Investigating lethal and sublethal effects of the ... - CSIRO Publishing

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Marine and Freshwater Research, 2014, 65, 551–561 http://dx.doi.org/10.1071/MF13195

Investigating lethal and sublethal effects of the trace metals cadmium, cobalt, lead, nickel and zinc on the anemone Aiptasia pulchella, a cnidarian representative for ecotoxicology in tropical marine environments Pelli L. Howe A, Amanda J. Reichelt-Brushett A,B and Malcolm W. Clark A A

Marine Ecology Research Centre, School of Environment, Science and Engineering, Southern Cross University, PO Box 417, Lismore, NSW 2480, Australia. B Corresponding author. Email: [email protected]

Abstract. The zooxanthellate sea anemone Aiptasia pulchella is found throughout the tropical and subtropical oceans of the Indo-Pacific and is easily maintained in aquaria, posing potential suitability as a standard tropical marine test organism for use in ecotoxicology. To gain an understanding of the sensitivity of A. pulchella to trace metals, 96-h static-renewal toxicity tests were conducted. Values of 96-h LC50 between 946 and 1196 mg L 1 were estimated for cadmium, between 595 and 1146 mg L 1 for zinc, 8060 and 12 352 mg L 1 for lead and 2209 and 5751 mg L 1 for nickel. In addition, preliminary assessment of rapid tentacle retraction was made. Six-hour EC50 values of 355 and 979 mg L 1 for cadmium, between 384 and 493 mg L 1 for zinc, between 2340 and 2584 mg L 1 for nickel, and 2610 mg L 1 for lead, were estimated for ‘severe’ tentacle retraction. Cobalt concentrations up to 1547 mg L 1 caused extreme zooxanthellae loss, but no more than 10% mortality and no rapid ‘severe’ tentacle retraction. The present study has provided important baseline information, enabling comparison of the acute sensitivity of A. pulchella to trace metals with other marine invertebrates, and guiding the development of sublethal endpoints. Additional keywords: cnidaria, toxicity tests, trace metals, tropical marine ecotoxicology. Received 22 July 2013, accepted 18 October 2013, published online 7 May 2014

Introduction The majority of marine ecotoxicological data used to derive water-quality guidelines comes from toxicity tests on temperate organisms (Markich and Camilleri 1997; ANZECC and ARMCANZ 2000; van Dam et al. 2008). Consequently, the level of protection from anthropogenic contaminants granted to ecologically and economically critical tropical marine environments is potentially unreliable (e.g. van Dam et al. 2008). The use of temperate data in risk assessment in tropical marine ecosystems continues despite several decades of recommendations from the scientific community (Markich and Camilleri 1997; Peters et al. 1997; van Dam et al. 2008), and evidence of the unreliability of extrapolating toxicity data from one climatic region to another (Chapman et al. 2006; Kwok et al. 2007; van Dam et al. 2008). As human activity in tropical regions intensifies, so does the need to develop reliable, sensitive and regionally relevant risk-assessment tools for tropical marine environments (van Dam et al. 2008; Reichelt-Brushett 2012). In 1997, it was concluded that there was ‘insufficient’ metaltoxicity data for tropical organisms in Australia, and several metals, including those tested in the present study, were identified to be of primary concern for aquatic ecosystems in northern Australia (Markich and Camilleri 1997). Journal compilation Ó CSIRO 2014

Cadmium, cobalt, lead, nickel and zinc pollution is of concern to the health of marine environments because of the wide use and distribution of such metals, along with their notable toxicity (Markich and Camilleri 1997; ReicheltBrushett and Harrison 2005; Negri and Hoogenboom 2011). Cadmium is largely associated with zinc extraction and phosphate fertilisers (Loganathan and Hedley 1997). Hence, cadmium is of particular concern for marine environments that receive runoff from agricultural and mining operations, the scale and frequency of which are increasing in many developing countries in the tropics (Reichelt-Brushett 2012). Zinc and copper both have sources from marine activities, namely sacrificial anodes in the case of zinc, and antifoulant products in the case of copper. Additionally, terrestrial sources, including mining, industrial, urban and/or agricultural activities, can result in the deposition of these metals, along with lead and cobalt, into the marine environment. Of further concern in regard to metal pollution is that toxicity has been shown to increase with temperature for some species (Chapman et al. 2006; Kwok et al. 2007), making use of temperate toxicity data for these metals unreliable for risk assessment in the tropics. With predictions of global warming, there is further concern that metal exposure may results in an increased vulnerability of www.publish.csiro.au/journals/mfr

