Arch. Environ. Contam. Toxicol. 23,440-452 (1992)
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The Toxicity of Selenium in Experimental Freshwater Ponds Mark Crane 1, Tim Flower, Dawn Holmes, and Samantha Watson Ecotoxicology and Biodegradability Group, Water Research Centre, Medmenham, United Kingdom
Abstract. Flue Gas Desulphurization equipment installed in coal-fired power stations to reduce sulphur dioxide emissions produces effluent containing several contaminants, including selenium. To assess the effects that this might have on fish reproduction and biological community richness, selenate and selenite were added to freshwater pond systems to achieve duplicated nominal concentrations of 0 (control), 2, 10 and 25 I~g Se/L. Perch (Perca fluviatilis), grass carp (Ctenopharyngodon idella Val.) and stickleback (Gasterosteus aculeatus) were placed in each pond in the early summer and left relatively undisturbed until the following spring. A spawning tray was then placed in each pond and monitored regularly for the appearance of perch egg ropes. When ropes were found, they were removed to the laboratory and either placed in uncontaminated well water or exposed to selenium. Almost half the perch exposed to 25 I~g Se/L died during the pond experiment, but there were no observed deaths at the lower concentrations. No grass carp were recovered from the 25 ~g Se/L ponds but all were recovered from the other ponds. Stickleback were missing from all treatments, presumably due to predation by the perch. Perch egg ropes were found in seven of the eight ponds, and all but one rope showed signs of successful fertilisation. Hatching success in the laboratory was highly variable for eggs obtained from the 0, 2, and 10 ixg Se/L ponds, but was always above zero. No eggs hatched from ropes obtained from the 25 Ixg Se/L ponds. Effects of selenium on plants, macroinvertebrates and zooplankton in the ponds were generally limited. Accumulation of selenium in fish was dose-related and comparable with results reported from other contaminated systems. The results from this experiment suggest that mean environmental concentrations of approximately 25 txg Se/L may seriously affect the reproductive capacity of perch, but no clear effects on reproduction are evident at mean concentrations of 10 Ixg Se/L and below. Selenium-induced effects occur during gametogenesis, but only become evident between fertilization and hatching.
power generators to install Flue Gas Desulphurization (FGD) equipment. Similar equipment operating in Germany produces a range of contaminants, originating mostly from coal, including selenium at a concentration of 100 p,g Se/L in treated FGD effluent (Taylor et al. 1989). The normal disposal route for this FGD effluent is by admixing with cooling water obtained from and subsequently returned to rivers, estuaries or coastal waters. Selenium has been recognized as a potential problem in aquatic systems for several years (Demayo et al. 1979; Adams and Johnson 1981), but it is only during the past decade that ecological studies have shown the extensive damage to fish communities that can be caused by environmental concentrations of this metalloid as low as 10 I~g Se/L. An important study of the bioaccumulation and toxicity of selenium in Belews Lake, a power plant cooling reservoir in North Carolina, indicated that food chain accumulation had led to the elimination of most fish species within two years (Cumbie and van Horn 1978; Finley 1985; Hodson 1988; Lemly 1985a, 1985b) apparently due to reproductive impairment. Subsequent investigations have shown that the sublethal effects of selenium on fish are often most pronounced when reproductive endpoints are measured (Gillespie and Baumann 1986; Woock et al. 1987; Schultz and Hermanutz 1990). This paper describes an experiment in which invertebrates and fish species were exposed to selenium in freshwater ponds to assess effects on fish survival and reproduction and biological community richness. Perch (Percafluviatilis) were selected as the primary test organism because they are a common and economically important species in the UK; they are members of the Perciforme group, the largest vertebrate order; and they lay their eggs in gelatinous ropes (Craig 1987) which can easily be seen and removed from pond systems.
Materials and Methods
Pond Construction Concerns over sulphur dioxide emissions from coal-fired power stations have led to proposals from United Kingdom (UK)
~Correspondence to Mark Crane, WRc plc, Henley Road, Medmenham, PO Box 16, Marlow, Buckinghamshire, SL7 2HD, United Kingdom.
