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Copper effects in early stages of the Kelp, Laminaria saccharina
Volume 17/Number 5/May 1986 Sciences and Technologies Grants Committee, Belconnen, A.CT., 2616, Australia. 162pp. Culkin, F. & Riley, J. P. (1958). The occurrence of gallium in marine organisms. J. mar. biol. Assoc. UK 37,607-615. Flores, S. E. & Huacuja, L. I. (1966). Elementos menores contenidos en algunas gorgonaceas del Gulfo de Mexico. Bol. Inst. Oceanog., Univ. Oriente, 5,105-127. Harriss, R. C. & Ahny, C. C. Jr. (1964). A preliminary investigation into the incorporation and distribution of minor elements in the skeletal material of scleractinian corals. Bull. mar. Sci. GulfCarrib. 14, 418423. Howard, L. S. & Brown, B. E. (1984). Heavy metals in reef corals. Oceanogr. Mar. Biol. Ann. R ~ 22, 195-210. Jones, G. B. (1974). The occurrence of some trace metals in organisms collected from Lundy. Rep. Lundy FieM Soc. 25, 49-52. Lasker, H. R. (1981). A comparison of the particulate feeding abilities of three species of gorgonian soft coral. Mar. Ecol. Prog. Ser. 5, 6167. Mullin, J. B. & Riley, J. P. (1956). The occurrence of cadmium in sea-
water and in marine organisms and sediments. Z Mar. Res. 15, 103-122. Riley, J. P. & Segar, D. A. (1970). The distribution of the major and some minor elements in marine animals. I. Echinoderms and coelenterates. J. mar. biol. Assoc., UK 50, 721-730. Schlichter, D. (1982). Epidermal nutrition of the Alcyonarian Heteroxenia fuscescens (Ehrb.): absorption of dissolved organic material and lost endogenous photosynthates. Oecologia 53, 40-49. St. John, B. E. (1973). Trace elements in corals of the Coral Sea: their relationship to oceanographic factors. In: Proc. Int. Symp. on Oceanography of the South Pacific, Wellington. R. Fraser (Ed.) p p . 149-158. UNESCO. St. John, B. E. (1974). Heavy metals in the skeletal carbonate of scleractinian corals. In: Proc. 2nd Int. Coral Reef Symp., A. M. Cameron et al. (Eds). Vol. 2, pp. 461-469. Great Barrier Reef Committee, Brisbane. Tapiolas, D. M. (1980). Natural products derived from soft corals, 115 pp. Unpublished B.Sc. (Hon.) thesis, James Cook University of North Queensland.
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MarinePollutionBulletin, Vol. 17,No. 5,213-218,1986
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Copper Effects in Early Stages of the Kelp, Laminaria saccharina IK KYO CHUNG and BOUDEWlJN H. BRINKHUIS Marine Sciences Research Center, State University of New York, Stony Brook, N Y 11794-5000, USA
The effects of copper on the Laminaria saccharina were examined by culturing plants in 5, 10, 50, 100 and 500 ~g Cu I-t filtered and filtered plus uv treated seawater media. Fertile sporophytes were used to determine effects of copper on meiospore release. Untreated meiospores also were obtained and cultured in seawater media, followed by copper additions at various stages of development through the early sporophyte stage. The release of meiospores from copper-treated sorus materials was reduced by concentrations ~ 50 pg I-t. Settlement and germination o f meiospores were not affected by concentrations up to 500 pg I-t. Development of gametophytes and gametogenesis were delayed in concentrations ~_50 pg !-t. Growth of sporophytes, in terms of length increase or relative blade area increase, was inhibited with increasing concentrations > 10 pg I-t.
The passive and/or active uptake of heavy metals show various effects in algae. At certain concentration levels, biological minor, or trace, elements may increase the growth rate (Huntsman & Sunda, 1980). In the case of non-biological heavy metals, organisms are tolerant to some concentrations, depending on the conditions of the organism and the form of the metal (Lepp, 1981). But at higher concentration levels, the organisms are likely to be affected in their growth, photosynthesis, mitochon-
drial respiration and other general metabolic pathways. Moreover, these effects can lead to death. For many years, copper sulphato has been widely used as an algicide to control and/or prevent undesirable algal growths, especially waterblooms. Copper inactivates the electron transport between the oxidizing side of the reaction center of photosystem II and electron donating side of 1,5-diphenyl-carbazide, affecting reaction rates of dark step in green algae (Shioi et aL, 1978). Toxicity of copper may cause the succession or change in the community structure due to the differential responses of organisms in the water column (Thomas & Siebert, 1977; Leland & Carter, 1984). Like other heavy metals, copper affects permeability of the plasma membrane, causing loss of potassium from algal cells (De Filippis, 1979). Excessive concentrations of copper cause the loss of photosynthetic pigment Chlorella (De Filippis & Pallaghy, 1976). Copper toxicity is believed to be related to the concentration of the free copper ion, as chelating agents detoxify copper-spiked seawater. This was subsequently confirmed by Jackson & Morgan (1978) using a chemical speciation model to estimate the concentration of free copper ions. Several artificial seawater media have been introduced based on the thermodynamic equilibrium calculated with computer programs (Morel et al., 1979; Kuwabara & North, 1980). 213
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Few studies on the effects of copper on reproduction and development have been reported for seaweeds. The present study was conducted to delineate the general growth, development and morphological effects of copper on the brown alga, Laminaria saccharina, especially during meiospore release, gametogenesis and early sporophyte stages. These stages are susceptible to high mortality rates throughout the life cycle of this kelp species (Chapman, 1984).
Materials and M e t h o d s
The present study followed the culture methods of Brinkhuis et al. (1983, 1984). An incubation chamber was maintained at 10 + I*C and illuminated by very high output cool-white fluorescent light providing cultures with 50 0E cm -2 s-l on 12:12 h photoperiod.
