Development of a chronic, early life-stage sub-lethal toxicity testand recovery assessment for the tropical zooxanthellatesea anemone Aiptasia pulchella

Pelli L. Howe n, Amanda J. Reichelt-Brushett, Malcolm W. ClarkMarine Ecology Research Centre, School of Environment, Science and Engineering, Southern Cross University, PO Box 417, Military Drive, Lismore,NSW 2480, Australia

a r t i c l e i n f o

Article history:Received 9 March 2013Received in revised form12 October 2013Accepted 16 October 2013Available online 12 November 2013

Keywords:Aiptasia pulchellaTrace metalsToxicityTropical marine ecotoxicologyPedal lacerate development

a b s t r a c t

There is an urgent need to identify additional tropical marine species and develop sensitive sub-lethaland chronic toxicity test methods for routine ecotoxicology. The tropical symbiotic sea anemone Aiptasiapulchella is a suitable species for use in ecotoxicology and here we have assessed the effects of tracemetal exposures on the development of asexually produced A. pulchella pedal lacerates to a juvenilestage. Concentrations of 55 mg/L for cadmium, 262 mg/L for cobalt, 5 mg/L for copper, and 269 mg/L for zincwere estimated to inhibit normal development by 50 percent after 8-d exposures, and are among themost sensitive available toxicity estimates for marine organisms. This work illustrates the potential valueof this species and sub-lethal toxicological endpoint for routine ecotoxicology in tropical marine environments.

& 2013 Elsevier Inc. All rights reserved.

1. Introduction

The paucity of tropical marine species used in routine ecotoxicologyhas been criticised for more than a decade and results in a relianceon temperate toxicity data for derivation of water quality guidelines(Lacher and Goldstein, 1997; Peters et al., 1997; Van Dam et al., 2008).This practise continues despite the effects that temperature mayhave on toxicity (Cairns et al., 1975; Chapman et al., 2006; Sokolovaand Lannig, 2008), biological differences in tropical compared totemperate marine species and ecosystems (Canning-Clode et al.,2011), and the overall unreliability of extrapolating toxicity databetween climatic regions (Chapman et al., 2006; Kwok et al., 2007). Alack of regionally-relevant toxicity data for commonly detectedcontaminants such as trace metals (Markich and Camilleri, 1997)and pesticides (GBRMPA, 2010) has been discussed, and the need foradditional tools for ecotoxicological assessment of an array of newly-emerging contaminants has been emphasised (e.g. engineered nano-particles; Matranga and Corsi, 2012; Van Dam et al., 2008). It isnecessary to identify a number of tropical marine species and toprovide representation from a range of ecologically-relevant taxa(Van Dam et al., 2008), and to ensure that both filter-feeding and

suspension-feeding organisms are included in routine ecotoxicologi-cal testing (Matranga and Corsi, 2012).

One ecosystem potentially at high risk due to these knowledgegaps are coral reefs (Van Dam et al., 2008). These ecosystems supportareas of the highest biodiversity on earth (Reaka-Kudla, 1997), affectglobal climate (Jones and Ristovskil, 2010) and provide invaluableeconomic, ecological and subsistence value for humans (Birkeland,1997). Globally, the greatest risks to coral reefs come from increasesin sea surface temperature (SST; Hoegh-Guldberg, 1999), and oceanacidity (Veron et al., 2009). However, contaminants have also beenrecognised as primary threats to coral reefs in Australasia and theIndo-Pacific (Australian Government, 2013; Reichelt-Brushett, 2012).Furthermore, there is evidence that global-stressors may act addi-tively, synergistically or sequentially with local-stressors (which aremuch more easily mitigated) including synthetic contaminants suchas biocides (Negri and Heyward 2001), and excess naturally occurringcontaminants (e.g. trace metals; Negri and Hoogenboom, 2011;Sokolova and Lannig, 2008; Yaroslavtseva and Sergeyeva, 2008).

Sea anemones are cnidarians which usually house intracellularphotosynthetic dinoflagellates from the genus Symbiodinium sp.(Birkeland, 1997). Anemones have been used in laboratory researchfor many decades, including in studies of cnidarian physiology (Muller-Parker, 1984), algal-host symbioses (Steen, 1986), metal uptake andaccumulation (Mitchelmore et al., 2003), and stress responses such aszooxanthellae loss (Dunn et al., 2002; Perez, 2007). In corals, highermetal accumulation has been reported in zooxanthellae and soft tissue

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Ecotoxicology and Environmental Safety

0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ecoenv.2013.10.024

n Corresponding author. Fax: þ61 266203650.E-mail address: pelli.howe@scu.edu.au (P.L. Howe).

Ecotoxicology and Environmental Safety 100 (2014) 138–147

compared to the skeleton (Reichelt-Brushett and McOrist, 2003),hence anemones may be useful proxies in studies of metal toxicity,uptake and accumulation (Main et al., 2010) and would at the veryleast provide some representation from the phylum Cnidaria. Unlikecorals, many anemone species are easy to culture in large numbers inlaboratory conditions, and reliably reproduce both sexually andasexually throughout the year (Howe et al., 2012; Ladriere et al.,2008b; Lehnert et al., 2012). Similarly to corals, anemones will appearbleached, produce excess mucus, and retract their tentacles and tissuein response to stressors such as extreme temperatures (Dunn et al.,2002) and contaminants (Bao et al., 2011; Howe et al., 2012;Main et al., 2010).

The tropical zooxanthellate anemone Aiptasia pulchella(Carlgren, 1943) has a wide range throughout the tropics andsub-tropics and is therefore a regionally-relevant species forecotoxicology throughout Australasia, including areas where mar-ine biodiversity ‘hotspots’ are increasingly exposed to anthropo-genic contaminants (Reichelt-Brushett, 2012). A. pulchella haspreviously been identified as a useful cnidarian species forlaboratory studies (Hoegh-Guldberg, 1988; Hong et al., 2009;Sawyer and Muscatine, 2001), and relatively simple and inexpen-sive methods of laboratory husbandry reliably produce sufficientnumbers of organisms to run replicated toxicity tests (Howe et al.,2012). However, prior to our recent work the sensitivity of A.pulchella to a range of contaminants, and hence its suitability as atoxicity test species, had not been investigated.

