Embed Size (px)
Citation preview
Exposure to chronic and high dissolved copper concentrations impedes meiospore
development of the kelps Macrocystis pyrifera and Undaria pinnatifida (Ochrophyta)
PABLO P. LEAL1*, CATRIONA L. HURD
1,2, SYLVIA G. SANDER3,4, BIRTHE KORTNER
4AND MICHAEL Y. ROLEDA
1,5
1Department of Botany, University of Otago, 479 Great King Street, Dunedin 9016, New Zealand2Institute for Marine and Antarctic Studies, University of Tasmania, 20 Castray Esplanade, Battery Point,
Hobart 7004, Tasmania, Australia3National Institute for Water and Atmospheric (NIWA)/University of Otago Research Centre for Oceanography,
Union Place West, Dunedin 9016, New Zealand4Marine and Freshwater Chemistry, Department of Chemistry, University of Otago, Union Place West, Dunedin 9016,
New Zealand5Norwegian Institute for Bioeconomy Research, Kudalsveien 6, 8049 Bodø, Norway
ABSTRACT: Copper in low natural concentrations is essential for cell metabolism but in excess it becomes extremely toxicto aquatic life, including to the early life stages of marine macroalgae. This work determined the effects of copperexposure on meiospore development of two kelp species, the nativeMacrocystis pyrifera and invasive Undaria pinnatifida.After settlement, meiospores were exposed to nominal copper concentrations of control (no added copper), 100, 200, 300and 400 lg L�1 Cu for 9 days. Inductively coupled plasma mass spectrometry of total dissolved copper (CuT)concentrations in the blanks showed that nominal copper concentrations were reduced to 54, 91, 131 and 171 lg L�1 CuT,respectively, indicating that . 50% of the dissolved copper was adsorbed onto the culture vessel walls. In the media withmeiospores, the dissolved copper concentrations decreased to 39, 86, 97 and 148 lg L�1 CuT in M. pyrifera and to 39, 65,97 and 146 lg L�1 CuT in U. pinnatifida, indicating that 6–15% of the dissolved copper was adsorbed by the cells. Forboth species, meiospores germinated in all copper treatments, with germination decreasing with increasing copperconcentration. However, gametophyte growth and sexual differentiation were arrested under all copper treatments. Theeffective copper concentration causing 50% of arrested germination (Cu-EC50) was 157 and 231 lg L�1 CuT for M.pyrifera and U. pinnatifida, respectively. The higher Cu-EC50 for U. pinnatifida suggests ecological success for the invasivespecies in copper-polluted environments; however, the subsequent inhibition of gametogenesis under all coppertreatments indicated no difference in copper tolerance between both kelp early life stages. We compare our results withthe literature available on the effects of copper on the development of early life stages of brown seaweed (Laminarialesand Fucales) and discuss the importance of reporting actual experimental dissolved copper concentrations and thenecessity of standardizing the response variables measured in macroalgal copper ecotoxicology.
KEY WORDS: Dissolved concentration, Effective concentration, Gametophyte, Germination, Invasive kelp, Nominalconcentration
INTRODUCTION
Copper is an essential microelement for algal metabolism,
required in trace amounts for the functioning of important
biological processes such as enzymatic cofactors (amine
oxidase and cytochrome c oxidase) and electron carriers in
photosynthesis, mitochondrial respiration, cell wall metab-
olism and hormone signalling (Gledhill et al. 1997; Raven et
al. 1999; Saez et al. 2015). Copper in excess, however,
becomes one of the most toxic metals, adversely affecting
cellular functions such as photosynthesis, fatty acid metab-
olism and enzyme activity. Copper can initiate the formation
and accumulation of highly toxic reactive oxygen species,
which cause oxidative damage of membrane lipids, nucleic
acids and proteins, which can lead to cell death (Rijstenbil et
al. 1994; Saez et al. 2015).
Copper concentrations in pristine coastal seawater varybetween 0.5 and 3 lg L�1 (Lewis 1995). The higherconcentrations (. 5 lg L�1) found in some coastal watersmay be attributed to both natural (e.g. rivers, atmosphereand hydrothermal vents) and anthropogenic sources (Nor1987). Human activities including industrial and domesticwastes, agricultural practices, copper mine drainage, copper-based pesticide application and antifouling paints have beenthe main contributor to a progressive increase in copperconcentrations in aquatic environments (Nor 1987; Gledhillet al. 1997; Callow & Callow 2002). As sessile organisms,macroalgae are readily exposed to pollutants from coastaldischarges (Littler & Murray 1975; Swartz et al. 1986). Forexample, in coastal areas impacted by the dumping ofuntreated mining tails with copper concentrations as high as. 200 lg L�1 (Correa et al. 1999), the biological diversitydeclines owing to the complete loss of invertebrates and mostmacroalgal species (Castilla 1983). This decline in macro-algal abundance in copper-impacted environments might bea consequence of failure in recruitment or mortality of theirearly life stages (Bellgrove et al. 1997; Lotze et al. 2001;Contreras et al. 2007), which are more vulnerable to abiotic
* Corresponding author ([email protected]).† Present address: Norwegian Institute for BioeconomyResearch, Kudalsveien 6, 8049 Bodø, Norway.DOI: 10.2216/15-87.1� 2016 International Phycological Society
Phycologia Volume 55 (1), 12–20 Published 14 December 2015
12
stress than the macroscopic phase (Roleda et al. 2007, 2009;Nielsen et al. 2014). In addition, the establishment ofopportunistic and stress tolerant macroalgae such as thegenus Ulva Linnaeus has been observed after a decline oflocal macroalgal communities (Borowitzka 1972; May 1985;Correa et al. 1996, 1999).
New Zealand’s coastal ecosystems consist of a rich anddiverse macroalgal flora: 770 macroalgal species are known, ofwhich 265 are endemic and 22 invasive (Hurd et al. 2004). Ofthe native species, the giant kelp Macrocystis pyrifera(Linnaeus) C. Agardh is one of the most studied macroalgae(Graham et al. 2007). In New Zealand, this kelp species growsin open coasts and harbours, from the south-west of the SouthIsland to the south-east of the North Island, and aroundCampbell and Auckland Islands (Hay 1990). Undaria pinna-tifida (Harvey) Suringar is one of the most successful invasivemacroalgae, with a global expansion from its native habitat innortheast Asia (Yamanaka & Akiyama 1993; Fletcher &Manfredi 1995; Floc’h et al. 1996; Campbell & Burridge 1998;Silva et al. 2002; Meretta et al. 2012). In New Zealand, U.pinnatifida has invaded all of the major ports and a number ofsecondary ports, and it is well established on rocky shorelines(Russell et al. 2008). Macrocystis pyrifera and U. pinnatifidacurrently cohabit in many areas along New Zealand’s coasts(Russell et al. 2008; Schiel & Thompson 2012).
