MARINE ECOLOGY PROGRESS SERIES

Mar Ecol Prog Ser Vol. 330: 127–137, 2007 Published January 25

INTRODUCTION

Reefs on a global scale are under threat from many

sources; one of the most insidious is land-based pollutants from current agricultural practices (Rawlins et al.

1998). Large areas of reef, in otherwise relatively unaffected regions, are threatened by the combined assault

of increasing turbidity and chemical contaminants

(Fabricius 2005). For example, in Australia, >80% of

the Great Barrier Reef (GBR) catchment area supports

some form of agriculture (Gilbert & Brodie 2001). The

scale of this threat has been recognised by the Australian and State Governments which recently initiated

a 10 yr, $40 million (USD) programme to improve land

management practices and to monitor the effectiveness of management on improving water quality on

adjacent inshore reefs.

Numerous herbicides, insecticides and fungicides

are used in catchments that flow into the GBR lagoon,

and up to 74 t of the insecticide chlorpyrifos has

been applied annually within catchments of the GBR

(Hamilton & Haydon 1996). Current pesticide concentrations in nearshore tropical environments are largely

unknown; however, given the considerable quantities

in use, it is not surprising that large accumulations

have occasionally been detected. Haynes et al. (2000)

recorded concentrations of a number of herbicides and

organochlorine insecticides at up to 10 μg kg–1 in sub-

*Corresponding author. Email: a.negri@aims.gov.au © Inter-Research 2007 · www.int-res.com

Insecticides and a fungicide affect multiple 

coral life stages

Kathryn L. Markey1, 2, Andrew H. Baird3, Craig Humphrey2, Andrew P. Negri 2,*

1School of Marine Biology and Aquaculture, and 3

ARC Centre of Excellence for Reef Studies, James Cook University, 

Townsville, Queensland 4811, Australia

2

Australian Institute of Marine Science, PMB 3, Townsville, Queensland 4810, Australia

ABSTRACT: Coral reefs are under threat from land-based agricultural pollutants on a global scale.

The vulnerability of early life stages of corals is of particular concern. Here, we compared the sensitivity of gametes, larvae and adult branches of the broadcast-spawning coral Acropora millepora

(Ehrenberg) to a number of common pollutants, including 4 classes of insecticides—2 organophosphates (chlorpyrifos, profenofos), an organochlorine (endosulfan), a carbamate (carbaryl) and a

pyrethroid (permethrin)—and a fungicide (2-methoxyethylmercuric chloride, MEMC). Fertilisation

of gametes was not affected by any of the insecticides at concentrations up to 30 μg l–1. In contrast,

settlement and metamorphosis were reduced by between 50 and 100% following 18 h exposure to

very low concentrations (0.3 to 1.0 μg l–1) of each insecticide class. The insecticides had few visible

effects on adult branches following 96 h exposure to a concentration of 10 μg l–1, with the exception

of profenofos, which caused polyp retraction, bleaching (i.e. algal symbiont densities were reduced)

and a slight reduction in photosynthetic efficiency of the algal symbionts. The fungicide MEMC

affected all life-history stages: both fertilisation and metamorphosis were inhibited at 1.0 μg l–1, and

polyps became withdrawn and photosynthetic efficiency was slightly reduced at 1.0 μg l–1. At 10 μg

l

–1 MEMC, branches bleached and some host tissue died. This high susceptibility of coral larvae to

pesticides at concentrations around their detection limit highlights the critical need to assess toxicity

against all life-history stages of keystone organisms: to focus on mature individuals may underestimate species sensitivity.

KEY WORDS: Coral · Reef · Pollution · Insecticide · Fungicide · Fertilisation · Metamorphosis ·

Pesticide · Settlement

Resale or republication not permitted without written consent of the publisher

OPEN

 ACCESS
Mar Ecol Prog Ser 330: 127–137, 2007

tidal sediments along the GBR. The insecticide endosulfan has been detected at concentrations as high as

840 μg kg–1 in sediment samples from Queensland

sugar cane and cotton farms (Müller et al. 2000), and

wet season runoff from such highly contaminated

farms poses the risk of insecticide transport to

nearshore waters of the GBR. In addition to runoff,

some reefs, such as those in the Florida Keys, are at

risk from exposure to organophosphates, such as

dibrom, via accidental mosquitocide overspray (Pierce

1998). Between 500 and 1000 kg of the fungicide 2-

methoxyethylmercuric chloride was applied each year

for 40 yr in a single catchment that flows into the GBR

(Johnson & Ebert 2000). Mercury concentrations in

sediment cores taken from the GBR identified concentrations of up to 100 μg kg–1, an order of magnitude

higher than background concentrations (Walker &

Brunskill 1997). These concentrations were attributed

to the contemporary application of mercury-based

fungicides on sugar cane farms (Walker & Brunskill

1997). The abundance of pollutants in the environment

is clearly patchy, and, while more comprehensive

sampling is needed to determine the full scale of the

problem, these figures demonstrate a clear threat to

key organisms such as corals.

