edta chelation and zinc antagonism with cadmium in sediment: effects on the behavior and mortality...

Marine Biology84, 125-130 (1984) Marine BiOlOgY �9 Springer-Verla9 1984

EDTA chelation and zinc antagonism with cadmium in sediment: effects on the behavior and mortality of two infaunal amphipods

J. M. Oakden*, J. S. Oliver and A. R. Flegal

Moss Landing Marine Laboratories; Moss Landing, California 95039, USA

Abstract

The two species of infaunal amphipod crustaceans Rhepoxynius abronius (Phoxocephalidae) and Eohaustorius sencitlus (HaustorHdae) are characteristic of nearshore sandy bottoms along the California (USA) coast, and are highly sensitive to moderate levels of heavy metals. In laboratory experiments, both zinc and the chelator EDTA increased the survival of amphipods in sediment containing otherwise lethal levels of cadmium (8.5~gg-1), which are representative of moderately polluted environments. In simple choice experiments, amphipods prefer sediment with complexed cadmium. The behavioral and survival patterns of both species were similar in the experiments, EDTA prevented about 50% of the added cadmium from initially being incorporated into the sediment, and increased the rate of cadmium released from the sediment. These data illustrate the limitations of operational definitions of chemical analyses, since weak-acid (0.5 N HC1) leaches that were intended to provide an estimate of the "biologically available" metal concentrations extracted both toxic and EDTA-complexed cadmium species and did not account for their antagonistic interactions with zinc.

Introduction

Nearshore marine sediments accumulate pollutants, in- cluding heavy metals, released into the environment (Hershelman et al., 1981). The acute and chronic effects of these chemicals on the benthos are poorly understood and are especially difficult to explore in the complex geo- chemical environments of sedimentary habitats (McIntyre,

* Present address: California Department of Fish and Game, State Mussel Watch, P.O. Box 223, Moss Landing, California 95039, USA

1977; Luoma and Bryan, 1978). The biological effects of metals are complicated by their synergistic and antagonistic interactions with other metals (Moulder, 1980; Wright and Frain, 1981; Foster and Morel, 1982) and by metal speciation (Zamuda and Sunda, 1982). For example, zirzc in- fluences the toxicity of cadmium (Dunlop and Chapman, 1981), and chelating agents such as Tris, NTA, and EDTA reduce the toxicity of metals by sequestering reactive species (Sunda el al., 1978; Engel and Sunda, 1979). How- ever, these and related studies (Lang etal., 1981; Verma etal., 1982; Crecelius etal., 1982; Sullivan etal., 1983) are usually performed in the laboratory on animals that live exposed to water currents (plankton, nekton, epifauna, etc.), and not on benthic animals that live in the sediment where the metals are deposited. Since interactions of chemicals with organisms in seawater may be very different from those in sedimentary environments, the results of most of those experiments may not be applicable to the benthos.

Amphipod crustaceans are among the most abundant and widespread benthic animals living in marine sediments. They are considered more sensitive to chemical changes in the environment than many other infaunal groups (Pearson and Rosenberg, 1978; Swartz etal., 1979; Word and Mearns, 1979). The Phoxocephalidae and Haustoriidae are the two most highly modified infaunal burrowing families among the Amphipoda (Bousfield, 1970). Since they often dominate nearshore marine communities along the California coast (Oliver etal., 1980), their reactions to environmental perturbations may have broad ecological implications. Therefore, the present paper concerns the effects of metals on the behavior and survival of Rhepoxynius abronius, a phoxocephalid, and Eohaustorius sencillus, a haustoriid amphipod.

Materials and methods

Two types of laboratory experiments were used to in- vestigate the effects of EDTA (ethylenediaminetetraacetic

126 J.M. Oakden et al.: Zinc and cadmium effects on amphipods

acid) and zinc on the availability of cadmium in sediment to the amphipods Rhepoxynius abronius and Eohaustorius sencillus. Non-choice experiments, in which amphipods were confined in a dish with a single type of sediment, measured amphipod survival. Behavioral-selection experiments, which allowed the amphipods to choose between two sediment types, indicated the relative acceptability of sediment to the organisms.