The aims of this study were to investigate the sensitivity andefficacy of a newly-developed chronic sub-lethal laboratory toxi-city test using the zooxanthellate tropical sea anemone A. pul-chella. The contaminants used to investigate this test method werecadmium, cobalt, copper, nickel and zinc, for which sub-lethalchronic toxicity estimates are provided. Results are compared totoxicity estimates for other tropical marine species, and demon-strate the relevance and suitability of this species and sub-lethalendpoint as a routine test method for ecotoxicology relevant totropical marine environments.

2. Methods

2.1. Test organisms

Original stocks of A. pulchella were obtained from the flow-through seawatersystem at the National Marine Science Centre, Charlesworth Bay, NSW, Australia andhoused at Southern Cross University, Lismore, NSW, Australia (Howe et al., 2012).This species reproduces asexually by ‘pedal laceration’, whereby a small piece ofparent pedal disc tissue separates from the adult and subsequently develops into agenetically-identical anemone (Clayton, 1985; Hunter, 1984; Lin et al., 1992).The offspring is termed a ‘lacerate’ until such time as eight tentacles have developedand it is defined as a ‘juvenile’, as described for Aiptasia pallida (Cary, 1911).

In this study new lacerates were collected from culturing tanks and lacerates withno tentacles were selected using a stereo microscope (Olympus SZ51s). Four lacerateswere placed in each of five replicate 2 M HCl washed and 75-mL seawater-agedpolyethylene test containers for all metals, with the exception of copper where twolacerates were used in each of five replicates. Each test container was filled with 73 mLof solution. Initial observations of lacerate development showed that the time for firsttentacle appearance was short (1–2 d) and that subsequent development to a juvenilestage was often complete within 3 d. Hence, a short 3-h acclimation period was usedto ensure that test organisms had no tentacles at test commencement; all laceratesadhered to test containers within 24 h.

2.2. Toxicity tests

Five metal concentrations and a seawater control were used in all tests. Metalstock solutions (500 mg/L) were prepared using Analar grade metal salts of CuCl2,CdCl2, CoCl2, NiCl2 and ZnCl2 (99 percent purity, Sigma-Aldrich©) and reverseosmosis water. All test solutions were prepared using these stocks and naturalseawater no more than 1 h prior to test commencement and 48-h test solutionrenewals. The seawater used as a diluent was collected from the same location(28.491S, 153.551E) on an incoming tide, and was aerated at ambient roomtemperature for use within 48 h of collection. Seawater had been collected from

this location for use in culturing and other experiments for approximately 18months prior to this study, during which time there was no evidence ofcontamination in culturing tanks, fresh dilution water, or associated with testprocedures (see Howe et al., 2012; Howe et al., in press; Howe et al., unpublisheddata). Regular water quality measurement within 48 h of collection showedvariations in pH and electrical conductivity less than 0.5 units and 5 percent,respectively, and a minimum of 85 percent DO saturation during this 18 monthperiod. In addition, periodic analysis of dissolved concentrations identified negli-gible background levels of cadmium, cobalt, copper, lead, nickel and zinc.

All tests were conducted in water baths at 2571 1C with a 12: 12-h photo-period (50–60 mE m2 s�1) using 30 W white (Sylvinia Aquastar©) and blue(Coralstar©) fluorescent aquarium lighting. Electrical conductivity, pH, and tem-perature were measured using a Eutech Cyberscan PC300 © combined metre atmidday every 24 h and dissolved oxygen (DO) concentrations were measured usinga YSI© metre at test commencement, and in old and new solutions at each 48-hrenewal. At test commencement and four renewal times, 10 mL aliquots of eachnew solution were acidified and filtered to 0.45 mm before measurements ofdissolved metal concentrations using inductively coupled plasma mass spectro-metry (ICP-MS) as per American Public Health Association method 3120 foranalysing metals (1 mg/L detection limit; APHA, 2005). Aliquots were also takenfrom spent test solutions and analysed to provide an indication of the changes inmetal concentrations after 48-h in static test conditions. As the metal concentra-tions of old solutions was only tested once, the mean measured dissolved metalconcentrations from new solutions at 0, 2, 4, 6 and 10 d were used to calculatetoxicity estimates.

The number of visible tentacles on each anemone was counted using a stereomicroscope (Olympus SZ51©) prior to solution renewal at 4, 6, 8, 10 and 14 d.A juvenile was recorded if eight tentacles of any length were visible (Cary, 1911).Mortality was recorded if necrotic tissue was observed, which occurs rapidlyfollowing death (authors personal observation). After 14 d, test solutions weredecanted and test containers were rinsed three times and filled with clean seawaterfor a 7-d recovery period with the same experimental conditions as the test period.

2.3. Data analyses

The probit method (SPSS© statistical software) was used to estimate 8-d and14-d EC10 and EC50 values using the mean metal concentrations. For tests wherelacerate mortality occurred, the probit method was used to calculate 8- and 14-dlethal metal concentration estimates for 10 percent (lethal concentration, 10percent; LC10) and 50 percent of the test population (lethal concentration, 50percent; LC50). Estimated LC/EC10 values are the preferred alternatives to no-observed effect concentrations (NOECs) or lowest-observed effect concentrations(LOECs) (Chapman et al., 1996).