General Culture Procedure Laminaria saccharina plants with mature sori were collected in Long Island Sound at Crane Neck, New York. A ripe portion of sorus was cut from the plant and wiped clean with 5% NaOCl (v/v in filtered seawater) and further rinsed with membrane filtered (Gelman) seawater. Cleaned sorus materials were allowed to dry for several hours prior to meiospore release (1-6 h). During that time, the meiospore release potential was checked periodically. Several drops of filtered seawater were pipetted on the surface of the sorus material and after 3 min the suspension on the surface was carefully picked up by pipette and examined under the microscope. When the number of meiospores exceeded about 10-20 meiospores mm -2 under × 100 magnification, the sorus material was immersed in the culture media. Meiospore Release in the Cu Media Small squares (1 cm 2) were excised from the dried sorus material which was ready for meiospore release using Teflon coated razor blades. Each excised specimen was immersed in a 24 ml glass vial containing 20 ml of media which had been 0.2 ltrn filtered, uv-treated and enriched with 883 ;xM NCO 3- (as NaNO3) and 36 PO43- (as NaH2PO4) (Sunda & Guillard, 1976), then supplemented with diluted copper stock solution (Cu DILUT-IT; 1 g Cu as CuSO4 to make 1 1.). For microscopic specimen cultures, the media were 0.2 lun filtered again for sterilization before copper augmentation. All surfaces were acid cleaned and soaked in Milli-Q water before use. The concentrations of copper were 5, 10, 50 and 100 0g 1-~. The glass vials were covered and wrapped with aluminium foil, placed in an incubator for 1 h, and shaken several times during the 1 h release period. Sorus materials were then removed from the vial. The number of meiospores was counted using a hemacytometer. Cu Treatment Before the Meiospore Settlement Similarly sized specimens were placed in the plastic Petri dishes (Nalgene 5500-0010) and allowed to release meiospores for 24 h. Copper treatment was then initiated before actual settlement had begun. 214
Cu Treatment after Meiospore Settlement The remainder of sorus material was placed in a beaker for bulk meiospore release. After examining the density of meiospores, volumetric inoculations (50 Ixi) were placed on the 18 x18 mm coverslips in glassquadrant Petri dishes, plexiglass boats or directly on plastic Petri dishes (Nalgene 5500-0010 and B-D 3006). Inoculated materials were kept in darkness in the incubator for 12 h, the control media was added; 9 ml for the glass quadrant Petri dishes, 4 ml for plastic Petri dishes and 125 ml for plexiglass boats. The media were renewed every 4 days. Developmental Stage Analysis Several significant stages can be identified during the incubation period of 3 weeks. They can be defined as six steps (modified from Burrows, 1971; Burrows & Pybus, 1971): Stage I: Meiospore settlement with germ tube production Stage I I: Early stage of developing gametophyte (movement of cytoplasmic content to germ tube and increase in cell size) Stage l/I: Later stages of developing gametophyte (morphogenesis) Stage IV: Mature gametophytes Stage V: Early stage of sporophyte (1-4 cells) Stage VI: Later stages of sporophyte One hundred plants were scored for stage development in each coverslip or culture dish. If a particular stage represented more than 50% of the population, the development status of that culture was described as representing that stage. Culture of Young Fronds For sporophytes sized above 1 cm long, 5 1. plastic planting boxes or 11. beakers which contained five to ten plants were immersed in the refrigerated aquarium at 12+1"C under cool-white fluorescent light with 20 0E cm -2 s -~ on 12:12 h photoperiod. The media were constantly agitated by wave plates in each planting box. Both boxes and beakers were aerated. The plants which had been cultured in the cold room were acclimated for 1 day and photographed before treatment. After 7 days of incubation, the plants were also photographed and hole punches (154 m m 2) were taken for chlorophyll analysis. The blade area was measured with computerprogrammed graphic analysis from the photographs. Growth rates were monitored by the relative blade area increase. Statistical Analysis All data were examined using statistical procedures described in Sokal & Rohlf (1981). Results
Meiospore Release The density of released meiospores in each vial is shown in Table 1. The mobility of released meiospores did not seem to be affected by the range of copper concentrations tested, but the densities were quite
Volume 17/Number 5/May 1986
remained in the early gametophyte stage, and plants in 50 and 100 pg Cu 1-~ did not show the progress compared to the Day 8 observation. By the 15th day, control plants reached the sporophyte stage, but those in 5 and Treatment Average*~ St. Dev.*~ n Sigma R*2 10 pg Cu 1-1 showed delayed sporophyte formation, and Control 23.0 4.27 10 330.5 the rest in 50 and 100 pg Cu 1-1 remained in the previous 5 lag 1-I 21.6 2.22 10 301.5 developing gametophyte stage. By the 18th day, plants in 10 pg 1-I 26.3 3.50 10 412.0 50 pg 1-I 16.6 3.50 10 162.0 5 pg Cu 1-1 began to produce zygotes and sporophytes, 100 pg 1-I 11.1 3.48 10 69.0 but those in 10 pg Cu 1-1 did not show this development, and some developing gametophytes in 50 pg Cu 1-1 lost *1 Unit: 5 X104 meiospores I-L *2 Sigma R: Pooled rank sum from Kruskal-Wallis Test. their cellular contents. Cells in 100 pg Cu 1-1 still *Note: Hadi.=39.63 > X20.1..= 18.467. remained in the developing gametophyte stage and the majority of plants showed retarded development. Three weeks after inoculation plants in control, 5 and 10 pg Cu TABLE 2 1-1 media all formed sporophyte stages (by the > 50% Nonparametric multiple comparison STP (an unplanned test for equal characterization), but those in 50 pg Cu 1-~ did not show sample size, values: Us from Mann-Whitney U-Test) for the meiospore release by Laminaria saccharina in copper added media as in sporophyte formation and those in 100 pg Cu 1-1 Table 1. remained in the developing gametophyte stage. The mean length of sporophytes was 492.6 + 73.37 Control pm (mean + S.D.) for control plants and 63.7 5:22.88 prn 5 pgl -I 58.5 10 pg 1-I 26.5 15.5 for those in 5 pg Cu 1-1 media on 26th day. These 50 pg 1-j 91.5" 90.0* 98.5* measurements were significantly different based on the 87.0* 100 pg !-I 99.5* 100.0" 100.0" test of equality of means of two samples whose variances *Significant at an experimentwise error rate of 0.05. are assumed to be unequal (a -= 0.05, ts,-16.80 > t0.05** Uo.0515.,01=86.09. 2.28). Sporophytes in 10 p.g Cu 1-1 media showed abnormal growth patterns and other plants in the higher concentrations remained in the same stage or were dead. different among treatments tested. Sorus tissues in 10 pg The summary of the above development is presented in Cu 1-1 media showed the best meiospore release and Fig. 1. those in over 50 pg Cu 1-1 exhibited severely reduced meiospore release. vl com~ / o," A Kruskal-Wallis test showed that meiospore release / / / .5 `06 CU.L"1 in different media was significantly different among = ""'_,.L_-.. J'' CU.L-1 treatments (Table 1). Nonparametric multiple Comparison STP (Table 2) showed that the set (control, 5 pg Cu 1-~, and 10 pg Cu 1-1) was not significantly heteroII ....................... . . cu.,.-1 geneous. Since the 50 and 100 pg Cu 1-1 were heterogeneous with any other treatment, any set containing either 50 and 100 pg Cu 1-1 (or both) would be signifiI cantly heterogeneous. The data therefore partition into the following: (control, 5 pg Cu 1-l, 10 pg Cu 1-1) (50 lag ; 17 1; Cu 1-l) (100 pg Cu 1-1). Meiospore release was reduced DAYS /~FTER INOCULATION at the higher copper concentrations (_~50 pg Cu 1-1). Fig. 1 Summary of the development of Laminaria saccharina gametoTABLE I
Meiospore release by Laminaria saccharina in copper supplemented media.
~ V
Gametophyte Development The developmental stages were examined on Day 3, 5, 8, 10, 15, 18, 21. Final observations were taken on Day 26. Identification of the development was based on the 6 stage steps described earlier. Copper Treatment before Meiospore Settlement. After inoculation, all meiospores began to settle and germinate. The Day 3 observation showed well-settled meiospores with germ tubes in all treatments. The early stage of gametophytes appeared on the 5th day, which can be identified by the movement of cytoplasmic content to the germ tube and increased cell size. Up to the 8th day, all the cells in the different concentrations of copper showed the same developmental pattern. On the 10th day, one-celled oogonia were easily found and several sporophytes began to appear in control media. However, plants in other media showed somewhat delayed development. Cells in 5 and 10 pg Cu 1-1
/J
"'/ "_.'//'lg
phytes. The copper treatment was applied before meiospore setdement (Day 0).
CONTROL
II1"
C
U
I
~ Igg, ~
(2)
(7)
58 ,06 CU.L"1 ,IJ6 CU.L-1
(12)
(17)
DAYS /~TER INOCULATION (DAYS N:'rER OJPP'ERTREATI~NT)
Fig. 2 Summary of the development of Laminaria saccharina gametophytes. The copper treatment was applied after meiospore settlement (Day 3).