Macrocystis pyrifera and U. pinnatifida each have a similarreproductive strategy: macroscopic sporophytes bear basalsporophylls that produce sori where microscopic meiosporesare formed. After their release and dispersal, meiospores settleon the substratum and germinate into microscopic male andfemale gametophytes that, after sexual fertilization, developinto the diploid sporophyte (Bartsch et al. 2008; Leal et al.2014). The survival of early microscopic life history stagesmay determine the population dynamics of subsequentmacroscopic algal assemblages (Lotze et al. 2001), and thehigh stress tolerance of the invasive species may promote theirexistence over native species (Borowitzka 1972; May 1985;Doblin & Clayton 1995; Castilla 1996; Bellgrove et al. 1997).
In M. pyrifera and U. pinnatifida, meiospore germinationand germ tube growth were found to be less sensitive tocopper concentrations , 100 lg L�1 Cu2þ than the later stagesof the life cycle such as gametogenesis and sporophyteproduction (Lee et al. 1989; Anderson et al. 1990). Therefore,this mechanistic study aimed to determine whether thedevelopment of meiospores under high and chronic copperconcentrations (. 100 lg L�1 Cu2þ) is different between thenative M. pyrifera and the invasive U. pinnatifida. Wehypothesized that (1) meiospore germination of both kelpspecies will not be inhibited by high copper concentrations,and (2) the invasiveU. pinnatifida will have higher tolerance tocopper exposure than M. pyrifera. We also conducted aliterature review on the effects of copper on the developmentof early life stage of members of Laminariales and Fucales tocompare with the results of the present study.
MATERIAL AND METHODS
To minimize metal contamination, all laboratory-ware usedfor seawater sampling and toxicity tests (including polysty-
rene tissue culture vessels) was cleaned as follows: (1)soaking in a 1% v/v Citranoxt (Alconox Inc. New York,New York USA ) bath for 1 week and then rinsing five timeswith distilled water; (2) soaking in a 10% v/v HCl (6 N AR-HCl) bath for at least 4 weeks and then rinsing five timeswith high-purity ultrapure water (resistivity � 18 MX cm);and (3) soaking in a 1% v/v HCl (8 N ultra clean quartzdistilled-HCl) bath for at least 4 weeks, then rinsing fivetimes with ultrapure water and stored (in sealed plastic bags)until used.
Sampling was performed during autumn 2013, in theupper sub-littoral of Hamilton Bay (458790S; 1708640E),Otago Harbour, New Zealand. During low tide, fertilesporophylls from 10 individuals of each of M. pyrifera andU. pinnatifida were collected and transported to thelaboratory in a cool box within 1 hour of collection. Atthe same time, 30 litres of seawater was collected, proximateto the sporophyte sampling area, and stored in a polystyrenecontainer. In the laboratory, the seawater was filtered (0.22lm, polyethersulfone membrane, MillextGP, Millipore Ire-land Ltd., Cork, Ireland), transferred to another polystyrenecontainer and stored in darkness at 48C until use in allsubsequent experiments. Fertile sporophylls were gentlycleaned of epibiota by brushing them under filtered (0.22lm) seawater, blotted dry, wrapped in moist tissue paper andstored overnight at 48C before meiospore release.
Meiospore release and cultivation was performed accord-ing to Leal et al. (2014). After overnight desiccation ofsporophylls, discs of 2 cm2 (a total of 45 g of 2 cm2 discs perspecies) were excised using a cork borer from sporophylls of10 individual sporophytes. The pool of excised sori wasimmersed in 500 ml of filtered (0.2 lm) natural seawater for15 minutes. Then, the sori were removed, and the number ofmeiospores released was counted using a haemocytometer(0.1 mm depth, Neubauer improved bright-line, Marienfeld,Germany). Meiospore densities were adjusted to 20,000–25,000 cells ml�1 and separately dispensed onto eachcompartment of a six-well polystyrene tissue culture vessels(Costar 3516; Corning Inc., Corning, New York USA)containing filtered seawater. After meiospore settlement (3hours), cultures were exposed to different nominal copperconcentrations, described below. Meiospores were cultivatedin a temperature-controlled room at 128C under a 12:12hours light:dark photoperiod of 50 6 2 lmol photons m�2
s�1 of photosynthetic active radiation (cool-white fluores-cent; Philips, Eindhoven, The Netherlands).
Copper stock solutions were prepared by dissolving CuCl2(anhydrous, powder, 99% trace metal basis, Sigma-AldrichCo. LLC, St. Louis, Missouri USA) in ultrapure water (2 gL�1). This stock solution was prepared in a 250 ml bottle(Durant laboratory glass bottle, Duran Group GmbH,Mainz, Germany) every 3 days and was used within 1 hourof preparation. The copper nominal concentrations used inexperiments were control (no added copper), 100, 200, 300and 400 lg L�1 Cu.
From each the replicates of each copper treatment and theanalytical blanks, 0.15 ml of culture medium was diluted in4.25 ml of ultrapure water and acidified with 0.09 ml ofHNO3 (acidified sample, 4.5 ml 2% HNO3) and stored untilanalysis. Total copper concentrations were quantified byinductively coupled plasma mass spectrometry (ICP-MS).
Leal et al.: Chronic copper exposure impedes kelp meiospore development 13
Owing to copper adsorption onto the experimentalpolystyrene culture vessels, the actual CuT concentrationsthat the meiospores were exposed to were determined using alinear regression analysis on the nominal and measureddissolved copper concentrations. The corresponding CuTconcentrations for the nominal concentrations given abovewere 11 (control), 54, 91, 131 and 171 lg L�1 (Fig. 1). Coppertaken up (adsorption and/or absorption) by cells of M.pyrifera and U. pinnatifida was computed as the differencebetween the dissolved copper concentrations measured foreach experimental treatment and the analytical blank.
After settlement, meiospores were exposed to five nominalconcentrations (n ¼ 6 independent replicates per copperconcentration). The culture medium with the appropriatecopper concentrations was renewed every 3 days to avoidinorganic carbon and nutrient depletion and to simulatechronic copper exposure. Analytical blanks corresponding toeach copper treatment were also prepared. Total dissolvedcopper concentrations in the treatments and blanks weremeasured as described above.
Every 3 days, at least five randomly chosen visual fieldsusing a 103 objective of an inverted microscope (Olympus
CK2; Olympus Optical Co. Ltd., Tokyo, Japan) were
photographed using a video camera (5.1 M CMOS camera,
UCMOS0510KPA). Photographs were viewed using the
digital camera software ToupView 3.5. Three hundred and
fifty meiospores were counted and classified into alive or
dead according to Leal et al. (2014). After 9 days, germling
size and gametophyte size were obtained by measuring the
area (lm2) of previously photographed individuals (n ¼ 10
per replicate). Meiospores with a visible germ tube that were
sexually ambiguous were classed as germlings. Upon sexual
differentiation, gametophytes were counted and classified as
either a male or female. Sex ratio was calculated as described
by Roleda et al. (2012): sex ratio¼number of males/(number
of males þ number of females).
