The potential role of the estuarine amphipod Gammarus duebeni in sub-lethal ecotoxicology testing

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Wal. Sci. Tech. Vol. 34. No. 7-8, pp. 93-100,1996. Copyright © 1996 1AWQ. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved.

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THE POTENTIAL ROLE OF THE ESTUARINE AMPHIPOD GAMMARUS DUEBENI IN SUB-LETHAL ECOTOXICOLOGY TESTING Andrew Lawrence and Carl Poulter Department ofApplied Biology, The University of Hull, Cottingham Road, Kingston upon Hull, Humberside HU6 7RX, UK

ABSTRACT The aim of this study was to assess the suitability of Gammarus duebeni as an indicator of estuarine pollution. This involved the development of sub-lethal polIution bioassays monitoring respiration rate, swimming efficinecy and precopula pairing; assessment of the sensitivity of the assays to copper pollution and comparison of the suitability of the assays. Significant impairment of respiration rate, as measured by changes in pleopod beat, was determined at a copper concentration of 600 Ilg I-I. Significant impairment to swimming ability was determined after exposure to copper polIution at a concentration of 600 Ilg I-I and precopula pairing was significantly reduced at a concentration of 600 Ilg P. Of the assays, swimming efficiency and pleopod beat frequency appear to offer the most potential for further development. The lower limit of sensitivity of the bioassays developed in this study is yet to be determined. However. the levels of copper shown to induce an effect can be experienced in the natural environment. The study has shown that Gammarus duebeni can be used in sub-lethal pollution assays, at relatively high Cu concentrations, and indicates that it is potentially a useful species with which to assess estuarine water and sediment polIution. Copyright © 1996 IAWQ. Published by Elsevier Science Ltd.

KEYWORDS Arnphipod; bioassay; copper; estuarine pollution; respiration rate; sub-lethal. INTRODUCTION Gammarus duebeni is widely distributed in North Sea estuaries including the Humber. It is often found in areas with a freshwater influence and can exhibit wide temperature, salinity and hypoxia tolerance (Bulnheim, 1979; Ritz, 1980; Sheader, 1983). Gammarus duebeni has an important ecological role in these estuarine areas. It is one of the major food species in the trophic web and acts as a shredder, particulating vegetation in the community (McLusky, 1989). It is therefore important both in the passage of food to other organisms and the natural regeneration of organic matter in underlying sediment. In additon to its ecological importance, Gammarus duebeni also fulfil the criteria, devised by Phillips (1980) for the selection of indicator species for the study of the bioaccumulation of pollutants. It is widespread, hardy, common and tolerant of low salinities. Furthermore, amphipods do not regulate trace metals 93

94

A. LAWRENCE and C. POULTER

(Rainbow and White. 1989; Rainbow et al. 1989). Metal levels in the animal will therefore reflect the bioavailability of the metal in the environment.

Gammarus duebeni is a truly estuarine species (McLusky. 1989). Its potential use in ecotoxicology is particularly important. therefore. because estimates of the effects of pollutants in estuaries have often b~en derived from experiments using marine species (eg Hunter 1949) and there is a need to use more estuanne animals in this type of study (Bryan and Langston. 1992). If. as suggested by McLusky et. al. (1986) estuarine species live near the limit of their tolerance range they are more likely to be susceptIble to any additional stress. Alternatively. by having a wide tolerance to abiotic factors they may be pre-adapted to tolerate pollution stress (Jernelov and Rosenberg. 1976; Gray. 1981). In either case. their response to any pollutant will be much more indicative of the impact of the pollutant in that environment than will tests using marine species. In the past this genus, but particularly the freshwater species Gammarus pulex, has been used in acute and sub-lethal ecotoxicology studies (Ritz, 1980; Stephenson, 1983; Hunte et al. 1985; Poulton and Pascoe, 1990; Borlakoglu and Kickuth, 1990; Crane and Maltby, 1991; Reish, 1993; Taylor et at. 1993 Taylor et al. 1994). In addition. several physiological parameters have been used to examine combined salinity/pollution effects on Gammarus duebeni (Wright, 1986; Tendengren and Nils Kautsky. 1988; Johnson and Jones, 1989; Jones and Johnson, 1992). This suggests that Gammarus duebeni should be a good species with which to develop sub-lethal toxicity bioassays. To date, however, a sub-lethal pollution bioassay has not been developed using this species. The aim of this study was to assess the suitability of Gammarus duebeni as an indicator of estuarine pollution. As a prerequisite, this involved the development of a suitable sub-lethal pollution bioassay. assessment of the sensitivity of the potential assay to copper pollution and comparison of the various assays developed. Metal concentrations on the Humber have been measured and all, except copper, fall below the Environmental Quality Standard (NRA. 1992). Copper has, therefore, been used in this study. MATERIALS AND METHODS