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marine organisms to increased sea-surface temperatures (Negri et al. 2011; Negri and Hoogenboom 2011). Marine cnidarians (including corals, anemones, jellies and hydra) are not represented in standard toxicity-test protocols (e.g. OECD and US EPA), despite their high sensitivity to a range of contaminants (e.g. Thompson et al. 1980; ReicheltBrushett and Harrison 2005; Negri and Hoogenboom 2011) and their wide distribution in tropical marine waters (Veron 1986). Furthermore, contaminant toxicity may act synergistically, additively or antagonistically on these organisms in combination with global stressors such as increased sea-surface temperatures (Negri and Hoogenboom 2011). The primary hindrance in developing routine coral toxicity tests is the difficulties in culturing sufficient numbers of test organisms. Work continues to overcome this (e.g. Vijayavel and Richmond 2012); the majority of coral toxicological work has used wildcollected specimens (e.g. Heyward 1988; Goh 1991; ReicheltBrushett and Harrison 1999; Reichelt-Brushett and Harrison 2004; Negri and Hoogenboom 2011), which is not a suitable method for routinely acquiring test organisms. The zooxanthellate anemone Aiptasia pulchella (Carlgren 1943) has been used in laboratory research for several decades (e.g. Muller-Parker 1984; Chen et al. 2008), and fulfills much of the criterion required of a standard laboratory test species for ecotoxicological study (Howe et al. 2012). Previous work showed a reasonably high sensitivity of A. pulchella to copper in acute and chronic tests (Howe et al. 2012). A. pulchella has a wide geographic range throughout the tropical and subtropical Indo-Pacific, and is therefore a potential representative species of this geographic area. A. pulchella is easily cultured in large numbers in a range of nutritional and symbiotic states (MullerParker 1984; Howe et al. 2012), and has intracellular associations with zooxanthellae of Clade B1, a clade associated with some reef-building corals (Jokiel and York 1982). The aims of the present study were to (1) assess the acute sensitivity of A. pulchella to a range of trace metals that have not been previously investigated using this species, (2) assess tentacle retraction as a potential rapid toxicity assessment tool, and (3) observe other common toxicological responses in A. pulchella that may be useful sublethal endpoints. Materials and methods Test organisms and rearing conditions Fifty A. pulchella individuals were obtained from the flowthrough seawater system at the National Marine Science Centre, Charlesworth Bay, New South Wales (NSW), Australia, and maintained as brood stock at Southern Cross University, Lismore, NSW, Australia, for 6 months before the study. A. pulchella individuals were cultured in 160-L aquaria in natural seawater at 25 28C, and were provided with a 12-h : 12-h photoperiod (50–60 mE m2 s 1) using 30-W white (Aquastar, Sylvania ) and blue (Coralstar, Sylvania) fluorescent aquarium lighting. Weekly 10% water exchanges were completed with freshly collected seawater and twice a week, anemones were provided with commercially produced adult Artemia sp. (AquaOne) as a food source, in addition to the food available in natural seawater during weekly water exchanges. One week before the commencement of each test, asexually produced

P. L. Howe et al.

offspring from brood anemones with a pedal disc diameter between 1 and 2 mm were collected from culture tanks and placed in separate acid-washed and seawater-aged 75-mL polyethylene test containers filled with natural seawater. Test containers were randomly placed in water baths at 25 18C and provided with a 12-h : 12-h photoperiod during a 7-day acclimation period, during which seawater was completely exchanged every 48 h. Test solutions Stock solutions (500 mg L 1) of cadmium, nickel and lead were prepared using Analar grade CdCl2, NiCl2, and Pb(NO3)2 (99% purity, Sigma-Aldrich) in reverse-osmosis water (milli-Q water delivered at 18.2 MOhms cm 1). For zinc and cobalt, Analar grade 1000 mg L 1 standards (99% purity, Sigma-Aldrich) were used as stocks. All test solutions were freshly prepared using stock solution and natural seawater at test commencement and for each 48-h renewal treatment. Each initial metal dose was subsampled, filtered to 0.45 mm, and analysed using inductively coupled plasma mass spectrometry (ICP–MS) as per American Public Health Association (APHA) Method 3120 for analysing metals (1 mg L 1 detection limit) at the Environmental Analysis Laboratory at Southern Cross University. Although there were limitations to the extent of analyses that could be completed in the study, preliminary assessment showed that reductions in metal concentrations during each 48 h, which are typical in static renewal tests, were largely offset by evaporation at test temperatures of 258C. Toxicity tests Five replicates of each metal treatment were used in one to three static-renewal range-finding tests on each metal. Five metal concentrations and a seawater control were used in all tests (except cobalt; four concentrations), with an individual preacclimatised anemone in each test container. For each replicate, 75-mL polyethylene test containers were used. The sensitivity of A. pulchella to each metal was also tested at least once using 10 replicates, and a single 20-replicate zinc test was conducted to determine the appropriate level of replication. Test durations were generally 96 h; however, where low mortalities occurred within 96 h in some tests using cobalt, nickel and lead toxicants, the tests were extended to 144 h, to allow observations after a longer exposure and to guide future studies. The pH, electrical conductivity and temperature of test solutions were measured using a Cyberscan PC300 (Eutech) combined meter at mid-day every 24 h. Electrical conductivity was converted to salinity. Dissolved oxygen was measured using a YSI meter (Xylem Analytics) at test commencement and at 96 h. Mortality and tentacle retraction were recorded at 0, 1, 6, 12, 18 and 24 h, and every 24 h thereafter. Mortality was recorded if no movement was observed under stereo microscopy (SZ51, Olympus) in response to gentle stimuli. A. pulchella were recorded as either normal or ‘severely’ retracted, where ‘severe’ retraction was recorded when tentacles were of equal or lesser length than the oral-disc diameter, measured using a transparent ruler (e.g. Fig. 1a, b). Various degrees of ‘mild’ to ‘moderate’ retraction were observed in A. pulchella during these experiments; however,

Aiptasia pulchella responses to metals


(a) Controls 1 : 1

553

‘Severe’ tentacle retraction 1 : 1 (b)

(c)

1000 µm

1000 µm

(d)

(e)

1000 µm

1000 µm

(f )

(g)

1000 µm

1000 µm

1000 µm

Fig. 1. Examples of (a) healthy Aiptasia pulchella and (b–g) severely retracted Aiptasia pulchella. ‘Severe’ tentacle retraction was defined by the ratio of the mean length of the three longest tentacles to the diameter of the oral disc as illustrated in (a) and (b). Note in (a) the newly-produced juvenile (circled).

these were not used as part of the data analyses because of difficulties associated with quantifying the extent of the retraction. Preliminary experiments illustrated that A. pulchella individuals that were retracted to the degree defined here as ‘severe’ were considerably or completely inhibited in their ability to feed heterotrophically (i.e. very slow or negligible response to food). Furthermore, once transferred to natural seawater following the experiments, no recovery of normal appearance (e.g. colouration, tentacle length) was observed for up to 2 weeks after metal exposure. A. pulchella recorded as ‘severely’ retracted in the present study had limited potential for full recovery. Furthermore, observations during our work with this species suggest that asexual reproduction is inhibited in completely retracted anemones. In addition, observations of various other responses were recorded during the toxicity tests and highlighted as potential future endpoints, including en masse zooxanthellae and/or pigment loss and associated tissue bleaching, and inhibited asexual reproduction and subsequent offspring development. Data analysis Measured metal concentrations were used in all data analysis. The linear interpolation method (ICp; US EPA 2002) was used to calculate the metal concentrations estimated to be lethal to