Fig. 1. Linear regression between nominal (added) copper concen-trations and total dissolved copper (CuT) concentrations in themedium of the analytical blanks and cultures of meiospores ofMacrocystis pyrifera and Undaria pinnatifida. Corresponding linearregression equations are shown. Points represent x� 6 s (n ¼ 3).
Fig. 2. Meiospore germination of Macrocystis pyrifera and Undariapinnatifida after 9 days of CuT exposure. Data are expressed as apercentage of the control culture. Bars represent x� 6 s (n ¼ 6).Different letters indicate significant differences between species andtreatments (Tukey test, P , 0.05).
Fig. 3. Germling size of Macrocystis pyrifera and Undariapinnatifida after 9 days of CuT exposure. Data are expressed as apercentage of the control culture. Bars represent x� 6 s (n ¼ 6).Different letters indicate significant differences between species andtreatments (Tukey test, P , 0.05).
Fig. 4. Gametophyte development after 9 days for Macrocystispyrifera and Undaria pinnatifida exposed to a range of CuTconcentrations. / indicates female and ? indicates male gameto-phytes. Scale bar¼ 20 lm.
14 Phycologia, Vol. 55 (1)
Effective copper concentration causing 50% of arrestedgermination (Cu-EC50) values represent the concentration ofcopper in each experimental treatment that caused theinhibition of 50% meiospore germination after 9 days ofculture under copper treatments. Cu-EC50 values werecalculated using the total dissolved copper concentrations.Cu-EC50 values were computed with the Dose EffectAnalysis (Probit model, P , 0.05) in the software XLSTAT(Addinsoft 2015).
Percentage data (meiospore germination and germlingsize) was logit transformed (Warton & Hui 2011). AKolmogorov–Smirnov test was used to test Normality, andthe Levene’s test was used to test homogeneity of variance. Atwo-way analysis of variance (ANOVA; P , 0.05) was usedto test the effects of copper concentration (four levels, fixed)on meiospore germination, germling size and gametophytesize of both kelps (species: two levels, fixed). When asignificant effect (single and/or two-way interaction) ofindependent variables was observed, a post hoc Tukey testwas applied. A Student’s t test (P , 0.05) was used tocompare the gametophyte size and sex ratio within eachspecies and between species, respectively. The non-paramet-ric Mann–Whitney U test was used to test EC50 differencesbecause the data did not meet the assumptions of normalityand homoscedasticity required for a parametric test.Statistical analysis was performed using the softwareSigmaPlot 12.0 (Systat Software, Inc., San Jose, California).
RESULTS
Total dissolved copper (CuT) concentrations
The coastal seawater used for the experiment had abackground CuT concentration of 9.5 6 3 lg L�1. After theexperiment, and due to adsorption and/or absorption into thecell walls, the dissolved background of CuT concentrationdecreased to 4.5 6 3 and 8.4 6 9 lg L�1 in meiospore culturesof M. pyrifera and U. pinnatifida, respectively (Fig. 1).
Analysis of CuT concentrations showed that in analyticalblanks, the nominal concentrations (100, 200, 300 and 400 lgL�1 Cu) were reduced to 54 6 10, 91 6 2, 131 6 3 and 171 6
10 lg L�1 CuT, respectively. In the culture media ofmeiospores, the nominal concentrations (100, 200, 300 and400 lg L�1 Cu) were reduced to 39 6 7, 86 6 32, 97 6 6 and148 6 5 lg L�1 CuT, respectively, in M. pyrifera and to 39 6
21, 65 6 2, 97 6 2 and 146 6 3 lg L�1 CuT, respectively, inU.pinnatifida (Fig. 1).
Effects of copper on meiospore germination and development
Germination under control conditions, i.e. without additionalcopper, was up to 95% in bothM. pyrifera and U. pinnatifida.Under increasing CuT concentrations (54, 91, 131 and 171 lgL�1 CuT), germination was 85 6 5, 75 6 3, 70 6 9 and 43 6
7% of control, respectively, inM. pyrifera (Fig. 2; Tukey, P ,
0.05; 54 . 91¼131 . 171 lg L�1 CuT) and 90 6 4, 72 6 8, 686 4 and 66 6 8% of control, respectively, in U. pinnatifida(Fig. 2; Tukey, P , 0.05; 54 . 91¼ 131¼ 171 lg L�1 CuT).Meiospore germination was significantly different betweenspecies and CuT treatments. However, a significant two-wayinteraction (species3Cu treatments) was also observed (Table1). Post hoc Tukey (P , 0.05) multiple comparison testsshowed that the effect of copper on meiospore germinationwas lowest at 54 lg L�1 CuT inU. pinnatifida followed by 54 lgL�1 CuT in M. pyrifera and highest at 171 lg L�1 CuT in M.pyrifera. The effect of the other concentrations (91 and 131 lgL�1 CuT) on both species was not significantly different (Fig.2).
Germling size under increasing CuT concentrations (54, 91,131 and 171 lg L�1 CuT) was 28 6 4, 28 6 9, 21 6 4 and 24 6
3% of control inM. pyrifera (Fig. 3; Tukey, P , 0.05; 54¼ 91. 131¼171 lg L�1 CuT) and 22 6 2, 23 6 4, 18 6 2 and 18 6
2% of control in U. pinnatifida (Fig. 3; no statistical differencebetween copper treatments). Germling size was statisticallydifferent between species (Table 1; Tukey, P , 0.05; M.pyrifera. U. pinnatifida under 54 and 171 lg L�1 CuT) and Cutreatments (Table 1; Tukey, P , 0.05; 54¼ 91 . 131¼ 171 lgL�1 CuT) but no interactive effect between the independentvariables was observed (Table 1).
Visual effects of dissolved copper treatments on thedevelopment of meiospores of M. pyrifera and U. pinnatifidaare summarized in Fig. 4. In the controls (no added copper),meiospores of both M. pyrifera and U. pinnatifida grew
Table 1. Two-way ANOVA and significance values for effects of CuT exposure on meiospore germination, germling area and gametophytearea of Macrocystis pyrifera and Undaria pinnatifida.
Variable Source of variation Degree of freedom Sum of squares Mean square F P1
Meiospore germination Species 1 0.208 0.208 8.477 0.006*(Cu treatments) Cu treatment 3 3.946 1.315 53.546 , 0.001*
Species 3 Cu treatment 3 0.444 0.148 6.032 0.002*Residual 40 0.983 0.025Total 47 5.581 0.119
Germlings size Species 1 0.140 0.140 13.750 , 0.001*(Cu treatments) Cu treatment 3 0.183 0.061 5.989 0.002*
Species 3 Cu treatment 3 0.011 0.004 0.358 0.784ns
Residual 72 0.407 0.010Total 79 0.740 0.016
Gametophyte size Species 1 937.806 937.806 0.266 0.612ns
(Control cultures) Gametophyte sex 1 19,900.829 19,900.829 5.648 0.028*Species 3 Gametophyte sex 1 421.988 421.988 0.120 0.733ns
Residual 36 70,466.696 3523.335Total 39 91,727.319 3988.144
1 Asterisk indicates significance; ns, not significant.
Leal et al.: Chronic copper exposure impedes kelp meiospore development 15
normally and developed into female and male gametophytesafter 9 days. Meiospore germination occurred under alldissolved copper concentrations but no gametophyte devel-opment was observed after 9 days for either species (Fig. 4).