Eight square ponds, 1-m deep and measuring 5 m x 5 m at the surface, sloping to 4 m x 4 m at the bottom, were excavated in April 1989. Each pond was lined with butyl rubber pond liner and filled with Medmenham River Thames water, which was allowed to stand until the leachate from the rubber no longer rendered the water toxic to Daphnia magna Strauss in an acute 48-h static test (OECD 1984). The
Selenium Toxicity in Freshwater Ponds
ponds were then drained and filled with a 5-10-cm depth of sediment obtained from established ponds on site, before refilling with river water. Plants and invertebrates present in the established ponds were introduced to each pond in the sediment and the water.
441
distribute the organisms evenly, and withdrawing 5 ml with a syringe. The animals present in these subsamples were then counted and identified in a Bogarov chamber under a 70× binocular microscope. A final set of zooplankton samples, replicated three times within each pond was taken on 2 April 1991 (Day 512).
Water Chemistry Fish Survival and Growth in Ponds Routine physical and chemical monitoring of the pond waters was performed at bimonthly and then monthly intervals. Dissolved oxygen levels and pH were measured in each pond using hand-held meters (EDT models GP 353 and 343). Temperature was measured in each pond with in situ maximum-minimum thermometers. Ammonia, nitrate, nitrite, soluble reactive phosphorus, and total hardness levels were determined from a single, 1-L water sample taken from each pond, using standard methods (Standing Committee of Analysts 1980, 1981a, 1981b, 1981c). Chlorophyll samples were collected from each pond at monthly intervals by pumping 25 L of water from each pond into a large tub using a submersible pump. Chlorophyll a levels were then determined in I-L subsamples by acetone extraction and spectrophotometric estimation (Golterman and Clymo 1969).
Selenium Treatments The ponds were first dosed with selenium in March 1990 after several months of naturalization, to achieve nominal concentrations of 0 (control), 2, 10 and 25 ixg Se/L in duplicate pond systems, selected at random. The concentrations were chosen to span the range expected in UK effluent and receiving waters and the selenium was dosed as a mixture of selenium VI (selenate) and selenium IV (selenite) in a ratio of 60:40, in order to mimic the ratio in FGD effluent. Application of the selenium was achieved by pumping a spray-free jet of stock solution onto the surface of each pond. Repeat doses were administered to maintain nominal values by using a computer program to calculate the amount of additional dose required from the rate of change of selenium concentrations between sampling dates. Unfiltered samples were taken from each pond bimonthly for total selenium determination by hydride generation/atomic absorption spectrometry (Gunn 1981 ) after allowing 1 week after each dose for mixing to occur.
Invertebrate Abundance The benthic invertebrates in each pond were sampled monthly along a randomly selected 2 m transect. A submersible pump with a rigid hose attachment was moved along the bottom of the ponds to pump sediment and invertebrates to the water surface over a 120-s period, where they were passed through a 500-1xm mesh net. The material remaining in the net was then hand-sorted and preserved in 70% ethanol for subsequent identification to the lowest practicable taxonomic level. A final set of benthic samples, replicated five times within each pond, was taken in the same way on 3 April 1991 (Day 513), live-sorted and identified. The animals in these final benthic samples were then divided into insects, molluscs, and crustaceans, bulked by pond, rinsed in well water, and dried at 60°C to constant weight before determination of selenium content by hydride generation/atomic absorption spectrometry (Standing Committee of Analysts 1985). Zooplankton were sampled monthly from each pond along another, randomly selected 2 m transect. A submersible pump was used to pump water from the water column through a 50-1xm mesh net for 120 s. After sampling, the zooplankton were washed into a sample jar and preserved in 90% ethanol. Two 0.5% subsamples were then withdrawn by making the sample up to 1-L with tap water, agitating the water to
Adult perch, seine-netted from a natural pool near Aylsham in Norfolk were treated with an antibiotic (oxytetracycline) to control Foecal Perch Ulcer Disease (FPUD) before addition to the ponds on 20 June 1990 (Day 226). Unfortunately, the fish could not be sexed before the experiment because they were obtained after the breeding season. Eight randomly selected perch were added to each pond to produce a total biomass per pond of between 421 g and 508 g. Two unsexed grass carp (Ctenopharyngodon idella Val.) obtained from Maidenhead Aquatics, Bourne End, Buckinghamshire, were selected at random and added to each pond on 5 July 1990 (Day 241) to produce a total biomass of between 24.6 g and 35.8 g per pond. These were included in the experiment to assess bioaccurnulation in an herbivorous species. One hundred and thirty unsexed stickleback (Gasterosteus aculeatus) obtained from National Rivers Authority filter beds at Fobney Mead, Reading, were also randomly selected and added to each pond on 29 June 1990 (Day 235) to produce a total biomass per pond of between 68.0 g and 100.0 g. The ponds were observed at least weekly, and visible fish mortality recorded. At the end of the experiment, the ponds were drained, surviving fish were netted, killed with the anaesthetic MS 222, and weighed. Muscle and gonad tissue samples were digested and selenium concentrations determined by hydride generation/atomic absorption spectrometry (Standing Committee of Analysts 1985).