50% (lethal concentration, 50%; LC50) of the test population after 24-, 48- and 96-h exposure, and 95% confidence limits. Additionally, concentrations predicted to cause a low effect within 96 h were estimated (96-h LC10; lethal concentration, 10%). The ICp method was also used to calculate sublethal effective concentrations (effective concentration, 50%; EC50, effective concentration, 10%; EC10) and 95% confidence limits for ‘severe’ tentacle retraction after 1-, 6- and 12-h exposure. All data analysis was conducted using Toxcalc version 5.0 (Tidepool Scientific Software, McKinleyville, CA, USA, 1996). Results All organisms in control treatments remained healthy throughout each experiment. Asexual reproduction was commonly observed in controls in experimental conditions within 96 h and the rate of reproduction and the condition of asexually reproduced offspring tended to decrease with increasing metal concentration, although this was not quantified in the present study. Physico-chemical water-quality parameters remained within ranges suitable for organism health and survival in all treatments. The pH ranged between 7.93 and 8.38, electrical conductivity varied between 52.6 and 59.1 mS cm 1 (34.7–39.5 salinity), the concentration of dissolved oxygen was between 7.79 and 8.39 mg L 1, and the temperature was maintained at

479 (164–591) 384 (98–596) 327 (209–556) 1000 (875–1160) 446 (312–867) ** 574 (NC) 448 (NC) 256 (135–287) 3280 (2910–3900)

*

5070 (4720–5340) 2580 (2210–3010) 2380 (2090–2670) *

3490 (NC) 2340 (459–4060) 1430 (1260–2800) **

**

355 (282–566) 276 (249–355)

**

3540 (1860–9650) 2610 (1860–9650) 1740 (1310–3850) * **

12-h EC50

6-h EC50

1260 (1140–1420) 979 (446–1290) 711 (535–1420) ‘Severe’ retraction 1-h EC50

Mean 96-h LC50

96-h LC50

2690 (2350–7120) **

5750 (NC) 12 400 (NC) 1 (49.4 mM) 8060 (0–11 700) 10 230 mg L 1196 (NC)

* * 48-h LC50

**

829 (766–937) 493 (369–639) ** 785 (719–810) **

1120 (NC) 1150 (NC) *

* * * * *

Test 4 (10)

* *

1000 (922–1460) * 856 (717–1181) * 595 (238–817) 955 mg L 1 (14.6 mM) * *

4700 (NC) * 3290 (3090–5050) * 2210 (1380–3790) 3980 mg L 1 (67.9 mM) * * * *

1940 (1760–2250) 1540 (1440–1790) 950 (590–1290) 1040 mg L 1 (9.3 mM) 1200 (1190–1290) 1200 (1190–1290) 979 (NC)

Lethal responses of Aiptasia pulchella to selected metals The overall toxicological responses of A. pulchella to the tested trace metals were time- and concentration-dependent. In terms of the average molar concentrations of metals, the lethality to A. pulchella after 96-h exposure decreased from cadmium $ zinc . lead . nickel (Table 2). Table 2 also provides the calculable 24-, 48- and 96-h LC50 values for these metals. However, no LC50 values could be calculated for cobalt as .50% mortality did not occur during exposure for up to 144 h at concentrations up to 1547 mg L 1. Cobalt concentrations between 38 and 1547 mg L 1 caused very minimal mortality of A. pulchella at 96 h, with only 10% mortality occurring at the second highest concentration (629 mg L 1) in Test 2. However, some additional mortality occurred with a longer exposure time; 20% at 1547 mg L 1 (Test 1), 629 mg L 1 and 1231 mg L 1 (Test 2) at 144 h. Values of 96-h LC50 of 946, 979 and 1196 mg L 1 were estimated for cadmium in three separate tests using five or 10 replicates per dose (Table 2). The 96-h LC50 values for lead exposure of 8060 and 12 352 mg L 1 were estimated and, in this instance, there was some variability in the LC50 values between separate tests using 5 or 10 replicates. Lead caused A. pulchella mortalities at concentrations $688 mg L 1 after 96-h exposure. In Test 1 (5 replicates), 60% mortality had occurred at 5070 mg L 1 at 96 h, whereas no mortality had occurred at 9460 mg L 1 at 96 h. The percentage mortality was lower in Test 2 (10 replicates), where between 20% and 60% mortality had occurred at 96 h at concentrations between 5093 and 15 530 mg L 1 lead. Similar variability was noted in the 96-h LC50 values for nickel exposure between tests (2210 and 5750 mg L 1 for Tests 2 and 3, respectively; Table 2). Because of a difference in dose range (Table 1) and slightly different organism response, no LC50 values could be estimated from the results of Test 1. The studies using zinc were completed with 5, 10 and 20 replicates for each treatment

24-h LC50

25 18C. Measured dissolved metal concentrations are in Table 1.

Zinc

1525 2775 1363 * * 12 730 15 530 5211 5061 5922 611 1551 896 1209 1167

Test 3 (5)

1209 2041 1196 1547 1231 10 070 12 352 1766 4343 5066 536 1221 665 957 986

Test 2 (5)

1176 1541 1050 762 629 7390 9647 859 3305 4489 360 1042 442 874 924

Test 1 (5)

781 1263 807 403 329 3890 5093 444 3258 3258 193 792 211 746 832

Test 3 (10)

361 946 497 42 38 688 2610 4 2651 1505 88 714 106 612 591

Test 2 (5)

0 0 0 0 0 1 0 0 2 2 0 0 0 11 0

Nickel

1 2 3 1 2 1 2 1 2 3 1 2 3 4 5

Test 1 (5)

5

Test 2 (10)

4

Lead

Zn

3

Test 1 (5)

Ni

2

Test 3 (10)

Pb

1

Test 2 (10)

Co

Control

Cadmium

Cd

Test no.

Test 1 (5)

Metal

Toxicity estimate

Table 1. Measured dissolved metal concentrations (lg L21) for test solutions used in repeated toxicity tests on Aiptasia pulchella. * four cobalt concentrations were tested.