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Copper Treatment after Meiospore Settlement. Treatments were applied from stage I (at day 3) when all the meiospores had germinated in the control media. Fiveday old cells in the all treatments showed the early stage of gametophyte development and some cells in 100 and 500 gg Cu 1-1 media began to show mortality, which was defined as empty cells and further confirmed with Evans blue staining--dark blue staining of whole cell (Taylor & West, 1980). Ceils in the control, 5 and 10 lag Cu 1-l media showed the same development pattern, but those in 50 gg Cu 1-1 media began to die by the 8th day of observation. By the 10th day, mature gametophytes dominated in control and 5 0g Cu 1-l media, but ceils in 10 gg Cu 1-I did not show maturity. Other plants in higher copper concentrations showed retarded development (50 og Cu 1-1 ), remained in the same stage (100 gg Cu 1-t ) and 100% mortality (empty cells and cell lysis, 500 0g Cu 1-I ). On the 15th day of observation, control sporophytes covered the whole coverslip, and gametophytes in 5 and 10 ~ Cu 1-1 showed maturity. Those in higher concentrations did not develop further. Mature gametophytes did not develop into sporophytes in 5 and 10 pg Cu 1-l media until the 18th day, but sporophytes were well developed in the control. Gametophytes in 50 0g Cu 1-l were still in the developing gametophyte stage. After 3 weeks, plants in the control and 5 Og Cu 1-1 media showed the later sporophyte stage, specimens in 10 0g Cu 1-l stayed inthe mature gametophyte stage and cells in 50 ltg Cu 1-l remained in the developing gametophyte stage. The length of fronds reached 940.5 + 23.32 pan for control plants, 111.7 + 24.60 ptm for those in 5 0g Cu 1-~ and 97.3 ± 23.32 grn for those 10 0g Cu 1-l on the 26th day. The others remained in the same stage or dead. Some sporophytes in 10 Og Cu 1-l media showed abnormal growth patterns. Multiple comparisons among pairs of means reveals that the length of control plants is significantly different from others (0t-" 0.05), but those of 5 and 10 pg Cu 1-l are not significantly different at 0t--0.05 level. These developmental patterns are summarized in Fig. 2.
TABLE 3 Relative blade area increase in Laminaria saccharina. R.B.A.I. ffi Blade area,2 -- Blade areatj × 100 Blade area, Relative Blade Area Increase (R.B.A.I.) Day 0-4 Day 4-9 Total
Treatment Control 10 pgCul -~ 50 0gCul -~ 100 0gCul -~ 500 0gCul -~
63.7+11.4 74.8±24.2 70.5±25.2 8.7+ 5.4 -5.2:1:4.1
54.2± 5.9 63.9+31.7 55.9±20.0 dead dead
blade area increase is summarized in Table 4. Growth rates in all treatments were significantly different from each other based on the comparison of means at 0t--0.05 level. Inhibition of growth increased with increasing copper concentration. During the incubation period, plants in 100 and 500 pg Cu l-I media exhibited chlorosis and disintegration in the distal and marginal blade portions.
TABLE 4 Relative blade area increase in Laminaria saccharina. Treatment
R.B.A.I.*
Control 10 ILgI-l 50 $tgI-~ I00 l~gl-l 500 ~g I-t
34.9 + 4.92 26.9 + 3.33 9.0 ± 3.82 2.8±4.93 -5.1 :I:4.78
216
% 1 O0 76.9 25.8 8.1 -14.8
*R.B.AJ.: same as Table 3.
TABLE 5 Comparison of developmental stages. Burrows (1971)
Burros & Pybus (1971)
1. Settled zoospore
1. Settled zoospore
2. Developing gametophyte
3. Fertile gametophyte
2. Settled zoospore with germ tube and/or vegetative cells 3. Fertile gametophyte
4. First sporophyte division
4. First sporophyte division
5. Later sporophyte stages
5.2-8 celled sporophyte
Growth of YoungPlants Growth of young plants which were grown in the cold room from the meiospore to 1-3 cm long sporophytes and then exposed to copper treatment is summarized in Table 3. All the values were tested by the multiple comparison among pairs of means at 0c-, 0.05 level. During the first 4 days, the relative blade area increase of plants in 10 ~g Cu 1-1 was highest among all treatments, but was not significantly different from those of the control and 50 0g Cu 1-l treatments. However, the above three values were significantly different from those of 100 and 500 gg Cu 1-l. During the next 5 days, plants in 100 and 500 0g Cu 1-l exhibited severe ehlorosis and total degradation and were not able to be photographed. The remaining three gi'oups showed continuous growth with decreasing relative blade area increase compared to the first 4-day period, but those were not significantly different from eachother. Growth of young sporophytes which were 8-10 cm long prior to copper exposure in terms of the relative
152.5±21.8 181.3+30.4 168.9±63.0
Present study 1, SeRled meiospore with germ tube 2. Early stages of developing gametophyte 3. Late stages of developing gametophyte 4. Mature gametophyte 5. Early stages of sporophyte
(1-4 celled) 6. First longitudinal division in sporophyte 7. All later stages
6. Later stages o f sporophyte
Discussion According to preliminary observations, i~ was necessary to modify the development stage system defined by
Volume 17/Number 5/May 1986 TABLE 6 Concentrations of copper in nearshore region. Location New York Bays, US La Rosiere, UK Norwegian Fjord, Norway La Rabida, Spain Ras Beirut, Beirut
Concentration (unit; p.g 1-j)
Reference
2.7-65
Waldhauer et al. (1978) 17.4 (seawater) Romeril (1977) 60.2 (effluent) 3.75-77.0 Stenner & Nickless (1974) 107 Stenner & Nickless (1975) 100-230 Shiber & Shatila (1979)
Burrows & Pybus (1971). As shown in Figs. 1 and 2, an inhibition of development began at a later stage of gametophyte development, which could not be described by the system of Burrows & Pybus (see Table
5). The plants which were incubated in 0.2 u filtered, uvtreated, copper supplemented media in this study exhibited effects of copper toxicity in terms of their. development and growth on the basis of total copper concentrations, which included the background ambient copper concentration that was assumed to be below 1 Og Cu 1-1 (Preston et aL, 1972; Brewer, 1975). During the course of incubation with copper, it was obvious that there were signs of delayed gametophyte development, inhibition of sporophyte formation and growth of sporophytes. Burrows & Pybus (1971) found delayed development, due to the suspended materials (silt) which decreased the light intensity, e.g. by 6-9 days of the fertile gametophyte stage and by 12 days of first sporophyte division. This is a greater delay than the results from the present study because their culture conditions (especially light was too low) were suboptimal for gametogenesis (Liining & Neushul, 1978; Liining, 1980). Similar results to those of Burrows & Pybus (1971) were made by Hopkins & Kain (1978) in their copper concentration range of 25-75 ~g dm -3, where sporophyte formation was delayed by up to 13 days. A l t h o uhg the meiospore release was done under artificial stress conditions, there seemed to be impacts of copper on meiospore release (Tables 1, 2). Generally, each stage seemed to have quite different responses to copper. There was no effect of copper on the first stage-meiospore settlement and germination--under copper concentrations up to 500 0g Cu 1-s. After movement of cytoplasmic content to germ tube, vegatative gametophyte growth was inhibited at the concentrations of 50 Itlg Cu l - l , and higher. Once the gametophytes were mature, the plants showed parallel development into sporophytes below copper concentrations of 50 0g 1-~, but growth rates of all copper treated plants were significantly lower than that of controls. Slight differences in the responses in terms of initiation and duration of the effects of copper toxicity between two experiments may be caused by the timing of copper application. Though meiospore settlement and germination were not affected, there might be differen-