Perch Reproduction in Ponds and Laboratory Flow-Through Systems The ponds were cleared of a dense growth of Elodea canadensis by raking the weed out on 4 April 1991 (Day 514). Spawning trays were constructed by attaching 100-120 strips of greenhouse shading, measuring approximately 50 cm x 5 cm, to polyethylene containers measuring 100 cm × 50 cm x 10 cm. One tray was placed in the centre of each pond and weighted with bricks, so that the strips of greenhouse shading floated vertically, to imitate pondweed. The spawning trays were monitored for the presence of egg ropes at least every 2 days. When egg ropes were observed, they were removed from the ponds and returned to the laboratory. Subsamples of approximately 50 eggs from every egg rope were placed in 250-ml jars containing well water and observed under a microscope daily for four days after removal from the ponds, to estimate fertilisation success. The first two ropes produced in each pond were also used in a laboratory experiment designed to investigate the effect of selenium on perch gametogenesis and larval survival. Subsamples of 200-500 eggs from each rope were prepared for each of the following experimental treatments:
l. Egg ropesfrom selenium-dosedponds: A subsample from each rope from each of the two ponds at each of the three dosing levels was placed in (i) Undosed control vessels to assess the effect of pond-dosed selenium on gametogenesis alone. (ii) Well water continuously dosed to the nominal concentration of selenium existing in the pond of origin. This was done to examine the effect of waterborne selenium on the gametogenesis and early life stages of perch.
M. Crane et al,
442
Table 1. Experimental design for laboratory perch early life stage test (each treatment duplicated) Nominal Se concentration in ponds (Ixg/L)
Pond numbers
Nominal Se concentration in laboratory (Ixg/L)
0 0 0 0 2 2 10 10 25 25
l&6 l&6 l&6 l &6 3&8 3&8 4&7 4&7 2& 5 2&5
0 2 10 25 0 2 0 10 0 25
nominal concentrations although there was considerable variability about these means (measured mean total selenium concentrations = < 1 . 0 Ixg/L, 2.0 Ixg/L (STD = 0.8), 8.0 Ixg/L (STD = 5.6) and 24.4 Ixg/L, (STD = 14.9)). Concentrations of selenium remained constant for several weeks after the initial dose, with levels in the 25 Ixg Se/L nominal concentration ponds generally measuring between 30 and 40 Ixg Se/L (Figure 2). Total selenium concentrations in the other ponds were nearer their nominal concentrations during this period. During the second half of the dosing period, however, selenium disappeared from the water column of the ponds more rapidly than before, and more frequent dosing was required to maintain mean nominal concentrations in the water column. This led to fluctuations in selenium concentrations around mean values.
Plants 2. Egg ropes from controlponds: Subsamples of eggs from each of the two ponds were placed in: (i) Undosed control vessels to assess hatching success. (ii) Well water continuously dosed to the nominal concentrations in the treated ponds (2, 10, and 25 Ixg Se/L). This was done to isolate the effects of waterborne selenium on perch early life stage development alone. Glass flow-through aquaria were set to contain 10 litres of test medium with a flow rate of 100 ml/min via single-axis siphon dosers. The egg subsamples were exposed in 500-ml Pyrex vessels with 500-1xm mesh collars, designed to allow the flow of medium over the eggs, but prevent the escape of hatching larvae. Two of these vessels were placed in each aquarium, each containing eggs from one of the duplicate pond treatments. Each laboratory treatment was also duplicated in a randomised block design, so that eggs from two ropes from each pond could be used, if available (Table 1). If only one rope was produced in a pond, then eggs from that rope were placed in all the vessels available for that pond. Water samples were taken from each test aquarium for total selenium analysis one week before and immediately after the addition of the first egg rope subsamples, and then at weekly intervals until the end of the experiment. Temperature, dissolved oxygen, pH and hardness were measured daily in each aquarium for the duration of the experiment. The eggs were observed daily, and the vesselsremoved when the larvae had successfully hatched. The number of live and dead larvae, deformities and unhatched eggs were then recorded.