Mercury (Rawlins et al. 1998) and banned organochlorine insecticides, such as chlordane, lindane and

DDT (Olafson 1978), can accumulate in coral tissue.

Preliminary work indicates that Pocillopora damicornis

larvae are moderately sensitive to the contemporary

insecticides chlorpyrifos and carbaryl, which caused

larval mortality only at high concentrations (100 mg l–1

and 1000 μg l–1, respectively) (Acevedo 1991). In contrast, adult P. damicornis branches were more sensitive

to chlorpyrifos in 96 h exposures, with 50% of individuals dying at 6 μg l–1 (LC50 = 6 μg l–1) (Te 1998). Some insecticides can also induce changes in gene expression

of corals at very low concentrations. For example, the

insecticide dibrom caused the up-regulation of 2 genes

of unknown function at concentrations of 0.5 μg l–1 in

adult branches of Acropora cervicornis (Morgan & Snell

2002). In contrast, neither permethrin nor lindane affected expression at higher concentrations. While the

effects of organomercurial insecticides on corals are unknown, inorganic mercury can cause reductions in both

polyp and symbiont biomass (Bastidas & García 2004).

Toxicants can affect corals in a variety of ways. For

example, herbicides have been shown to reduce the

photosynthesis of coral symbionts (the dinoflagellates

Symbiodinium spp.) at low concentrations (Jones &

Kerswell 2003, Negri et al. 2005). Higher concentrations of herbicide can damage the partnership between host and symbiont, resulting in the expulsion (or

loss) of symbionts from the coral host (bleaching).

However, the specific effects of pesticide groups such

as insecticides and fungicides on coral remain virtually

unknown. Early events in life, such as fertilisation and

larval metamorphosis, may be more sensitive to pollutants than are adults (Mercurio et al. 2004). Most

scleractinian corals reproduce by broadcast spawning,

with many species releasing gametes for external

fertilisation in often highly synchronised seasonal

mass spawning events (Harrison & Wallace 1990). The

resulting planula larvae have an obligate planktonic

stage of between 2 and 4 d, after which they are competent to settle on the reef (Baird 2004). Larvae of

many coral species require a biochemical inducer

from crustose coralline algae to trigger metamorphosis

(Heyward & Negri 1999, Baird & Morse 2004, Harrington et al. 2004). All these developmental stages and

events are critical points in the life history of corals,

and the failure of any one event will reduce the capacity of populations to be replenished. In the present

study, gametes, larvae and adult branches of the

broadcast-spawning coral Acropora millepora were

exposed to 4 common classes of insecticides and a

fungicide (summarised in Table 1) in a series of laboratory experiments to determine the threshold concentrations at which they became toxic to key life-history

events and stages.

MATERIALS AND METHODS

Toxicants. Toxicants were chosen to represent 4 of

the main insecticide groups: organophosphates (chlorpyrifos, profenofos), organochlorines (endosulfan), carbamates (carbaryl) and pyrethroids (permethrin) plus a

fungicide (2-methoxyethylmercuric chloride, MEMC).

Table 1 summarises the usages and modes of action for

each of the toxicants. The insecticides and MEMC

were sourced from Sigma-Aldrich, and chlorpyrifos

and chlorpyrifos oxon were obtained from Dow AgroSciences. Stock solutions of chlorpyrifos, profenofos,

endosulfan and permethrin were prepared in GF/C

(Whatman) filtered seawater (FSW) using 0.1% acetone (Merck) to improve dissolution. A stock solution

representing the acetone control was prepared in the

same way. Treatment concentrations were prepared

from dilutions of this stock solution.

Coral collection, broadcast spawning and larval

cultivation. Gravid colonies of the broadcast-spawning

coral Acropora millepora (Ehrenberg) were collected

from 3 to 5 m depths at High Island, central GBR

(17° 09.60’ S, 146° 00.34’ E) and maintained in flowthrough outdoor aquaria (27°C) at the Australian Institute of Marine Science until spawning on 2 November

2004, at 21:30 h. The symbiont-free gametes were

collected and cultured in indoor flow-through aquaria

(27°C) using methods described in Negri & Hey128
Markey et al.: Pesticides affect multiple coral life stages

ward (2000). In the adult exposure treatments, small

branches (4 to 5 cm) were cut from mature colonies of

A. millepora and attached upright to 2 cm2 glass tiles

using Loctite instant adhesive (Henkel). The branch

clones were allowed to heal and acclimate in an outdoor flow-through tank for 2 wk prior to treatment.

Fertilisation assays. Gametes from 3 colonies were

selected for fertilisation experiments, 1 colony providing the eggs and 2 others the sperm. The egg–sperm

bundles from each colony were gently agitated to separate the eggs and sperm into 2 layers. The buoyant

eggs were then isolated from the sperm by gentle suction and washed several times in sperm-free, 0.2 μm

filtered seawater. Sperm concentrations were measured from 2 further colonies using a haemocytometer,

and a concentrated sperm mixture was prepared from

equal sperm concentrations of each colony. Eggs and

sperm were then transferred into 20 ml, acetonewashed glass scintillation vials (100 eggs and 106 sperm

per 10 ml FSW) (Negri & Heyward 2000). Four replicate vials were used for each toxicant treatment (0.3 to

30 μg l–1), and control treatments consisted of a no toxicant treatment and an acetone treatment (equivalent

volume of acetone stock as added in the maximum

toxicant treatment). Vials were loosely lidded and

maintained under low light at 27°C. The eggs and

embryos were fixed with 5% Bouins solution after 3 h,

when early cell cleavage had reached a maximum

endpoint (Heyward 1988). Fertilisation levels were

quantified using a dissecting microscope.