tial responses of each stage and chronic effects in later vegetative gametophyte development and the early 1-4 celled sporophyte stage. There have been several studies describing growth inhibition by metals in seaweeds (see Rai et al., 1981 for review). Str6mgren (1979a, b, 1980a, b) presented the relationship between the percentage reduction (z) in growth rate and the product of time (x) and concentration (y) in metal (Pb, Cd, Hg, Cu and Zn) treated fucoid algae (Fucus, Ascophyllum and Pelvetia); z "= kxy He found at least 60% reduction in growth rates at copper concentrations over 33 0g 1-~ during the course of an 11 day incubation. Here, young sporophytes (1-3 cm long) in 100 and 500 0g Cu 1-~ media showed 100% mortality within 4 days, a rate higher than the results of Str6mgren. The 5-10 cm long fronds showed higher reduction at a given value of product of x and y The k value varies with increasing value of product of x and y: up to 100-=0.3; 100-500--0.2; 500-1000---0.1. These are much higher than Str6mgren's values. The differences may be ascribed to the type of incubated materials; only growing apicies were used in his study but whole plants were used in the present experiments. The present information of the effect of copper can be applied to the ecosystem level. The effect of copper on the early stages of L. saccharina may decrease the total primary production in the kelp-based system due to failure in the recruitment and decreased growth. Abnormalities in the early life cycle stage, reduction in chlorophyll content and change in the fine structure may have potential deleterious impacts on the primary production in the kelp ecosystem. The concentrations applied in the present study were similar to those found in the natural environment. Copper is present in the open ocean at a concentration of approximately 1.0 Og 1-~ (Brewer, 1975). However, concentrations increase up to 230 lxg 1-~ in nearshore waters which are highly susceptible Io metal pollution (see Table 6). This is the zone inhabitated by kelps. Even though there are high concentrations of copper, the toxicity and bioavallability of copper is dependent on its chemical form and the metal-organic complexes (Davey et aL, 1973; Hirose & Sugimura, 1985; Piotrowicz et al., 1984; Wood, 1983). Due to metal organic complexing agents like humic acid, the activities of free copper ion can be maintained at a native constant level against relatively small fluctuations by the addition or uptake in the marine environment (Hirose & Sugimura, 1985). The ability to concentrate ions of metals from the surrounding seawater has been well documented for the brown algae and make these seaweeds useful indicators of marine heavy metal pollution (Bryan, 1983; Bryan and Hummerstone, 1973; Haug et al., 1974;MYklestad et al., 1978). The bioaccumulated metals in seaweed tissue may be transferred to the detrital food web. The process of the microbial activity in the sediment may be decreased by the metals accumulated in algal detritus. Finally, the total production of the system may be affected by the decreased microbial activity which provides the regenerated nutrients to the water column (Babich & Stotzky, 1985). 217
Marine PollutionBulletin
Summary The present study demonstrates the following results: 1. The density of meiospore release was reduced in higher concentrations of copper _ 50 ptg 1-I in treated sorus materials. 2. Settlement and germination of meiospores were not affected by the range of copper concentrations up to 5 0 0 ~tg 1-1.
3. Development of gametophytes and gametogenesis were delayed in copper concentrations ~ 50 lag 1-m. 4. Growth of sporophytes in terms of length increase or relative blade area increase was inhibited with increasing copper concentrations > 10 ~g l-~. 5. The critical concentrations of copper which inhibit the development of gametophytes and young sporophytes is between 10 and 50 l~g l-~.
This research was supported by the Gas Research Institute, New York State Energy Be.search and Development Authority and the New York Gas Group by contracts to B.H.B. Marine Science Research Center Contribution No. 502.
Babich, H. & Stotzk-y, G. (1985). Heavy metal toxicity to microbemediated ecologic processes: A review and potential application to regulatory poficies. Environ. Res. 36, 111-137. Brewer, E G. (1975). Minor elements in seawater. In Chemical Oceanography Vol. 1, (J. E Riley & G. Skirrow, eds.), pp. 415-496. Academic Press, New York. Brinkhuis, B. H., Breda, V. A., Tobin, S. & Macler, B. A. (1983). New "York Marine Biomass Program--Culture of Laminaria saccharina. J. Wid Maricult. Soc. 14, 360-379. Brinkhuis, B. H., Mariani, E. C., Breda, V. A. & Brady-Campbell, M. M. (1984). Cultivation of Laminaria saccharina in New York Marine Biomass Program. Hydrobiologica. 116/117, 266-271. Bryan, G. W. (1976). Some aspects of heavy metal tolerance in aquatic organisms. In Effects of Pollutants on Aquatic Organisms. (A. P. M. Lockwood, ed.) pp. 7-34. Cambridge University Press, Cambridge. Bryan, G. W. (1983). Brown seaweed, Fucus vesiculosus, and the gastropod, Littorina littomlis, as indicators of trace-metal availability in estuaries. Sci. Tot. Environ. 28, 91-104. Bryan, G. W. & Hummerstone, L G. (1973). Brown seaweed as an indicator of heavy metals in estuaries in south-west England. J. Mar. B/or U.K. 53, 705-720. Burrows, E. M. (1971). Assessment of pollution effects by the use of algae. Proc. Roy. Soc. Lond. B. 177, 295-306. Burrows, E. M. & Pybus, C. (1971). Laminaria saccharina and marine pollution in North-East England. Mar. Pollut. Bull 2, 53-56. Chapman, A. R. O. (1984). Reproduction, recruitment and mortality in two species of Lam/natia in Southwest Nova Scotia. J. Exp. Mar. BioL Ecol. 78, 99-109. Davey, E. W., Morgan, M. J. & Erickson, S. J. (1973). A biological measurement of copper complexation capacity of seawater. LiranoL Oceano~. 18, 993-997. De Filippis, L. F. (1979). The effect of heavy metal compounds on the permeabifity of Chlorella cells. Z. Pflanzenphysiol. 92, 39-49. De Filippis, L. E & PaUaghy, C. K. (1976). The effects of sublethal concentrations of mercury and zinc on Chiorella. II. Photosynthesis and pigment composition. Z. Pflanzenphysiol, 78, 314-322. Haug, A., Melsom, S. & Omang, S. (1974). Estimation of heavy metal pollution in two Norwegian 0ord areas by analysis of the brown alga AscophyUum nodosurn. Environ. PoUut. 7, 179-192. I-lirose, K. & Sugimura, Y. (1985). Role of metal--organic complexes in the marine environment. Mar. Chent 16, 239-247. Hopkins, R. & Kain, J. M. (1978). The effects of some pollutants on the survival, growth and respiration of Laminaria hyperborea. Estuar. Coastl Mar. Sci. 7, 531-553.