Results Water Chemistry Dissolved oxygen, pH, temperature, total hardness, total ammonia, nitrite, nitrate, and soluble reactive phosphorus levels are presented as the mean of all eight ponds on each sampling date (Figure 1). There were few unusual features about these measurements, except that pH remained high for much of the study, apparently due to the photosynthetic activity of pondweed. There were also a number of peaks in the concentration of total ammonia measured in the ponds, but these were at levels below those likely to cause damage to freshwater systems (Seager et al. 1988). Mean measured selenium levels in the ponds averaged over the course of the experiment were close to
The pondweed Elodea canadensis rapidly colonized the experimental systems, forming dense mats in every pond. There were no apparent differences in percentage cover between any of the ponds. No other macrophytes established populations over the course of the experiment. Chlorophyll a levels oscillated independently of each selenium concentration during the experiment, reflecting the seasonally-related abundance of phytoplankton species (Figure 3). Although chlorophyll levels dropped to zero in both of the ponds dosed at 25 Ixg Se/L immediately after dosing began, levels had risen by the next sampling date, and thereafter remained similar to those at the other concentrations.
Invertebrate Abundance There were no major differences over the course of the study in the benthic macroinvertebrate communities found in ponds dosed at different selenium concentrations. The species richness was similar in all the ponds, with 20 to 24 different taxa present in each. The abundance of the most common species at each selenium concentration are plotted in Figure 4. These results suggest that seasonal factors were a more important determinant of benthic invertebrate abundance than the concentration of selenium within each pond. Table 2 shows the mean abundance of commonly occurring invertebrates (i.e., those taxa found in more than 50% of the samples taken from control ponds) in the benthic samples taken from ponds at each concentration at the end of the experiment. These data indicate the patchy occurrence of pond invertebrates: many samples did not include representatives from even these four most common taxa, and variation among samples was high. Chironomid larvae were the most abundant organisms in this set of samples, and significant differences were apparent among ponds dosed at different selenium concentrations. A Newman-Keuls test (Zar 1984) showed that fewer chironomids were found in ponds dosed at 10 Ixg Se/L compared with those dosed at 0 or 2 Ixg Se/L (q = 6.8 and 6.3, respectively, p < 0.001), but there were no significant differences between low dose ponds and those dosed at 25 Ixg Se/L. This suggests that the difference in chironomid abundance was not directly related to selenium toxicity.
SeleniumToxicityin FreshwaterPonds
443 Dlssolved Oxygen
30
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4.66, p < 0.01) and Simocephalus expinosus was more abundant in the control ponds compared with those dosed at 10 and 25 Ixg Se/L (q = 4.04 and 3.85, p < 0.025 and p < 0.05, respectively). This apparent response to selenium was not evident in populations of Eurycereus lamellatus and Acroperus harpae. The former showed a
Selenium Toxicity in Freshwater Ponds
445
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Fig. 4. Abundance of c o m m o n benthic macroinvertebrate taxa in ponds (mean for two ponds at each concentration)