Gammarus duebeni were collected between mid to high water from a rocky area of the south shore of the Humber estuary near Immingham. Animals were maintained in shallow tanks of sea water diluted to 1.66% salinity and kept in an aquarium at 11°C. They were fed Enteromorpha collected from the site with the animals. An initial range finding LC 50 acute toxicity test was perfomed. Animals were maintained in 0, 1, 2, 3,4 and 5 mg 1-1 copper solutions, prepared from anhydrous copper sulphate in 1,66% sea water. Deaths were monitored over the following 120 hours. The acute test was repeated over the following months using 0, 0.2, 0.6, 1 and 3 mg I-I Cu solutions. An acute toxicity test was also performed to assess the combined effects of salinity and Cu toxicity on survival. A Cu solution of 1 mg 1-1 was prepared using seawater of 0, 1.6 and 3.18% salinity. Percentage deaths were recorded over 96 hours. The first sub-lethal bioassay developed examined the effect of Cu on Gammarus duebeni pleopod beat freq~ency. The pleopods are abdominal appendages used both in swimming and to facilitate the exchange of respuatory gases across the animals' integument. As such, pleopod beat frequency has been used as a direct measure of respiration rate (Clark, 1966). Five groups of animals, with a regular pleopod beat, were seperated and maintained in test solutions of 0, 0.2, 0.6 .1 an~ 3 ~g 1- I Cu. The method for counting pleopod beat described by Clark (1966) was adopted. At appropnate tIme mtervals, individual animals were placed into glass tubes filled with test solution. Pleopod ?eats we~e counted and timed and converted to beats min-I. Counts were recorded immediately after mtroductIOn to the test solution and then at convenient time intervals over the next 96 hours. Test solutions were replaced every 48 hours.

Sub-lethal ecotoxicology testing

95

This protocol was repeated to examine the effect of salinity on the rate of pleopod beat. The effect of test solutions of 0, 1.01, 1.66 and 3.16% salinity was determined. The instantaneous pleopod beat frequency was recorded for each individual and subsequently monitored over the following 72 hours. Test solutions were renewed every 48 hours. The effect of sub-lethal copper pollution on swimming efficiency was also determined. A system was developed in which the animals ability to move against a head flow of water could be examined. Plastic tubing was connected to the inlet and outlet of a peristaltic pump. The pump was used to adjust the flow rate of the test solution. A scale was marked on the outlet pipe giving a distance of 100 mm each side of a zero mark. The inlet hose was immersed in the relevant test solution and the system filled. Using a pipette, individual anaimals were then placed into the outlet pipe at the zero position on the marked scale. At a flow rate of 110 ml min-I the movements of individual Gammarus were recorded when maintained in Cu solutions of 0, 1, and 3 mg I-I. Recordings were taken at intervals between 2 and 27 hours after introduction to the test solution, If an animal moved beyond the scale, its position was marked and measured. The test was then repeated at a flow rate of 145 ml min-I. Movement was converted to mm moved min- 1 and the average at each flow rate determined. Finally, the effect of Cu exposure on precopula pairing in Gammarus duebeni was determined. Ten pairs of animals were placed in solutions of 0, 0.2, 0.6 and 1 mg I-I Cu. The separation of pairs was then recorded over the next 76 hours. RESULTS Initial toxicity tests were evaluated using Probit analysis, the results of which are shown in Table 1. The LT 50 was determined for each test solution. In the first, range finding, test the LT 50 wa~ calculated as over 70 hours for each test solution. In the following tests the LT 50 ranged from 135 hours 10 0.2 mg I-I Cu to 37 hours in 3 mg 1-1 Cu. In addition, Probit analysis was used to determine the 48 h LC 50 ad the 96 h LC 50 from the follow-up tests. These were calculated as 2.596 and 0.893 mg I-I, respectively. Table 1. LT 50 values determined by probit analysis from acute toxicity tests Cu

LT 50 .-

Concentrationi mg 1-1

o

I

...