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Huntsman, S. A. & Sunda, W. G. (1980). The role of trace metals in regulating phytoplankton growth. In The Physiological Ecology of Phytoplankton. (I. Morris, ed.). pp. 285-328. University of California Press, Berkeley. Jackson, G. A. & Morgan, J. J. (1978). Trace metal-chelator interactions and phytoplankton growth in seawater media: Theoretical analysis and comparison with reported observations. Limnol. Oceanogr. 23, 268-282. Kuwabara, J. S. & North, W. J. (1980). Culturing microscopic stages of Macrocysits pyrifem (Phaeophyta) in Aqufl, a chemically defined medium. J. Phycol. 16, 546-549. Lepp, N. W. (1981). Effects of Heavy Metal Pollution on Plants. Vol. 1. Effects of Trace Metals on Plant Function. Applied Science Publishers. London & New Jersey. 352 p. Leland, H. V. & Carter, J. L. (1984). Effects of copper on species composition of periphyton in a Sierra Nevada, California, stream. Fmshwat. Biol. 14, 281-296. Liining, K. (1980). Critical levels of light and temperature regulating the gametogenesis of three Laminaria species (Phaeophyceae). J. Phycol. 16, 1-15. Liining, K. & Neushul, M. (1978). Light and temperature demands for growth and reproduction of Laminarian gametophytes in southern and central California. Mar. BioL 45, 297-309. Morel, F. M. M., Rueter, J. G., Anderson, D. M. & Guillard, R. R. L. (1979). Aquih A chemically defined phytopiankton culture medium for trace metal studies. J. Phycol. 15, 135-141. Myklestad, S., Edie, I. & Melsom, S. (1978). Exchange of heavy metals in Ascophyllum nodosum (L.) Le Jol. in situ by means of transplanting experiments. Environ. Pollut. 16, 277-284. Piotrowicz, S. R., Harvey, G. R., Boran, D. A., Weisel, C. P. & SpringerYoung, M. (1984). Cadmium, copper and zinc interactions with marine humus as a function of ligand structure. Mar. Chem. 14, 333346. Preston, A., Jeffries, D. F., Dutton, J. W., Harvey, B. R. & Steele, A. K. (1972). British Isles coastal waters: The concentrations of selected heavy metals in seawater, suspended matter and biological indicators--a pilot survey. Environ. PoUut. 3, 69-82. Rai, L. C., Guar, J. P. & Kumar, H. D. (1981). Phycology and heavy-metal pollution. BioL Reu. 56, 99-151. Romefil, M. G. (1977). Heavy metal accumulation in the vicinity of a desalination plant. Mar. Poilut. BuR 8, 84-87. Shiber, J. G. & Shatila, T. (1979). Certain metals in three coastal algae from Ras Beriut waters. Hydrobiologia 63, 105-112. Shioi, Y., Tamai, H. & Sasa, T. (1978). Inhibition of photosystem II in the green alga Ankistrodesmus falcatus by copper. Physiol. Pl. 44, 434438. Sokal, R. IL & Rohlf, E J. (1981). Biometry. W. H. Freeman, San Francisco. Stenner, R. D. & Nickless, G. (1974). Distribution of some heavy metals in organisms in Hardangerfjord and Skjerstadfjord, Norway. War.Air Soil Pollut. 3,279-291. Stermer, R. D. & Nickless, G. (1975). Heavy metals in organisms of the Atlantic coast of S. W. Spain and Portugal. Mar. PoUut. Bull 6, 89-92. Strtmgren, T. (1979a). The effect of zinc on the increase in length of five species of intertidal Fucales. J. Exp. Mar. Blot Ecol. 40, 95-102. Strtmgren, T. (1979b). The effect of copper on length increase in Ascophyllum nodosum (L.) Le Jolis. J. Exp. Mar. BioL Ecol. 37, 153159. Strtmgren, T. (1980a). The effect of dissolved copper on the increase in length of four species of intertidal fucoid algae. Mar. Environ. Res. 3, 5-13. Strtmgren, T. (1980b). The effect of lead, cadmium, and mercury on the increase in length of five intertidal Fucales. J. Exp. Mar. Biol. Ecol. 43, 107-119. Sanda, W. & Guillard, R. R. L. (1976). The relationship between cupric ion activity and the toxicity of copper to phytoplankton. J. Mar. Res. 34, 511.-52.9. Taylor, J. A. & West, D. W. (1980). The use of Evans Blue stain to test the survival of plant cells after exposure to high osmotic pressure. J. Exp. Bot. 31, 571-576. Thomas, W. H. & Seibert, D. L. R. (1977). Effects of copper on the dominance and the diversity of algae: Controlled ecosystem pollution experiment. Bull Mar. ScL 27.23-33. Waldhauer, R, Matte, A. & Tucker, R. E. (1978). Lead and copper in the waters of Raritan and lower New York Bays. Mar. Pollut. BuR 9, 3842. Wood, A. M. (1983). Available copper ligands and the apparent bioavailability of copper to natural phytoplankton assemblages. Sci. Tot. Environ. 2g, 51-64.
water and in marine organisms and sediments. Z Mar. Res. 15, 103-122. Riley, J. P. & Segar, D. A. (1970). The distribution of the major and some minor elements in marine animals. I. Echinoderms and coelenterates. J. mar. biol. Assoc., UK 50, 721-730. Schlichter, D. (1982). Epidermal nutrition of the Alcyonarian Heteroxenia fuscescens (Ehrb.): absorption of dissolved organic material and lost endogenous photosynthates. Oecologia 53, 40-49. St. John, B. E. (1973). Trace elements in corals of the Coral Sea: their relationship to oceanographic factors. In: Proc. Int. Symp. on Oceanography of the South Pacific, Wellington. R. Fraser (Ed.) p p . 149-158. UNESCO. St. John, B. E. (1974). Heavy metals in the skeletal carbonate of scleractinian corals. In: Proc. 2nd Int. Coral Reef Symp., A. M. Cameron et al. (Eds). Vol. 2, pp. 461-469. Great Barrier Reef Committee, Brisbane. Tapiolas, D. M. (1980). Natural products derived from soft corals, 115 pp. Unpublished B.Sc. (Hon.) thesis, James Cook University of North Queensland.
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MarinePollutionBulletin, Vol. 17,No. 5,213-218,1986
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Copper Effects in Early Stages of the Kelp, Laminaria saccharina IK KYO CHUNG and BOUDEWlJN H. BRINKHUIS Marine Sciences Research Center, State University of New York, Stony Brook, N Y 11794-5000, USA
The effects of copper on the Laminaria saccharina were examined by culturing plants in 5, 10, 50, 100 and 500 ~g Cu I-t filtered and filtered plus uv treated seawater media. Fertile sporophytes were used to determine effects of copper on meiospore release. Untreated meiospores also were obtained and cultured in seawater media, followed by copper additions at various stages of development through the early sporophyte stage. The release of meiospores from copper-treated sorus materials was reduced by concentrations ~ 50 pg I-t. Settlement and germination o f meiospores were not affected by concentrations up to 500 pg I-t. Development of gametophytes and gametogenesis were delayed in concentrations ~_50 pg !-t. Growth of sporophytes, in terms of length increase or relative blade area increase, was inhibited with increasing concentrations > 10 pg I-t.
