PH: S0273-1223(96)00729-9

~ PergamonWal. Sci. Tech. Vol. 34. No. 7-8, pp. 93-100,1996.

Copyright © 1996 1AWQ. Published by Elsevier Science LtdPrinted in Great Britain. All rights reserved.

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THE POTENTIAL ROLE OF THEESTUARINE AMPHIPOD GAMMARUSDUEBENI IN SUB-LETHALECOTOXICOLOGY TESTING

Andrew Lawrence and Carl Poulter

Department ofApplied Biology, The University ofHull, 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 estuarinepollution. 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 pollutionand comparison of the suitability of the assays. Significant impairment of respiration rate, as measured bychanges in pleopod beat, was determined at a copper concentration of 600 Ilg I-I. Significant impairment toswimming ability was determined after exposure to copper polIution at a concentration of 600 Ilg I-I andprecopula pairing was significantly reduced at a concentration of 600 Ilg P. Of the assays, swimmingefficiency and pleopod beat frequency appear to offer the most potential for further development. The lowerlimit of sensitivity of the bioassays developed in this study is yet to be determined. However. the levels ofcopper shown to induce an effect can be experienced in the natural environment. The study has shown thatGammarus duebeni can be used in sub-lethal pollution assays, at relatively high Cu concentrations, andindicates 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 inareas 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 theseestuarine areas. It is one of the major food species in the trophic web and acts as a shredder, particulatingvegetation in the community (McLusky, 1989). It is therefore important both in the passage of food to otherorganisms 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 thebioavailability of the metal in the environment.

Gammarus duebeni is a truly estuarine species (McLusky. 1989). Its potential use in ecotoxicology isparticularly important. therefore. because estimates of the effects of pollutants in estuaries have often b~enderived from experiments using marine species (eg Hunter 1949) and there is a need to use more estuanneanimals 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 anyadditional stress. Alternatively. by having a wide tolerance to abiotic factors they may be pre-adapted totolerate pollution stress (Jernelov and Rosenberg. 1976; Gray. 1981). In either case. their response to anypollutant 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 andsub-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/pollutioneffects 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 todevelop sub-lethal toxicity bioassays. To date, however, a sub-lethal pollution bioassay has not beendeveloped using this species.

The aim of this study was to assess the suitability of Gammarus duebeni as an indicator of estuarinepollution. 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 assaysdeveloped. Metal concentrations on the Humber have been measured and all, except copper, fall below theEnvironmental 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 theHumber 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 theanimals.

An initial range finding LC 50 acute toxicity test was perfomed. Animals were maintained in 0, 1, 2, 3,4 and5 mg 1-1 copper solutions, prepared from anhydrous copper sulphate in 1,66% sea water. Deaths weremonitored 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 ofsalinity and Cu toxicity on survival. A Cu solution of 1 mg 1-1 was prepared using seawater of 0, 1.6 and3.18% salinity. Percentage deaths were recorded over 96 hours.

The first sub-lethal bioassay developed examined the effect of Cu on Gammarus duebeni pleopod beatfreq~ency. The pleopods are abdominal appendages used both in swimming and to facilitate the exchange ofrespuatory gases across the animals' integument. As such, pleopod beat frequency has been used as a directmeasure 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. Atappropnate 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 aftermtroductIOn to the test solution and then at convenient time intervals over the next 96 hours. Test solutionswere 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 testsolutions of 0, 1.01, 1.66 and 3.16% salinity was determined. The instantaneous pleopod beat frequency wasrecorded for each individual and subsequently monitored over the following 72 hours. Test solutions wererenewed every 48 hours.

The effect of sub-lethal copper pollution on swimming efficiency was also determined. A system wasdeveloped in which the animals ability to move against a head flow of water could be examined. Plastictubing was connected to the inlet and outlet of a peristaltic pump. The pump was used to adjust the flow rateof the test solution. A scale was marked on the outlet pipe giving a distance of 100 mm each side of a zeromark.