We added metals to the experimental sediment by dissolving CdC12 or ZnSO4 in 100 ml of seawater, then adding sediment and stirring to homogenize. When both a metal and the organic chelating agent, EDTA, were used, they were equilibrated in seawater before being added to sediment. NaEDTA was added slightly in excess of the amount necessary to bind all of the metal added. The cadmium and zinc levels used in the non-choice experiments are representative of concentrations in moderately polluted marine environments (Hershelman etal., 1981), and were chosen because previous studies had demonstrated that they cause significant mortality in Rhepoxynius abronius (Oakden et al., 1984). The zinc levels in the metal interaction experiments were chosen to cause minimal mortality based on earlier studies (Oakden et al., 1984), but to be high enough to provide several times the molar quantity of zinc compared to cadmium. The natural control sediment was treated in the same manner as the metal-enriched sediment, except that no chemicals were added.

Amphipods and sediment were collected in water depths of 15 m in Monterey Bay, California (USA) in 1983 by SCUBA divers using plastic implements. The sediment, a clean well-sorted quartz sand (mean phi= 3.22), was sieved in the laboratory through a 0.5 mm Nitex screen, and held for 1 to 10 d in running seawater until used in experiments. Amphipods were sorted with an eyedropper and held at least 24 h before use to reduce collection-related mortality. Experiments were run on a water-table with flowing seawater from Monterey Bay, so temperature and salinity were at ambient levels. Trace- metal-clean procedures were utilized, as described previously (Oakden et al., 1984).

Each treatment was performed in triplicate. In the non-choice experiments, 15 amphipods were added to bowls containing a 3 cm-deep layer of sediment. In the behavioral-choice experiments, bowls were divided in half by a removable plastic barrier, and contrasting sediments were placed on each side. A cylindrical screen of 0.5 mm Nitex which reached above the water surface was fitted around the outside of each dish to prevent amphipod escape. Treatment bowls were oriented on the table so that both sediment choices had the same water-flow and control sediments were not contaminated by metals desorbed from the enriched sediments. After 15 min, 1 cm surface cores were taken for elemental analysis of the initial elemental concentrations, 10 amphipods were added to each side, and the barrier between the sediments was removed. Experiments ran for 72 h, which is the optimal period for observing habitat-selection behavior without

possible starvation effects (Oakden, 1984). The amphipods on the sediment surface were then removed, sediment cores were taken for analysis, and the sediment from each half was sieved to remove all remaining amphipods.

Two extraction techniques were used in the metal analyses. The sediment was digested with concentrated acid (HNO3) to determine the total elemental concentrations, or leached with dilute (0.5 N HC1) acid (Flegal, 1984) to extract the relatively labile species of metals thought to be "biologically available". Elemental analyses were made by atomic absorption spectrophotometry, using a Perkin-Elmer 603 with a deuterium-arc background corrector.

Results

Amphipod mortality

In the non-choice experiments, both additions of zinc and EDTA reduced the mortality of amphipods exposed to otherwise lethal concentrations of cadmium (Fig. 1). The responses of the two species of amphipods were very similar. With elevated cadmium concentrations (8.5 #g g-l) alone, there was 76% mortality in Rhepoxynius abronius and 98% mortality in Eohaustorius sencillus. With concur- rent additions of sublethal concentrations of zinc, amphipod mortality dropped significantly for both species (chi- square test, ] )<0 .05) to 33% for R. abronius and 70% for E. sencillus, which were not significantly different (chi- square test, p <0.05) from control treatments. There was little mortality (none > 7%) in the zinc only, EDTA only, and natural sediment treatments.

Although the same amount of cadmium was mixed with the sediment in all cadmium treatments, 50% less cadmium absorbed onto the sediment in the presence of EDTA. Therefore, the decreased mortality in the CdEDTA treatments may have been due to the relatively lower concentration of cadmium in the sediment, not to the reduced bioavailability of that cadmium. To test this hypothesis, sediment was prepared by adding 2.5 times more cadmium than in previous cadmium treatments, along with a corresponding increase in EDTA. The increase in cadmium caused the sediment cadmium levels to be 30% higher than in the other cadmium treatments (Fig. 1). Cadmium complexation with EDTA still reduced amphipod mortality significantly (chi-square test, p<0.05), from 98 to 24%. Therefore, the lower mortality in the presence of EDTA could not be solely explained by the presence of less cadmium.

Behavioral-choice experiments

The behavioral-choice experiments (Fig. 2) demonstrate that amphipods prefer sediment containing cadmium complexed with EDTA over that without EDTA. Again, the results for the two species of amphipods were very similar.

