Responses of the brackish-water amphipod Gammarus duebeni (crustacea) to saline sewage

141

Netherlands Journal of Sea Research 30:141-147 (1992)

RESPONSES OF THE BRACKISH-WATER AMPHIPOD GAMMARUS DUEBENI (CRUSTACEA) TO SALINE SEWAGE M.B. JONES and I. JOHNSON* Department of Biological Sciences, University of Plymouth, Plymouth, Devon PL4 8AA, UK

ABSTRACT

Soon after the opening of the Looe sewage treatment works (Cornwall, southwest England) in 1973, it became colonized by the brackish-water amphipod Gammarus duebeni Liljeborg. The works is unusual as it operates with saline sewage and has a tidally-based pattern of salinity fluctuation (S=13 to 34). Various responses of this unique amphipod population (sewage amphipods) have been compared with G. duebeni from the adjacent Looe River estuary (estuarine amphipods) in an attempt to identify long-term responses to sewage. Sewage amphipods were significantly smaller than their estuarine equivalents; the sewage population was biased significantly to males, whereas the sex ratio of the estuarine population significantly favours females. Compared with the estuary, the consistently lower oxygen levels in the works were reflected in significant differences in metabolism. Sewage amphipods maintained high levels of activity under hypoxia (e.g. swimming), and had higher survival and lower rates of lactic acid accumulation under anoxia than estuarine individuals. In addition, sewage amphipods recovered more rapidly from anoxia and had a lower critical oxygen tension (Pc) than estuarine amphipods. Sewage amphipods are exposed to higher levels of heavy metals associated with the domestic sewage and zinc concentrations are particularly elevated in the works. Exposure to elevated zinc concentrations resulted in similar patterns of body zinc uptake for sewage and estuarine Gammarus at high (30) and low (10) salinity, with zinc regulation apparently occurring to an external threshold of 200 i~gZn'dm "3. No consistent interpopulational differences in the effect of zinc on zinc uptake or on osmoregulation have been identified. However, sewage amphipods had higher survival at all zinc/salinity combinations compared with estuarine individuals. These findings indicate that sewage amphipods are adapted to the unusual combination of conditions prevailing in the treatment works and, if reproductive isolation is confirmed, suggest that the speciation process may have commenced.

1. INTRODUCTION Gammarus duebeni Liljeborg (Crustacea, Amphipoda) has an extensive geographical distribution in the North Atlantic Ocean, ranging from southern Labrador to Cape Cod on the western coastline and from northern Siberia to northern France on the eastern coastline (LINCOLN,1979; SHEADER,1983). It is a brackish-water species with wide tolerances to salinity and hypoxia (FORSMAN,1951; SUTCLIFFE,1967; PINKSTERet al., 1970; BULNHEIM,1979; RITZ, 1980). Within the confines of a typical peracarid life cycle, G. duebeni has a plastic reproductive biology, making it an opportunistic species, well suited to life in demanding and unpredictable environments (SHEADER, 1983). Since 1973, a permanent breeding population of G. duebeni

has become established within the sewage treatment works at Looe, Cornwall (southwest England), colonizers presumably being recruited from the Looe River estuary adjacent to the works (Fig. 1). It appears that immigration of G. duebeni into the works has ceased as none was collected at the inlet to the works during a monthly survey over twelve months (JONES & WlGHAM, 1988). Various physiological responses of this unique population of sewage amphipods to hypoxia/anoxia and elevated zinc concentrations have been compared with those of G. duebeni from the Looe River estuary (AGNEW& JONES, 1986; JOHNSON& JONES, 1989, 1990). The present paper describes differences in population structure and summarizes this published work in an attempt to isolate possible long-term effects of sewage on life-

*Present address: Water Research Centre, Henley Road, Medmenham, Marlow, Bucks SL7 2HD, UK

142

M.B. JONES & I. JOHNSON

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to that in the estuary (JONES & JOHNSON, 1991). Although salinity and temperature conditions are broadly similar in the works and estuary, sewage gammarids experience lower oxygen concentrations and higher concentrations of heavy metals (as a consequence of the domestic sewage), with zinc being particularly elevated in the works (30 to 144.5 ~tgZn-dm °) compared with the estuary (14 to 28.5 ~tgZn.dm 3) (JONES & JOHNSON, 1991). The same two collection sites were used throughout this study; sewage amphipods were taken from one of the secondary settlement tanks (humus tank) and estuarine amphipods were sampled at Terras Bridge, East Looe River (Fig. 1). 3. MATERIALS AND METHODS 3.1. POPULATION STUDIES

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From June to December 1989, Gammarus duebeni were collected from each site at approximately monthly intervals (LOVELL, 1990). Sewage individuals were sampled by scraping the straight edge of an FBA net (0.5 mm mesh size) against the humus tank wall at the air-water boundary where gammarids often congregate. Estuarine amphipods were searched for under algae (Fucus spp.) and rocks within a restricted area of the shore. A sample size of approximately 100 individuals was aimed for at each site, but this was not always possible at the estuary. In the laboratory, amphipods were preserved in 4% seawater formalin, sorted into males, females and 4~w ovigerous females, and measured (from the anterior margin of the head to the tip of the telson) with the Fig. 1. Location of the Looe sewage treatment works and estuarine sampling site (Terras Bridge) in the East Looe animal straightened along a measuring rule. River. Representative salinity ranges are given in parenthe3.2. LABORATORY STUDIES ses. lkm

history traits and on physiology, and to gain some insight into the mechanisms of the evolution of tolerance to sewage pollution.