M. Crane et al.
446
Table 2. Abundance (number per sample) of common benthic invertebrate taxa in samples taken from ponds on 3 April 1991, Day 513 (n = 10) Abundance Taxon
Concentration (pog Se/L)
Median
Range
Kruskal-Wallis H
Probability
Chironomids
0 2 10 25 0 2 10 25 0 2 10 25 0 2
33.5 31.0 4.5 13.5 1.5 1.0 3.5 0.5 2.5 3.5 1.0 4.0 1.5 2.0
11-62 0-85 0-14 0-77 0-9 0-5 0-10 0-8 0-39 0-13 0-11 1-17 0-9 0-7 0-5 0-21
15.02
0.05). The concentrations of selenium found in perch muscle and gonads at the end of the experiment are also shown in Table 4. Levels of selenium were similar in the two types of tissue and increased significantly with environmental concentrations up to approximately ten times control values. (ANOVA on log-transformed data: muscle, F = 49.73, P < 0.001; gonad F = 85.38, P < 0.001). Tukey's Least Significant Difference test (Zar 1984) showed that gonad selenium levels were significantly different at p < 0.025 between all treatments. Muscle selenium levels were significantly different at p < 0.05 between all treatments, with the exception of the difference be-
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tween the 10 Ixg Se/L and 25 Ixg Se/L ponds which was not statistically significant. Two grass carp (the initial number added) were recovered from all of the ponds dosed below 25 txg Se/L. One dead carp was recovered from one of the 25 ixg Se/L ponds (pond 2 on 7 January 1991, Day 427), and the remaining three carp from these ponds were missing at the end of the experiment. The concentrations of selenium found in grass carp muscle at the end of the experiment are shown in Table 5. Concentrations increased ten-fold compared with controls in those carp exposed to 10 p~g Se/L. Only two sticklebacks were recovered from the entire set of ponds at the end of the experiment. Both sticklebacks were recovered dead from pond 2, on 25 February 1991 (Day 476) and 4 March 1991 (Day 483), respectively.
Perch Spawning and Hatching Success and Larval Survival The perch began to spawn on the trays placed in the ponds on 13 April 1991 and continued until 3 May 1991 (Table 6). Egg ropes were found in all of the ponds, with the exception of pond 2 (25 Ixg Se/L). Fertilization success was greater than 90% of eggs in subsamples taken from all of the ropes, except for the rope recovered from pond 3 (2/xg Se/L) on 13 April 1991 (Day 523), in which there was no evidence of fertilization success. Water quality during the perch early life stage test was maintained at 15°C -+ 1.3°C, pH 7.86 + 0.15 and 95-110% ASV. Water hardness was 300 mg/L as CaCO 3. Selenium concentrations were maintained near their nominal values (range of measured values (ixg S e / L ) =
Archives
of
E nvironmental c o n t a ma nidn a t i o n
Iloxicology
© 1992 Springer-Verlag New York Inc.
The Toxicity of Selenium in Experimental Freshwater Ponds Mark Crane 1, Tim Flower, Dawn Holmes, and Samantha Watson Ecotoxicology and Biodegradability Group, Water Research Centre, Medmenham, United Kingdom
Abstract. Flue Gas Desulphurization equipment installed in coal-fired power stations to reduce sulphur dioxide emissions produces effluent containing several contaminants, including selenium. To assess the effects that this might have on fish reproduction and biological community richness, selenate and selenite were added to freshwater pond systems to achieve duplicated nominal concentrations of 0 (control), 2, 10 and 25 I~g Se/L. Perch (Perca fluviatilis), grass carp (Ctenopharyngodon idella Val.) and stickleback (Gasterosteus aculeatus) were placed in each pond in the early summer and left relatively undisturbed until the following spring. A spawning tray was then placed in each pond and monitored regularly for the appearance of perch egg ropes. When ropes were found, they were removed to the laboratory and either placed in uncontaminated well water or exposed to selenium. Almost half the perch exposed to 25 I~g Se/L died during the pond experiment, but there were no observed deaths at the lower concentrations. No grass carp were recovered from the 25 ~g Se/L ponds but all were recovered from the other ponds. Stickleback were missing from all treatments, presumably due to predation by the perch. Perch egg ropes were found in seven of the eight ponds, and all but one rope showed signs of successful fertilisation. Hatching success in the laboratory was highly variable for eggs obtained from the 0, 2, and 10 ixg Se/L ponds, but was always above zero. No eggs hatched from ropes obtained from the 25 Ixg Se/L ponds. Effects of selenium on plants, macroinvertebrates and zooplankton in the ponds were generally limited. Accumulation of selenium in fish was dose-related and comparable with results reported from other contaminated systems. The results from this experiment suggest that mean environmental concentrations of approximately 25 txg Se/L may seriously affect the reproductive capacity of perch, but no clear effects on reproduction are evident at mean concentrations of 10 Ixg Se/L and below. Selenium-induced effects occur during gametogenesis, but only become evident between fertilization and hatching.