3. Results

3.1. Quality assurance and control

The temperature of 2571 1C was maintained, and the pH rangedbetween 7.98 and 8.4 and the DO concentration between 7.6 and8.5 mg/L. Electrical conductivity varied between 52.1 and 53.8 mS/cmin new test solutions, and increased by up to 15 percent in 48-h oldsolutions. Analysed dissolved metal concentrations of new solutionsvaried by o20 percent over the duration of the test. Metal concentra-tions in 48-h old solutions were at least 88 percent of the originalconcentrations, and there was a general tendency for concentrationsto increase after 48 h in test conditions (Table 1). This was likely due toevaporation of test solutions and the fact that test containers were notwashed between renewals, and was generally more pronounced inlower treatment concentrations.

3.2. General observations

There was no mortality or obvious loss of condition in anycontrol in any test, and all had developed to a juvenile stage within8 d (Figs. 1A and 2A). Since the exact time since separation fromthe parent was unknown, variability was seen in the tentacledevelopment rate in controls during the initial 8 d (Fig. 1A). Table 2provides the concentrations estimated to cause 10 percent (EC10)and 50 percent (EC50) inhibition of normal lacerate developmentto a juvenile stage, and 10 and 50 percent mortality (LC10 andLC50 values). The order of toxicity for inhibition of normal lacerate

P.L. Howe et al. / Ecotoxicology and Environmental Safety 100 (2014) 138–147 139

development in A. pulchella was copper4cadmium4zincZcobalt4nickel.

3.3. Cadmium

Cadmium elicited a similar toxicity to zinc, a higher toxicity thancobalt or nickel, but was much less toxic than copper. An 8-d EC50

value of 55 mg/L was estimated, and the higher concentration of166 mg/L estimated after 14 d illustrates that cadmium delayedlacerate development (Table 2). Exposure to cadmium concentra-tions between 17 and 476 mg/L inhibited tentacle developmentin the initial 96 h, with little difference between concentrations.At 6 d, however, there was a clear distinction in tentacle develop-ment between controls, those exposed to cadmium concentrations

Table 1Mean dissolved metal concentrations (n¼5) of new test solutions and 48-h old spent solutions (seawater controls and treatments 1–5; T1–T5). Standard deviations are inbrackets.

Metal Cont. T1 T2 T3 T4 T5

New old Old New old Old New old Old New old Old New old Old

Cd 0 (70) 17 (73) 25 29 (74) 33 52 (73) 64 106 (75) 118 476 (725) 567Co 0 (70) 30 (72) 36 140 (713) 170 282 (77) 324 551 (717) 549 820 (724) 933Cu 0 (70) 2 (71) 7 6 (72) 10 10 (71) 15 18 (71) 15 37 (72) 35Ni 3 (71) 11 (72) 26 57 (76) 75 114 (712) 132 201 (710) 238 491 (733) 566Zn 19 (77) 48 (74) 87 163 (723) 156 323 (747) 292 592 (736) 576 871 (760) 826

Fig. 1. (A) Mean percentage of Aiptasia pulchella pedal lacerates in seawater control treatments that had developed to a juvenile stage over 14 d (n¼90), and the percentagewhich survived and developed to a juvenile stage within 14-d exposure to; (B) cadmium, (C) cobalt, (D) copper, (E) nickel and (F) zinc (n¼20 except copper where n¼10).Error bars show the standard error.

P.L. Howe et al. / Ecotoxicology and Environmental Safety 100 (2014) 138–147140

between 17 and 106 mg/L, and those exposed to 476 mg/L cadmium(data not shown). After 14-d exposure fewer lacerates in the 17 mg/Lcadmium treatment had developed to a juvenile stage than at 29, 52or 106 mg/L (Fig. 1B), while effects on survival were concentration-dependant (Fig. 1B).

Dark areas in the pedal disc which were never observed incontrols were often seen in anemones exposed to 17 and 52 mg/Lcadmium (Fig. 2B). Tentacle retraction (Fig. 2C) and/or bleaching(Fig. 2D and E) occurred within 6 d in all replicates at higherconcentrations, and in all concentrations within 11 d. Deformitieswere observed in up to 50 percent of anemones exposed to 52 and106 mg/L cadmium, including the development of two oral discs(Fig. 2F), split tentacles (Fig. 2G), and column bloating (Figs. 2Dand 2E). Despite these abnormalities, only 5 percent mortalityoccurred within 11-d exposure to 106 mg/L cadmium, whilst thesame exposure to 476 mg/L resulted in 65 percent mortality and nonormal lacerate development.

Within 14 d all lacerates in all treatments were severely dis-coloured, and 15 percent mortality had occurred at 52 mg/L cad-mium; 8- and 14-d LC50 values of 433 and 396 mg/L were estimatedrespectively (Table 2). Anemones previously exposed to cadmiumremained pale throughout the 7-d recovery period. Two individualswhich had not developed to a juvenile stage during exposure to476 mg/L cadmium began to develop tentacles during the recoveryperiod and no further mortalities occurred, however 470 percentdid not show signs of recovery of normal appearance.

3.4. Cobalt

Cobalt concentrations between 30 and 820 mg/L also inhibitedA. pulchella juvenile development. In this test, less laceratesexposed to the lowest concentration (30 mg/L) had developed toa juvenile stage within 14 d than at two higher concentrations(140 and 282 mg/L; Fig. 1C). 8- and 14-d EC50 values of 262 and561 mg/L cobalt were estimated respectively (Table 2). Laceratesexposed to 282, 551 and 820 mg/L cobalt also produced excessmucus and were discoloured within 96 h, though aggregatedzooxanthellae (e.g. Fig. 2H) was rarely observed in test containers.Split tentacles (Fig. 2G), tentacle retraction (Fig. 2C), discoloura-tion, bloating, elongation, and/or contortion of the column (Fig. 2Dand E) were observed in several individuals within 14-d cobaltexposure. In addition, the epidermis of lacerates exposed to 551and 820 mg/L cobalt often exhibited a degraded appearance(Fig. 2E), as was also described in Aiptasia sp. exposed to hightemperatures (Dunn et al., 2002).