*

P. L. Howe et al.

Test 5 (20)


Table 2. Toxicity estimates (LC50 and EC50 values; lg L-1) for different exposure times during repeated toxicity tests exposing Aiptasia pulchella to selected trace metals The values in parentheses following the test number are the number of replicates used in each test. 95% confidence limits are in parentheses following LC and EC values. *Not calculable because of low (,50%) effect; **not calculable because of 100% effect; NC, confidence limits not calculable

554



(a) 2500

(b)

Cadmium

555

Lead

12 000 2000 9000 1500 6000

Mean LC/EC50 (µg L1)

1000

3000

500 0

0 0

12

24

36

(c)

48

60

72

84

96

0

(d )

Nickel

12

24

36

1500

48

60

72

84

96

Zinc

6000 1000 4000 500

2000

0

0 0

12

24

36

48

60

72

84

96

0

12

24

36

48

60

72

84

96

Hours of exposure Fig. 2. Mean 1-, 6- and 12-h EC50 values for ‘severe’ tentacle retraction ( ) and mean 24-, 48- and 96-h LC50 values ( ) for Aiptasia pulchella during exposure to: (a) cadmium, (b) lead, (c) nickel, and (d ) zinc. Error bars show the standard deviation. The absence of error bars indicates that only a single estimate was calculable at that assessment time.

and respective LC50 values 595, 1150 and 1120 mg L 1 were estimated (Table 2), showing that both 10 and 20 replicates resulted in very similar LC50 values. These results highlight the importance of defining suitable replication. Sublethal responses to selected metals Tentacle retraction (Fig. 1) was a common and rapid response in A. pulchella during exposure to all the tested metals except cobalt, where ‘severe’ retraction was not observed until after 72-h exposure. Fig. 2 shows that the mean 6- and 12-h EC50 values for cadmium, lead, nickel and zinc were consistently lower than the mean 24-, 48- and 96-h LC50 values, highlighting the sensitivity of tentacle retraction as an endpoint. After 24-h exposure to cadmium, nickel and zinc, all A. pulchella individuals were ‘severely’ retracted (e.g. Fig. 1b–g), whereas controls in each of these experiments were consistently healthy and maintained extended tentacles (e.g. Fig. 1a). The early 1-h EC50 value of 1262 mg L 1 for cadmium was similar to the 24-h LC50 value of 1196 mg L 1 (Table 2). The 6-h and 12-h EC50s for ‘severe’ retraction were somewhat lower, and some variability was noted between tests (Table 2, Fig. 2a). In addition to these measured responses, $80% of surviving anemones exposed to cadmium concentrations between 497 and 1525 mg L 1 exhibited en masse zooxanthellae loss within 48 h. This was evident by zooxanthellae aggregations in test containers, and the bleaching of anemone tissue, which were not observed in controls, or in the 361 mg L 1 cadmium treatments. Tentacle retraction was observed in all lead treatments after only 1-h exposure, resulting in mean 1-h EC50s values that were

lower than the mean 96-h LC50 values (Fig. 2b, Table 2). In addition, all surviving A. pulchella individuals appeared completely bleached by 144-h exposure. When this test was repeated using a slightly higher concentration range (Table 1), retraction was similarly rapid (within 1 h) at lead concentrations between 3890 and 12 730 mg L 1. En masse zooxanthellae loss, similar to the one observed for cadmium-exposed anemones, occurred within 6-h exposure. By 96 h, en masse zooxanthellae loss was observed in between 80% and 90% of A. pulchella individuals exposed to lead concentrations .688 mg L 1 in all three tests. Often a thick zooxanthellae aggregation was expelled when these anemones were very gently prodded. The EC50 values for nickel were similar to LC50 values after longer exposures, specifically the 6- and 12-h EC50 values were similar to the 48- and 96-h LC50 values (Table 2, Fig. 2c). In Tests 1 and 2 (5 replicates), ‘severe’ retraction occurred within 1-h exposure to 5211 and 5061 mg L 1 nickel, respectively, and within 24 h in all A. pulchella individuals exposed to concentrations $1766 mg L 1. In addition, 50% of A. pulchella individuals exhibited thin, dark and limp column and tentacles within 6-h exposure to .5000 mg L 1 nickel, which was unique to nickel tests. Zinc also caused ‘severe’ and, in many cases, complete tentacle and column retraction (e.g. Fig. 1). The 1-h EC50 values for ‘severe’ retraction were similar to 24-h LC50 values and the 6- and 12-h EC50 values were much lower (Table 2, Fig. 2d). A. pulchella individuals exposed to zinc concentrations of .442 mg L 1 also exhibited darkened tentacle tips, single limp tentacles and occasionally en masse zooxanthellae loss within 6 h, and at higher concentrations within 1 h. Similar to

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nickel, and in contrast to cadmium, lead and cobalt exposures, the majority of dead zinc-exposed anemones retained zooxanthellae and maintained a dark appearance, which was exacerbated by tissue and tentacle retraction. There was limited tentacle retraction observed after shortterm cobalt exposure (i.e. 1–12 h). However, in cobalt Test 2, between 70% and 100% of A. pulchella individuals exposed to concentrations of $38 mg L 1 were completely retracted after 96-h exposure, and a 96-h EC50 of 27 mg L 1 was estimated for ‘severe’ retraction. Furthermore, bloating and detachment of the pedal disc from test containers was observed after 24-h exposure and was observed in between 40% and 100% of A. pulchella individuals exposed to cobalt at concentrations of $629 mg L 1. In addition, $60% of anemones at all cobalt concentrations were extremely bleached by 96 h, often to the degree that intracellular zooxanthellae could not be seen using a stereo microscope. By 144 h, complete bleaching was observed in all surviving cobalt-exposed A. pulchella individuals.