power generators to install Flue Gas Desulphurization (FGD) equipment. Similar equipment operating in Germany produces a range of contaminants, originating mostly from coal, including selenium at a concentration of 100 p,g Se/L in treated FGD effluent (Taylor et al. 1989). The normal disposal route for this FGD effluent is by admixing with cooling water obtained from and subsequently returned to rivers, estuaries or coastal waters. Selenium has been recognized as a potential problem in aquatic systems for several years (Demayo et al. 1979; Adams and Johnson 1981), but it is only during the past decade that ecological studies have shown the extensive damage to fish communities that can be caused by environmental concentrations of this metalloid as low as 10 I~g Se/L. An important study of the bioaccumulation and toxicity of selenium in Belews Lake, a power plant cooling reservoir in North Carolina, indicated that food chain accumulation had led to the elimination of most fish species within two years (Cumbie and van Horn 1978; Finley 1985; Hodson 1988; Lemly 1985a, 1985b) apparently due to reproductive impairment. Subsequent investigations have shown that the sublethal effects of selenium on fish are often most pronounced when reproductive endpoints are measured (Gillespie and Baumann 1986; Woock et al. 1987; Schultz and Hermanutz 1990). This paper describes an experiment in which invertebrates and fish species were exposed to selenium in freshwater ponds to assess effects on fish survival and reproduction and biological community richness. Perch (Percafluviatilis) were selected as the primary test organism because they are a common and economically important species in the UK; they are members of the Perciforme group, the largest vertebrate order; and they lay their eggs in gelatinous ropes (Craig 1987) which can easily be seen and removed from pond systems.
Materials and Methods
Pond Construction Concerns over sulphur dioxide emissions from coal-fired power stations have led to proposals from United Kingdom (UK)
~Correspondence to Mark Crane, WRc plc, Henley Road, Medmenham, PO Box 16, Marlow, Buckinghamshire, SL7 2HD, United Kingdom.
Eight square ponds, 1-m deep and measuring 5 m x 5 m at the surface, sloping to 4 m x 4 m at the bottom, were excavated in April 1989. Each pond was lined with butyl rubber pond liner and filled with Medmenham River Thames water, which was allowed to stand until the leachate from the rubber no longer rendered the water toxic to Daphnia magna Strauss in an acute 48-h static test (OECD 1984). The
Selenium Toxicity in Freshwater Ponds
ponds were then drained and filled with a 5-10-cm depth of sediment obtained from established ponds on site, before refilling with river water. Plants and invertebrates present in the established ponds were introduced to each pond in the sediment and the water.
441
distribute the organisms evenly, and withdrawing 5 ml with a syringe. The animals present in these subsamples were then counted and identified in a Bogarov chamber under a 70× binocular microscope. A final set of zooplankton samples, replicated three times within each pond was taken on 2 April 1991 (Day 512).
Water Chemistry Fish Survival and Growth in Ponds Routine physical and chemical monitoring of the pond waters was performed at bimonthly and then monthly intervals. Dissolved oxygen levels and pH were measured in each pond using hand-held meters (EDT models GP 353 and 343). Temperature was measured in each pond with in situ maximum-minimum thermometers. Ammonia, nitrate, nitrite, soluble reactive phosphorus, and total hardness levels were determined from a single, 1-L water sample taken from each pond, using standard methods (Standing Committee of Analysts 1980, 1981a, 1981b, 1981c). Chlorophyll samples were collected from each pond at monthly intervals by pumping 25 L of water from each pond into a large tub using a submersible pump. Chlorophyll a levels were then determined in I-L subsamples by acetone extraction and spectrophotometric estimation (Golterman and Clymo 1969).