Less than 10 percent mortality occurred within 8-d exposure tocobalt concentrations r820 mg/L. However, 25, 55 and 60 percent

mortality had occurred at 282, 551 and 820 mg/L respectivelywithin 14 d, and a 14-d LC50 value of 601 mg/L cobalt wasestimated (Table 2). More than half of the test organisms whichsurvived the 14-d exposure to 551 mg/L cobalt died during the 7-drecovery period, while only one of the surviving eight anemonesfrom the 820 mg/L treatment died during this time. At the end ofthe recovery period, 80 percent and 65 percent mortality hadoccurred in the 551 and 820 mg/L treatments respectively. Mostanemones exposed to r282 mg/L cobalt rapidly recovered normalmorphology and colouration within 7 d. However, 50 percent ofsurviving lacerates from the 551 and 820 mg/L cobalt treatments,and 410 percent of lacerates from the 30, 182 and 240 mg/Ltreatments remained completely retracted at the end of the 7-drecovery period (Fig. 2J).

3.5. Copper

Lacerate development was inhibited by very low copper con-centrations, with estimated 8- and 14-d EC50 values of 5 and 7 mg/L,and 8- and 14-d EC10 values of 1 and 2 mg/L (Table 2). There wassome tentacle development in all copper treatments up until 48 h.At 48 h the number of emerged tentacles was higher in anemonesexposed to 37 mg/L compared to 18 mg/L copper, and by 96 hlacerates exposed to both these concentrations were less devel-oped than controls (data not shown). However at 96 h all laceratesexposed to 18 and 37 mg/L copper were either not developed orhad completely retracted tentacles; no further normal develop-ment occurred in these treatments. Exposure to 6 and 10 mg/Lcopper also caused reductions in the number of tentacles, whilst aslight increase in the rate of tentacle development was evident inanemones exposed to 18 mg/L between 4 and 6 d, after which timethis effect disappeared (data not shown). All the tested copperconcentrations increased the time taken for lacerates to develop toa juvenile stage.

Tentacle retraction and thickening of the epidermis (Fig. 2D, K,and L), en masse zooxanthellae loss (Fig. 2H and I), excess mucusproduction (Fig. 2I) and dark aggregations in the pedal disc (Fig. 2Band K) were observed in all copper treatments within 96 h.At lower concentrations some mild tissue discolouration, particu-larly at the tentacle tips (Fig. 2C, K and L), was observed, althoughanemones generally appeared darker due to tissue retraction.

Within 48-h and 6-d exposure to 37 mg/L copper, laceratemortality was 5 and 100 percent respectively, whilst exposure to18 mg/L caused 100 percent mortality within 8 d. An LC50 value of23 mg/L copper was estimated at 8 and 14 d, with only oneadditional mortality occurring between these assessment times.During the recovery period, 95 percent of anemones exposed to

Table 2Toxicity estimates for Aiptasia pulchella exposed to different trace metals in chronic laboratory toxicity tests. Data are metal concentrations (mg/L) estimated to cause 10 and50 percent mortality (LC10/50), or inhibition of normal lacerate development (EC10/50 values). 95 percent confidence limits are in brackets. n.c. – not calculable.

Metal Inhibited development Lethality

EC10 EC50 LC10 LC50

8-d 14-d 8-d 14-d 8-d 14-d 8-d 14-d

Cd n.c. n.c. 55 166 229 153 433 396(36–83) (110–415) (118–303) (54–214) (360–535) (319–519)

Co n.c. n.c. 262 561 n.c. n.c. n.c. 601(469–832)(161–362) (399–961)

Cu 1 2 5 7 18 12 23 23(0––3) (0–4) (4–8) (5–10) (n.c.) (5–16) (n.c.) (18–31)

Ni n.c. 265 4 491 4 491 n.c. n.c. n.c. n.c.(37–736)

Zn 48 84 269 166 262 89 573 251(0–112) (22–118) (211–353) (131–232) (145–348) (7–138) (493–676) (206–311)

P.L. Howe et al. / Ecotoxicology and Environmental Safety 100 (2014) 138–147 141

copper concentrations between 2 and 10 mg/L appeared to recover,and no further mortalities occurred among anemones exposed to18 mg/L copper, although less than 35 percent had developed to ajuvenile stage within 7 d.

3.6. Nickel

The tested nickel concentrations caused minimal effects on laceratedevelopment or survival (Fig. 1E), and hence reliable 8- and 14-dtoxicity estimates were not calculable. However, exposure to 491 mg/L

nickel inhibited normal development in 25 percent of lacerates,and a 14-d EC10 of 265 mg/L was estimated (Table 2). Assessmentof development at 8 d compared to 14 d, however, showed thatnickel delayed development; only 45 percent of lacerates exposedto 491 mg/L nickel had developed to a juvenile stage within 8 d,whereas 75 percent had reached the developmental endpoint by14 d. Nickel-exposed lacerates exhibited only mild tentacle retrac-tion and discolouration within 96 h, after which time the severityof these symptoms increased with time and concentration. Mor-phological changes such as thinning, darkening, and loss of rigidity

Fig. 2. Common responses of Aiptasia pulchella to 14-d trace metal exposures. (A) Control juvenile, (B) dark aggregations in the pedal disc, (C) tentacle retraction, epidermisthickening, mucus production, (D) abnormal tentacle development, column bloating and severe bleaching, (E) abnormal tentacle development, column bloating and extremebleaching, (F) development of two oral discs, (G) split tentacle, (H) en masse zooxanthellae loss, (I) expulsion of clumped zooxanthellae, mucus production and no tentacledevelopment, (J) thin limp tentacles, dark areas in pedal disc and bleached tentacle tips and (K) thin tentacles and bleached tentacle tips.