Expelled zooxanthellae did not form aggregates around each anemone, like during the cadmium and lead exposures, rather the bleaching occurred more gradually by the loss of individual zooxanthellae. Discussion Effects of metals on the survival of Aiptasia pulchella Cadmium, lead, nickel and zinc caused considerable mortality of A. pulchella within 96-h exposure at concentrations that are relevant in the context of available LC50 toxicity data for tropical marine invertebrate species. These concentrations are several magnitudes higher than the concentrations in marine environments, which is typical of results of acute lethal toxicity tests. Table 3 provides data for a range of marine organisms from studies reported on in published literature and documented in the United States Environmental Protection Agency ECOTOX database (US EPA2013).

Table 3. Comparison of the 96-h LC50 values for cadmium, lead, nickel and zinc for Aiptasia pulchella from the present study with LC50 estimates from ecotoxicological studies on a range of tropical marine invertebrates NR, not reported Metal

Species

Type of organism

Life stage

Time (h)

LC50 (mg L 1)

Reference

Cd

Aiptasia pulchella Meretrix meretrix Gammarus aequicauda Hydroides elegans Amphiascus tenuiremis Portunus sanguinolentus Charybdis feriatus Portunus pelagicus Anadara granosa Donax faba Litopenaeus vannamei Penaeus monodon Penaeus indicus Varuna litterata Aiptasia pulchella Meretrix meretrix Penaeus monodon Hydroides elegans Perna viridis Goniastrea aspera Cerithedia cingulata Aiptasia pulchella Americamysis bahia Mysidopsis bigelowi Acartia pacifica Pocillopora damicornis Apocyclops borneoensis Tigriopus japonicus Aiptasia pulchella Haliotis diversicolor texta Hydroides elegans Litopenaeus vannamei Penaeus monodon Donax faba Perna viridis Anadara granosa

Anemone Mollusc Mollusc Annelid Crustacean Crustacean Crustacean Crustacean Mollusc Mollusc Crustacean Crustacean Crustacean Crustacean Anemone Mollusc Crustacean Annelid Mollusc Coral Mollusc Anemone Crustacean Mollusc Crustacean Coral Crustacean Crustacean Anemone Mollusc Annelid Crustacean Crustacean Mollusc Mollusc Mollus

1–2 mm Larva Adults Adults Adults Larva Larva Larva Spats Adults Post-larva Post-larva Adults NR 1–2 mm Larva Post-larva Adults Adults Adults Adults 1–2 mm Adults Juveniles Neonates Adults Adults Adults 1–2 mm Post-larva Adults Post-larvae Adults Adults Adults Spats

96 96 96 96 96 48 96 96 96 96 96 96 96 96 96 72 96 96 96 96 96 96 96 96 48 12 48 96 96 96 96 96 96 96 96 96

946–1196 68 100 230 224 250 250 430 940 990 1070 1720 2070 32 200 8090–12 400 353 410 946 8820 9890 15 507 2210–5750 508 634 2360 9000 13 050 26 560 595–1150 1200 1235 1350 2360 3650 6090 7760

Present study Wang et al. 2009 Prato et al. 2006 Gopalakrishnan et al. 2008 Greene et al. 1993, cited in US EPA 2013 Greenwood and Field 1993 Greenwood and Field 1993 Greenwood and Field 1993 Ong and Din 2001 Ong and Din 2001 Wu and Chen 2004 Rajkumar et al. 2011 McClurg 1984 Kalkarni 1983, cited in U.S. EPA 2013 Present study Wang et al. 2009 Rajkumar et al. 2011 Gopalakrishnan et al. 2008 Chan 1988 Reichelt-Brushett and Harrison 1999 Ramakritinan et al. 2012 Present study Gentile and Cardin 1982, cited in U.S. EPA 2013 Lussier and Walker 1985, cited in U.S. EPA 2013 Mohammed et al. 2010 Goh 1991 Mohammed et al. 2010 Mohammed et al. 2010 Present study Liao and Lin 2001 Gopalakrishnan et al. 2008 Wu and Chen 2004 Rajkumar et al. 2011 Ong and Din 2001 Chan 1988 Ong and Din 2001

Pb

Ni

Zn


Cadmium was more toxic to A. pulchella than to the tropical crustaceans Penaeus monodon (96-h LC50 of 1720 mg L 1; Rajkumar et al. 2011), Penaeus indicus (96-h LC50 of 2070 mg L 1; McClurg 1984) and Veruna litterata (96-h LC50 of 32 200mg L 1; (US EPA 2013); yet it was generally less toxic to A. pulchella than to tropical annelids, arthropods and molluscs (Table 3). The 96-h LC50 values for cadmium for A. pulchella showed a very similar sensitivity to early life stages of the crustacean Litopenaeus vannamei (96-h LC50 of 1070 mg L 1; Wu and Chen 2004), and the tropical bivalve molluscs Anadara granosa and Donax faba for which the LC50 were determined at 940 and 990 mg L 1, respectively (Ong and Din 2001; Table 3). Some tropical species are more sensitive to cadmium than A. pulchella include the annelid Hydroides elegans (96-h LC50 of 230 mg L 1; Gopalakrishnan et al. 2008), larvae of the crabs Carybdis feriatus (96-h LC50 of 250 mg L 1), Portunus pelagicus (96-h LC50 of 380 mg L 1) and Portunus sanguinolentus (48-h LC50 of 250 mg L 1) (Greenwood and Field 1983) and the mollusc Meretrix meretrix (96-h LC50 of 68 mg L 1; Wang et al. 2009). Aiptasia pulchella mortality was low during the early stages of the zinc tests, inhibiting the estimation of some LC50 values (Table 2). This is likely to have been due to different toxicological responses and potential detoxification mechanisms associated with essential metals (e.g. zinc), as opposed to non-essential metals (e.g. cadmium) (Pe´rez and Beiras 2010). However, similar concentrations of cadmium and zinc caused mortality after 96-h exposure (Table 2, Fig. 2a, d ). The 96-h LC50 values for zinc from the present study are within the range found in other toxicity studies using tropical marine invertebrates (Table 3). The tropical mollusc Haliotis diversicolor texta (Liao and Lin 2001), annelid Hydroides elegans (Gopalakrishnan et al. 2008) and crustacean Litopenaeus vannamai (Wu and Chen 2004) have LC50 values similar to the upper end of values determined for A. pulchella in the present study (Table 3). In general, the survival of A. pulchella was more sensitive to zinc than was the survival of other tropical marine invertebrates. Mortality of A. pulchella varied considerably among tests using nickel (Table 2, Fig. 2c). No other marine toxicity studies could be found in the literature in which the LC50 results of repeated tests using nickel were reported; thus, the repeatability of tests using nickel on other species is not clear. However, relatively high variability in fertilisation success was also reported in the coral Goniastrea aspera following nickel exposure (Reichelt-Brushett and Harrison 2005). Variability in replicate responses to nickel should be investigated further. It is notable that all the LC50 estimates for nickel for A. pulchella (Table 2, Fig. 2c) indicate that this species is more sensitive to nickel than are the coral Pocillopora damicornis (Goh 1991) and the tropical copepods Tigriopus japonicus and Apocyclops borneoensis (Mohammed et al. 2010; Table 3). However, the tropical mysids Americamysis bahia (96-h LC50 of 508 mg L 1) and Mysidopsis bigelowi (96-h LC50 of 634 mg L 1) and temperate species such as the mysid Mysidopsis intii (96-h LC50 of 149mg L 1) and the mollusc Haliotis rufenscens (96-h LC50 of 226 mg L 1) (Hunt et al. 2002) are much more sensitive to acute nickel exposure than is A. pulchella. Variability in A. pulchella survival was also observed in several tests using lead as the stressor (Table 2). Although these