Gametophyte development and sexual differentiation wereobserved only under control conditions (Fig. 4). The size ofmale and female gametophytes was 506 6 48 and 440 6 46lm2 for M. pyrifera and 485 6 66 and 436 6 74 lm2 for U.pinnatifida (Fig. 5). Regardless of species, gametophyte sizewas significantly different between sexes (Table 1; Tukey, P ,
0.05; male . female). However, within each species, malegametophytes were only significantly larger than femalegametophytes in M. pyrifera (Student’s t test, t10 ¼ 2.439, P¼ 0.035). There was no two-way interaction betweenindependent variables (species 3 gametophyte sex; Table 1).Gametophyte sex ratio (0.46 6 0.06,M. pyrifera; 0.47 6 0.02,U. pinnatifida; Fig. 5) was not significantly different betweenspecies (Student’s t test, t10¼�0.182, P¼ 0.859).
Cu-EC50 for meiospore germination
Cu-EC50 values for meiospore germination were 157 and 231lg L�1 CuT for M. pyrifera and U. pinnatifida, respectively(Table 2), and they were statistically different (Mann–WhitneyU¼ 4.5, n1¼ n2¼ 6, P¼ 0.026).
DISCUSSION
Effect of copper on meiospore germination and development
The first hypothesis that copper will not inhibit meiosporegermination was supported because meiospore germinationoccurred under all Cu treatments. We found that meiosporesof both M. pyrifera and U. pinnatifida germinated undercopper concentrations (54 to 171 lg L�1 CuT) as high as thosefound in highly polluted coastal areas (e.g. . 5 to 250 lg L�1
Cu) (Correa et al. 1999); although, germination declined withincreasing CuT. These results may be explained by therelatively high concentrations that Dickson & Hunter (1981)found in Otago Harbour surface seawaters (0.15 to 3.26 lgL�1 Cu). In addition, the CuT concentration in the seawater
obtained for this experiment was around 20-fold higher thanthe previously reported concentration for Hamilton Bay (0.48lg L�1 Cu) and around 40-fold higher than the average (0.84lg L�1 Cu) for OtagoHarbour (Dickson &Hunter 1981). Thissuggests that the pollution in this area has increasedsignificantly since the 1980s. Therefore, the constant exposureof both kelp populations to increasing copper pollution insideOtago Harbour might allow them to overcome the toxiceffects of copper during meiospore germination. Accordingly,copper-tolerant ecotypes have been reported in macroalgalpopulations inhabiting copper-polluted environments becauseof selective pressure (Reed & Moffat 1983; Saez et al. 2015).Under control conditions, meiospores of M. pyrifera and U.pinnatifida germinated within 3 days and developed to formhealthy microscopic male and female gametophytes as isexpected for members of the order Laminariales (Bartsch et al.2008; Leal et al. 2014).
The completion of meiospore germination (i.e. germ tubeformation and cell growth) found for bothM. pyrifera and U.pinnatifida under all CuT treatments was not surprising giventhat this process is tolerant to copper stress (Anderson et al.1990). However, other metals (e.g. zinc) are thought to inhibitmeiospore photosynthesis and thus disrupt germination(Anderson & Hunt 1988). Anderson et al. (1990) suggestedthat calcium ion transport across the cell membrane isessential for germ tube formation, and the process may beinhibited by exposure to metals (Anderson et al. 1990). Forexample, copper disrupts specific developmental processes inFucus serratus embryos where elevated copper concentrationsinhibit rhizoid formation and elongation; the inhibitory effectis primarily due to the blockage of fucoidan secretion (acomponent of the cell wall) and reduction of intracellularcalcium gradient signals (Nielsen et al. 2003a). Germ tubeformation can occur in darkness (Han et al. 2011), whichsuggests that cells are able to mobilize their endogenousnitrogen and carbohydrate reserves to initiate the germinationprocess; this may explain the findings observed in this study.We know little of the mechanisms involved in the responses ofearly life stages of Laminariales meiospores to copper, andmore physiological work is needed to better understand thehigh tolerance of meiospore germination and the highsensitivity of gametogenesis to copper in M. pyrifera and U.pinnatifida.
The second hypothesis, that U. pinnatifida will be moretolerant to copper than M. pyrifera, was only supported bysome of the response variables measured. Meiospore germi-nation was significantly higher for U. pinnatifida than M.pyrifera under 54 and 171 lg L�1 CuT. Similarly, Cu-EC50 formeiospore germination in U. pinnatifida was higher than thatfor M. pyrifera, indicating that the invasive kelp may have anadvantage on ecological success in copper-polluted environ-ments over the native giant kelp. However, germlings of M.pyrifera were bigger than those of U. pinnatifida under 54 and171 lg L�1 CuT. In addition, gametogenesis in both kelpspecies was arrested under all CuT treatments, indicating thatthere would not be a difference in copper tolerance betweenmicrostages of M. pyrifera and U. pinnatifida in a copper-polluted environment. These results suggest that meiosporegermination is not the most sensitive response variable fordetermining copper ecotoxicology of M. pyrifera and U.pinnatifida, and long-term experiments that also assess
Fig. 5. Area (lm2) and sex ratio of gametophytes of Macrocystispyrifera and Undaria pinnatifida after 9 days cultivation undercontrol (no added copper) conditions. Bars and circles represent x�
6 s (n ¼ 6). The asterisk indicates a significant difference ingametophyte size within M. pyrifera (Student’s t test, P , 0.05).
16 Phycologia, Vol. 55 (1)
Table2.Effectofcopper
concentrationonearlylife
stages
ofmem
bersoftheOrdersFucalesandLaminariales.Copper
concentrationsandexposure
timeatwhichanegativeeffect
on
theresponse
variable
wasobserved
are
given.Concentrationsreported
are
either
nominal(i.e.added)ordissolved
(i.e.measured).
