BIOMARKERS IN EEL TO EVALUATE EFFECTS OF DIQUAT IN A CHRISTCHURCH RIVER
L A TremblayLandcare Research, CENTOX, PO Box 69, Lincoln 8152, New Zealand. E-mail: [email protected]
Manuscript received, 30/5/03; resubmitted, 4/12/03; accepted, 8/12/03.
ABSTRACTChristchurch City Council uses herbicides for the control of exotic plant species in and around the waterways throughout ChristchurchCity. This study evaluated the acute effects on fish associated with the use of the herbicide diquat to control the water weed Egeriadensa. Shortfin eels (Anguilla australis) were caged at locations in the Avon River that received diquat treatments. Following a three-weekexposure, a series of physiological responses (biomarkers) was measured and compared to responses in eels caged upstream from thetreatment areas. The biomarkers measured included hepatic mixed-function oxygenase, plasma lysozyme, and vitellogenin. Resultsshowed the diquat treatments caused no observable signs of acute toxicity in the shortfin eel and did not significantly alter biomarkerresponses. Key words: biomarkers, herbicide, shortfin eel, vitellogenin, cytochrome P450.
Previous studies have reported that diquat has moderate to
There is concern over the effects on non-target species in New
practically non-toxic effects on fish and aquatic invertebrates (see
Zealand where herbicides are being used in large quantities to
Campbell et al. 2000). Due to the low toxicity of diquat to fish and
control the growth of exotic plant species in and around waterways.
the lack of knowledge about any potential mechanism of toxicity, a
Diquat (1,1’-ethylene-2, 2’-dipyridine, CAS registry number 2764-
variety of specific and non-specific biomarkers were measured.
72-9), molecular formula C H N , has a molecular weight of
186.26 (Howard 1991; Worthing and Hance 1991). It is a quick-
acting herbicide and plant desiccant with some translocation
properties and little residual activity. It is used to control floating
All manipulations were approved by the Landcare Research Animal
and submerged weeds in water. Diquat has been shown to be
Ethics Committee. An in situ protocol using shortfin eel has been
moderately toxic via ingestion in mammals (Stevens and Sumner
developed and used to evaluate the effects of diquat. The use of the
1991) and to have very little to no effects on fish and invertebrates
eel in this study was warranted by concerns over the potential
(Campbell et al. 2000). In Christchurch, diquat treatments are used
adverse effects of diquat on native fish. The eels were obtained
in the Avon River, a spring-fed stream, to control the growth of
from Lake Ellesmere, located south of Christchurch and where
Egeria densa, a water weed that rapidly forms dense strands
eels are harvested commercially, and acclimatised in the Lincoln
crowding out other plants and fish. The treated area of the Avon
University aquatic laboratory facility for 21 days. The fish were
River is particularly used by a rowing club, where the high density
held in groups of 10 in 350-L black polyethylene tanks with constant
of Egeria interferes with the club’s normal operations. The treated
aerated freshwater flow at 18°C. They were fed fresh veal liver
area is lightly under tidal influence.
every two days. Fish weights and lengths were recorded before the
The in situ approach, whereby fish are confined to treated areas by
experiment began. During the exposure, the fish were not fed as
placing them in cages, was taken to ensure exposure to diquat under
they could obtain sufficient food from the environment.
normal application (treatment) conditions. The fish can be exposed
The Avon River was treated with diquat (Reglone‚ Zeneca Ltd,
to the waterborne diquat and also through limited contact with
UK) on 15 January 2001. The area treated was a 2.0-km length at
sediment. Following a series of public consultations by the
Kerr’s Reach with an average width of 50 m. The formulation was
Christchurch City Council, concerned Maori (the New Zealand
applied at an application rate of 30 L/hectare as recommended by
native people) and other interest groups have identified several
the manufacturer. The formulation contained 200 g/L of diquat as
potential adverse effects on the environment resulting from the
the bromide salt in the form of a soluble concentrate. Council staff
herbicide use. To address these concerns, the council initiated a
were responsible for the treatments at two locations, which were
monitoring programme including measurements of diquat levels
carried out from boats and lasted a few hours. Stainless steel cages
in water and sediments (R. Vaughan, pers. comm.), effects on
with a nylon mesh insert containing 10 fish were deployed at four
invertebrates (McMurtrie 2001), and toxicity to fish. The current
locations on the Avon River on 12 January 2001, three days prior
research evaluated the potential of diquat to cause adverse effects
to diquat treatment, for a 3-week period. Sites for placement of the
to the shortfin eel (Anguilla australis), a New Zealand native fish
cages were chosen to reflect the condition of the river. The Avon
species. Eels are resident in the Avon River and therefore likely to
River is an urban stream that receives contamination from a series
of point and non-point sources. Therefore, it was important toinclude a site that was located near the source of the Avon River, todifferentiate effects related to diquat from those of contaminants
arising from other sources. Two cages were placed downstreamfrom the diquat treatment sites (one was lost) and two referencecages were placed upstream from the treatment sites (1 km upstreamfrom treatment area) and close to the source of the Avon River(Table 1). Cages were monitored daily for fish mortality and toclear debris from around the cage. Dissolved oxygen levels weremeasured twice a week using a 6920 multi-parameter data sonde(underwater probe) (YSI, USA).