All physiological work was carried out at 20°C (as this approximated a summer temperature at each site) using only adult intermoult males and non-ovigerous females >15.0 mg wet body weight (>3 mg dry body 2. STUDY SITE weight; ~ 1.0 to 1.5 cm body length). A. Exposure to hypoxia: tolerance to anoxia was The Looe sewage treatment works (4°27'W, 50°21'N) measured over 10 h using groups of five individuals, is situated in a small valley above the town near the and metabolic responses to hypoxia were assessed confluence of the West and East Looe Rivers (Fig. 1). based on lactate levels (the total L-lactate content The works is conventional in design and sewage was determined by the method of GUTMANN& WAHLprocessing, and operates with biological filters (JONES EFELD, 1974) produced after different exposure times & JOHNSON,1991). The works is rather unusual, how- using groups of 10 animals (for full experimental ever, as it treats saline rather than the more conven- details see AGNEW& JONES,1986). tional freshwater sewage. Saline water enters mainly B. Exposure to elevated zinc concentrations: through seepage into the open-jointed pipes embed- amphipods were acclimated for seven days to two ded in the mud of the Looe River estuary which carry experimental salinities (S=10 and 30, chosen raw sewage to the works. This seawater intrusion because they approximated the salinity extremes causes a tidally-based, cyclical pattern of salinity fluc- recorded in the humus tank (JONES& JOHNSON,1991) tuation within the works (S=13 to 34) which is similar and experienced by estuarine individuals). At each

RESPONSES OF GAMMARUS TO SEWAGE

4o i salinity, three replicates of 10 animals each were exposed to a range of zinc concentrations (<5, 100, 200, 500, 1000 ~tgZndm3). Amphipods were held in ! 30 !i individual mesh containers in plastic tanks filled with 1.5 dm 3 of test media. They were fed twice weekly up c~ to 72 h prior to the experiment after which they were "d starved. Survival was measured daily, over seven 8 £ days, and the total body zinc levels of surviving amphipods were determined by atomic absorption 10 I ' spectrophotometry (JOHNSON& JONES, 1989). Haemol5' ymph osmotic concentration (using the RAMSAY & BROWN (1955)freezing-point technique), haemolymph 04 sodium (using atomic absorption spectro-photometry) and cuticle apparent water permeability (measured 801 using the tritiated water outflux method of LOCKWOOD et al. (1973)) were also measured from surviving 70 amphipods (JOHNSON& JONES, 1990).

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4. RESULTS AND DISCUSSION 4.1. POPULATION STUDIES

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M.B. JONES & I. JOHNSON

TABLE 1 Summary of metabolic responses to hypoxia of Gammarus duebenifrom the East Looe River Estuary and the Looe Sewage Treatment Works. After AGNEW& JONES(1986). estuary sewage parameter (tidal hypoxia) (continuous hypoxia) oxygen tensions in water ca. 150 Torr 0-50 Torr critical oxygen tension (Pc) < 20 Torr <10 Torr LTsofor exposure to anoxia (<1 Torr) 2.7 h 6.5 h lactate concentration (tlM.g -~) under normoxia 1.6 3.5 lactate accumulation during 6-h exposure to anoxia (pM.g ~) 20.2 14.5 lethal lactate concentration OiM.g4) 20.2 23 recovery time from 5-h exposure to anoxia >8h 5.611 food supply for G. duebeni (removing intraspecific competition for food as a possible mechanism leading to reduced body size) compared with the estuary. It may well be that such high food availability results in rapid growth and smaller maturity size, as reported for another amphipod species (SIEGFRIED, 1985). Based solely on field measurements, it is obviously not possible to determine which of these factors (or combinations of factors) is responsible for a reduced size of Gammarus in sewage waters compared with estuarine, and confirmation must await laboratory studies. Sex ratios (calculated from numbers collected over the entire sampling period) deviated significantly from 1:1 (;(2, p
ratio in the Looe estuary is a sign of the decline in this population. 4.2. EXPOSURE TO HYPOXlA

Differences in response to hypoxia and anoxia are summarized in Table 1. Sewage amphipods have 100