J. M. Oakden et al.: Zinc and cadmium effects on amphipods 127

PgCd g-1 8.5 8.2 4.0 0 0 0

pgZn g-1 14 79 10 79 12 15

lOO

80

o 60

4 0

g 20

0

+

Cd Cd +

Zn

R. abronius

r - ~ r - h

Cd Zn +

E D T A

T r e a t m e n t

E D T A N a t u r a l

pgCd g-1 8.4 7.4 3.2 0 0 0 11.6

pg Zn g-1 9 67 11 76 14 12 -

lOO

80

o 60

40 r

r 20

o

- . p

- r h Cd Cd CD Zn

+ + Zn E D T A

T r e a t m e n t

E. sencillus

E D T A N a t u r a l 2 . 5 X Cd

E D T A

Fig. 1. Rhet)oxynius abronius and Eohaustorius sencillus. Percent mortality of amphipods in sediment non-choice treatments; means of 3 replicates; error bars are ranges. Cadmium and zinc levels ~ g g-~ dry wt) for each treatment, determined by weak- acid extraction, are means of the initial and final concentrations

% D e a d 20%

pgCd g-1 6.s o

r ._=' 1 O0

80

60 0~ L.

4o

20

o 7 Cd Nat

2% 10%

3.4 0 0 0

CD Nat EDTA Nat +

EDTA

% D e a d

pgCd g-1

1oo

i~ - ~ 80

60

L- 40

cL 20 o~

o

8 %

5.4 0

r

I

Cd Nat

5% 2%

2.7 0 0 O

~r

Cd Nat EDTA Nat + E D T A

Fig. 2. Rhepoxynius abronius and Eohaustorius sencillus. Percent- age of amphipods in each of two sediment choices in behavioral- choice experiments; means of 3 replicates; error bars are ranges. Also shown are percent mortality and cadmium concentration in each sediment type (mean of initial and final concentrations of the weak-acid leach). Treatments which had significant differences between the numbers of amphipods in each choice are indicated by asterisks

When amphipods were offered a choice between sediment with elevated cadmium concentrat ions and unt rea ted sediment, 84% of the Rhepoxynius abronius and 98% of the Eohaustorius sencillus preferred the na tura l sediment. When the elevated cadmium was complexed with EDTA, fewer amphipods avoided the sediment, a l though the natural sediment was still slightly preferred by both species (60 vs 40%: Fig. 2). Fifty-eight percent o f the R. abronius and 74% of the E. sencillus also preferred natural sediment to sediment containing only EDTA (Fig. 2). When offered a choice between sediment with elevated cadmium concentrations, 85% of the R. abronius and 86% of the E. sencillus were found in the sediment where the cadmium was complexed with EDTA (Fig. 3).

As in the survival experiments, the addi t ion of EDTA reduced the amount o f cadmium in the sediment. Since behavioral avoidance is posit ively correlated with metal concentration (Oakden etal., 1984), the observed prefer- ence for sediment containing EDTA-complexed cadmium over uncomplexed cadmium might also have been a reflection of the differences in metal concentrat ion. ~ e r e - fore, this exper iment was repeated, adding 2.5 times more

R. abronius E. sencillus

%Dead

.. pg Cd g-1

E 100 0~ r

80 $

60 O ._~ 40 Q.

E 2 0

a~ r 0 19

26% 2%

7.7 3.9 5.0 2,9

+

Cd Cd Cd c+d EDTA EDTA

2%

8.4 10.2

ct C d 2 . 5 X

C d

ED+TA

Fig. 3. Rhe?oxynius abronius and Eohaustorius sencillus. Percent- age of amphipods in each sediment choice; means of 3 replicates; error bars are ranges. Also shown are percent mortality in each experiment, and cadmium concentration in each sediment type (mean of initial and final concentrations of the weak-acid leach). Asterisks indicate significant differences between numbers of amphipods in the respective sediment types


Table 1. Rhepoxynius abronius. Amount ~ug g-1 dry wt) of cadmium and zinc in six non-choice treatments. Weak aIld strong extraction techniques are compared. Initial samples were taken at start experiment and final samples 72 h later. Percent loss for added metals for each of the extraction techniques is shown at bottom of table. ND: not detected; -: metal not added

Extraction Time Cd Cd + Zn Cd + EDTA Zn EDTA Natural

Cd Zn Cd Zn Cd Zn Cd Zn Cd Zn Cd Zn

Weak Initial 8.7 13 8.8 81 5.2 9 ND 78 ND 12 ND 16 Final 8.2 15 7.5 77 2.7 10 ND 80 ND 11 ND 15

Strong Initial 8.5 29 8.2 98 4.3 37 ND 88 ND 31 ND 34 Final 6.9 39 6.8 94 2.6 32 ND 99 ND 33 ND 34

% metal loss Weak 6 - 15 5 48 - - + 2 . . . . Strong 19 - I7 4 40 - - + l I . . . .