Classificationoftest
alga1
Copper
concentration
range(lgL�1)
Reported
concentration
Response
variable
measured
Effectof
copper
(lgL�1)
Exposure
time
Reference
Laminariales
Ecklonia
radiata
(C.Agardh)
J.Agardh
0–10002
Nominal
Germination
EC50¼
330
48h
Burridgeet
al.(1999)
Growth
EC50¼
480
48h
0–3402
Nominal
Germination
EC50¼
300
48h
Ross
&Bidwell(1999)
Laminariahyperborea
(Gunnerus)
Foslie
0–1002
Nominal
Germination
Reducedby�
100
14d
Hopkin
&Kain
(1978)
Gametophyte
survival
Reducedby�
100
14d
Lessonia
nigrescensBory
deSaint-Vincent
0–1003
Dissolved
Meiospore
release
Reducedby�
88h
Contreraset
al.(2007)
Meiospore
settlement
Reducedby�
848h
Germination
Reducedby�
200
48h
Gametophyte
production
Reducedby�
45
40d
Macrocystispyrifera
(Linnaeus)
C.Agardh
0–1002
Nominal
Germination
Inhibited
by�
100
48h
Andersonet
al.(1990)
Germ
tubegrowth
Inhibited
by�
18
48h
Sporophyte
development
Inhibited
by�
18
20d
0–1602
Dissolved
Germination
Reducedby�
20
24h
Garm
anet
al.(1994)
Germ
tubegrowth
Reducedby�
40
24h
Nuclearmigration
Reducedby�
20
42h
0–1714
Dissolved
Germination
EC50¼
157
69
9d
Presentstudy
Saccharinajaponica(A
reschoug)
C.E.Lane,
C.Mayes,Druehl&
G.W
.Saunders
0–4002
Nominal
Germination
EC50¼
120
24h
Hanet
al.(2011)
Germ
tubegrowth
EC50¼
81
24h
Saccharinalatissim
a(Linnaeus)
C.E.Lane,
C.Mayes,Druehl&
G.W
.Saunders[¼
Laminariasaccharina(Linnaeus)
J.V.Lamouroux]
0–1002
Nominal
Meiospore
release
Reducedby�
50
12h
Chung&
Brinkhuis
(1986)
Germination
Noeffect
12h
Gametogenesis
Inhibited
by�
10
7d
Sporophyte
development
Inhibited
by�
50
20d
0–2004
Nominal
Germlinggrowth
Inhibited
by�
50
14d
Thompson&
Burrows(1984)
0–200,0003
Dissolved
Gametophyte
survival
Inhibited
by�
50,000
14d
Yeet
al.(2005)
Undariapinnatifida(H
arvey)Suringar
0–1005
Nominal
Germination
Reducedbyallconcentrations
21d
Lee
etal.(1989)
Gametophyte
development
Reducedby�
543d
0–1714
Dissolved
Germination
EC50¼
231
668
9d
Presentstudy
Fucales
FucusserratusLinnaeus
0–553
Nominal
Rhizoid
elongation
Inhibited
by�
13
10d
Nielsen
etal.(2003a)
0–12803
Nominal
Rhizoid
elongation
Reducedby�
13
8d
Nielsen
etal.(2003b)
0–1275
Nominal
Rhizoid
elongation
Reducedby�
32
10d
Nielsen
etal.(2005)
0–645
Nominal
Rhizoid
elongation
Reducedby�
66d
Nielsen
etal.(2014)
FucusspiralisLinnaeus
0–1282
Dissolved
Germlingsize
Reducedby�
12
10d
Bondet
al.(1999)
FucusvesiculosusLinnaeus
0–202
Nominal
Rhizoid
elongation
Reducedby�
2.5
24h
Andersson&
Kautsky(1996)
0–3202
Nominal
Embryolength
Reducedby�
20
14d
Brookset
al.(2008)
0–644
Dissolved
Germlinggrowth
Inhibited
by�
32
19d
Gledhillet
al.(1999)
Horm
osira
banksii(Turner)Decaisne
0–5002
Nominal
Rhizoid
initiation
EC50¼
130
48h
Myerset
al.(2006)
EC50¼
90
72h
Germination
EC50¼
170
48h
EC50¼
220
72h
1Currentsynonymsobtained
from
AlgaeB
ase
(Guiry&
Guiry2015).
2Culture
medium
renew
al:notspecified.
3Culture
medium
renew
al:daily.
4Culture
medium
renew
al:every3days.
5Culture
medium
renew
al:every2days.
Leal et al.: Chronic copper exposure impedes kelp meiospore development 17
gametogenesis are needed for more accurate interpretations ofresponses of macroalgal early life stages to copper.
Standardization of the data reported is also important tofacilitate comparison between studies on different species. Themost common form of standardization is to calculate, from adose–response curve, the metal concentration at which 50% ofthe response variable is affected (i.e. EC50) (Chapman 1995).However, few studies on the early life history stages ofseaweed have reported EC50, and in most of the studies, theassay results were expressed as a reduction or inhibition of thevarious response variables (Table 2). Including the presentstudy, Cu-EC50 values for germination have been calculated inonly five ecotoxicological studies for four macroalgal speciesof the Orders Laminariales and Fucales (Table 2). Compar-ison of Cu-EC50 for germination suggest that the most coppersensitive Laminarian species were Saccharina japonica (Are-schoug) C.E. Lane, C. Mayes, Druehl & G.W. Saunders withan EC50¼120 lg L�1 (Han et al. 2011) andM. pyriferawith anCu-EC50¼ 157 lg L�1 (present study), while the most coppertolerant were U. pinnatifida with an Cu-EC50 ¼ 231 lg L�1
(present study) and Ecklonia radiata (C. Agardh) J. Agardhwith an Cu-EC50¼ 300 lg L�1 (Burridge et al. 1999; Ross &Bidwell 1999). The Cu-EC50 for germination for the FucaleanHormosira banksii (Turner) Decaisne was 170 and 220 lg L�1
after 48 and 72 hours of copper exposure, respectively (Myerset al. 2006), indicating that copper tolerance can change withexposure time.
The response variables that have been often used in coppertoxicity assays are the germination and growth of the germtube (Laminariales) or rhizoid (Fucales) (Table 2). Differentcopper concentrations have been reported to affect differentphysiological processes; although, these are mostly nominalconcentrations rather than measured (Table 2). In theLaminariales, copper concentrations � 40 lg L�1 significantlyinhibit meiospore germination, reduce germ tube growth andinterrupt nuclear migration in M. pyrifera (Anderson et al.1990; Garman et al. 1994). In U. pinnatifida, meiosporegermination significantly decreased at copper concentrations. 5 lg L�1 (Lee et al. 1989); 100 lg L�1 of copper reducedmeiospore germination and gametophyte survival in Lami-naria hyperborea (Gunnerus) Foslie (Hopkin & Kain 1978).Copper (8 lg L�1) considerably reduced meiospore release andcompletely interrupted post settlement development in Lesso-nia nigrescens Bory de Saint-Vincent (Contreras et al. 2007).Meiospore release by Saccharina latissima (Linnaeus) C.E.Lane, C. Mayes, L.D. Druehl & G.W. Saunders [¼Laminariasaccharina (Linnaeus) J.V. Lamouroux] was reduced and thegametogenesis delayed by 50 lg L�1 copper but germinationwas not affected by concentrations � 500 lg L�1 (Thompson& Burrows 1984; Chung & Brinkhuis 1986; Ye et al. 2005). Inmembers of the Fucales, copper (� 3 lg L�1) reduced zygotegermination and rhizoid elongation in F. serratus Linnaeus,Fucus spiralis Linnaeus and Fucus vesiculosus Linnaeus(Andersson & Kautsky 1996; Bond et al. 1999; Gledhill etal. 1999; Nielsen et al. 2003a, 2003b, 2005, 2014; Brooks et al.2008).