P.L. Howe et al. / Ecotoxicology and Environmental Safety 100 (2014) 138–147142

in some tentacles (Fig. 2K and L) and collapse of the column, wereobserved in several individuals at all nickel concentrations whichwere unique responses to nickel contamination.

Only two mortalities occurred during 14-d exposure to nickelconcentrations between 11 and 491 mg/L, inhibiting the calculationof LC50 values (Fig. 1E). During the 7-d recovery period, anadditional mortality occurred from the 491 mg/L nickel treatment,and less than 50 percent of anemones from this treatment haddeveloped to a juvenile stage by the end of the recovery period,compared to 85–95 percent of those previously exposed tor114 mg/L nickel.

3.7. Zinc

In contrast to other metals, zinc caused a time-dependantinhibition of lacerate development, with an 8-d EC50 of 269 mg/Land a 14-d EC50 of 166 mg/L, and 8- and 14-d EC10 values of 48 and84 mg/L (Table 2). Tentacle development was completely inhibited

by exposure to concentrations 4592 mg/L zinc at all assessmenttimes, and inhibited tentacle development and mortality wasobserved in zinc treatments within the first 48 h (data not shown).Tentacle retraction and thickening of the epidermis (Fig. 2C), enmasse zooxanthellae loss (Fig. 2H and I), excess mucus production(Fig. 2I) and dark areas in the pedal disc (Fig. 2B and K) wereobserved to some degree in all zinc treatments within 96 h.Individual and aggregated zooxanthellae were seen in test con-tainers (Fig. 2B, H and I), although as for copper there was minimaltissue discolouration due to retraction. After 8-d exposure to323 mg/L zinc, one lacerate had developed two oral discs, and at10 d this abnormality was also observed in the 163 mg/L treatment(Fig. 2F).

Mortalities occurred in all zinc treatments, and 8- and 14-d LC50values of 573 and 251 mg/L were estimated (Table 2). Furthermore,recovery from zinc toxicity was poor during the 7-d recovery period,with a further mortality from the 48 and 323 mg/L treatments andfive from 163 mg/L treatment. Furthermore, only 10 percent of

Table 3Toxicity estimates (mg/L) for selected trace metals for survival and early life-stages in a range of marine species. Endpoints: S¼survival, D¼early life-stage development,G¼growth, LS¼ larval settlement, R¼asexual reproduction, M¼metamorphosis, E¼embryogenesis.

Metal Species Exp. time LC/EC50 NOEC(LOEC)

Endpoint Reference

Cd Aiptasia pulchella (anemone) 14 d 396 106 (476) S(lacerates)

This study

8 d 55 o17 D This study96 h 973–1256 o946 S (1-2 mm) Howe et al. (2012)

Montipora verrucosa (isolatedzoox.)

8 d - 100 G Goh and Chou (1992)

Hydroides elegans (polycheate) 96 h 47 – LS Gopalakrishnan et al. (2008)Meretrix meretrix (bivalve) 96 h 68–84 – S/G Wang et al. (2009)Tigriopus japonicas (copepod) 96 h 25,200 10,000 S Lee et al. (2007)Chanos chanos (fish) 96 h 62,800 – S Biuki et al. (2010)

Co Aiptasia pulchella(anemone) 14 d 604 282 (551) S(lacerates)

This study

8 d 265 o30 D This study28 d 583 25 (131) R Howe et al, unpublished data96 h 1547 – S (1-2 mm) Howe et al. (2012)

Palaemon serratus (crustacean) 9 d 45 – S Cited in ANZECC and ARMCANZ (2000)Ulva pertusa (algae) 96 h 100 – G Cited in ANZECC and ARMCANZ (2000)Phaeodactylium sp. (algae) o 5 d 23,600 – G Cited in ANZECC and ARMCANZ (2000)Monhystera sp. (nematode) 4 d 94,000 – S Cited in ANZECC and ARMCANZ (2000)

Cu Aiptasia pulchella (anemone) 14 d 23 18 (37) S(lacerates)

This study

8 d 5 2 (6) D This study28 d 22 11 (19) R Howe et al, unpublished data96 h 30–90 – S (1–2 mm) Howe et al. (2012)

Actinia sp. (anemone) 96 h 157–347 – S Hughes et al. (2005)Acropora tenuis (coral) 6–48 h 32–35 20–34 LS/M Reichelt-Brushett and Harrison (2000), Negri and Hoogenboom (2011)Acropora millepora (coral) o 24 h 26 – M Negri and Hoogenboom (2011)Ruditapes decussates (bivalve) 24 h 46.3 16 (32) E Fathallah et al. (2011)

Ni Aiptasia pulchella (anemone) 8 d 501 201(491) D This study28 d 552 513 (1014) R Howe et al., unpublished data96 h 2556–

13,3633258–5066 S (1–2 mm) Howe et al. (2012)

Poccilopora damicornis (coral) 52 h – (1000) LS Goh (1991)Mysidopdis intii (mysid) 28 d 22 o 12.6 S Hunt et al. (2002)Haliotis rufenscens (abalone) 48 h 146 – D Hunt et al. (2002)Hydroides elegans (polycheate) 96 h 1472 – S Gopalakrishnan et al., 2008

162 – LS Gopalakrishnan et al., 2008Apocyclops borneoensis (copepod) 48 h 13,050 – S Mohammed et al. (2010)

Zn Aiptasia pulchella(anemone) 14 d 251 48 (163) S(lacerates)

This study

8 d 269 19 D This study28 d 278 140 (270) R Howe et al., unpublished data96 h 707–1164 714–986 S (1-2 mm) Howe et al. (2012)

Ruditapes decussates (bivalve) 24 h 43.4 64 (128) E Fathallah et al. (2011)Hydroides elegans (polycheate) 96 h 1235 – S Gopalakrishnan et al. (2008)

119 – LS Gopalakrishnan et al. (2008)Pagrus major (fish) 10 d – (300) D Huang et al, (2010)Perna viridis (fish) 96 h 6090 – S Chan (1988)Tigriopus japonicas (copepod) 96 h 7800 4700 S Lee et al. (2007)

P.L. Howe et al. / Ecotoxicology and Environmental Safety 100 (2014) 138–147 143

anemones exposed to 323 mg/L had developed to a juvenile stageafter 7-d recovery.