Metamorphosis assays. Larvae 6 d old were preexposed to toxicants or controls for 18 h in 20 ml,

acetone-washed scintillation vials. Each vial contained

approximately 15 larvae and 10 ml of 0.2 μl filtered

seawater along with toxicants (30 to 300 μg l–1) and/or

seawater and acetone controls. After 18 h the larvae

were transferred to sterile 6 well polystyrene cell

culture plates (12 ml, Nunc) at 27°C and 120 μmol

quanta m–2 s–1 under a 12:12 h light:dark cycle. Stock

methanolic extract prepared from the crustose coralline

alga species Neogoniolithon fosliei and Hydrolithon

onkodes (15 μl) was added to each well to induce metamorphosis, and percent metamorphosis was assessed

after 18 h (Heyward & Negri 1999). Metamorphosis was

defined as the initiation of deposition of the basal plate,

which is clearly visible through the juvenile coral tissue

within 12 h of settlement in the Acropora (Babcock et

al. 2003). This assay procedure was repeated using 7 d

old larvae, with toxicant concentrations ranging

from 1.0 to 30 μg l–1, and again with 8 d old larvae,

at concentrations ranging from 0.1 to 1.0 μg l–1.

Adult exposures. Adult exposure experiments were

carried out in replicate 12 l glass flow-through chambers with dual peristaltic pumps (16 channel Masterflex L/S) delivering both 1 μm FSW and toxicant stock

to each tank. Water velocity of 5 cm s–1 was maintained

in the O-shaped tanks using submerged propellers.

Metal halide lamps provided illumination of 140 to

160 μmol quanta m–2 s–1 during 12:12 h light:dark

cycles over 96 h. Each tank contained 6 branchlets

129

Pesticide group Representative Primary usage Mode of action

Organochlorine Endosulfan Primarily: Cotton Antagonises the action of the neurotransmitter

(OC) Also: Fruit & vegetables, GABA, which induces the uptake of chloride

grains ions by neurons resulting in uncontrolled excitation

Organophosphate Chlorpyrifos Primarily: Sugarcane Inhibits acetylcholinesterase (AChE) in nerve

(OP) (Chlorpyrifos Also: Cotton, fruit & vegetables, synapses, leading to build-up of acetylcholine

oxon) grains, termites, domestic pests, (ACh) at nerve junctions, producing constant

mosquitos, veterinary nervous stimulation. While profenofos is able

Profenofos Primarily: Cotton to inhibit AChE directly, chlorpyrifos needs to 

undergo metabolic conversion to the active 

form, chlorpyrifos oxon

Carbamate Carbaryl Primarily: Sugarcane, cotton Reversible acetylcholinesterase inhibition, again 

Also: Fruit & vegetables, leading to build-up of ACh

grains, termites, domestic pests, 

veterinary

Pyrethroids Permethrin Primarily: Cotton Inactivation of the nerve junction by prolonged 

Also: Mosquitos, fruit & vege- opening of the sodium channels in synapses

tables, grains, domestic pests

Fungicide MEMC Primarily: Sugarcane seed Denaturation of membranous and intra-cellular 

proteins and inactivation of enzymes

Table 1. Modes of action and primary usages for the pesticides examined in this study (van Emden & Service 2004) (MEMC: 

2-methoxyethylmercuric chloride)
Mar Ecol Prog Ser 330: 127–137, 2007

(2 branchlets from each colony), and duplicate tanks

were used for each treatment. Branches were monitored at 96 h to check on tentacle condition (feeding or

retracted) and ability to respond to disturbance (gentle

prodding with stainless steel forceps). Visible bleaching (paling), tissue sloughing and partial or whole

branchlet mortality were also recorded at 96 h.

The photosynthetic efficiency of symbiotic dinoflagellates was examined using a pulse-amplitude modulation (PAM) fluorometer (Jones et al. 2003). Maximum

effective quantum yield of light-adapted symbionts

([Fm’ – F ]Fm’ = ΔF/Fm’) was measured using a MINIPAM (Walz). Eight measurements were taken per

branch, using a 2 mm fibre optic sensor held perpendicular to the coral tissue. ΔF/Fm’ measurements were

taken 6 h into each light cycle under constant illumination of 140 μmol quanta m–2 s–1. Dinoflagellate densities were measured for each branchlet as per Jones

(1997) and expressed on the basis of surface area determined using the wax method of Stimson & Kinzie

(1991).