The inlet hose was immersed in the relevant test solution and the system filled. Using a pipette, individualanaimals were then placed into the outlet pipe at the zero position on the marked scale. At a flow rate of 110ml 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 testsolution, If an animal moved beyond the scale, its position was marked and measured. The test was thenrepeated at a flow rate of 145 ml min-I. Movement was converted to mm moved min- 1 and the average ateach flow rate determined.

Finally, the effect of Cu exposure on precopula pairing in Gammarus duebeni was determined. Ten pairs ofanimals were placed in solutions of 0, 0.2, 0.6 and 1 mg I-I Cu. The separation of pairs was then recordedover the next 76 hours.

RESULTS

Initial toxicity tests were evaluated using Probit analysis, the results of which are shown in Table 1. The LT50 was determined for each test solution. In the first, range finding, test the LT 50 wa~ calculated as over 70hours for each test solution. In the following tests the LT 50 ranged from 135 hours 10 0.2 mg I-I Cu to 37hours 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

LT 50...

~ . -

Dec '94 Jan '95 Feb '95- -

0 0 0

135.6

114.9 96 88.1

74.7 56.1 63.1

60.8 37.5 45.1

92.2

139.3

89.1

72.9

86.3

Oct '94

omg 1-1

o0.2

0.6

1

2

3

4

5

Cu ConcentrationiI

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

96

100

25

A. lAWRENCE and C POULTER

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

A 3.18 % salinity, 1 mg I-I Cu

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 ANOVA. 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 TwoWay ANOVA and Least Significant Difference multiple range test. There was a significant differencebetween treatments with time (F = 303.45, P < 0.05). This was supported by a One Way ANOVA whichshowed 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 fromeach other. Those Cu concentration series underlined show no significant difference, those not on the sameline 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

- 200I

t:·8

ISOC/J....ro~

.D"00 1000..0Q)

0:50

• 0 mg I-I Cu controlo O. 2 mg I-I Cu• O. 6 mg I-I Cuo 1.0 mg I-I Cu.A. 3.0 mg I-I Cu

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 asignificant difference between the treatments with time as there was with salinity alone (F =1394.7, P <0.05).

250

- 200I

c:'6

<J:J 150.....C':lel).0"e0 100g-el)-c..

50

• 0 % salinityo 3.16 % salinity• 1.66 % salinityo 1.01 % salinity

20 40Time (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 sameline do not differ significantly. Salinity stress at either extreme of the range, therefore, also reduces pleopodbeat 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 at110 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 ANOVAconfirmed the significant difference between concentration series. A Least Significant Difference testshowed that each treatment was significantly different from the next and that Cu reduced swimmingefficiency in a dose related manner.

Cu concentration (mg 1-1) _1_

30

50

.0 mg 1-1 Cu

01 mg 1-1 Cu

.3 mg 1-1 Cu

-150...1-------,-----,.-------,.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.

98 A. LAWRENCE and C. paULTER

50

-,c: 0E2E

"'d -50~

~(1) -100uc:~

<-J(/)

o -150

.0 mg I-I Cu

o 0.6 mg I-I Cu01 mg I-I Cu.3 mg I-I Cu

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

10 20 30

Time (Hours)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 swimmingefficiency between treatments at a flow rate of 145 m1 min-I. In this case there were both significant timeand Cu concentration effects (F = 13.26, P < 0.05). The Least Significant Difference test showed which ofthe treatments were significantly different. Those concentration series underlined were not significantlydifferent annd again indicate a dose related copper effect.

Cu concentration (mg 1-1) _0_ 0.6 1.0

The effect of sub-lethal Cu pollution on Gammarus pair formation is shown in Figure 6. A One WayANOVA showed that there were significant differences between treatments (F = 14.71, P < 0.05). LeastSignificant Difference tests identified which of the treatments differed. Those series underlined were notsignificantly different.