Fish samplingAll chemicals used were from Sigma. Fish were collected from thecages and transported to the Landcare Research laboratory in icedwater to minimise handling and transport stress. At the laboratory,
fish were anaesthetised by immersion in a solution of ice and
The plasma egg yolk protein precursor vitellogenin was measured
benzocaine (250 mg L-1, ethyl p-aminobenzoate) as modified from
by a specific enzyme-linked immunosorbent assay (ELISA)
Arukwe et al. (1997). The fish weights and lengths were recorded
following the methodology described in Ataria et al. (in print).
and 1-mL blood samples taken from the caudal vein, using aheparinised syringe. The plasma was collected by centrifugation at
4°C for 10 min at 1000 x g, and stored at -80∞C until analysed for
Group means were compared using a one-way analysis of variance
vitellogenin content and lysozyme activity. Spleen and liver weights
with P-values for the hypothesis of no difference calculated using
were recorded and livers stored at -80∞C until analysed for hepatic
an F-test. Although some measurements had rather skewed
distributions, transformations had little effect on P-values. The leastsignificant difference (LSD) needed for significance with P = 0.05
was also calculated from the analysis of variance to illustrate whether
The hepatosomatic index (HSI) was calculated by the following
the experiment was precise enough to detect biologically important
equation: (liver weight (g)/body weight (g)) x 100. The condition
factor (K) of each fish was calculated by the following equation:
Because there was no replication of sites, it must be assumed that
(weight (g) x 100)/(length (cm))3. This index provides an overall
the cause of differences between the measurements was exposure
to diquat or other contaminants present in the river, and not otherconditions related to site.
Liver mixed-function oxygenasesThe induction of hepatic mixed-function oxygenases (MFO) was
evaluated by measuring the enzyme cytochrome P450 (CYP1A)
Chemical analyses revealed that the river water contained a peak
activity by the ethoxyresorufin-O-deethylase (EROD) assay.
concentration of 3.51 mg L-1 of diquat dibromide that rapidly
Activity was measured in the S-9 fraction using a fluorometric
decreased after 1 h to undetectable levels (R. Vaughan, Christchurch
technique described by Burke and Mayer (1974), as adapted for
City Council, pers. comm.). Three of the four cages were
96-well plate format and simultaneous protein measurement by
successfully deployed on the Avon River. Unfortunately, one of the
Kennedy and Jones (1994). Protein concentrations were evaluated
cages located downstream from the diquat treatment area was lost.
by the fluorescamine method of Bridges et al. (1986) using bovine
At the end of the 3-week exposure period no fish had died but one
fish escaped from Reference 1 (site 1). Observations of fishmorphology showed no sign of common external stress such as fin
rot or lesions. However, all fish, independently of site, showed slight
Plasma lysozyme activity was determined by a method adapted
loss of body weight after three weeks, resulting in a lower condition
from Lie et al. (1986). Briefly, heat-killed Micrococcus lysodeikticus
factor (K) (not statistically significant among sites; before P = 0.2,
was dispersed into 1% agar made up in Phosphate Buffer Salt (PBS)
after P = 0.3, Figure 1). K is an indication of the overall health of
buffer (0.13 M NaCl, 1 mM KH PO , 8 mM Na HPO , 2 mM KCl,
the fish as calculated by a ratio between length and weight. Drop in
0.1 mM CaCl , 1 mM MgCl , pH = 6.3) at a concentration of 1 mg
body weight is a normal response in fish being caged, as they had
M. lysodeikticus/mL. Aliquots of 25 mL were poured into 90-mm
to adapt to a new feeding regime different from that under laboratory
Petri dishes and allowed to set overnight at 4°C. Small regular holes
conditions. Measurement of dissolved oxygen indicated that the
were made in the agar using a plastic straw. The wells were filled
water was saturated at near maximum at all sites throughout the
with 20-µL aliquots of diluted eel plasma or hen egg white (HEW)
lysozyme standards (0.001 to 10 mg mL-1). Plates were incubatedfor 24 h at 37°C. Lysozyme concentration was quantified by
Following the 3-week exposure, biomarkers were measured in the
measuring the diameter of lysis around the wells and comparing it
fish to evaluate the biological effects of contaminants. Biomarkers
can be described as functional measures of exposure to stressorsusually expressed at the suborganism level of biologicalorganisation, and the information generated can then be used inecological risk assessment of diquat (Adams et al. 2001). Somebiomarkers are specific while others are less specific in their power
to establish cause-and-effect relationships between an exposure tocontaminants and biological responses.