4. Discussion

4.1. Sensitivity of A. pulchella lacerates to trace metals

Overall, assessment of normal A. pulchella lacerate develop-ment to a juvenile stage provided sensitive sub-lethal toxicityestimates compared to other marine species (Table 3). The resultsprovide encouragement for the use of this species and sub-lethalendpoint in ecotoxicology and as a toxicological risk assessmenttool for tropical marine environments.

A. pulchella was previously reported to be highly sensitive tocadmium compared to cobalt, lead, nickel or zinc in acute andchronic tests (Howe et al., 2012; Howe et al., in press). The 8-dEC50 value of 55 mg/L estimated here reiterates the previouslyreported high sensitivity of this species to cadmium. Cadmium ishighly toxic to many other marine organisms (e.g. Gopalakrishnanet al., 2008; Wang et al., 2009; Xie et al., 2005) and accumulates insome zooxanthellate cnidarians (Mitchelmore et al., 2003;Mitchelmore et al., 2007; Reichelt-Brushett and McOrist, 2003).For some species cadmium is more toxic than copper (e.g.Pocillopora damicornis; Mitchelmore et al., 2007). The delayeddevelopment observed in cadmium-exposed A. pulchella lacerateshas also been observed in urchins (Vaschenko et al., 1999).Cadmium causes oxidative stress (Mitchelmore et al., 2007),inhibits cell differentiation, and affects cell membrane permeabil-ity interfering with calcium cation exchange, which plays anessential role in early development (Swann and Whitaker, 1990;Vaschenko et al., 1999).

Inhibited lacerate development in A. pulchella provides themost sensitive cadmium toxicity estimate for a tropical marinecnidarian (Table 3). Lower cadmium concentrations inhibited A.pulchella lacerate development than have been reported to inhibitreproduction in some corals. In Acropora tenuis, successful fertili-sation (Z50 percent) occurred following exposure to 10,000 mg/Lcadmium, and a NOEC of 2000 mg/L was estimated for fertilisationsuccess (Reichelt-Brushett and Harrison, 2005). Similarly, highercadmium concentrations did not significantly affect fertilisation inthe corals Goniastrea aspera (200 mg/L) and Oxypora lacera(1000 mg/L) (Reichelt-Brushett and Harrison, 1999). However, highsensitivities to cadmium have been reported in some zooxanthel-late cnidarians and associated zooxanthellae, such as zooxanthel-lae isolated from Montipora verrucosa (NOEC of 100 mg/L; Goh andChou, 1992), and Pocillopora damicornis where host mortality occurredwithin 3-d exposure to 50 mg/L (Mitchelmore et al., 2007).

The responses of A. pulchella to cadmium support several otherstudies which illustrated that some zooxanthellate cnidarians,and the symbiosis itself, are highly sensitive to cadmium andmust be represented in routine ecotoxicology for tropical marineenvironments (Table 3). The greater effect of the lowest testedcadmium concentration than higher concentrations (Fig. 1B) issimilar to results of a 28-d test using reproductive A. pulchella,where the less offspring were produced by adults exposed to 28 mg/Lcadmium than to 58 or 107 mg/L (Howe et al., unpublished data).The effects on lacerate development and observations of muchmore extreme lacerate bleaching at the higher cadmium concen-trations, suggests that the degree of bleaching induced by higherconcentrations resulted in more effective metal detoxification thanlower concentration. This is supported by evidence of higher metalaccumulation in zooxanthellae than in host cells in some corals(Reichelt-Brushett and McOrist, 2003).

The sensitivity of A. pulchella lacerate development to cobalt isrelevant in the context of toxicity data for a range of other marine

organisms (Table 3). The 8-d EC50 for juvenile development(262 mg/L; Table 2) and the breakdown of the symbiosis duringexposure to concentrations between 30 and 820 mg/L highlight thepotential underestimation of cobalt toxicity to zooxanthellatemarine organisms due to the acknowledged lack of relevantstudies (Markich et al., 2002).

There were less developed juveniles in the 140 and 282 mg/Lcobalt treatments compared to 30 mg/L. This result combined withthe more extreme bleaching at the higher concentrations, suggestthat the higher cobalt concentrations induced more extremebleaching, and hence more effective detoxification, as was alsoseen in for cadmium. Severe effects of cobalt on algae have beenreported (El-Sheekh et al., 2003) and cobalt was the only metal toinduce an azooxanthellate state (Fig. 2E), although extremebleaching A. pulchella also occurred in cadmium treatments.Severe bleaching was observed at much lower cobalt concentra-tions than caused anemone mortality, indicating that A. pulchellaare particularly efficient at utilising zooxanthellae expulsion as ameans of cobalt detoxification, or that cobalt causes severedamage to zooxanthellae and/or disruption of the intracellularsymbioses itself. In addition, higher mortality at 551 mg/L cobalt(80 percent) compared to 820 mg/L (65 percent) at the end of therecovery period further suggests that zooxanthellae loss, whichwas greater at the higher concentration, was somewhat protectivefor the host. El-Sheekh et al. (2003) reported increased growth inthe algae Monoraphidium minutum and the diatom Nitzschiaperminuta during exposure to similar cobalt concentrations asused here (El-Sheekh et al., 2003), and in this study it is alsopossible that excessively rapid zooxanthellae growth resulted intheir expulsion from the host A. pulchella.