Table 2. Eohaustorius sencillus. Amount ~ug g-~ dry wt) of cadmium and zinc in seven non-choice treatments. Weak and techniques are compared. Initial samples were taken at start of experiment and final samples 72 h later. Percent loss for each of the extraction techniques is shown at bottom of table. NA: not analysed; ND: not detected; -: metal not added

strong extraction added metals for

Extraction Time Cd Cd+Zn Cd-EDTA Zn EDTA

Cd Zn Cd Zn Cd Zn Cd Zn Cd Zn

Natural 2.5 Cd + EDTA

Cd Zn Cd Zn

Weak initial 9.2 7 8.6 66 4.6 12 ND 78 ND 15 ND 12 14,1 NA Final 7.7 13 6.2 67 1.7 10 ND 73 ND 12 ND 11 9.0 NA

Strong Initial 9.5 29 8.3 89 4.3 31 0.5 98 ND 31 ND 40 NA NA Final 8.8 33 5.5 75 2.8 27 ND 73 ND 36 ND 33 NA NA

% metal loss Weak 16 - 16 +1 63 - - 6 . . . . 36 - Strong 7 - 34 15 35 - - 15 . . . . . .

Table 3. Rhepoxynius abronius. Amount ~ug g-1 dry wt) of cadmium in 4 non-choice treatments using high levels of zinc and ED- TA. Weak and strong extractions are compared. Initial samples were taken at start of experiment and final samples 72 h later. Per- cent amphipod mortality is shown for each treatment

Extraction Time Zn Zn + EDTA EDTA Natural

Weak Initial 559 394 16 15 Final 435 183 13 13

Strong Initial 536 418 31 32 Final 444 198 31 34

% mortality 100 95 97 0

cadmium and EDTA, thus raising the sediment cadmium concentration above that in the cadmium sediment without EDTA. Ninety-four percent of the Eohaustorius sencillus still preferred the sediment with added cadmium and EDTA over uncomplexed cadmium sediment (Fig. 3). This indicates that cadmium complexed with EDTA not only causes lower mortality in these amphipods, but is less avoided by them than sediment containing comparable concentrations of cadmium which is not complexed with EDTA.

Metal extraction

Both the weak and the near-total metal extractions removed essentially all of the cadmium and zinc that were

added to the experimental sediments (Tables 1 and 2). Since the resolution of the analysis was 0 . 5 # g g -1 for cadmium, differences between the two extractions were within the precision of the analysis. However, there was a significant difference (chi-square test, p <0.05) between the two extractions for background zinc, with about 12 ktg g-1 for the weak and about 33 ktg g-1 for the total digestion.

As in previous experiments (Oakden et al., 1984), there was generally a decrease in the added metal concentrations over the 72 h experimental duration. For the four treatments where cadmium was added without EDTA, the loss ranged from 6 to 16%, which is similar to previously reported results (Oakden et aL, 1984). When EDTA was present, the cadmium loss over 72 h was much greater, 38 to 55%.

The addition of EDTA reduced the initial incorpora- tion of the added metal in the sediment, In the non-choice experiments, only 55% as much cadmimn was initially present in the sediment with EDTA (Tables 1 and 2). This effect of EDTA was further demonstrated in a set of non- choice experiments with Rhepoxynius abronius using high levels of zinc and EDTA (Table 3). Little can be said of the latter responses of the amphipods, since there was almost total mortality in both the zinc and zinc complexed with EDTA treatments, and the amount of EDTA necessary to complex 600/zg Zn g-1 caused 97% mortality in the EDTA control. Again, the addition of EDTA caused 25% less metal to be initially associated with the sediment, and the

J. M. Oakden et al.: Zinc and cadmium effects on amphipods 129

72h metal loss in the ZnEDTA treatment was 53% as opposed to 20% in the zinc only treatment. EDTA did not remove detectable amounts of zinc initially present in the sediment, as demonstrated by the normal background zinc concentrations in the EDTA treatment (Table 3). However, metals added to sediment in a complexed form were less likely to enter the sediment, and were mobilized out of the sediment more readily.