Cu concentration (mg I-I) o 0.2 1.0 0.6

"'d 75(1)

~0•(1)

~ 50l-<.~

0•4-0o~ 25<-Jc:(1)

8~

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

806020 40

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 toassess toxic impacts within the estuarine environment. Each of the bioassays developed showed a directresponse to copper pollution. Significant impairment of respiration rate, as measured by changes in pleopodbeat, 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 pairingbetween male and female Gammarus duebeni was significantly impaired at a concentration of 600 ~g 1-1. Ofthe assays, swimming efficiency and pleopod beat frequency appear to offer the most potential for furtherdevelopment. In both a dose related effect to copper was observed. Precopula pairing shows a response tocopper but appears to be less dose dependent. In addition, it is difficult to standardise for animal size in thisassay. 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 detenninedusing the freshwater species. Drift response of Gammarus puiex was affected by copper treatment at aconcentration of 702 ~g 1-1 (Taylor et ai. 1994) whilst feeding rate was significantlt reduced by 3 hoursexposure 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, thecopper concentration shown to induce an effect can be experienced in the natural environment. Solublecopper 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 maybe important in assessing pollution impacts (Edwards et al. 1991). This highlights the potential significanceof 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 bioassayfor 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 thebioavailability and toxicity of the pollutant. In addition, heavy metal concentrations in sediments can bemany times greater than that in the overlying water. Copper levels in Humber sediments can be as high as206 ~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 watersand 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 levelsrelated to those of the sediment indicating that they can accumulate copper absorbed on particles ofsuspended sediment (Luoma et ai. 1982). As a detritivore Gammarus deubeni feeds on surface sediment andplant 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 theanimal. 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 andprecopula 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 detoxificationhas not been characterised in Gammarus duebeni, work on other crustacea indicate that it probably involvestemporary 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 andthis has previously been shown in a population from a saline sewage treatment works subjected to a highzinc load (Jones and Johnson, 1992).

These reults indicate that Gammarus duebeni is potentially a very useful species with which to assessestuarine pollution. The lower limit of sensitivity of the bioassays developed in t~is study are yet t.o bedetermined. However, the study has shown that they can be used in sub-lethal pollutIOn assays at relatIvelyhigh Cu concentrations. In ad~ition, ~ts ecology .and de~ri.tiv~ry. suggest t~at ~t may be us~ful in ~hedevelopment 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

\00 A. lAWRENCE and C. POULTER

role of Gammarus duebeni in each of these aspects of pollution biology. This work is currently beingundertaken in our laboratory.

REFERENCES

Borlakoglu. 1. T. and Kickuth. R. (1990) Behavioural changes in .Gammarus pulex .and its significance in the toxicity assessmentof very low levels of environmental pollutants. Bull. Environ. Contam. Toxlcol. 45, 258-263... . .Bryan. G. W. and langston. W. J. (1992). Bioavailability, accumulation and effects of heavy metals 10 sediments wIth specialreference to United Kingdom estuaries: a review. Env. Poll. 76. 89-131. ..Bulnheim, H.-P. (1979). Comparative studies on the physiological ecology of five euryhaline Gammarus specIes. OeeologlG(Bert) 44.80-86.