The passive and/or active uptake of heavy metals show various effects in algae. At certain concentration levels, biological minor, or trace, elements may increase the growth rate (Huntsman & Sunda, 1980). In the case of non-biological heavy metals, organisms are tolerant to some concentrations, depending on the conditions of the organism and the form of the metal (Lepp, 1981). But at higher concentration levels, the organisms are likely to be affected in their growth, photosynthesis, mitochon-
drial respiration and other general metabolic pathways. Moreover, these effects can lead to death. For many years, copper sulphato has been widely used as an algicide to control and/or prevent undesirable algal growths, especially waterblooms. Copper inactivates the electron transport between the oxidizing side of the reaction center of photosystem II and electron donating side of 1,5-diphenyl-carbazide, affecting reaction rates of dark step in green algae (Shioi et aL, 1978). Toxicity of copper may cause the succession or change in the community structure due to the differential responses of organisms in the water column (Thomas & Siebert, 1977; Leland & Carter, 1984). Like other heavy metals, copper affects permeability of the plasma membrane, causing loss of potassium from algal cells (De Filippis, 1979). Excessive concentrations of copper cause the loss of photosynthetic pigment Chlorella (De Filippis & Pallaghy, 1976). Copper toxicity is believed to be related to the concentration of the free copper ion, as chelating agents detoxify copper-spiked seawater. This was subsequently confirmed by Jackson & Morgan (1978) using a chemical speciation model to estimate the concentration of free copper ions. Several artificial seawater media have been introduced based on the thermodynamic equilibrium calculated with computer programs (Morel et al., 1979; Kuwabara & North, 1980). 213
MarinePollutionBulletin
Few studies on the effects of copper on reproduction and development have been reported for seaweeds. The present study was conducted to delineate the general growth, development and morphological effects of copper on the brown alga, Laminaria saccharina, especially during meiospore release, gametogenesis and early sporophyte stages. These stages are susceptible to high mortality rates throughout the life cycle of this kelp species (Chapman, 1984).
Materials and M e t h o d s
The present study followed the culture methods of Brinkhuis et al. (1983, 1984). An incubation chamber was maintained at 10 + I*C and illuminated by very high output cool-white fluorescent light providing cultures with 50 0E cm -2 s-l on 12:12 h photoperiod.
General Culture Procedure Laminaria saccharina plants with mature sori were collected in Long Island Sound at Crane Neck, New York. A ripe portion of sorus was cut from the plant and wiped clean with 5% NaOCl (v/v in filtered seawater) and further rinsed with membrane filtered (Gelman) seawater. Cleaned sorus materials were allowed to dry for several hours prior to meiospore release (1-6 h). During that time, the meiospore release potential was checked periodically. Several drops of filtered seawater were pipetted on the surface of the sorus material and after 3 min the suspension on the surface was carefully picked up by pipette and examined under the microscope. When the number of meiospores exceeded about 10-20 meiospores mm -2 under × 100 magnification, the sorus material was immersed in the culture media. Meiospore Release in the Cu Media Small squares (1 cm 2) were excised from the dried sorus material which was ready for meiospore release using Teflon coated razor blades. Each excised specimen was immersed in a 24 ml glass vial containing 20 ml of media which had been 0.2 ltrn filtered, uv-treated and enriched with 883 ;xM NCO 3- (as NaNO3) and 36 PO43- (as NaH2PO4) (Sunda & Guillard, 1976), then supplemented with diluted copper stock solution (Cu DILUT-IT; 1 g Cu as CuSO4 to make 1 1.). For microscopic specimen cultures, the media were 0.2 lun filtered again for sterilization before copper augmentation. All surfaces were acid cleaned and soaked in Milli-Q water before use. The concentrations of copper were 5, 10, 50 and 100 0g 1-~. The glass vials were covered and wrapped with aluminium foil, placed in an incubator for 1 h, and shaken several times during the 1 h release period. Sorus materials were then removed from the vial. The number of meiospores was counted using a hemacytometer. Cu Treatment Before the Meiospore Settlement Similarly sized specimens were placed in the plastic Petri dishes (Nalgene 5500-0010) and allowed to release meiospores for 24 h. Copper treatment was then initiated before actual settlement had begun. 214
Cu Treatment after Meiospore Settlement The remainder of sorus material was placed in a beaker for bulk meiospore release. After examining the density of meiospores, volumetric inoculations (50 Ixi) were placed on the 18 x18 mm coverslips in glassquadrant Petri dishes, plexiglass boats or directly on plastic Petri dishes (Nalgene 5500-0010 and B-D 3006). Inoculated materials were kept in darkness in the incubator for 12 h, the control media was added; 9 ml for the glass quadrant Petri dishes, 4 ml for plastic Petri dishes and 125 ml for plexiglass boats. The media were renewed every 4 days. Developmental Stage Analysis Several significant stages can be identified during the incubation period of 3 weeks. They can be defined as six steps (modified from Burrows, 1971; Burrows & Pybus, 1971): Stage I: Meiospore settlement with germ tube production Stage I I: Early stage of developing gametophyte (movement of cytoplasmic content to germ tube and increase in cell size) Stage l/I: Later stages of developing gametophyte (morphogenesis) Stage IV: Mature gametophytes Stage V: Early stage of sporophyte (1-4 cells) Stage VI: Later stages of sporophyte One hundred plants were scored for stage development in each coverslip or culture dish. If a particular stage represented more than 50% of the population, the development status of that culture was described as representing that stage. Culture of Young Fronds For sporophytes sized above 1 cm long, 5 1. plastic planting boxes or 11. beakers which contained five to ten plants were immersed in the refrigerated aquarium at 12+1"C under cool-white fluorescent light with 20 0E cm -2 s -~ on 12:12 h photoperiod. The media were constantly agitated by wave plates in each planting box. Both boxes and beakers were aerated. The plants which had been cultured in the cold room were acclimated for 1 day and photographed before treatment. After 7 days of incubation, the plants were also photographed and hole punches (154 m m 2) were taken for chlorophyll analysis. The blade area was measured with computerprogrammed graphic analysis from the photographs. Growth rates were monitored by the relative blade area increase. Statistical Analysis All data were examined using statistical procedures described in Sokal & Rohlf (1981). Results
Meiospore Release The density of released meiospores in each vial is shown in Table 1. The mobility of released meiospores did not seem to be affected by the range of copper concentrations tested, but the densities were quite
Volume 17/Number 5/May 1986