Discussion

The survival of the infaunal amphipods Rhepoxynius abronius and Eohaustorius sencillus in sediment containing otherwise lethal concentrations of cadmium was increased when zinc was also present. EDTA, a strong chelating agent, simi- larly increased amphipod survival in cadmium-emiched sediment. Sediment containing EDTA-chelated cadmium was preferred to sediment containing unchelated cadmium in behavioral selection experiments.

The literature concerning interactions between cadmium and zinc is contradictory and difficult to interpret, with the results often expressed in terms of "toxic units" or other dubious mechanistic assumptions (Brown, 1968). Thus, cadmium and zinc in seawater have been reported to interact synergistically (Hutchinson and Czyrska, 1972; Eaton, 1973; Eisler and Gardner, 1973; Negilski etaL, 1981), additively, and antagonistically (Dunlop and Chap- man, 1981). The present study demonstrates antagonistic interactions between cadmium and zinc in marine sediment. Cadmium and zinc are thought to act on similar sites in crustaceans (Negilski etal., 1981). Thus, elevated concentrations of zinc may reduce the toxic effects of cadmium by acting as a competing ion.

By decreasing the amount of labile cadmium ions, EDTA reduces cadmium toxicity to crustaceans (Sunda etal., 1978). The amount of labile cadmium may also cause the changes in behavioral responses, Although crustaceans can detect very low concentrations of pollutants (Erdem and Meadows, 1980; Pearson, 1980), cadmium bound to EDTA may be relatively sequestered from most biochemical reactions, and so not detectable by the amphipods.

Amphipod behavioral responses could also be based on physiological reactions. In the early stages of metal poisoning, there is often an increase in activity rates of crustaceans (Sullivan etal., 1983). Differential activity rates are a possible mechanism behind habitat selection (orthokinesis), as by speeding up in unfavorable environments and slowing down in favorable environments, in- vertebrates can concentrate in favorable habitats (Weiser, 1956). Increased activity in the uncomplexed cadmium sediment might cause amphipods to concentrate in the EDTA-complexed cadmium sediment, where activity rates are lower.

Selection behavior is widespread among benthic in- vertebrates (Gray, 1974). Such behavior can lead to spurious results in bioassay experiments where a layer of

test sediment is placed on a substrate of uncontaminated sediment (Tatem and Baker, 1980), because the animals simply burrow through the test sediment into the uncontaminated sediment. Since the distribution of heavy metals is patchy in the environment (Swartz et al., 1982), selection behavior would enable the infauna to locate small-scale layers or patches with lower metal concentrations. Fur- thermore, by concentrating in patches where the metals are present in complexed form, the toxic effects of the metals would be reduced.

The effects of complexing agents on metal toxicity in the environment are poorly understood. In our experiments, EDTA not only reduced the toxic effects of cadmium, but also prevented about 50% of the cadmium and 25% of the zinc from entering the sediment. In fresh water, Gardiner (1974) found almost no adsorption of EDTA- complexed metals onto sediment. The higher rate of loss of EDTA-complexed metals over our 72b experimental durations also indicates that the complexed metals are less strongly sorbed onto the sediment. Since EDTA is not biodegradable (Bunch and Ettinger, 1967), and enters the environment in large quantities (170 ktg1-1) in domestic sewage effluent (Gardiner, 1976), it may keep metals in the water column, and prevent them from entering the sediment at the discharge site. Other complexing agents may also play similar roles in metal detoxiflcation.

Geochemists have developed operational definitions of bioavailability for analysis purposes. These often consider the labile metals, those extracted using a weak acid digestion, to be relatively "bioavailable". In our study, the weak digestion invariably extracted all of the cadmium added to the sediment, even when it was complexed with EDTA and did not affect the amphipods in the laboratory experiments. It also extracted roughly half of the background zinc from the sediment, although this fraction had no apparent effect on the amphipods. Thus, while operational measurements of lability provide a more realistic representation of bioavailability than measurements of total elemental concentrations, they provide a limited in- dication of metal bioavailability in sediment.

Acknowledgements. The authors would like to thank D. Smith and L. Dalaba, who performed the metal analyses. P. Slattery, L. McMasters, G. Gillingham, and K. Heath of the Moss Landing Marine Laboratories; M. Martin and M. Stevenson of California Department of Fish and Game; and J. Stull of the Los Angeles County Sanitation District, provided additional assistance. This research was funded by EPA Grant No. 808772-01, and the California Depart- ment ofFish and Game.

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Date of final manuscript acceptance: September 27, 1984. Communicated by N. D. Holland, La Jolla

edta chelation and zinc antagonism with cadmium in sediment: effects on the behavior and mortality...

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