. huttoni gained mass across pH 4–6⋅5 treatments, A. dieffenbachii
lost mass across all these pH treatments. A loss of mass in A. dieffenbachii may be
partially explained by a lack of feeding for the 4 days prior to their final weighing.
Significant percentage changes in mass across the pH 4–6⋅5 treatments were observed
for S. trutta (F 3,16 = 27⋅0, P < 0⋅001) and G. brevipinnis (F 3,16 = 8⋅0, P < 0⋅01). For S.
trutta, the loss of mass in the pH 4 treatment was significantly different from fish mass
gains in the pH 4⋅5–6⋅5 treatments (Table II). The mean percentage change in mass
for G. brevipinnis in the pH 4 treatment was 0⋅3% which was significantly less than
the mass gains observed in the pH 4⋅5 and 5⋅5 treatments (Table II).
Extremes of pH can be a major factor determining abundance and composition of
fish communities in lakes and streams (Hesthagen et al., 1999; Greig et al., 2010).
The effect of low pH on aquatic biota can differ markedly depending on whether the
acidity is natural due to organic acids (pH usually >4) or the result of human impacts
(pH <3⋅5) (Petrin et al., 2008; Greig et al., 2010). This study revealed that no species
would survive in the highly acidic waters (pH 3–3⋅5) of streams affected by mining
activities in New Zealand. Instead, the five species tolerated pH more characteristic of
naturally acidic water, with all juvenile and adult fishes surviving a two-week exposure
Table II. Mean ± s.e. change in percentage fish mass (based on initial wet masses) for the
waters of varying pH. Due to rapid mortality in the pH 3 and 3⋅5 treatments (and pH 4 for Gobiomorphus huttoni), no changes in mass were recorded. Superscript lowercase letters denote significant mass differences between pH treatments as indicated by analysis of variance (ANOVA)
followed by post hoc Tukey tests. Sample size for pH 4 varied based on fish mortality (see
Fig. 1), n = 5 for pH ≥4⋅5
Salmo trutta
Gobiomorphus huttoni
Galaxias brevipinnis
Anguilla dieffenbachii
Galaxias maculatus
pH 4
pH 4⋅5
pH 5⋅5
pH 6⋅5
−18⋅0 ± 2⋅6a
6⋅7 ± 1⋅7b
15⋅3 ± 10⋅5
24⋅3 ± 2⋅0b
−3⋅8 ± 0⋅6
15⋅5 ± 4⋅1
10⋅6 ± 3⋅1b
12⋅3 ± 3⋅4
18⋅2 ± 2⋅7b
−4⋅7 ± 0⋅5
17⋅5 ± 4⋅2
12⋅4 ± 1⋅8b
19⋅8 ± 6⋅5
12⋅7 ± 5⋅2ab
−4⋅3 ± 1⋅2
15⋅7 ± 3⋅1
0⋅3 ± 3⋅8a
−7⋅2 ± 1⋅2
9⋅9 ± 5⋅9
© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 85, 1746–1752
to pH 4⋅5. Whilst some species were able to survive, or even gain mass, in water of pH 4
after 2 weeks, medium to long-term survival in water of this acidity would appear to be
unlikely for most species given the range of pH waters they are known to occupy (Greig
et al., 2010). Had the experiment been conducted using a standard 96 h ecotoxicology
approach, the effect of pH 4 water would have appeared markedly different, since two
species had 100% survival after 96 h but started to die after a week or so.
This study suggests that a pH of at least 4⋅5 is required for optimal fish survival.
This corroborates the field survey findings of Greig et al. (2010), which suggested
that pH should be raised above 4⋅5 to remediate fish communities in naturally acidic,
mine-affected streams (in combination with reducing concentrations of dissolved metals). Some species may tolerate lower-pH waters if they are slowly acclimated (Trojnar,
1977) but acclimation does not always improve fish survival in low-pH waters (Audet
& Wood, 1988). The survival of <25% of larval G. maculatus at pH 4⋅5 compared with
100% survival of adults at pH 4 is consistent with previous studies that have suggested
that pH sensitivity is highest in early life stages (Fromm, 1980). Mass fish mortalities
and extinctions have occurred in countries that have experienced gradual acidification of lakes and rivers during the 20th century (e.g. Sweden, Norway, Canada and the
U.S.A.), and the common mechanism has been recruitment failure due to high mortality
in early life cycle stages (i.e. eggs, alevins and smolt) (Gjedrem & Rosseland, 2012).
From a fisheries perspective, determining an appropriate pH for stream remediation
will depend on whether a waterway is suitable for fish spawning and larval rearing or
whether the stream habitat is suitable only for juvenile and adult fishes. Since early life
stages are generally more sensitive to acidification than larger fishes (Rosseland et al.,
2001), the pH needed to restore fish communities may vary depending on whether larvae migrate out to sea or if they develop entirely in fresh water. Prior to any remediation
of stream pH, the acidity inherent to the system should be considered because it may
be detrimental to try and restore an affected stream to circum-neutral pH when the fish
communities are adapted to live in naturally acidic waters.
M. Marinov is thanked for assistance with fish collection and H. Stoddart provided laboratory
support. Experiments were performed under the University of Canterbury animal ethics permit
number 2009/2R. This research was funded by the Foundation for Research Science & Technology (contract CRLX0401) with funding to P.G.J. from NIWA Core Funding (project SA125085)
assisting with manuscript preparation.
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© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 85, 1746–1752

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