Toxicity estimates for A. pulchella lacerate development areamong the lowest available EC50 values for copper for a range ofmarine species (Table 3), and are more sensitive than toxicityestimates based on 28-d asexual reproduction in this species(Howe et al., unpublished data). Lacerate development was com-pletely inhibited at18 mg/L copper, and concentrations Z6 mg/Ldelayed development and caused several other toxic effects. Thehigh toxicity of copper to marine organisms is well documented,and the sub-lethal toxicity estimates for A. pulchella are relevant inthe context of other toxicological data for copper (Table 3).

Some hormesis was evident in A. pulchella lacerates exposed to18 mg/L copper in the early stages of the test (data not shown), aswas observed in the coral Goniastrea retiformis where higherfertilisation rates were reported at 50 and 100 mg/L coppercompared to 20 mg/L (Reichelt-Brushett and Harrison, 2005).Within 14 d, the majority of A. pulchella lacerates exposed to18 mg/L copper had completely retracted tentacles (Fig. 2J), whichis a common response to contaminants in anemones (Howe et al.,in press; Main et al., 2010; Mitchelmore et al., 2003) and corals (e.g. Negri et al., 2005). Tentacle retraction may provide someprotection during times of oxidative stress by reducing lightexposure (Negri et al., 2005), or may be due to retraction ofintracellular zooxanthellae (Ladriere et al., 2008a).

Bleaching was evident by the discoloration of A. pulchellatentacles, particularly at the tips (Fig. 2K and L), and zooxanthellaeaggregations in test containers (Fig. 2H and H), as was alsoobserved in A. pallida following copper exposure (Main et al.,2010). However, bleaching was not as common or as severe incopper-exposed A. pulchella compared to cobalt or cadmiumtreatments in this study, and many dead anemones retainedconsiderable intracellular zooxanthellae densities, as was alsoobserved in Anthropleura elegantissima (Mitchelmore et al.,2003). This suggests that these hosts are more sensitive to copperthan the symbiont or the symbiosis.

Tentacle development was observed in A. pulchella laceratesfrom the r6 mg/L copper treatment during the 7-d recovery

P.L. Howe et al. / Ecotoxicology and Environmental Safety 100 (2014) 138–147144

period. During this time progressive en masse zooxanthellae losswas also observed, which is a common detoxification strategyamong symbiotic cnidarians (Harland and Nganro, 1990; Mainet al., 2010; Mitchelmore et al., 2003). There is evidence ofheterotrophy in A. pulchella zooxanthellae (Steen, 1986), and ahigher copper sensitivity of zooxanthellate compared to azoox-anthellate anemones (Main et al., 2010), suggesting that bleachingmay be an energy efficient way to reduce metal stress within theanemone.

Similarly to cobalt and cadmium, the 14-d EC50 value for nickelis higher than at 8 d because although lacerate development at 8 dwas delayed compared to controls, by 14 d many lacerates hadreached a juvenile stage. The 8-d EC50 for inhibited A. pulchellalacerate development shows a similar sensitivity to nickel asasexual reproduction in this species, for which a 28-d EC50 of552 mg/L was estimated to reduce the number of asexuallyreproduced offspring by 50 percent (Howe et al., unpublisheddata). Bloating during nickel exposure suggests a disruption inionic balance (Fig. 2D and E), and is a common cnidarian stressresponse (Dunn et al., 2002).

This study shows that A. pulchella lacerate development is lesssensitive to nickel than fertilisation in Goniastrea aspera (5.5-h EC50of 100 mg/L; Reichelt-Brushett and Harrison, 2005). Some endpointsin marine organisms are more sensitive to nickel, such as the abaloneHaliotis rufenscens (48-h EC50 value of 146 mg/L for larval develop-ment and 22-d EC50 value of 26 mg/L for juvenile growth; (Huntet al., 2002), and the mysid Mysidopdis intii (28-d LC50 of 22 mg/L;(Hunt et al., 2002). Conversely, Goh (1991) determined a LOEC of1000 mg/L nickel for effects on Pocillopora damicornis larval settle-ment (Goh, 1991), showing a lower sensitivity than A. pulchellalacerate development. The inhibited lacerate development duringexposure to 11 and 57 mg/L nickel compared to responses at 106 and201 mg/L, suggests that detoxification mechanisms were more effi-cient at the higher concentrations (Fig. 1E), as also suggested forcadmium and cobalt. The wide 95 percent confidence limits (Table 2)show that the 14-d EC10 for nickel should be treated with cautionand further tests are recommended.

Zinc was at least an order of magnitude less toxic to A. pulchellalacerates than copper, of similar toxicity to cadmium (because ofsimilar chemistries; McConchie et al., 1988) and cobalt, and moretoxic than nickel (Table 2). The 8-d EC50 of 269 mg/L illustrates asimilar sensitivity to zinc as the EC50 value of 278 mg/L estimatedfor inhibition of asexual reproduction in this species (Howe et al.,unpublished data),. However, zinc was the only tested metal thatelicited a more typical time-dependant toxic effect and the 14-dEC50 of 166 mg/L is more sensitive than at 8 d (Table 2). Whilst the14-d LC50 for zinc (251 mg/L) is lower than for cadmium (396 mg/L),the order of toxicity is reversed for the 8-d EC50 values for laceratedevelopment (Table 2). However, the same zinc and cadmiumconcentrations (166 mg/L) were estimated to inhibit developmentin 50 percent of lacerates after 14-d (EC50 values; Table 2).In general, a similar toxicity of zinc and cadmium was seen,although zinc caused higher mortality and did not delay laceratedevelopment to the same extent as cadmium.

The effects of zinc on A. pulchella test differ to several tests on coralearly life-stages, where zinc was considerably more toxic thancadmium. For example, Acropora tenuis fertilisation success wasinhibited at 10 mg/L zinc, (Reichelt-Brushett and Harrison, 2005),whilst for cadmium 450 percent fertilisation success occurred atconcentrations up to 10,000 mg/L (Reichelt-Brushett and Harrison,2005; Table 3). Growth rates of zooxanthellae isolated from the coralMontipora verrucosa were significantly inhibited following 8-d expo-sure to 100 mg/L zinc (Goh and Chou, 1992), also showing a higher zincsensitivity than A. pulchella.