Cholinesterase assay. Organophosphates such as

chlorpyrifos and profenofos inhibit cholinesterase

(ChE) activity (see Table 1), so attempts were made to

measure ChE activity in corals and apply this to

organophosphate-treated branches. Branches from 3

Acropora millepora colonies were used in a preliminary study to test for ChE activity. Coral tissue was

obtained from branches using 3 methods (crushing,

tissue stripping into phosphate buffer and tissue stripping into FSW) to determine which method would

yield higher ChE activity. One set of branches from

each colony was ground to a fine powder in liquid

nitrogen and extracted in 4 ml of phosphate buffer

(pH 7.0) containing 1% Triton X. The samples were

then centrifuged at 4000 × g at 4°C for 30 min, and the

supernatant was stored at –80°C. An airbrush was

used to strip the coral tissue from the remaining

branches into either 4 ml of ice-cold FSW or 4 ml of

phosphate buffer. The samples stripped into FSW were

homogenised and then centrifuged at 1600 × g for

5 min to remove the symbionts. Then, 0.1% Triton X

solution was added to both FSW and phosphate buffer

samples, which were subsequently homogenised and

sonicated before being centrifuged at 4000 × g at 4°C

for 30 min; the supernatant was stored at –80°C.

ChE activity was determined spectrophotometrically

by the method of Ellman et al. (1961) as modified for

microplate reading by Bocquene & Galgani (1998). To

each well of the microplate, 300 μl of 0.02 M sodium

phosphate buffer (pH 7.0), 20 μl of dithiobisnitrobenzoic acid (DTNB 0.01 M) and 10 μl of sample were

added successively. After a 5 min incubation, 10 μl

of either acetylthiocholine (ACTC 0.1 M), propioylthiocholine (PrSCh 0.1 M), or butyrylthiocholine (BuSCh

0.1 M) was added to start the reaction. Enzyme kinetics were monitored on a microplate reader (Wallac

Spectra II) at 405 nm for 2 min. Each of the 3 tissue

preparation techniques was tested at 4 tissue concentrations (5, 10, 20 and 50 μl of the volume of sample,

the amount of sodium phosphate buffer was adjusted

accordingly) and with the 3 substrate types mentioned

above.

Data analysis. Percentage data were arcsine transformed to improve normality and homogeneity of variance; the mean percent fertilisation and metamorphosis,

dinoflagellate density, or ΔF/Fm’ was compared among

treatments using a series 1-way ANOVA. The Tukey

Honestly significant Difference test was used to determine which means were different. Statistical analyses

were performed using Statistica 6.0 (StatSoft). The

lowest observed effect concentrations (LOEC) were the

lowest contaminant concentrations that elicited significant differences (ANOVA, p < 0.05) from the control

treatment; 3- and 4-parameter sigmoidal regressions

were fitted to the concentration–percent response data

using Sigmaplot V7 (Jandel Scientific). The EC50s (concentrations which caused 50% inhibition) were calculated from the ensuing equations.

RESULTS

Fertilisation

High levels (±SE) of fertilisation were achieved in

controls with 97 ± 1% and 98 ± 1% fertilisation after

3 h in no toxicant and acetone control treatments,

respectively. None of the insecticide treatments significantly affected fertilisation at concentrations up to

30 μg l–1 (Fig. 1, Table 2). The fungicide MEMC, how130

Toxicant concentration (μg l–1)

1 10

Fertilization success (%)

0

20

40

60

80

100

MEMC

Chlorpyrifos

Fig. 1. Acropora millepora. Fertilisation success (%) for chlorpyrifos and 2-methoxyethylmercuric chloride (MEMC) treatments. Error bars (hidden by symbols) represent SE, n = 4.

The other insecticide results were identical to those for chlorpyrifos (see Table 2 for lowest observed effect concentration 

and estimated EC50 values)
Markey et al.: Pesticides affect multiple coral life stages

ever, reduced fertilisation to 90 ± 3% at 1.0 μg l–1 and

1.1 ± 0.8% at 3 μg l–1. The EC50 for MEMC was 1.68 ±

0.04 μg l–1. At 3.0 μg l–1 most eggs remained intact, but

all eggs had ruptured at 30 μg l–1.

Metamorphosis

Larval metamorphosis in uncontaminated seawater

was 77.8 ± 0.2% for 6 d old larvae and this increased to

97.8 ± 0.9% and 96.7 ± 0.9% for 7 and 8 d old larvae,

respectively. The acetone treatments did not affect

metamorphosis significantly (Table 2); 6 d old larvae

were exposed to 30, 100 and 300 μg l–1 of each toxicant.

All toxicants inhibited metamorphosis completely at

these concentrations (Fig. 2, Table 2). While most

larvae remained swimming at high concentrations of

insecticides, 30 μg l–1 of the fungicide MEMC caused

all larvae to rupture and die.