remained in the early gametophyte stage, and plants in 50 and 100 pg Cu 1-~ did not show the progress compared to the Day 8 observation. By the 15th day, control plants reached the sporophyte stage, but those in 5 and Treatment Average*~ St. Dev.*~ n Sigma R*2 10 pg Cu 1-1 showed delayed sporophyte formation, and Control 23.0 4.27 10 330.5 the rest in 50 and 100 pg Cu 1-1 remained in the previous 5 lag 1-I 21.6 2.22 10 301.5 developing gametophyte stage. By the 18th day, plants in 10 pg 1-I 26.3 3.50 10 412.0 50 pg 1-I 16.6 3.50 10 162.0 5 pg Cu 1-1 began to produce zygotes and sporophytes, 100 pg 1-I 11.1 3.48 10 69.0 but those in 10 pg Cu 1-1 did not show this development, and some developing gametophytes in 50 pg Cu 1-1 lost *1 Unit: 5 X104 meiospores I-L *2 Sigma R: Pooled rank sum from Kruskal-Wallis Test. their cellular contents. Cells in 100 pg Cu 1-1 still *Note: Hadi.=39.63 > X20.1..= 18.467. remained in the developing gametophyte stage and the majority of plants showed retarded development. Three weeks after inoculation plants in control, 5 and 10 pg Cu TABLE 2 1-1 media all formed sporophyte stages (by the > 50% Nonparametric multiple comparison STP (an unplanned test for equal characterization), but those in 50 pg Cu 1-~ did not show sample size, values: Us from Mann-Whitney U-Test) for the meiospore release by Laminaria saccharina in copper added media as in sporophyte formation and those in 100 pg Cu 1-1 Table 1. remained in the developing gametophyte stage. The mean length of sporophytes was 492.6 + 73.37 Control pm (mean + S.D.) for control plants and 63.7 5:22.88 prn 5 pgl -I 58.5 10 pg 1-I 26.5 15.5 for those in 5 pg Cu 1-1 media on 26th day. These 50 pg 1-j 91.5" 90.0* 98.5* measurements were significantly different based on the 87.0* 100 pg !-I 99.5* 100.0" 100.0" test of equality of means of two samples whose variances *Significant at an experimentwise error rate of 0.05. are assumed to be unequal (a -= 0.05, ts,-16.80 > t0.05** Uo.0515.,01=86.09. 2.28). Sporophytes in 10 p.g Cu 1-1 media showed abnormal growth patterns and other plants in the higher concentrations remained in the same stage or were dead. different among treatments tested. Sorus tissues in 10 pg The summary of the above development is presented in Cu 1-1 media showed the best meiospore release and Fig. 1. those in over 50 pg Cu 1-1 exhibited severely reduced meiospore release. vl com~ / o," A Kruskal-Wallis test showed that meiospore release / / / .5 `06 CU.L"1 in different media was significantly different among = ""'_,.L_-.. J'' CU.L-1 treatments (Table 1). Nonparametric multiple Comparison STP (Table 2) showed that the set (control, 5 pg Cu 1-~, and 10 pg Cu 1-1) was not significantly heteroII ....................... . . cu.,.-1 geneous. Since the 50 and 100 pg Cu 1-1 were heterogeneous with any other treatment, any set containing either 50 and 100 pg Cu 1-1 (or both) would be signifiI cantly heterogeneous. The data therefore partition into the following: (control, 5 pg Cu 1-l, 10 pg Cu 1-1) (50 lag ; 17 1; Cu 1-l) (100 pg Cu 1-1). Meiospore release was reduced DAYS /~FTER INOCULATION at the higher copper concentrations (_~50 pg Cu 1-1). Fig. 1 Summary of the development of Laminaria saccharina gametoTABLE I
Meiospore release by Laminaria saccharina in copper supplemented media.
~ V
Gametophyte Development The developmental stages were examined on Day 3, 5, 8, 10, 15, 18, 21. Final observations were taken on Day 26. Identification of the development was based on the 6 stage steps described earlier. Copper Treatment before Meiospore Settlement. After inoculation, all meiospores began to settle and germinate. The Day 3 observation showed well-settled meiospores with germ tubes in all treatments. The early stage of gametophytes appeared on the 5th day, which can be identified by the movement of cytoplasmic content to the germ tube and increased cell size. Up to the 8th day, all the cells in the different concentrations of copper showed the same developmental pattern. On the 10th day, one-celled oogonia were easily found and several sporophytes began to appear in control media. However, plants in other media showed somewhat delayed development. Cells in 5 and 10 pg Cu 1-1
/J
"'/ "_.'//'lg
phytes. The copper treatment was applied before meiospore setdement (Day 0).
CONTROL
II1"
C
U
I
~ Igg, ~
(2)
(7)
58 ,06 CU.L"1 ,IJ6 CU.L-1
(12)
(17)
DAYS /~TER INOCULATION (DAYS N:'rER OJPP'ERTREATI~NT)
Fig. 2 Summary of the development of Laminaria saccharina gametophytes. The copper treatment was applied after meiospore settlement (Day 3).
215
Marine Pollution Bulletin
Copper Treatment after Meiospore Settlement. Treatments were applied from stage I (at day 3) when all the meiospores had germinated in the control media. Fiveday old cells in the all treatments showed the early stage of gametophyte development and some cells in 100 and 500 gg Cu 1-1 media began to show mortality, which was defined as empty cells and further confirmed with Evans blue staining--dark blue staining of whole cell (Taylor & West, 1980). Ceils in the control, 5 and 10 lag Cu 1-l media showed the same development pattern, but those in 50 gg Cu 1-1 media began to die by the 8th day of observation. By the 10th day, mature gametophytes dominated in control and 5 0g Cu 1-l media, but ceils in 10 gg Cu 1-I did not show maturity. Other plants in higher copper concentrations showed retarded development (50 og Cu 1-1 ), remained in the same stage (100 gg Cu 1-t ) and 100% mortality (empty cells and cell lysis, 500 0g Cu 1-I ). On the 15th day of observation, control sporophytes covered the whole coverslip, and gametophytes in 5 and 10 ~ Cu 1-1 showed maturity. Those in higher concentrations did not develop further. Mature gametophytes did not develop into sporophytes in 5 and 10 pg Cu 1-l media until the 18th day, but sporophytes were well developed in the control. Gametophytes in 50 0g Cu 1-l were still in the developing gametophyte stage. After 3 weeks, plants in the control and 5 Og Cu 1-1 media showed the later sporophyte stage, specimens in 10 0g Cu 1-l stayed inthe mature gametophyte stage and cells in 50 ltg Cu 1-l remained in the developing gametophyte stage. The length of fronds reached 940.5 + 23.32 pan for control plants, 111.7 + 24.60 ptm for those in 5 0g Cu 1-~ and 97.3 ± 23.32 grn for those 10 0g Cu 1-l on the 26th day. The others remained in the same stage or dead. Some sporophytes in 10 Og Cu 1-l media showed abnormal growth patterns. Multiple comparisons among pairs of means reveals that the length of control plants is significantly different from others (0t-" 0.05), but those of 5 and 10 pg Cu 1-l are not significantly different at 0t--0.05 level. These developmental patterns are summarized in Fig. 2.
TABLE 3 Relative blade area increase in Laminaria saccharina. R.B.A.I. ffi Blade area,2 -- Blade areatj × 100 Blade area, Relative Blade Area Increase (R.B.A.I.) Day 0-4 Day 4-9 Total
Treatment Control 10 pgCul -~ 50 0gCul -~ 100 0gCul -~ 500 0gCul -~
63.7+11.4 74.8±24.2 70.5±25.2 8.7+ 5.4 -5.2:1:4.1
54.2± 5.9 63.9+31.7 55.9±20.0 dead dead
blade area increase is summarized in Table 4. Growth rates in all treatments were significantly different from each other based on the comparison of means at 0t--0.05 level. Inhibition of growth increased with increasing copper concentration. During the incubation period, plants in 100 and 500 pg Cu l-I media exhibited chlorosis and disintegration in the distal and marginal blade portions.
TABLE 4 Relative blade area increase in Laminaria saccharina. Treatment
R.B.A.I.*
Control 10 ILgI-l 50 $tgI-~ I00 l~gl-l 500 ~g I-t
34.9 + 4.92 26.9 + 3.33 9.0 ± 3.82 2.8±4.93 -5.1 :I:4.78
216
% 1 O0 76.9 25.8 8.1 -14.8
*R.B.AJ.: same as Table 3.