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tests differed in their number of replicates and specific dose concentrations, this variable response is likely to have been largely induced by the precipitation of lead mostly as cerussite (PbCO3) from the high carbonate and bicarbonate buffer of seawater. The Ksp of cerussite at 258C is 7.40 10 4, which under ideal conditions allows a maximum lead concentration of 0.272 mM to remain in solution. The highest concentration used in these experiments of 15 540 mg L 1 (75 mM) is well above the calculated solubility maximum. Hence, variability in the dose– response (Table 2) is most likely from the ‘hit and miss’ of precipitated lead contacting an individual A. pulchella, particularly in the semi-static test conditions used in the present study. Precipitated lead may also be entering the gut of the anemone, where acidic conditions (Nicol 1959) could cause enhanced lead solubility and explain why the sublethal endpoint is relatively insensitive. There was lower mortality of A. pulchella in Test 2 of the lead-exposure studies (96-h LC50 of 12 352 mg L 1) than in Test 1, in which the 96-h LC50 value was 8060 mg L 1 (Table 2). However, even the less sensitive LC50 value for lead from Test 2 is within a range similar to that determined for the survival of larvae of the coral Goniastrea aspera, which was between 10 000 and 20 000 mg L 1 lead, depending on the age of larvae (Reichelt-Brushett and Harrison 2004), and more sensitive than survival of the mollusc Cerithedia cingulata (96-h LC50 value of 15 507 mg L 1; Ramakritinan et al. 2012). A. pulchella demonstrated a similar sensitivity to lead as did the mollusc Perna viridis (Chan 1988), whereas it was less sensitive than the crustacean Penaeus monodon, the polychaete Hydroides elegans and the mollusc Meretrix meretrix (Table 3). Tentacle retraction as a sublethal endpoint The ‘severe’ tentacle retraction endpoint used in the present study was developed from initial observations of the behavioural responses of A. pulchella to metal exposure during early tests on survivorship. Furthermore, tentacle retraction has previously been reported in copper-exposed A. pulchella (Howe et al. 2012) and A. pallida (Main et al. 2010). Several time points of 1, 6 and 12 h were assessed, and EC50 values for each of these times showed an increasing sensitivity with longer duration of exposure for all the tested metals (Table 2). The 6- and 12-h EC50 values determined in the present study for A. pulchella were more sensitive than 96-h LC50 values for cadmium, lead, nickel and zinc (Fig. 2), highlighting a rapid sublethal response to these metals. During the initial tests, various degrees of tentacle retraction were used to rank the severity of the response. However, these were not easy to quantify clearly into distinct categories and the decision to use the ‘severe’ retraction criteria (defined as when tentacles were of equal or lesser length than the oral disc diameter; Fig. 1a, b) provided a standardised endpoint for this behavioural response. This degree of tentacle retraction is likely to considerably reduce the absorption of photosynthetically active radiation (PAR) by zooxanthellae, and hence the nutrients provided to the host. In addition, observations showed that the ability to catch prey is considerably or completely hindered in A. pulchella in a ‘severely’ retracted state, by both a reduction in surface area and an observed considerable increase in the time