The 7 and 8 d old larvae were exposed to successively lower concentration ranges of each toxicant (1.0,

3.0, 10 and 30 μg l–1 for 7 d and 0.1, 0.3 and 1.0 μg l–1

for 8 d larvae). Larval settlement was extremely sensitive to the insecticides, and was reduced by between

60 and 100% following 18 h pre-exposures to very low

concentrations (0.3 to 1.0 μg l–1). The LOEC was 0.3 μg

l

–1 for chlorpyrifos oxon and profenofos, 1.0 μg l–1 for

permethrin, endosulfan, chlorpyrifos and MEMC, and

3.0 μg l–1 for carbaryl (Fig. 2, Table 2). The insecticides

did not affect the swimming behaviour of larvae at any

concentration, but larvae exposed to MEMC were

often motionless at 3 μg l–1 and above.

Adult branches

Adult branches of Acropora millepora were exposed

to each of the toxicants in 3 successive 96 h experiments. All branches in no toxicant and acetone controls

survived, and no polyp retraction, bleaching, or partial

mortality was observed. The tentacles in most of the

insecticide treatments were feeding; however, tentacles on branches exposed to 10 μg l–1 profenofos

and MEMC were permanently retracted after 96 h

131

Day LOEC EC50 ± SE SS df F p Effect

(μg l–1) (μg l–1)

Fertilisation

Acetone 0.0015 1 0.444 0.530 n.s.

Endosulfan >30 >30 0.0032 5 0.466 0.796 n.s.

Chlorpyrifos >30 >30 0.0275 5 1.502 0.238 n.s.

Chlorpyrifos oxon >30 >30 0.0107 5 0.701 0.630 n.s.

Profenofos >30 >30 0.0047 5 0.517 0.760 n.s.

Carbaryl >30 >30 0.0139 5 1.088 0.400 n.s.

Permethrin >30 >30 0.0368 5 1.405 0.270 n.s.

MEMC 1.0 1.68 ± 0.04 10.7721 5 416.179 0.000 con = 0.1 = 0.3 < 1 = 3 = 10 = 30

Metamorphosis

Acetone 7 0.0041 1 0.212 0.651 n.s.

8 0.0009 1 0.046 0.833 n.s.

Endosulfan 7 1.0 1 ± 3a 15.6557 4 36.101 0.000 con < 1 < 3 = 10 = 30

8 2.5242 3 37.523 0.000 con = 0.1 = 0.3 < 1

Chlorpyrifos 7 1.0 1 ± 2a 16.3572 4 95.803 0.000 con < 1 = 3 = 10 = 30

8 0.9299 3 8.404 0.000 con = 0.1 = 0.3 < 1

Chlorpyrifos oxon 7 0.3 0.39 ± 0.01 17.4122 4 384.252 0.000 con < 1 = 3 = 10 = 30

8 5.9087 3 64.847 0.000 con = 0.1 < 0.3 < 1

Profenofos 7 0.3 0.6 ± 2 15.7026 4 91.570 0.000 con < 1 = 3 = 10 = 30

8 2.1884 3 42.753 0.000 con = 0.1 < 0.3 = 1

Carbaryl 7 3.0 1 ± 2a 18.7020 4 321.258 0.000 con = 1 < 3 = 10 = 30

8 0.0784 3 1.049 0.387 n.s.

Permethrin 7 1.0 1 ± 1 15.5260 4 44.289 0.000 con < 1 = 3 = 10 = 30

8 3.2129 3 35.944 0.000 con = 0.1 = 0.3 < 1

MEMC 7 1.0 2.5 ± 0.3 11.2758 4 24.861 0.000 con < 1 = 3 = 10 < 30

8 0.1186 3 1.962 0.144 n.s.

aHigh standard errors due to steep Hill slopes caused by a few datapoints ~50% metamorphosis

Table 2. Acropora millepora. Lowest observed effect concentration (LOEC), the concentration that inhibited fertilisation by 50%

(EC50) and summary of ANOVA results for fertilisation and the 7 and 8 d old metamorphosis assays. n.s.: no significant 

differences (p > 0.05). con: control
Mar Ecol Prog Ser 330: 127–137, 2007

(Table 3, Fig. 3). Tentacles in the endosulfan, chlorpyrifos, permethrin and carbaryl treatments all retracted

normally when contacted gently with stainless steel

forceps.

Paling (apparent bleaching) was observed visually

on the tips of many branches in 10 μg l–1 contaminant

treatments, and paling over the entire surface was

observed with endosulfan, permethrin and profenofos

at this concentration (Table 3). Dinoflagellate densities, however, were only reduced in the presence of

10 μg l–1 profenofos and MEMC in comparison with the

controls (Tables 3 & 4). MEMC caused tissue mortality

(Tables 3 & 4), with lesions exposing underlying coral

skeleton in 7 of the 12 replicate

branches (Fig. 3). The maximum effective quantum yields of light-adapted

symbionts (ΔF/Fm’) in Acropora millepora branches were between 0.71 and

0.72 in uncontaminated seawater.

Slight but significant reductions in

ΔF/Fm’ were observed following 96 h

exposures to 10 μg l endosulfan and

chlorpyrifos (Tables 3 & 4). MEMC

reduced ΔF/Fm’ at both 1 and 10 μg l–1

(Tables 3 & 4). No esterase activity

was detectable in coral tissue using 3

common substrates (acetylthiocholine,

propioylthiocholine, or butyrylthiocholine—data not shown).