TABLE 5 Comparison of developmental stages. Burrows (1971)
Burros & Pybus (1971)
1. Settled zoospore
1. Settled zoospore
2. Developing gametophyte
3. Fertile gametophyte
2. Settled zoospore with germ tube and/or vegetative cells 3. Fertile gametophyte
4. First sporophyte division
4. First sporophyte division
5. Later sporophyte stages
5.2-8 celled sporophyte
Growth of YoungPlants Growth of young plants which were grown in the cold room from the meiospore to 1-3 cm long sporophytes and then exposed to copper treatment is summarized in Table 3. All the values were tested by the multiple comparison among pairs of means at 0c-, 0.05 level. During the first 4 days, the relative blade area increase of plants in 10 ~g Cu 1-1 was highest among all treatments, but was not significantly different from those of the control and 50 0g Cu 1-l treatments. However, the above three values were significantly different from those of 100 and 500 gg Cu 1-l. During the next 5 days, plants in 100 and 500 0g Cu 1-l exhibited severe ehlorosis and total degradation and were not able to be photographed. The remaining three gi'oups showed continuous growth with decreasing relative blade area increase compared to the first 4-day period, but those were not significantly different from eachother. Growth of young sporophytes which were 8-10 cm long prior to copper exposure in terms of the relative
152.5±21.8 181.3+30.4 168.9±63.0
Present study 1, SeRled meiospore with germ tube 2. Early stages of developing gametophyte 3. Late stages of developing gametophyte 4. Mature gametophyte 5. Early stages of sporophyte
(1-4 celled) 6. First longitudinal division in sporophyte 7. All later stages
6. Later stages o f sporophyte
Discussion According to preliminary observations, i~ was necessary to modify the development stage system defined by
Volume 17/Number 5/May 1986 TABLE 6 Concentrations of copper in nearshore region. Location New York Bays, US La Rosiere, UK Norwegian Fjord, Norway La Rabida, Spain Ras Beirut, Beirut
Concentration (unit; p.g 1-j)
Reference
2.7-65
Waldhauer et al. (1978) 17.4 (seawater) Romeril (1977) 60.2 (effluent) 3.75-77.0 Stenner & Nickless (1974) 107 Stenner & Nickless (1975) 100-230 Shiber & Shatila (1979)
Burrows & Pybus (1971). As shown in Figs. 1 and 2, an inhibition of development began at a later stage of gametophyte development, which could not be described by the system of Burrows & Pybus (see Table
5). The plants which were incubated in 0.2 u filtered, uvtreated, copper supplemented media in this study exhibited effects of copper toxicity in terms of their. development and growth on the basis of total copper concentrations, which included the background ambient copper concentration that was assumed to be below 1 Og Cu 1-1 (Preston et aL, 1972; Brewer, 1975). During the course of incubation with copper, it was obvious that there were signs of delayed gametophyte development, inhibition of sporophyte formation and growth of sporophytes. Burrows & Pybus (1971) found delayed development, due to the suspended materials (silt) which decreased the light intensity, e.g. by 6-9 days of the fertile gametophyte stage and by 12 days of first sporophyte division. This is a greater delay than the results from the present study because their culture conditions (especially light was too low) were suboptimal for gametogenesis (Liining & Neushul, 1978; Liining, 1980). Similar results to those of Burrows & Pybus (1971) were made by Hopkins & Kain (1978) in their copper concentration range of 25-75 ~g dm -3, where sporophyte formation was delayed by up to 13 days. A l t h o uhg the meiospore release was done under artificial stress conditions, there seemed to be impacts of copper on meiospore release (Tables 1, 2). Generally, each stage seemed to have quite different responses to copper. There was no effect of copper on the first stage-meiospore settlement and germination--under copper concentrations up to 500 0g Cu 1-s. After movement of cytoplasmic content to germ tube, vegatative gametophyte growth was inhibited at the concentrations of 50 Itlg Cu l - l , and higher. Once the gametophytes were mature, the plants showed parallel development into sporophytes below copper concentrations of 50 0g 1-~, but growth rates of all copper treated plants were significantly lower than that of controls. Slight differences in the responses in terms of initiation and duration of the effects of copper toxicity between two experiments may be caused by the timing of copper application. Though meiospore settlement and germination were not affected, there might be differen-
tial responses of each stage and chronic effects in later vegetative gametophyte development and the early 1-4 celled sporophyte stage. There have been several studies describing growth inhibition by metals in seaweeds (see Rai et al., 1981 for review). Str6mgren (1979a, b, 1980a, b) presented the relationship between the percentage reduction (z) in growth rate and the product of time (x) and concentration (y) in metal (Pb, Cd, Hg, Cu and Zn) treated fucoid algae (Fucus, Ascophyllum and Pelvetia); z "= kxy He found at least 60% reduction in growth rates at copper concentrations over 33 0g 1-~ during the course of an 11 day incubation. Here, young sporophytes (1-3 cm long) in 100 and 500 0g Cu 1-~ media showed 100% mortality within 4 days, a rate higher than the results of Str6mgren. The 5-10 cm long fronds showed higher reduction at a given value of product of x and y The k value varies with increasing value of product of x and y: up to 100-=0.3; 100-500--0.2; 500-1000---0.1. These are much higher than Str6mgren's values. The differences may be ascribed to the type of incubated materials; only growing apicies were used in his study but whole plants were used in the present experiments. The present information of the effect of copper can be applied to the ecosystem level. The effect of copper on the early stages of L. saccharina may decrease the total primary production in the kelp-based system due to failure in the recruitment and decreased growth. Abnormalities in the early life cycle stage, reduction in chlorophyll content and change in the fine structure may have potential deleterious impacts on the primary production in the kelp ecosystem. The concentrations applied in the present study were similar to those found in the natural environment. Copper is present in the open ocean at a concentration of approximately 1.0 Og 1-~ (Brewer, 1975). However, concentrations increase up to 230 lxg 1-~ in nearshore waters which are highly susceptible Io metal pollution (see Table 6). This is the zone inhabitated by kelps. Even though there are high concentrations of copper, the toxicity and bioavallability of copper is dependent on its chemical form and the metal-organic complexes (Davey et aL, 1973; Hirose & Sugimura, 1985; Piotrowicz et al., 1984; Wood, 1983). Due to metal organic complexing agents like humic acid, the activities of free copper ion can be maintained at a native constant level against relatively small fluctuations by the addition or uptake in the marine environment (Hirose & Sugimura, 1985). The ability to concentrate ions of metals from the surrounding seawater has been well documented for the brown algae and make these seaweeds useful indicators of marine heavy metal pollution (Bryan, 1983; Bryan and Hummerstone, 1973; Haug et al., 1974;MYklestad et al., 1978). The bioaccumulated metals in seaweed tissue may be transferred to the detrital food web. The process of the microbial activity in the sediment may be decreased by the metals accumulated in algal detritus. Finally, the total production of the system may be affected by the decreased microbial activity which provides the regenerated nutrients to the water column (Babich & Stotzky, 1985). 217
Marine PollutionBulletin
Summary The present study demonstrates the following results: 1. The density of meiospore release was reduced in higher concentrations of copper _ 50 ptg 1-I in treated sorus materials. 2. Settlement and germination of meiospores were not affected by the range of copper concentrations up to 5 0 0 ~tg 1-1.
3. Development of gametophytes and gametogenesis were delayed in copper concentrations ~ 50 lag 1-m. 4. Growth of sporophytes in terms of length increase or relative blade area increase was inhibited with increasing copper concentrations > 10 ~g l-~. 5. The critical concentrations of copper which inhibit the development of gametophytes and young sporophytes is between 10 and 50 l~g l-~.
This research was supported by the Gas Research Institute, New York State Energy Be.search and Development Authority and the New York Gas Group by contracts to B.H.B. Marine Science Research Center Contribution No. 502.
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