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RESPONSES OF GAMMARUS TO SEWAGE

lower critical oxygen tensions (Pc) than estuarine individuals. Previous work has shown that Gammarus duebeni has a high tolerance to hypoxia and that there is some variation in the Pc between different populations of G. duebeni (BULNHEIM, 1979; RITZ, 1980). Reduction in Pc is interpreted as an adaptation to life in permanently low oxygen tensions, which are a feature of the humus tank, for it allows high levels of activity at low pO 2. The apparent flexibility in Pc measured for different populations of the same species, however, suggests that acclimation to low pO 2 rather than genetic change is the mechanism of adaptation involved (e.g. BAYNE & LIVINGSTONE, 1977). Sewage amphipods survived exposure to anoxia significantly longer (t-test, p
h, whereas estuarine animals did not survive beyond 6 h. The lethal lactate concentration was lower in estuarine than sewage individuals (Table 1 ). These differences in responses to hypoxia are clearly adaptive and can be related directly to differences in oxygen availability between the two study habitats. Intertidal estuarine amphipods are exposed to short periods of anoxia only at times of low tide and are able to recover in normoxic conditions during each high-tide period. Animals in the humus tank, however, experience continuous hypoxia (AGNEW & JONES, 1986; JONES & JOHNSON, 1991) and must emerge from the water to encounter high oxygen tensions. Thus, it is not surprising that sewage animals display responses that improve their ability to survive anoxic conditions. 4.3. EXPOSURE TO ELEVATED ZINC In response to elevated zinc concentrations, sewage gammarids have significantly higher survival (ANOVA, p
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M.B. JONES & I. JOHNSON

seven days at 1000 p.gZn.dm 3 in combination with low salinity (Fig. 3A). Irrespective of amphipod body size and salinity, total body zinc levels did not differ significantly from control levels (ANCOVA, p>O.05) up to 200 ~gZn.dm 3 (Fig. 4), and this is interpreted as indicating that both populations regulate body zinc to this external threshold. At each salinity, estuarine and sewage amphipods showed size-dependent differences in zinc uptake, such that small individuals (Fig. 4A and 4C) have more body zinc than larger animals (Fig. 4B and 4D), but there were no significant differences (ANCOVA, p>O.05) in body zinc levels between the two populations. Indeed, the only major difference to emerge from this aspect of the work was the inabili~ of estuarine gammarids to survive in 1000 ~gZn-dm°in combination with low salinity (Fig. 4A and 4B). Estuarine and sewage Gammarus duebeni showed no reduction in osmoregulatory response when exposed to elevated zinc concentrations (to 1000 p.gZn.dm -3) at high salinity (S=30) (JOHNSON& JONES, 1990). There was osmoregulatory impairment at low salinity, although only at >500 pgZn'dm -3, and the responses are summarized in Table 2. The haemolymph osmotic concentration of sewage and estuarine amphipods was reduced by the same amount (60 mOsmol.kg-1), but the reduction was relatively more severe for estuarine animals due to their smaller initial gradient between the body fluids and external medium (Table 2). The mechanism underlying this osmotic reduction was due to significant (t-test, p
TABLE 2 Summary of osmoregulatory responses to elevated zinc of Gammarus duebeni maintained at S=10 from the East Looe River Estuary and the Looe Sewage Treatment Works. After JOHNSON& JONES (1990). *Control <5 p_gZn'dm3. parameter estuary sewage dissolved zinc concentration 14.0- 28.5 30.0-144.5 (~g.dm-3) in water lowest zinc concentration (pg-dm-3) causing significant 500 500 osmotic reduction haemolymph osmotic concentration (mOsmol.kg-1) *Control 523 593 500 ~gZn-dm-3 463 533 haemolymph sodium concentration (mM-dm-3) *Control 295 302 500 ~gZn-dm-3 257 250 cuticle apparent water permeability (rain) *Control 10.3 12.1 500 p.gZn'dm-3 9.6 10.5 gest that the sewage gammarids are adapted to the novel combination of conditions which occur in a sewage treatment works. The question remains as to whether such differences reflect phenotypic acclimation by an opportunistic species or whether genetic change has occurred, presumably leading to speciation. For Gammarus spp. it is well documented that habitat specialization leads to speciation (KOLDING, 1985), albeit over a much longer time scale than has been available to G. duebeni since it colonized the sewage works in 1973. The sewage population appears to be reproductively isolated (JONES& WlGHAM, 1988) and, if restricted gene flow is confirmed, the populational and physiological differences summarized here suggest the intriguing possibility that the speciation process has commenced. Acknowledgements.--We thank David Lovell for the length measurements of Gammarus, and the staff at the Looe sewage treatment works for continued interest and assistance. 5. REFERENCES AGNEW, D.J. & M.B. JONES, 1986. Metabolic adaptations of Gammarus duebeni Liljeborg (Crustacea, Amphipoda) to hypoxia in a sewage treatment plant.--Comp. Biochem. Physiol. 84A: 475-478. BAYNE,B.L. & D.R. LIVINGSTONE,1977. Responses of Mytilus edulis L. to low oxygen tension: acclimation of the rate of oxygen consumption.~, comp. Physiol. Bl14: 129-142. BROWN,B. & M. AHSANULLAH,1971. Effect of heavy metals on mortality and growth.--Mar. Pollut. Bull. 2:182-188. BULNHEIM,H.P., 1978. Interaction between genetic, external and parasitic factors in sex determination of the crusta-

RESPONSES OF GAMMARUS TO SEWAGE

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