DISCUSSION

Acropora millepora was highly susceptible to insecticides, with larval settlement and metamorphosis being at

least 10-fold more sensitive to 4 insecticide groups than are mature coral

branchlets or oocyte fertilisation. The

fungicide MEMC exhibited broader

toxicity at very low concentrations

across each critical life transition and

stage.

The organomercurial fungicide

MEMC denatures membrane and

intra-cellular proteins and inactivates

enzymes causing general metabolic

disruption (WHO 1996). Fertilisation is

a complex sequence of events, reliant

on the action of specific enzymes and

proteins that control essential processes such as sperm motility, egg

recognition, sperm attachment and

penetration of the vitelline layer (Pinto

et al. 1990). MEMC would therefore

be expected to affect coral fertilisation success, but the

LOEC for Acropora millepora of 1.0 μg l–1 was surprising as the concentrations required to inhibit external

fertilisation of fish eggs are usually orders of magnitude higher (>400 μg l–1) (Akiyama 1970). Inorganic

mercury (which has a similar mode of action to MEMC;

WHO 1996), has been shown to inhibit the fertilisation

and development success of ascidian and sea urchin

embryos (Franchet et al. 1997), but again at very high

concentrations (>400 μg l–1). The concentration of

MEMC that inhibits coral fertilisation is also an order

of magnitude lower than that reported for copper

(Negri & Heyward 2001).

132

Chlorpyrifos oxon

0

20

40

60

80

Chlorpyrifos 100

0.1 0 10 100

0.1 0 10 100

Metamorphosis (%)

0

20

40

60

80

100

Permethrin

0

20

40

60

80

100

Endosulfan

0

20

40

60

80

Profenofos 100

0

20

40

60

80

100

Carbaryl

0

20

40

60

80

100

MEMC

Toxicant concentration (μg l–1) 

0

20

40

60

80

100

8 day old larvae

7 day old larvae

6 day old larvae 

Fig. 2. Acropora millepora. Metamorphosis success (%) for insecticide and treatments. Bars represent standard errors, n = 6 (see Table 2 for lowest observed 

effect concentration and estimated EC50 values)
Markey et al.: Pesticides affect multiple coral life stages

Insecticides did not inhibit coral fertilisation within

the concentration range used in this study. This is perhaps not surprising, because neurons do not develop in

Acropora millepora until approximately 50 h postfertilisation (Ball et al. 2002). Consequently, the inactivity of the neurotoxic insecticides may be due to the

lack of nerve-like cells in gametes. However, neurotoxins can affect neuro-transmitters such as AChE in

organisms prior to the formation of the nervous system,

and there is evidence to suggest

they regulate developmental processes such as cell division and differentiation (Buznikov et al. 2001).

AChE is found in ascidian eggs and

sperm (Pesando et al. 2003) and in the

eggs (Piomboni et al. 2001) of sea

urchins. The organophosphate diazinon decreases AChE activity and

lectin binding in urchin eggs at 30 μg

l

–1, resulting in a reduction in fertilisation (Pesando et al. 2003). If AChE is

present in 3 h A. millepora embryos its

function was not affected by chlorpyrifos exposures of up to 30 μg l–1.

The fungicide MEMC was a potent

inhibitor of coral settlement and metamorphosis, with an LC50 of 2.5 ± 0.3 μg

l

–1, second only to the antifoulant TBT

(Negri & Heyward 2001) and more

potent than copper (Negri & Heyward 2001). This

toxicity is likely to result from MEMC’s ability to denature proteins and inactivate a range of enzymes that

are critical to metamorphosis in cnidarians (Leitz 1997).

Coral larval metamorphosis was sensitive to all 4

classes of neurotoxic insecticides. Metamorphosis is an

intricate process involving detection of a chemical cue

and attachment to a surface by the larvae, and finally

cellular differentiation into a sessile juvenile polyp

133

Treatment Conc. Expt Polyp Visual Tissue Dinoflagellate ΔF/Fm’

(μg l–1) no. retraction bleaching mortality density (× 104) (cells cm–2)

Control 0 1 – – – 142 ± 15 0.72 ± 0.02

0 2 – – – 136 ± 7 0.71 ± 0.02

0 3 – – – 163 ± 12 0.71 ± 0.01

Acetone control 0 1 – – – 153 ± 13 0.69 ± 0.03

Endosulfan 1 1 – – – – 0.67 ± 0.03

10 1 – 6/12 (H) – 114 ± 13 *0.63 ± 0.04*

Chlorpyrifos 1 1 – – – – 0.69 ± 0.03

10 1 – 5/12 (L) – 127 ± 9 *0.65 ± 0.03*

Profenofos 1 3 – – – – 0.69 ± 0.01

10 3 12/12 12/12 (H) – *120 ± 18* 0.71 ± 0.01

Permethrin 1 2 – – – – 0.70 ± 0.02

10 2 – 7/12 (H) 119 ± 6 0.69 ± 0.02

Carbaryl 1 2 – – – – 0.70 ± 0.02

10 2 – – – 129 ± 8 0.71± 0.02

MEMC 1 3 1/12 2/12 (T) 1/12 – *0.61 ± 0.01*

10 3 12/12 5/12 (T) 7/12 *118 ± 9* *0.65 ± 0.01*

Table 3. Acropora millepora. Effect of each toxicant on polyp retraction, bleaching, tissue mortality (>2% of surface area),

dinoflagellate density (±SE) and light (ΔF/Fm’) adapted photosynthetic yields of symbionts. Results for adult branchlet exposures