A. pulchella lacerate survival and development were completelyinhibited at zinc concentrations of 592 and 323 mg/L respectively

(Fig. 1F). This shows a higher sensitivity than fertilisation successin Goniastrea aspera, where no significant effect was reported at500 mg/L zinc (Reichelt-Brushett and Harrison, 1999). In the coralPlatygyra ryekyuensis, 55 percent fertilisation success occurredduring exposure to 4500 mg/L zinc (Heyward, 1988), also showinga lower sensitivity than A. pulchella lacerate development. Overall,sub-lethal effects of zinc on A. pulchella lacerate development arerelevant in the context of toxicity data for a range of other taxa(Table 3).

4.2. Quality assurance and control

This study suggests that reasonably strict quality control (QC)criteria may be used in toxicity tests with A. pulchella. Mortalityand/or exhibition of a defined sub-lethal effect in a small percen-tage of the test population in control treatments (e.g. 10 percent) isconsidered acceptable in standardised toxicity test protocols (e.g.OECD, ASTM, U.S.EPA). However, no control mortality occurred inthis study, or in previous toxicity tests with this species (Howeet al., 2012; Howe et al., in press; Howe et al., unpublished data).Therefore, it is recommended that results from toxicity tests usingA. pulchella are omitted from analysis if any control mortalityoccurs.

The normal development of all lacerates in controls treatmentsto a juvenile stage within 8 d suggests that it may be appropriateto use zero control response as a QC criterion. However, variabilityin the early parts of the tests suggests that a slightly longerdevelopmental time may not necessarily indicate toxicity, andfurther tests are needed to determine a suitable criterion fordevelopment time variability. It is possible that control variabilitywas due to differences in test organism age at test commence-ment; lacerates were selected on the basis on having no tentaclesbut the exact time since separation from the parent tissue wasunknown and may have varied by several days. The use oflacerates which have started to develop tentacles (e.g. with 1–2tentacles) is worthy of consideration. The increased variability indevelopment observed with exposure to increasing metal concen-trations may also be a reliable indicator of stress, as has previouslybeen suggested (Underwood, 1989). There is also potential torefine the endpoint used in this study and develop test methodswhich assess the contaminant concentration estimated to increasethe time taken for development to a juvenile stage.

Monitoring of water quality throughout this study showed thatpH variations up to 0.5 of a unit, and increases of up to 15 percent(8 mS/cm) in electrical conductivity caused no observable effectson controls. Therefore, it is recommended that pH ranges between7.9 and 8.4, and a range in electrical conductivity between 51.0 and61.0 mS/cm are used as preliminary water quality criteria intoxicity tests using A. pulchella. The DO concentration remainedabove 95 percent saturation (7.6 mg/L), similar to reported in otherstudies with this species (Howe et al., 2012; Howe et al., in press;Howe et al., unpublished data). Hence, 490 percent DO saturationmay be appropriate for QC control of test solutions. In addition,copper has been illustrated as a suitable reference toxicant intoxicity tests with A. pulchella (Howe et al., 2012), and could betested along all future tests to provide assurance of consistent testorganism health.

The change in dissolved metal concentrations after 48 h in testconditions (Table 1) is typical of static-renewal toxicity tests. Staticconditions create the potential for both increases and decreases incontaminant concentrations. For example, metal concentrationsmay vary due to absorption to test containers, evaporation of testsolutions, or an accumulation of metals in test containers due toincomplete removal of test solutions between renewals. These poten-tial effects may be minimised, for example by using test containerswith a larger volume, by topping up solutions with pure water to

P.L. Howe et al. / Ecotoxicology and Environmental Safety 100 (2014) 138–147 145

compensate for evaporative losses, or by transferring test organismsinto new test containers at each solution renewal. Variability indissolved metal concentrations by up to 20 percent is accepted instandard test protocols (e.g. OECD) and this criterion is consideredappropriate in tests using A. pulchella.

4.3. Directions for future research

The focus of this study was to ascertain whether A. pulchellalacerate development was affected by exposure to relevant con-centrations of a range of trace metals in the context of data forother species. However, it is important that to gain an under-standing of the reliability of results and it is recommended thatthe tests are repeated several times. The suitability of A. pulchellafor laboratory culturing and the large numbers of test organismsthat can reliably be produced allows for a large number ofreplicates, and the effect of increased replication on test resultsalso warrant further study.

The lack of mortality and the normal development of alllacerates in control treatments within 8 d show the suitability ofA. pulchella lacerates for use in chronic static-renewal toxicitytests. This study illustrates the potential to develop other chronicsub-lethal toxicological endpoints with this species. For example,tests could be developed which investigate population effects oflong-term contaminant exposures over multiple generations, orassess metal accumulation and uptake in host tissue and zoox-anthellae after long-term exposure to environmentally-relevantconcentrations.

5. Conclusions

This work responds to the urgent need to develop sub-lethalchronic toxicity tests using tropical marine species. The sensitivityof A. pulchella lacerate development to trace metals is demon-strated with estimated 8-d EC50 values of 55 mg/L for cadmium,262 mg/L for cobalt, 5 mg/L for copper, and 269 mg/L for zinc.All controls had developed to a juvenile stage within 8 d, andfuture tests can hence be reduced to improve the practicality ofusing this test as part of routine toxicological risk assessment. Thischronic sub-lethal toxicity test is simple, low cost and sensitive tothe tested trace metals. Overall, the results support the use ofA. pulchella in tropical marine ecotoxicology. Refinement andstandardisation of the test methods would contribute a suspension-feeding, zooxanthellate cnidarian to the limited number of tropicalmarine species currently used in toxicity testing and water qualityguideline derivation relevant to tropical marine environments.

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