~

Oct '94

o

Dec '94 0

Jan '95 0

-

Feb '95 -

0

0.2

135.6

0.6

114.9

96

88.1

74.7

56.1

63.1

60.8

37.5

45.1

1

92.2

2

139.3

3

89.1

4

72.9

5

86.3

The combined effects of salinity and copper pollution on survival are shown in Figure 1.

A. lAWRENCE and C POULTER

96

100

• 0 % salinity, 1 mg I-I Cu • 1.6 % salinity, 1 mg 1-1 Cu

25

3.18 % salinity, 1 mg I-I Cu

A

20

40

60

80

100

Time (Hours) Figure 1. The combined effects of salinity and copper pollution on Gammarus duebeni survival.

The effects of salinity and copper were examined using a Two Way ANOV A. despite observed differences, there was no significant difference between the treatments (F =2.78, P> 0.05). The effect of sub-lethal copper pollution on repiration rate are shown in Figure 2. Data was analysed by Two Way ANOVA and Least Significant Difference multiple range test. There was a significant difference between treatments with time (F = 303.45, P < 0.05). This was supported by a One Way ANOVA which showed significant differences between the concentration series (F = 11.54, P < 0.05). The Least Significant Difference test was used to determine which of the concentration series differed from each other. Those Cu concentration series underlined show no significant difference, those not on the same line are significantly different. Cu concn (mg I-I)

o

0.2

0.6

3

Copper, therefore, significantly reduces pleopod beat frequency in a dose related manner. 250

-

200

·8 ....ro

ISO

I

t:

C/J

~

.D "0

0

0..

0

•

0 mg I-I Cu control o O. 2 mg I-I Cu • O. 6 mg I-I Cu o 1.0 mg I-I Cu .A. 3.0 mg I-I Cu

100

Q)

0:

50

20

40

60

80

100

Time (Hours) Figure 2. The effect of Sub-lethal Copper Pollution on the Respiration Rate of Gammarus duebeni.

Sub-lethal ecotoxicology testing

97

The effect of salinity on repiration rate is shown in Figure 3. Two Way ANOVA showed that there was a significant difference between the treatments with time as there was with salinity alone (F 1394.7, P < 0.05).

=

250

c: '6 I


200 150

el)

.0

"e 0

g-

-

100

el)

c..

•

0 % salinity

•

1.66 % salinity

o 3.16 % salinity

50

o 1.01 % salinity 20

40 Time (Hours)

60

80

Figure 3. The effect of salinity on the respiration rate of Gammarus duebeni.

Least Significant Difference tests showed which treatments were different. Those treatments on the same line do not differ significantly. Salinity stress at either extreme of the range, therefore, also reduces pleopod beat frequency. Salinity (%)

1.01

1.66

3.16

o

The effects of copper pollution on Gammarus duebeni swimming efficiency at flow rates of 110 ml min- 1 and 145 ml min- 1 are shown in Figures 4 and 5, respectively. Two Way ANOVA of the assay performed at 110 ml min- l showed that there was a significant concentration effect on swimming efficiency (F = 6.18, P < 0.05). There was no significant time effect or concentration/time interaction. A One Way ANOVA confirmed the significant difference between concentration series. A Least Significant Difference test showed that each treatment was significantly different from the next and that Cu reduced swimming efficiency in a dose related manner. Cu concentration (mg 1-1)

_1_

50

.0 mg 1-1 Cu 01 mg 1-1 Cu .3 mg 1-1 Cu

-150...1-------,-----,.-------,. 30 10 20 Time (Hours) Figure 4. The effect of copper on the swimming efficiency of Gammarus duebeni against a flow rate of 110 ml min - 1.