(n = 12). Treatments were performed in 3 consecutive experiments. *Values significantly different from those of the control treatment (p < 0.05, see Table 4). Low-concentration treatments of dinoflagellate density (1.0 μg l–1) not calculated. Polyp retraction:

majority of polyps withdrawn without disturbance, remaining in this state for at least 24 h (effects scored per branchlet). H: heavy 

bleaching, >80% surface; L: light bleaching, <10% surface; T: tip bleaching

Fig. 3. Acropora millepora. Adult branches. Tentacles are out and feeding on

control branchlet, while branchlet exposed to 10 μg l–1 MEMC exhibits tissue 

retraction around the polyps and tissue death exposing underlying skeleton
Mar Ecol Prog Ser 330: 127–137, 2007

(Harrison & Wallace 1990, Leitz 1997). Coral larvae

possess nerve cells after 2 to 3 d, which transform into

a diffuse nerve net as the larvae become competent to

settle (Ball et al. 2002). RFamide staining of Acropora

millepora larvae has revealed the presence of a nerve

net spreading along the base of the ectoderm, with distinct sensory neurons projecting from the net to the

surface of the ectoderm (Hayward et al. 2001). It is

thought that metamorphosis in cnidarians is induced,

at least in part, by the release of neuropeptides that

spread through the larvae following the stimulation of

sensory cells by external cues (Müller & Leitz 2002).

AChE appears to play an integral role in the metamorphosis of some marine larvae, such as ascidians, where

AChE activity is sometimes localised on the surface of

adhesive papillae prior to settlement (Coniglio et al.

1998). Cholinesterase activity was not confirmed in

adult A. millepora branches in this study. Low levels of

AChE activity, however, have been identified in soft

coral (Horiuchi et al. 2003), using the same type of

assay as adopted in the present study. A more sensitive

assay may be needed to accurately detect the presence

of ChE activity in scleractinian corals.

Small differences in LOECs for metamorphosis were

observed among the insecticide treatments (see

Table 2). The metabolic conversion of chlorpyrifos to

chlorpyrifos oxon is usually required to ‘activate’ AChE

inhibition by this organophosphate (van Emden & Service 2004). In the present study, chlorpyrifos oxon and

profenofos, the 2 organophosphates that are considered toxic without need of oxidative activation, were

slightly more potent than the other insecticides. Chlorpyrifos was also toxic, indicating that if the mode of

action is related to AChE, then some conversion to

chlorpyrifos oxon may have occurred during the 18 h

exposure period. However, studies on the development of sea urchin embryos indicate that chlorpyrifos

itself is toxic and may be acting through a different

pathway such as the disruption of cholinergic receptors (Buznikov et al. 2001). Although insecticides

inhibited metamorphosis at very low concentrations,

the larvae were still actively swimming, indicating that

the mode(s) of action is much more subtle than MEMC.

Larvae of the brooding coral Pocillopora damicornis

were also active following exposure to high concentrations of chlorpyrifos and carbaryl (Acevedo 1991). It is

likely, therefore, that the insecticides act upon relatively specific neural pathway(s) such as the detection

of an external morphogen by sensory cells or one of the

internal signalling pathways involved in this critical

life-history transition.

In this study, adult coral branches were exposed to

only 2 concentrations of each toxicant; however, it was

apparent that MEMC was more potent against adults

than the insecticides (Table 3). MEMC exposure resulted in polyp retraction and tissue damage at 10 μg

l

–1, indicating a toxic effect on the host animal. The

expulsion of dinoflagellates (bleaching) observed at

10 μg l–1 plus a reduction in photosynthetic efficiency

(ΔF/Fm’) at 1.0 μg l–1 also demonstrates effects on the

symbiotic dinoflagellates. These observations are consistent with the broad mode of action of MEMC. The

effects on adult corals are similar to those of the

antifoulant TBT, which can cause both tissue damage

and a slight reduction in photosynthesis at low concentrations (Smith et al. 2003).

The insecticides had little effect on adult branches

over the 96 h exposure periods (Table 3). The most

noticeable effect was that of profenofos at 10 μg l–1,

where polyp retraction and slight bleaching in all

branches were observed. Many of the branches in other

treatments appeared to have paled; however, this may

have been due to tissue retraction and not a reduction in

dinoflagellate density. The organochlorine endosulfan

was the only insecticide to have a significant effect on

photosynthetic efficiency (~10% drop at 10 μg l–1). The

mechanism for this is unclear, but it may be a direct

effect on Photosystem II or alternatively an effect on the

host, which results in unfavourable conditions for the

symbiont. This response contrasts with that of herbicides, which have been shown to consistently inhibit

photosynthesis in coral symbionts by ~20% at 1.0 μg l–1

or lower (Jones & Kerswell 2003, Negri et al. 2005). Adult

Acropora millepora appear to be less sensitive to chlorpyrifos than Pocillopora damicornis, which was shown

to have an LC50 of 6 μg l–1 in 96 h exposures (Te 1998).