These developmental processes are ecologically importantbecause any interruption or delay in meiospore developmentcan expose the cryptic microscopic early life history stages tosustained grazing pressure and recurrent abrasion andsediment burial (Devinny & Volse 1978). Such a developmen-
tal delay may also favour other competing algal species (Reed& Foster 1984; Dean et al. 1989; Reed 1990) including invasiveor opportunistic species (Castilla 1996). Furthermore, a delay,in germ tube growth and germination may decrease theprobability of gametophyte maturation when the optimalenvironmental conditions are present (Anderson et al. 1990;Reed 1990; Garman et al. 1994). Comparison between studieson copper toxicological effects on early life history stages ofmacroalgae is difficult because, in most of them, only thenominal copper concentrations were reported (Table 2). Ourresults suggest that both the nominal and dissolved copperconcentrations need to be detailed in toxicological studies toprove that the desired exposure concentrations did not changeduring the experiment (Table 2), due to adsorption into thecontainer walls and macroalgal cells.
Analytical procedures of copper ecotoxicology of algae
We found that the dissolved copper concentrations weresubstantially reduced compared with the nominal copperconcentrations that were added to the experimental treat-ments. The concentrations of copper measured in theanalytical blanks were about 50% lower than that of thenominal concentrations added to the culture media. Atnatural pH, metals (e.g. copper) in the solution may havebeen adsorbed by the walls of the glass bottle containing thestock solution and by the plastic walls of the six-well plateculture vessels used in the experiment (both the analyticalblanks and dose–response experiments) (Gledhill et al. 1999).The metal adsorption by the stock solution container wallscan be avoided by acidification (to pH 2) of the stock solutionusing HCl or HNO3 (Moody 1982; Parr 1986). This step wasomitted in this study to avoid acidification of the culturemedia, which could affect spore germination and cell division(Sorokin 1962; Roleda et al. 2012).
The reduction of total dissolved copper concentration in themeiospore cultures of both kelps was even higher (6–15%)than in the analytical blanks. This result may be related to twomechanisms that algae have developed for metal homeostasis:biosorption and bioaccumulation. Biosorption is the removalof trace metals by a passive binding into the algal cell wallfrom the seawater (Davis et al. 2003; Chojnacka 2010). Underlaboratory conditions, metal biosorption can be performed byexposing living or non-living biomass to a range of metalconcentrations (Davis et al. 2003; Chojnacka 2010). Incontrast, bioaccumulation is performed by living biomassand refers to an active transport of the metal ions from the cellwall to the inside of the cell that occurs after the biosorption(Davis et al. 2003; Chojnacka 2010). The inherent capacity ofmacroalgae to bioabsorb and bioaccumulate trace metalsmakes them an important biotechnological tool to bioremedi-ate polluted marine environments and to treat industrial wastewater (Chojnacka 2010).
In summary, Cu-EC50 values for meiospore germinationwere greater for the invasive U. pinnatifida than the native M.pyrifera to copper exposure. However, the subsequentdevelopment of gametophytes was equally arrested by copperin both kelp species, and therefore we cannot suggest that U.pinnatifida will have a competitive advantage overM. pyriferain marine coastal areas with high copper concentrations.Ecotoxicological studies on microscopic life stages of M.
18 Phycologia, Vol. 55 (1)
pyrifera and U. pinnatifida have rarely been performed, whichis surprising given that they are considered the most sensitivelife history stage to environmental stress. In addition to ourwork, only two publications on M. pyrifera and only one onU. pinnatifida were found. Furthermore, comparison betweenstudies is difficult because there is an inconsistency inreporting relevant protocol steps such as nominal and/ordissolved copper concentrations and response variable effects(e.g. EC50). Such inconsistencies may result in misinterpreta-tion of the effects of copper on the ecophysiology of the earlylife history stages of kelps.
Finally, our results demonstrate the importance of technicalprocedure in toxicity experiments. The actual dissolved copperconcentrations were substantially lower than the nominaladdition to the analytical blanks andmeiospore culture media.This decrease in dissolved copper concentrations may beattributed to adsorption to container walls or extra- andintracellular association of copper with the kelp germlings. Incopper ecotoxicological studies, it is thus important to reportboth the nominal concentrations and the actual concentra-tions measured within culture vessels.
ACKNOWLEDGEMENTS
We thank BECASCHILE-CONICYT, the Royal Society ofNew Zealand Mardsen grant (UOO0914) and the NewZealand MBIE programme C01X1005 for funding. Wethank Pamela Fernandez and Rocio Suarez for the assistanceduring field sampling. We thank the two anonymousreviewers for their thoughtful and constructive comments.
REFERENCES
ADDINSOFT. 2015. XLSTAT 2015, Data analysis and statisticssoftware for Microsoft Excel. Addinsoft, Paris, France.
ANDERSON B.S. & HUNT J.W. 1988. Bioassay methods for evaluatingthe toxicity of heavy metals, biocides and sewage effluent usingmicroscopic stages of giant kelp Macrocystis pyrifera (Agardh): apreliminary report. Marine Environmental Research 26: 113–134.
ANDERSON B.S., HUNT J.W., TURPEN S.L., COULON A.R. & MARTIN
M. 1990. Copper toxicity to microscopic stages of giant kelpMacrocystis pyrifera: interpopulation comparisons and temporalvariability. Marine Ecology Progress Series 68: 147–156.
ANDERSSON S. & KAUTSKY L. 1996. Copper effects on reproductivestages of Baltic Sea Fucus vesiculosus. Marine Biology 125: 171–176.
BARTSCH I., WIENCKE C., BISCHOF K., BUCHHOLZ C.M., BUCK B.H.,EGGERT A., FEUERPFEIL P., HANELT D., JACOBSEN S., KAREZ R.,KARSTEN U., MOLIS M., ROLEDA M.Y., SCHUBERT H., SCHUMANN
R., VALENTIN K., WEINBERGER F. & WIESE J. 2008. The genusLaminaria sensu lato: recent insights and developments. EuropeanJournal of Phycology 43: 1–86.
BELLGROVE A., CLAYTON M.N. & QUINN G.P. 1997. Effects ofsecondarily treated sewage effluent on intertidal macroalgalrecruitment processes. Marine and Freshwater Research 48: 137–146.
BOND P.R., BROWN M.T., MOATE R.M., GLEDHILL M., HILL S.J. &NIMMO M. 1999. Arrested development in Fucus spiralis(Phaeophyceae) germlings exposed to copper. European Journalof Phycology 34: 513–521.
BOROWITZKA M.A. 1972. Intertidal algal species diversity and theeffect of pollution. Australian Journal of Marine and FreshwaterResearch 23: 73–84.