A. LAWRENCE and C. paULTER

98

50

-,

0

c:

E

2 E

-50

"'d

~

~

(1)

u

c: ~

.0 mg I-I Cu

-100

o 0.6 mg I-I Cu 01 mg I-I Cu

<-J (/)

o -150

.3 mg I-I Cu

-200-L-----..,..----~----_r

10 Time (Hours)

20

30

Figure 5. The effect of copper on the swimming efficiency of Gammarus duebeni against a flow rate of 145 ml min-I.

A Two Way ANOVA showed that there was a significant difference in Gammarus duebeni swimming efficiency between treatments at a flow rate of 145 m1 min-I. In this case there were both significant time and Cu concentration effects (F = 13.26, P < 0.05). The Least Significant Difference test showed which of the treatments were significantly different. Those concentration series underlined were not significantly different annd again indicate a dose related copper effect.

_0_

Cu concentration (mg 1-1)

0.6

1.0

The effect of sub-lethal Cu pollution on Gammarus pair formation is shown in Figure 6. A One Way ANOVA showed that there were significant differences between treatments (F = 14.71, P < 0.05). Least Significant Difference tests identified which of the treatments differed. Those series underlined were not significantly different. Cu concentration (mg I-I)

"'d (1)

o

0.2

1.0

0.6

75

~0• (1)

~ 50 l-<

.~

• 0 mg I-I Cu o 0.2 mg 1- 1 Cu • 0.6 mg 1-1 Cu o I mg 1-1 Cu

0• 4-0

o

~ 25

c: (1)

<-J

8

~

20

40

60

80

Time (hours) Figure 6. The effect of sub-lethal copper pollution on Gammarus duebeni pair separation.

DISCUSSION Preliminary observations suggest that Gammarus duebeni might provide a useful species with which to assess toxic impacts within the estuarine environment. Each of the bioassays developed showed a direct response to copper pollution. Significant impairment of respiration rate, as measured by changes in pleopod beat, was determined at a copper concentration of 600 ~g I-I. Significant impairment to swimming ability

Sub-lethal ecotoxicology testing

99

was determined after exposure to copper pollution at a concentration of 600 ~g 1-1 and precopula pairing between male and female Gammarus duebeni was significantly impaired at a concentration of 600 ~g 1-1. Of the assays, swimming efficiency and pleopod beat frequency appear to offer the most potential for further development. In both a dose related effect to copper was observed. Precopula pairing shows a response to copper but appears to be less dose dependent. In addition, it is difficult to standardise for animal size in this assay. Size, condition, and reproductive state are known to influence the impact of pollutants on an amimal (Rainbow and Moore, 1986; Rainbow and White, 1989). The effects levels determined in the assays described here are within the effects range for copper detennined using the freshwater species. Drift response of Gammarus puiex was affected by copper treatment at a concentration of 702 ~g 1-1 (Taylor et ai. 1994) whilst feeding rate was significantlt reduced by 3 hours exposure to 101 ~g 1-1 copper (Taylor et ai. 1993). The precise sensitivity of each of the assays, described here, to copper is still to be detennined. However, the copper concentration shown to induce an effect can be experienced in the natural environment. Soluble copper concentrations have been recorded at a maximum of 300 ~g 1-1 at Spurn Head on the Humber estuary (NRA, 1992) and as high as 600 ~g 1-1 in the Camon River and Restronguet Creek (Bryan and Langston, 1992). In addition, it has been shown that the effect of peak rather than the mean toxicant concentration may be important in assessing pollution impacts (Edwards et al. 1991). This highlights the potential significance of episodic pollution events in an environment (Taylor et al. 1994). Gammarus duebeni may also provide a suitable species with which to develop a sensitive sediment bioassay for the estuarine environment. There is increasing need for sediment bioassays (McCarthy and Shugart, 1990). Sediments can quickly sequester heavy metals from solution (Taylor et ai. 1994) affecting the bioavailability and toxicity of the pollutant. In addition, heavy metal concentrations in sediments can be many times greater than that in the overlying water. Copper levels in Humber sediments can be as high as 206 ~g g-1 (Grant and Middleton, 1990) and up to 2400 ~g g-1 at Restronguet Creek (Bryan and Langston, 1992). Furthermore, sediment bound metals may be quickly remobilised into interstitial and surface waters and sediment/metal interactions can be affected by pH, salinity and oxygen tension (Bryan and Langston, 1992). Some species including Zostera marina, Fucus versicuiosus and Nereis diversicoior contain copper levels related to those of the sediment indicating that they can accumulate copper absorbed on particles of suspended sediment (Luoma et ai. 1982). As a detritivore Gammarus deubeni feeds on surface sediment and plant particles including Fucus (McClusky, 1989). In addition, amphipods do not regulate body copper (Rainbow and White, 1989) highlighting the potential role of Gammarus duebeni in this type of assay. When body metal burden is not regulated, it has to be sequested, with an associated metabolic cost to the animal. In Gammarus duebeni this is supported by evidence from the bioassays developed in this study. Each of the assays indicate a reduction or redirection of available energy from respiration, swimming and precopula pairing. Physiological effects of pollutants on Gammarus duebeni also support this (Wright, 1986; Tendengren and Nils Kautsky, 1988; Johnson and Jones, 1989). Whilst the specific method of detoxification has not been characterised in Gammarus duebeni, work on other crustacea indicate that it probably involves temporary storage and excretion of metals or the production of binding proteins (Rainbow et al.. 1990). Furthermore, this would suggest that Gammarus duebeni might also develop tolerance to metal pollutIOn and this has previously been shown in a population from a saline sewage treatment works subjected to a high zinc load (Jones and Johnson, 1992). These reults indicate that Gammarus duebeni is potentially a very useful species with which to assess estuarine pollution. The lower limit of sensitivity of the bioassays developed in t~is study are yet t.o be determined. However, the study has shown that they can be used in sub-lethal pollutIOn assays at relatIvely high Cu concentrations. In ad~ition, ~ts ecology .and de~ri.tiv~ry. suggest t~at ~t may be us~ful in ~he development of an estuarine sedIment bIOassay and Its plaStiCIty mdicates that It ffilght be useful m st~dymg the development of tolerance mechanisms to estuarine pollution. Further studies are needed to determme the