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taken to engulf prey, which could readily be quantified in future studies. Reduced nutrition during sustained retraction is likely to result in effects on growth and reproduction, and assessment of rapid tentacle retraction may be reliably predictive of chronic population-level effects. Further study is recommended to refine the endpoint, and to investigate whether reliable correlations can be made between tentacle retraction and more commonly used sublethal endpoints. Sublethal endpoints are valuable as they highlight impacts of pollutants on the test population that inhibit and/or affect the capacity an organism to function in a normal manner and therefore place the longevity and the long-term recruitment success of the individuals at risk. ‘Severe’ tentacle retraction provides an endpoint that is simple to assess, cost-effective and rapid, and which is also relatively sensitive compared with other sublethal endpoints identified in the literature for other marine invertebrates, including fertilisation success (Reichelt-Brushett and Harrison 2005), larval motility (Reichelt-Brushett and Harrison 2004), metamorphosis (Martin et al. 1981; King and Riddle 2001), cellular function (Gilbert and Guzman 2001) and growth (Martin et al. 1981). Tissue retraction is a cnidarian stress response that has been observed in several studies (e.g. Thompson et al. 1980; Hughes et al. 2005; Negri et al. 2005), and has been explored as a sublethal toxicological endpoint for corals (Thompson et al. 1980; Negri et al. 2005). Retraction may be a behavioural response to reduce the exposed tissue area, and/or to limit zooxanthellae photosynthesis by shading (Dykens and Shick 1984), and may result in tissue detachment and the escape of coral polyps from the skeleton (e.g. Negri et al. 2005). Although some studies have highlighted tissue retraction as an obvious stress response in corals, there are often difficulties associated with quantifying the degree of retraction or tissue thickness because the tissue retracts into the skeleton of corals and there are complications associated with the fact that tissue thickness varies with the lunar cycle and changes in light conditions (e.g. Rotmann and Thomas 2012). A. pulchella, like all anemones, does not have a skeletal structure and, in this species, tentacles are normally extended 24 h a day (P. Howe, A. ReicheltBrushett, pers. obs.). Therefore, tentacle length and the degree of tentacle retraction represent a reliable endpoint that can easily be measured and quantified. Sensitive and ecologically relevant sublethal toxicological endpoints using tropical marine species are notably lacking in the literature (van Dam et al. 2008) and the results of the present study show that ‘severe’ tentacle retraction in A. pulchella may be a valuable rapid endpoint for toxicological risk assessment. ‘Severely’ retracted A. pulchella individuals did not recover a normal appearance for up to 2 weeks after removal from the metal solutions, and it has been demonstrated that reproduction is reduced in A. pulchella when heterotrophic feeding is limited, with potential long-term effects on population structure (Howe et al. 2012). The lack of recovery of normal tentacle appearance observed in the present study contrasts with recovery in coral tissue retraction noted in Jones et al. (2003), which was reported to usually occur within hours. Further work on recovery would be valuable in determining whether a correlation can be made between ‘severe’ tentacle retraction during short-term metal-exposure and long-term health and

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reproduction, as well as understanding the biological relevance of this response. The 6-h EC50 values for zinc for A. pulchella are more sensitive than fertilisation success in the corals Goniastrea aspera (Reichelt-Brushett and Harrison 1999), Favites chinensis and Platygyra ryukyensis (Heyward 1988; Table 3). However, several studies have illustrated a much higher sensitivity of some symbiotic cnidarians to zinc. For example, exposure to only 10 mg L 1 zinc significantly inhibited fertilisation success in Acropora tenuis (Reichelt-Brushett and Harrison 2005). Also, the growth rate of zooxanthellae isolated from the coral Montipora verracosa was significantly reduced by a 96-h exposure to $100 mg L 1 zinc (Goh and Chou 1992). ‘Severe’ tentacle retraction in A. pulchella was a consistent response to cadmium and occurred within hours of exposure, resulting in the EC50 values shown in Table 2. Sublethal toxicity estimates based on fertilisation success showed that the urchin Diadema setosum (20-min EC50 of 6280 mg L 1; Thongra-ar 1997), and the corals Oxypora lacera (5.5-h EC50 of .1000 mg L 1; Reichelt-Brushett and Harrison 1999) and Acropora tenuis (5.5-h EC50 of .2000 mg L 1; Reichelt-Brushett and Harrison 2005), are less sensitive to cadmium than is A. pulchella survival and ‘severe’ tentacle retraction. The greater sensitivity of the rapid sublethal endpoint in A. pulchella than fertilisation success of other marine species for both cadmium and zinc indicated that these metals are interacting with cellular functions associated with more mature life stages. Indeed, the effects of some metals are seen at subcellular levels. For example, whereas tentacle retraction showed sublethal responses during nickel exposure in A. pulchella (EC50 values between 1430 and 3280 mg L 1; Table 2), the inhibition of carbonic anhydrase activity in the anemones Condylactis gigantea and Stichodactyla helianthus has been reported after exposure to only 40 mg L 1 nickel (Gilbert and Guzman 2001). Ideas for method development and future opportunities Chronic absence of symbionts may inhibit growth and reproductive success in cnidarians (e.g. corals; Cantin et al. 2007), and is recognised as an ecologically relevant toxicological endpoint worthy of further investigation (Jones 1997). Cobalt was the only metal tested in the present study that did not cause considerable mortality or rapid ‘severe’ tentacle retraction in A. pulchella at the tested concentrations. Survival and a lack of rapid tentacle retraction was observed despite extreme and often complete bleaching of $80% of A. pulchella individuals during exposure to cobalt concentrations as low as 38 mg L 1. These ‘bleached’ anemones looked healthy and their extended tentacles were similar to those seen in controls. However, zooxanthellae loss from anemones was slow compared with responses to other metals and only slight discolouration was observed after 72-h exposure to cobalt concentrations $629 mg L 1. Extreme zooxanthellae loss may have been due to a host response to a rapid growth rate of cobalt-exposed zooxanthellae. Such increased growth rate has been reported in marine algae and photosynthetic diatoms during exposure to concentrations of cobalt similar to those used in the present study (El-Sheekh et al. 2003). In A. pulchella, this reaction may place metabolic burden on the host, leading to zooxanthellae expulsion. This