BROOKS S.J., BOLAM T., TOLHURST L., BASSETT J., LA ROCHE J.,WALDOCK M., BARRY J. & THOMAS K.V. 2008. Dissolved organiccarbon reduces the toxicity of copper to germlings of themacroalgae, Fucus vesiculosus. Ecotoxicology and EnvironmentalSafety 70: 88–98.
BURRIDGE T.R., CAMPBELL S.J. & BIDWELL J. 1999. Use of the kelpEcklonia radiata (Laminariales: Phaeophyta) in routine toxicitytesting of sewage effluents. Australasian Journal of Ecotoxicology5: 133–140.
CALLOW M.E. & CALLOW J.A. 2002. Marine biofouling: a stickyproblem. Biologist 49: 10–14.
CAMPBELL S.J. & BURRIDGE T.R. 1998. Occurrence of Undariapinnatifida (Phaeophyta: Laminariales) in Port Phillip Bay,Victoria, Australia. Marine and Freshwater Research 49: 379–381.
CASTILLA J.C. 1983. Environmental impact in sandy beaches ofcopper mine tailings at Chanaral, Chile. Marine Pollution Bulletin14: 459–464.
CASTILLA J.C. 1996. Copper mine tailing disposal in northern Chilerocky shores: Enteromorpha compressa (Chlorophyta) as asentinel species. Environmental Monitoring and Assessment 40:171–184.
CHAPMAN P.M. 1995. Ecotoxicology and pollution – key issues.Marine Pollution Bulletin 31: 167–177.
CHOJNACKA K. 2010. Biosorption and bioaccumulation – theprospects for practical applications. Environment International36: 299–307.
CHUNG I.K. & BRINKHUIS B.H. 1986. Copper effects in early stagesof the kelp, Laminaria saccharina. Marine Pollution Bulletin 17:213–218.
CONTRERAS L., MEDINA M.H., ANDRADE S., OPPLIGER L.V. &CORREA J.A. 2007. Effects of copper on early developmentalstages of Lessonia nigrescens Bory (Phaeophyceae) EnvironmentalPollution 145:75–83.
CORREA J.A., GONZALEZ P., SANCHEZ P., MUNOZ J.A. & ORELLANA
M.C. 1996. Copper-algae interactions: inheritance or adaptation?Environmental Monitoring and Assessment 40: 41–54.
CORREA J.A., CASTILLA J.C., RAMIREZ M.A., VARAS M., LAGOS
N.A., VERGARA S., MOENNE A., ROMAN D. & BROWN M.T. 1999.Copper, copper mine tailings and their effect on marine algae inNorthern Chile. Journal of Applied Phycology 11: 57–67.
DAVIS T.A., VOLESKY B. & MUCCI A. 2003. A review of thebiochemistry of heavy metal biosorption by brown algae. WaterResearch 37: 4311–4330.
DEAN T.A., THIES K. & LAGOS S.L. 1989. Survival of juvenile giantkelp: the effects of demographic factors, competitors, and grazers.Ecology 70: 483–495.
DEVINNY J.S. & VOLSE L.A. 1978. Effects of sediments on thedevelopment of Macrocystis pyrifera gametophytes. MarineBiology 48: 343–348.
DICKSON R.J. & HUNTER K.A. 1981. Copper and nickel in surfacewaters of Otago Harbour. New Zealand Journal of Marine andFreshwater Research 15: 475–480.
DOBLIN M.A. & CLAYTON M.N. 1995. Effects of secondarily-treatedsewage effluent on the early life-history stages of two species ofbrown macroalgae: Hormosira banksii and Durvillaea potatorum.Marine Biology 122: 689–698.
FLETCHER R.L. & MANFREDI C. 1995. The occurrence of Undariapinnatifida (Phaeophyceae, Laminariales) on the south coast ofEngland. Botanica Marina 38: 355–358.
FLOC’H J.Y., PAJOT R. & MOURET V. 1996. Undaria pinnatifida(Laminariales, Phaeophyta) 12 years after its introduction intothe Atlantic Ocean. Hydrobiologia 326/327: 217–222.
GARMAN G.D., PILLAI M.C. & CHERR G.N. 1994. Inhibition ofcellular events during early algal gametophyte development:effects of select metals and an aqueous petroleum waste. AquaticToxicology 28: 127–144.
GLEDHILL M., NIMMO M., STEPHEN J.H. & BROWN M.T. 1997. Thetoxicity of copper(II) species to marine algae, with particularreference to macroalgae. Journal of Phycology 33: 2–11.
GLEDHILL M., NIMMO M., HILL S.J. & BROWN M.T. 1999. Therelease of copper-complexing ligands by the brown alga Fucusvesiculosus (Phaeophyceae) in response to increasing total copperlevels. Journal of Phycology 35: 501–509.
Leal et al.: Chronic copper exposure impedes kelp meiospore development 19
GRAHAM M.H., VASQUEZ J.A. & BUSCHMANN A.H. 2007. Globalecology of the giant kelp Macrocystis: from ecotypes toecosystems. Oceanography and Marine Biology: An Annual Review45: 39–88.
GUIRY M.D. & GUIRY G.M. 2015. AlgaeBase. World-wideelectronic publication, National University of Ireland, Galway.http://www.algaebase.org; searched on 14 August 2015.
HAN T., KONG J.A., KANG H.G., KIM S.J., JIN G.S., CHOI H. &BROWN M.T. 2011. Sensitivity of spore germination and germtube elongation of Saccharina japonica to metal exposure.Ecotoxicology 20: 2056–2068.
HAY C.H. 1990. The distribution of Macrocystis (Phaeophyta:Laminariales) as a biological indicator of cool sea surfacetemperature, with special reference to New Zealand waters.Journal of The Royal Society of New Zealand 20: 313–336.
HOPKIN R. & KAIN J.M. 1978. The effects of some pollutants on thesurvival, growth and respiration of Laminaria hyperborea.Estuarine and Coastal Marine Science 7: 531–553.
HURD C.L., NELSON W.A., FALSHAW R. & NEILL K.F. 2004. History,current status and future of marine macroalgal research in NewZealand: taxonomy, ecology, physiology and human uses.Phycological Research 52: 80–106.
LEAL P.P., HURD C.L. & ROLEDA M.Y. 2014. Meiospores producedin sori of non-sporophyllous laminae of Macrocystis pyrifera(Laminariales, Phaephyceae) may enhance reproductive output.Journal of Phycology 50: 400–405.
LEE J.A., SUNWOO Y.I., LEE H.J., PARK I.H. & CHUNG I.K. 1989.The effects of copper on the early stages of Undaria pinnatifida(Harv.) Suringar (Laminariales, Phaeophyta) under temperature-irradiance gradient. The Korean Journal of Phycology 41: 41–53.
LEWIS A.G. 1995. Copper in water and aquatic environments.International Copper Association Ltd. New York, New York.65 pp.
LITTLER M.M. & MURRAY S.N. 1975. Impact of sewage on thedistribution, abundance and community structure of rockyintertidal macro-organisms. Marine Biology 30: 277–291.