A. lAWRE NCE and C. POULT ER

\00

role of Gammarus duebeni in each of these aspects of pollution biology. This work is curren tly being undertaken in our laboratory.

REFERENCES Borlakoglu. 1. T. and Kickuth. R. (1990) Behavio ural changes in .Gamm arus pulex .and its signific ance in the toxicity assessm ent of very low levels of environ mental pollutants. Bull. Environ. Contam . Toxlcol. 45, 258- 263. .. . . Bryan. G. W. and langsto n. W. J. (1992). Bioavailability, accumu lation and effects of heavy metals 10 sedime nts wIth special reference to United Kingdo m estuaries: a review. Env. Poll. 76. 89-131. . . Bulnheim, H.-P. (1979). Compar ative studies on the physiol ogical ecology of five euryhal ine Gammarus specIes. OeeologlG (Bert) 44.80-8 6. Clalk, R. B. (1966). A Practical Course in Experimental Zoology. John Wiley, london . . .. Crane, M. and Maltby. L. (1991). The lethal and subletha l reponse s of Gammarus pulex to stress: sensItIvIty and sources of variation in an in situ bioassay. Env. Toxieo!. and Chem. 10, 1331-13 39. Edwards, R. W., Ormerod. S. J. and Turner. C. (1991). Field experim ents to assess biologic al effects of pollutio n episode s in streams. Verh. Int. Ver. Limnol. 24,1734 -1737. Grant, A. and Middleton. R. (1990). An assessm ent of metal contam ination of sedimen ts in the Humbe r estuary , U.K. Est. Coast. and Shelf Sci. 31. 71-85. Gray. 1. S. (1981). The Ecology of Marine Sediments. Cambri dge Univers ity Press, Cambri dge. Hunte, W., Myers. R. A. and Doyle. R. W. (1985). Bayesia n mating decision s in an amphip od. Gammarus lawrencianus (Bousfield) Anim. Behav. 33. 356-372 . Hunter. W. R. (1949). The poisoni ng of Marinogammarus marinus by cupric sulphat e and mercuri c chloride . J. Exp. Biol. 26, 113-124. Jemelov , A. and Rosenberg. R. (1976). Stress toleranc e of ecosyst ems. Environ. Conserv. 3. 43-46. Johnson, I. and Jones, M. B. (1989). Effects of zinc/salinity combin ations on zinc regulati on in Gamma rus dueben i from the estuary and the sewage treatme nt works at looe, Cornwa ll. J. Mar. Bio!. Assoc. U.K. 69, 249-260 . Jones. M. B. and Johnson. 1. (1992). Respon ses of the brackish water amphip od Gammarus duebeni (Crusta cea) to saline sewage. Neth.1. Sea Res. 30. 141-147. luoma, S. N.• Bryan, G. W. and langsto n. W. J. (1982). Scaven ging of heavy metals from particul ates by brown seawee d. Mar. Pollut. Bull. 13. 394-396. McCarthy, J. F. and Shugart, L. R. (1990). Biomarkers of Environmental Contamination. CRC Press, Boca Raton, Florida . McClusky, D. (1989). The Estuarine Ecosystem. 2nd Edn. Blackie and Sons ltd, london . McClusky, D .