The risk posed by a toxicant to an organism is the

product of sensitivity and potential exposure. Information on the environmental concentrations of insecticides and fungicides in nearshore tropical waters

where corals may be exposed is scarce; however, the

results from this study suggest that all 4 groups of

insecticides and the organomercurial fungicide are

among the most potent inhibitors of coral fertilisation

and/or metamorphosis reported thus far. Therefore,

the risks that these pesticides pose to corals, especially

reproduction, are potentially significant.

134

Parameter Expt SS df F p

ΔF/Fm’ 1 0.068 5 5.481 0.000

2 0.005 4 1.195 0.323

3 0.080 4 10.296 0.000

Dinoflagellate density 1 1.170 3 3.745 0.018

2 1.190 2 1.469 0.261

3 1.719 2 9.129 0.001

Table 4. Acropora millepora. Summary of ANOVA results for

ΔF/Fm’ and dinoflagellate densities for adult coral branchlet 

exposures (3 experiments)
Markey et al.: Pesticides affect multiple coral life stages

Commonly used sampling techniques, such as taking integrated water samples from various depths and

filtering, may result in an underestimation of the likely

exposure faced by multi-life-stage organisms such as

corals. For instance, many pesticides, such as chlorpyrifos and endosulfan, are lipophilic and likely to

adsorb to particulates, accumulate in sediment and

bioaccumulate in planktonic organisms to higher concentrations than the dissolved levels in the water column (Müller et al. 2000). Zooplankton, in particular,

can bioaccumulate insecticides to concentrations up

to 3000 times higher than ambient water (De Lorenzo

et al. 2002). As both autotrophic and heterotrophic

organisms, corals may accumulate insecticides directly

across membranes, as well as through feeding upon

contaminated zooplankton, suspended particulate

matter and sediment particles. The sea-surface microlayer (upper 50 μm) is also a concentration point for a

range of anthropogenic contaminants, including pesticides, which can be found at concentrations 100s of

times higher than in the water column (Hardy et al.

1987). Coral eggs are highly buoyant, and therefore

fertilisation may suffer from unrecognised pesticide

accumulation on the ocean surface. At settlement,

coral larvae are in close contact with marine sediment

and biofilms. Pesticides bound to sediments are often

present in higher concentrations and persist for longer

periods than pesticides in the water column and may

be as equally bioavailable as those in solution (Müller

et al. 2000). Similarly, biofilms that form on marine

surfaces and are critical triggers of coral larval settlement (Webster et al. 2004) can sequester pesticides

and prevent settlement in other invertebrates (Labare

et al. 1997).

The timing of pollution events can also be a critical

factor that may increase the risk to corals. The early

life-history stages and transitions of corals, such as

spawning, fertilisation, larval dispersal and metamorphosis (recruitment), occur during a finite time period

for many species (Harrison & Wallace 1990). Mass

spawning on the GBR generally occurs during November and December each year and may coincide with

the first rains of the wet season in tropical North

Queensland—the time during which the risk of contamination via terrestrial runoff is nearing its peak for

the GBR (Fabricius 2005). Levels of pesticides in

suspended sediments and in solution can increase dramatically following the first rainfall of the wet season.

For example, chlorpyrifos concentrations in suspended

sediment rose from below detection limits to 344 μg l–1

in the Lourens River, following the first heavy rains of

the South African wet season (Schultz et al. 2001).

Successful reproduction and recruitment is essential

for the maintenance of coral reefs, particularly following heavy disturbances, where recovery of coral abundance and diversity relies largely on recruitment by

larvae or propagules from other reefs (Connell et al.

1997). The early life stages of coral exhibit some of the

most sensitive biological responses to pesticide contamination in the marine environment. The extremely

high sensitivity of Acropora millepora settlement to a

range of insecticides and a fungicide indicates that

pesticide contamination of the reef habitat, even by

barely detectable concentrations, could have profound

consequences for population replenishment. The

examination of toxic thresholds provides valuable

information that can help management agencies

assess risk to key marine organisms. The high sensitivity of corals also raises questions as to whether current sampling and analysis techniques are capable of

detecting these low pesticide concentrations and

whether water quality guidelines adequately protect

all coral life stages.

Acknowledgements. The authors thank J. Mieog for field and

laboratory assistance, the AIMS Water Quality Team for

providing some of the corals and S. Delean for assistance

with statistical interpretation. We thank Dow AgroSciences

(Frenchs Forest, Australia) for donating chlorpyrifos and

chlorpyrifos oxon.

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Editorial responsibility: Howard Browman (Associate Editorin-Chief), Storebø, NorwaySubmitted: March 27, 2006; Accepted: May 31, 2006

Proofs received from author(s): January 9, 2007