extreme bleaching response within 96-h exposure to cobalt showed a relatively high sensitivity of this species and/or its symbiont in the context of the toxicity data used to derive ANZECC and ARMCANZ (2000) trigger values. Toxicity data for cobalt are particularly limited (Markich and Camilleri 1997; Markich et al. 2002), and there is considerable variability in the data that are available. For example, EC50 values of 300 and 23 600 mg L 1 were estimated on the basis of inhibited growth rates in the marine algae Dytilum sp. and Phaeodactylium sp. (cited in ANZECC and ARMCANZ 2000). Zooxanthellae loss (bleaching) was observed in various modes in all metal-exposed A. pulchella individuals. Bleaching may effectively reduce concentrations of metals in tissue in symbiotic cnidarians because of the tendency for zooxanthellae to accumulate high loads of some metals (Reichelt-Brushett and McOrist 2003). The low mortality and retraction observed in cobalt tests, in combination with the most severe bleaching seen in the present study, suggested that zooxanthellae accumulate high metal concentrations, and that their expulsion effectively reduces tissue concentrations in the anemone host. For cadmium, A. pulchella individuals exposed to higher concentrations often appeared less bleached than they did at lower concentrations. Despite, and perhaps attributable to, rapid en masse zooxanthellae and/or pigment loss from cadmium-exposed A. pulchella individuals, mortality was low during exposure to #1000 mg L 1. This may suggest that the ability of A. pulchella to rid itself of metal-laden zooxanthellae was overwhelmed by exposure to higher cadmium concentrations. Such reductions in the efficiency of detoxification mechanisms were reported in the oyster Crasstostrea virginica following exposure to cadmium at 600 mg L 1, compared with 100 or 300 mg L 1 (Engel and Fowlert 1979). The tested nickel concentrations caused mortality and ‘severe’ retraction, although only minimal bleaching was observed compared with cobalt-, cadmium- and lead-exposed A. pulchella. The different responses to nickel and cobalt, which are chemically quite similar, are likely to have been due to the higher tested nickel concentrations (42–1547 mg L 1 cobalt, 4–5922 mg L 1 nickel; Table 1), and, potentially, the different effects of cobalt on photosynthetic organisms (ElSheekh et al. 2003). Lead exposure caused a response different from that of other metals; in these tests, large amounts of zooxanthellae were expelled from the oral disc when anemones were gently stimulated. This may have been due to the ingestion of particulate lead, although this unique response requires further study. The observations in the present study showed that the development of zooxanthellae loss or a measure of such loss as a sublethal toxicological endpoint in A. pulchella would provide sensitive toxicity estimates for cobalt, cadmium, lead and zinc. No mortality or obvious loss of condition occurred in any control, demonstrating that the test conditions were suitable. The number of replicates in each test potentially influenced the results of nickel, lead and zinc tests, whereas the results of tests using cadmium showed good repeatability among tests with 5 or 10 replicates. Results for zinc exposures using 5, 10, and 20 replicates suggested that 10 replicates provided responses that were similar to those with 20 replicates, and it is recommended that future studies should use at least 10 replicates. Furthermore,


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it is possible that the health of test organisms varied slightly among tests and contributed to the variation in results. The development of a reference toxicant test using copper has been suggested for toxicity studies using A. pulchella (Howe et al. 2012), which would provide quality assurance of consistent testorganism health. Measured water-quality parameters were generally stable throughout the tests. The slight increases in electrical conductivity (#8 mS cm 1; 14%) between dose renewals each 48 h could be mitigated by 24-h rather than 48-h renewal. Alternatively, adding pure water to test containers may compensate for evaporative loss that occurs at the test temperatures of 258C. Our observations during the present study with A. pulchella and the lack of stress noted in any control across these tests suggested that this species is unaffected by these increases in electrical conductivity over a 48-h period. However, consideration should be given to the shifts in metal speciation that may result from such physico-chemical changes and potentially influence species sensitivity. The present study showed that sensitive toxicity tests using ‘severe’ tentacle retraction as an endpoint could be completed within a working day, with benefits for routine toxicity testing on an increasing number of contaminants (van Dam et al. 2008). The results suggested that 6 h is an appropriate duration of exposure for ‘severe’ tentacle retraction and could potentially be reduced to 4 h. Because this endpoint is not invasive, these experiments could be continued for 96 h to gain both 6-h EC50 values and 96-h LC50 values, as was conducted here. For more detailed investigation of this response, tentacle retraction in A. pulchella could easily be quantified in several stages of severity using image analyses, which has been used in coral toxicology (Negri et al. 2005). The degree and manifestation of A. pulchella bleaching was variable among metals; however, this was a notable stress response that occurred to some degree during all metal exposures. Further investigations on bleaching, and effects on Photosystem II efficiency in zooxanthellae, could provide other reliable toxicological endpoints. Reductions in Photosystem II efficiency have previously been used to determine sublethal responses of corals to the herbicides diuron and atrazine (Jones et al. 2003; Negri et al. 2005). The different mode of bleaching associated with different metals observed in the present study warrants further investigation. However, consideration must be given to potential difficulties in quantifying zooxanthellae responses in retracted anemones. Another observation made throughout the present study was that the number of asexually reproduced offspring was affected by increasing metal concentrations, and this may be a sensitive and ecologically relevant sublethal endpoint in A. pulchella and warrants further study. Conclusions The present study has provided 96-h lethal toxicity data for the tropical symbiotic anemone A. pulchella. These data showed the acute sensitivity of this species to a range of trace metals, and hence its potential suitability as a routine toxicity test organism. Estimated 96-h LC50 values between 946 and 1196 mg L 1 for cadmium, between 595 and 1146 mg L 1 for zinc, between 2209 and 5751 mg L 1 for nickel and 8090 mg L 1 and 12 352 mg L 1

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for lead were determined. Assessment of ‘severe’ tentacle retraction provided rapid (1–12 h) and sensitive sublethal toxicity estimates, and is a toxicological endpoint worthy of optimisation and testing using other contaminants. Six-hour EC50 values of 355 and 979 mg L 1 for cadmium, between 384 and 493 mg L 1 for zinc, of between 2340 and 2584 mg L 1 for nickel, and of 2610 mg L 1 for lead, were estimated for ‘severe’ tentacle retraction. Cobalt concentrations between 38 and 1547 mg L 1 caused insufficient mortality within 96 h, or rapid ‘severe’ tentacle retraction within 12 h to enable the calculation of the same toxicity estimates. However, an extremely low 96-h EC50 value of 27 mg L 1 cobalt was estimated for ‘severe’ tentacle retraction, and extreme bleaching of A. pulchella exposed to cobalt concentrations as low as 38 mg L 1 illustrated a high toxicity to the symbiont and/or the symbioses. Overall, the results warrant the further investigation of A. pulchella as a tropical representative from a very important phylum for use in routine ecotoxicology and risk assessment relevant to marine environments in the tropics. Acknowledgements Financial support was provided by the School of Environment, Science and Engineering, Southern Cross University, and by the Environmental Analysis Laboratory at Southern Cross University.

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