• Bryant, V. and Campbe ll, R. (1986). The effects of tempera ture and salinity on the toxicity of heavy metals to marine and estuarine inverteb rates. Oceanogr. Mar. Bio!. Ann. Rev. 24, 481-520 . National Rivers Authority (1992). The Water Quality of the Humbe r Estuary 1991. A Report of the Humbe r Commi ttee of the NRA. NRA, U.K. Poulton. M. and Pascoe. D. (1990). Disrupt ion of precopu la in Gamma rus pulex (l) - develop ment of a behavio ural bioassay for evaluating pollutan t and parasite induces stress. Chemosphere 20,403 -415. Rainbow, P. S. and Moore. P. G. (1986). Compar ative metal analysis in amphip od crustace ans. Hydrobiologia 141, 273-289 . Rainbow, P. S. and White. S. L. (1989). Compar ative strategi es of heavy metal accumu lation by crustace ans: zinc, copper and cadmiu m in a decapod , and amphip od and a barnacle. Hydrobiologia 174, 245-262 . Rainbow, P. S., Moore, P. G. and Watson , D. (1989). Talitrid amphip ods (Crusta cea) as biomon itors for copper and zinc. Est. Coast. Shelf Sci. 28, 567-582 . Rainbow, P. S., Phillips. J. H. and Depled ge, M. H. (1990). The signific ance of trace metal concent rations in marine invertebrates - a need for laboratory investig ation of accumu lation strategies. Mar. Poll. Bull. 21. 321-324 . Reish, D. J. (1993). Effects of metals and organic compou nds on surviva l and bioaccu mulatio n in two species of marine gammar idean amphip od, togethe r with a summar y of toxicolo gical research on this group. J. Nat. Hist. 27, 781-794 . Ritz, D. A. (1980). Toleran ce of intertid al amphip ods to fluctuating conditio ns of salinity , oxygen and copper. J. Mar. Biol. Ass. U.K. 60. 489-498 . Sheader. M. (1983). The reprodu ctive biology and ecology of Gamma rus dueben i (Crusta cea: Amphip oda) in souther n England. 1. Mar. Biol. Assoc. UK 63, 517-540 . Stephenson, R. (1983). Effects of water hardnes s, water tempera ture and size of the test organis m on the suscept ibility of the freshwater shrimp Gammarus pulex (L) to toxicants. Bull. Environ . Contam. Toxieol. 31, 459-466 . Taylor, E., Jones, D.• Maund. S. and Pascoe, D. (1993). A new method for measur ing the feeding activity of Gamma rus pulex (L) Chemosphere 26, 1375-1381. Taylor, E., Rees, E. and Pascoe. D. (1994). Mortali ty and drift related respons e of the freshwa ter amphip od Gammarus pulex (L) exposed to natural sedimen ts, acidific ation and copper. Aquat. Toxieol . 29, 83-101. Tedengren •. M. and Nils Kautsky , M. A. (1988). Ecophy siology and stress respons e of marine and brackis h water Gammarus species (Crustacea, Amphip oda) to changes in salinity and exposur e to cadmiu m and diesel-oil. Mar. Eco!. Prog. Ser. 47. 107-116. Wright. D. A. (1986). Trace metal uptake and sodium regulati on in Gammarus marinus from metal polluted estuarie s in England. J. Mar. Biol. Assoc. U.K. 66, 83-92.