Clalk, R. B. (1966). A Practical Course in Experimental Zoology. John Wiley, london. . ..Crane, M. and Maltby. L. (1991). The lethal and sublethal reponses of Gammarus pulex to stress: sensItIvIty and sources ofvariation in an in situ bioassay. Env. Toxieo!. and Chem. 10, 1331-1339.Edwards, R. W., Ormerod. S. J. and Turner. C. (1991). Field experiments to assess biological effects of pollution episodes instreams. Verh. Int. Ver. Limnol. 24,1734-1737.Grant, A. and Middleton. R. (1990). An assessment of metal contamination of sediments in the Humber estuary, U.K. Est. Coast.and ShelfSci. 31. 71-85.Gray. 1. S. (1981). The Ecology ofMarine Sediments. Cambridge University Press, Cambridge.Hunte, W., Myers. R. A. and Doyle. R. W. (1985). Bayesian mating decisions in an amphipod. Gammarus lawrencianus(Bousfield) Anim. Behav. 33. 356-372.Hunter. W. R. (1949). The poisoning of Marinogammarus marinus by cupric sulphate and mercuric chloride. J. Exp. Biol. 26,113-124.Jemelov, A. and Rosenberg. R. (1976). Stress tolerance of ecosystems. Environ. Conserv. 3. 43-46.Johnson, I. and Jones, M. B. (1989). Effects of zinc/salinity combinations on zinc regulation in Gammarus duebeni from theestuary and the sewage treatment works at looe, Cornwall. J. Mar. Bio!. Assoc. U.K. 69, 249-260.Jones. M. B. and Johnson. 1. (1992). Responses of the brackish water amphipod Gammarus duebeni (Crustacea) to saline sewage.Neth.1. Sea Res. 30. 141-147.luoma, S. N.• Bryan, G. W. and langston. W. J. (1982). Scavenging of heavy metals from particulates by brown seaweed. Mar.Pollut. Bull. 13. 394-396.McCarthy, J. F. and Shugart, L. R. (1990). Biomarkers ofEnvironmental Contamination. CRC Press, Boca Raton, Florida.McClusky, D. (1989). The Estuarine Ecosystem. 2nd Edn. Blackie and Sons ltd, london.McClusky, D.• Bryant, V. and Campbell, R. (1986). The effects of temperature and salinity on the toxicity of heavy metals tomarine and estuarine invertebrates. Oceanogr. Mar. Bio!. Ann. Rev. 24, 481-520.National Rivers Authority (1992). The Water Quality of the Humber Estuary 1991. A Report of the Humber Committee of theNRA. NRA, U.K.Poulton. M. and Pascoe. D. (1990). Disruption of precopula in Gammarus pulex (l) - development of a behavioural bioassay forevaluating pollutant and parasite induces stress. Chemosphere 20,403-415.Rainbow, P. S. and Moore. P. G. (1986). Comparative metal analysis in amphipod crustaceans. Hydrobiologia 141, 273-289.Rainbow, P. S. and White. S. L. (1989). Comparative strategies of heavy metal accumulation by crustaceans: zinc, copper andcadmium in a decapod, and amphipod and a barnacle. Hydrobiologia 174, 245-262.Rainbow, P. S., Moore, P. G. and Watson, D. (1989). Talitrid amphipods (Crustacea) as biomonitors for copper and zinc. Est.Coast. ShelfSci. 28, 567-582.Rainbow, P. S., Phillips. J. H. and Depledge, M. H. (1990). The significance of trace metal concentrations in marine invertebrates- a need for laboratory investigation of accumulation strategies. Mar. Poll. Bull. 21. 321-324.Reish, D. J. (1993). Effects of metals and organic compounds on survival and bioaccumulation in two species of marinegammaridean amphipod, together with a summary of toxicological research on this group. J. Nat. Hist. 27, 781-794.Ritz, D. A. (1980). Tolerance of intertidal amphipods to fluctuating conditions of salinity, oxygen and copper. J. Mar. Biol. Ass.U.K. 60. 489-498.Sheader. M. (1983). The reproductive biology and ecology of Gammarus duebeni (Crustacea: Amphipoda) in southern England. 1.Mar. Biol. Assoc. UK 63, 517-540.Stephenson, R. (1983). Effects of water hardness, water temperature and size of the test organism on the susceptibility of thefreshwater 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 measuring the feeding activity of Gammarus pulex (L)Chemosphere 26, 1375-1381.Taylor, E., Rees, E. and Pascoe. D. (1994). Mortality and drift related response of the freshwater amphipod Gammarus pulex (L)exposed to natural sediments, acidification and copper. Aquat. Toxieol. 29, 83-101.Tedengren•. M. and Nils Kautsky, M. A. (1988). Ecophysiology and stress response of marine and brackish water Gammarusspecies (Crustacea, Amphipoda) to changes in salinity and exposure to cadmium and diesel-oil. Mar. Eco!. Prog. Ser. 47.107-116.Wright. D. A. (1986). Trace metal uptake and sodium regulation in Gammarus marinus from metal polluted estuaries in England.J. Mar. Biol. Assoc. U.K. 66, 83-92.