The liver, the main detoxifying organ involved in eliminatingcontaminants from the body, will often increase in mass whenchallenged with heavy contamination. There was no difference inliver weights as indicated by HSI (data not shown, P = 0.6). Thelevel of the hepatic detoxifying enzyme biomarker cytochrome P4501A (CYP1A) activity was measured by the EROD assay (Figure2). This biomarker has previously been used successfully in eelcaging studies to measure the effects of pulp mill effluents (Joneset al. 1995). The induction of P450 enzymes is modulated by avariety of chemicals including dioxins, PAHs, and PCBs. The fish
Figure 1. Average condition factors (K) of the fish at the beginning of the
caged downstream (Site 3) and at one site upstream (Site 2) from
exposure and on sampling day. Sites are described in Table 1. Each bar
the treatment area had levels of CYP1A activity that were
represents the mean ± standard errors (S.E.M.). LSD = 0.017.
significantly higher than site 1 (P = 0.04). Site 2 was not exposedto diquat, this strongly suggests that the increase in P450 activitywas caused by contaminants from other sources. The Avon Riverdrains an urban catchment, therefore there are many sources thatmay be contributing to chemical contamination and other types ofstressors.
Lysozyme is an enzyme with bacteriolytic activity that acts as anon-specific component of innate immunity. It is present in serumand within cells with immune function. Lower plasma lysozymeactivities were present in fish from Sites 2 and 3 compared to fishfrom Site 1, and the difference in activity between Site 1 and 2exceeded the calculated LSD for this parameter (Figure 3). However,the three groups were not significantly different (P = 0.1.). A similarprofile was also seen with the spleen weights (data not shown)
Figure 2. Hepatic ethoxyresorufin-O-deethylase (EROD) activity in fishfollowing a 3-week exposure in the Avon River. Sites are described in Table
although these trends were also not significantly different (P = 0.09).
1. Each bar represents the mean ± standard errors (S.E.M.).
Further investigations should look at the effects of diquat and other
* indicates a significant difference (P = 0.05). LSD = 5.63
contaminants present in the Avon River on other components ofthe fish immune system.
Vitellogenin is a specific biomarker of exposure to contaminantswith oestrogenic activities, a family of endocrine-disruptingchemicals (Jones et al. 2000). Plasma vitellogenin was measuredby a specific eel ELISA, but no trace was detected in the exposedfish, suggesting that if compounds with oestrogenic activity arepresent in the Avon River, they would be at concentrations unlikelyto modulate vitellogenin production in the eel (data not shown).
The current study demonstrates the low acute toxicity of diquat tothe shortfin eel. Similarly, a study conducted concurrently on aquaticinvertebrates also found no adverse effects arising from diquattreatment (McMurtrie 2001). These results support conclusions
Figure 3. Average plasma lysozyme concentrations in fish following the 3-
from overseas studies that found diquat was successful in killing
week exposure in the Avon River. Sites are described in Table 1. Each bar
weeds without causing any fish mortality (Olaleye et al. 1993) and
represents the mean ± standard errors (S.E.M.). LSD = 0.340.
that diquat posed minimal risk to aquatic systems (Bartell et al. 2000; Campbell et al. 2000; Ritter et al. 2000).
Although diquat exposures are relatively transient for fish, potential
diquat accumulation in the sediments should be monitored, as it
Diquat had no significant effects on the physiological endpoints
binds tightly to upper layers of soil where it may remain for extended
measured in eels caged downstream from a treated area on the Avon
periods of time (Howard 1991). Investigations of the sensitivity of
River. The higher activities of P450 detoxifying enzymes appear to
other fish species both in the field and under controlled laboratory
be at least in part due to other stressors present in the Avon River.
conditions should be undertaken, as eel might not be representative
Overall, diquat had no statistically significant toxicity to the short-
finned eel under the application conditions used to control Egeriain the Avon River.
Kennedy SW and Jones SP. 1994. Simultaneous measurement of
This work was funded by the Christchurch City Council and the
cytochrome P4501A catalytic activity and total protein
Foundation for Research, Science and Technology, New Zealand.
concentration with a fluorescence plate reader. Anal. Biochem.222,
The author thanks Greg Arnold, Christopher Bailey, Leslie Brown,
Vanessa Hepplethwaite, and Anjoeka Pronk for their technical
Lie O, Syed M and Solbu H. 1986. Improved agar plate assays of
assistance; Rachel Vaughan, City Solution, for her assistance with
bovine lysozyme and haemolytic complement activity. Acta Vet.
the work planning and providing diquat data; Clem Smith for
providing the eels; Ngai Tahu for approving the eel work; Mikevan den Heuvel, Forest Research, for his help with the EROD assay;
McMurtrie S. 2001. Assessment of the effects of diquat on
James Ataria, Jo Cavanagh, and Kathryn O’Halloran for comments
invertebrates in the Avon River. A report prepared for the Water
on early drafts; Christine Bezar for editorial comments; and Wendy
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