LOTZE H.K., WORM B. & SOMMER U. 2001. Strong bottom-up andtop-down control of early life stages of macroalgae. Limnologyand Oceanography 46: 749–757.
MAY V. 1985. Observations on algal floras close to two sewerageoutlets. Cunninghamia 1: 385–394.
MERETTA P.E., MATULA C.V. & CASAS G.N. 2012. Occurrence of thealien kelp Undaria pinnatifida (Laminariales, Phaeophyceae) inMar del Plata, Argentina. BioInvasions Records 1: 1–5.
MOODY J.R. 1982. The sampling, handling and storage of materialsfor trace analysis. Philosophical Transactions of the Royal Societyof London A 305: 669–680.
MYERS J.H., DUDA S., GUNTHORPE L. & ALLINSON G. 2006.Assessing the performance of Hormosira banksii (Turner)Desicaine germination and growth assay using four referencetoxicants. Ecotoxicology and Environmental Safety 64: 304–11.
NIELSEN H.D., BROWN M.T. & BROWNLEE C. 2003a. Cellularresponses of developing Fucus serratus embryos exposed toelevated concentrations of Cu2þ. Plant, Cell and Environment26: 1737–1747.
NIELSEN H.D., BROWNLEE C., COELHO S.M. & BROWN M.T. 2003b.Inter-population differences in inherited copper tolerance involvephotosynthetic adaptation and exclusion mechanisms in Fucusserratus. New Phytologist 160: 157–165.
NIELSEN H.D., BURRIDGE T.R., BROWNLEE C. & BROWN M.T. 2005.Prior exposure to Cu contamination influences the outcome oftoxicological testing of Fucus serratus embryos. Marine PollutionBulletin 50: 1675–1680.
NIELSEN S.L., NIELSEN H.D. & PEDERSEN M.F. 2014. Juvenile lifestages of the brown alga Fucus serratus L. are more sensitive tocombined stress from high copper concentration and temperaturethan adults. Marine Biology 161: 1895–1904.
NOR Y.M. 1987. Ecotoxicity of copper to aquatic biota: a review.Environmental Research 43: 274–282.
PARR R.M. 1986. Technical considerations for sampling and samplepreparation of biomedical samples for trace element analysis.Journal of Research of the National Bureau of Standards 91: 51–57.
RAVEN J.A., EVANS M.C.W. & KORB R.E. 1999. The role of tracemetals in photosynthetic electron transport in O2-evolvingorganisms. Photosynthesis Research 60: 111–149.
REED D.C. 1990. The effects of variable settlement and earlycompetition on patterns of kelp recruitment. Ecology 71: 776–787.
REED D.C. & FOSTER M.S. 1984. The effects of canopy shadings onalgal recruitment and growth in a giant kelp forest. Ecology 65:937–948.
REED R.H. & L. MOFFAT. 1983. Copper toxicity and coppertolerance in Enteromorpha compressa (L.) Grev. Journal ofExperimental Marine Biology and Ecology 69: 85–103.
RIJSTENBIL J.W., DERKSEN J.W.M., GERRINGA L.J.A., POORTVLIET
T.C.W., SANDEE A., BERG M., DRIE J. & WIJNHOLDS J.A. 1994.Oxidative stress induced by copper: defense and damage in themarine planktonic diatom Ditylum brightwellii, grown in contin-uous cultures with high and low zinc levels. Marine Biology 119:583–590.
ROLEDA M.Y., WIENCKE C., HANELT D. & BISCHOF K. 2007.Sensitivity of the early life stages of macroalgae from the northernhemisphere to ultraviolet radiation. Photochemistry and Photobi-ology 83: 851–862.
ROLEDA M.Y., CAMPANA G.L., WIENCKE C., HANELT D., QUARTINO
M.L. & WULFF A. 2009. Sensitivity of Antarctic Urosporapenicilliformis (Ulotrichales, Chlorophyta) to ultraviolet radiationis life-stage dependent. Journal of Phycology 45: 600–609.
ROLEDA M.Y., MORRIS J.N., MCGRAW C.M. & HURD C.L. 2012.Ocean acidification and seaweed reproduction: increased CO2
ameliorates the negative effect of lowered pH on meiosporegermination in the giant kelp Macrocystis pyrifera (Laminariales,Phaeophyceae). Global Change Biology 18: 854–864.
ROSS K.E. & BIDWELL J.R. 1999. Comparative response of Eckloniaradiata zoospores and other marine species to complex effluents.Australasian Journal of Ecotoxicology 5: 113–122.
RUSSELL L.K., HEPBURN C.D., HURD C.L. & STUART M.D. 2008.The expanding range of Undaria pinnatifida in southern NewZealand: distribution, dispersal mechanisms and the invasion ofwave-exposed environments. Biological Invasions 10: 103–115.
SAEZ C.L., RAMESH K., GRECO M., BITONTI M.B. & BROWN M.T.2015. Enzymatic antioxidant defences are transcriptionallyregulated in Es524, a copper-tolerant strain of Ectocarpussiliculosus (Ectocarpales, Phaeophyceae). Phycologia 54: 425–429.
SCHIEL D.R. & THOMPSON G.A. 2012. Demography and populationbiology of the invasive kelp Undaria pinnatifida on shallow reefsin southern New Zealand. Journal of Experimental MarineBiology and Ecology 434–435: 25–33.
SILVA P.C., WOODFIELD R.A., COHEN A.N., HARRIS L.H. &GODDARD J.H.R. 2002. First report of the Asian kelp Undariapinnatifida in the northeastern Pacific Ocean. Biological Invasions4: 333–338.
SOROKIN C. 1962. Effects of acidity on cell division. ExperimentalCell Research 27: 583–584.
SWARTZ R.C., COLE F.A., SCHULTS D.W. & DEBEN W.A. 1986.Ecological changes in the Southern California Bight near a largesewage outfall: benthic conditions in 1980 and 1983. MarineEcology Progress Series 31: 1–13.
THOMPSON R.S. & BURROWS E.M. 1984. The toxicity of copper, zinc,and mercury to the brown macroalga Laminaria saccharina. In:Ecotoxicological testing for the marine environment (Ed. by E.Jaspers & C. Claus), pp. 259–269. University of Ghent andInstitute for Marine Scientific Research, Bredene, Belgium.
WARTON D.I. & HUI F.K.C. 2011. The arcsine is asinine: theanalysis of proportions in ecology. Ecology 92: 3–10.
YAMANAKA R. & AKIYAMA K. 1993. Cultivation and utilization ofUndaria pinnatifida (wakame) as food. Journal of AppliedPhycology 5: 249–253.
YE N.H., WANG G.C. & TSENG C.K. 2005. Effect of heavy metals(Cd, Cu) on the gametophytes of Laminaria japonica Aresch.Journal of Integrative Plant Biology 47: 942–951.
Received 14 August 2015; accepted 25 October 2015Associate Editor: Ricardo A. Scrosati
20 Phycologia, Vol. 55 (1)