Selenium Treatments The ponds were first dosed with selenium in March 1990 after several months of naturalization, to achieve nominal concentrations of 0 (control), 2, 10 and 25 ixg Se/L in duplicate pond systems, selected at random. The concentrations were chosen to span the range expected in UK effluent and receiving waters and the selenium was dosed as a mixture of selenium VI (selenate) and selenium IV (selenite) in a ratio of 60:40, in order to mimic the ratio in FGD effluent. Application of the selenium was achieved by pumping a spray-free jet of stock solution onto the surface of each pond. Repeat doses were administered to maintain nominal values by using a computer program to calculate the amount of additional dose required from the rate of change of selenium concentrations between sampling dates. Unfiltered samples were taken from each pond bimonthly for total selenium determination by hydride generation/atomic absorption spectrometry (Gunn 1981 ) after allowing 1 week after each dose for mixing to occur.
Invertebrate Abundance The benthic invertebrates in each pond were sampled monthly along a randomly selected 2 m transect. A submersible pump with a rigid hose attachment was moved along the bottom of the ponds to pump sediment and invertebrates to the water surface over a 120-s period, where they were passed through a 500-1xm mesh net. The material remaining in the net was then hand-sorted and preserved in 70% ethanol for subsequent identification to the lowest practicable taxonomic level. A final set of benthic samples, replicated five times within each pond, was taken in the same way on 3 April 1991 (Day 513), live-sorted and identified. The animals in these final benthic samples were then divided into insects, molluscs, and crustaceans, bulked by pond, rinsed in well water, and dried at 60°C to constant weight before determination of selenium content by hydride generation/atomic absorption spectrometry (Standing Committee of Analysts 1985). Zooplankton were sampled monthly from each pond along another, randomly selected 2 m transect. A submersible pump was used to pump water from the water column through a 50-1xm mesh net for 120 s. After sampling, the zooplankton were washed into a sample jar and preserved in 90% ethanol. Two 0.5% subsamples were then withdrawn by making the sample up to 1-L with tap water, agitating the water to
Adult perch, seine-netted from a natural pool near Aylsham in Norfolk were treated with an antibiotic (oxytetracycline) to control Foecal Perch Ulcer Disease (FPUD) before addition to the ponds on 20 June 1990 (Day 226). Unfortunately, the fish could not be sexed before the experiment because they were obtained after the breeding season. Eight randomly selected perch were added to each pond to produce a total biomass per pond of between 421 g and 508 g. Two unsexed grass carp (Ctenopharyngodon idella Val.) obtained from Maidenhead Aquatics, Bourne End, Buckinghamshire, were selected at random and added to each pond on 5 July 1990 (Day 241) to produce a total biomass of between 24.6 g and 35.8 g per pond. These were included in the experiment to assess bioaccurnulation in an herbivorous species. One hundred and thirty unsexed stickleback (Gasterosteus aculeatus) obtained from National Rivers Authority filter beds at Fobney Mead, Reading, were also randomly selected and added to each pond on 29 June 1990 (Day 235) to produce a total biomass per pond of between 68.0 g and 100.0 g. The ponds were observed at least weekly, and visible fish mortality recorded. At the end of the experiment, the ponds were drained, surviving fish were netted, killed with the anaesthetic MS 222, and weighed. Muscle and gonad tissue samples were digested and selenium concentrations determined by hydride generation/atomic absorption spectrometry (Standing Committee of Analysts 1985).
Perch Reproduction in Ponds and Laboratory Flow-Through Systems The ponds were cleared of a dense growth of Elodea canadensis by raking the weed out on 4 April 1991 (Day 514). Spawning trays were constructed by attaching 100-120 strips of greenhouse shading, measuring approximately 50 cm x 5 cm, to polyethylene containers measuring 100 cm × 50 cm x 10 cm. One tray was placed in the centre of each pond and weighted with bricks, so that the strips of greenhouse shading floated vertically, to imitate pondweed. The spawning trays were monitored for the presence of egg ropes at least every 2 days. When egg ropes were observed, they were removed from the ponds and returned to the laboratory. Subsamples of approximately 50 eggs from every egg rope were placed in 250-ml jars containing well water and observed under a microscope daily for four days after removal from the ponds, to estimate fertilisation success. The first two ropes produced in each pond were also used in a laboratory experiment designed to investigate the effect of selenium on perch gametogenesis and larval survival. Subsamples of 200-500 eggs from each rope were prepared for each of the following experimental treatments:
l. Egg ropesfrom selenium-dosedponds: A subsample from each rope from each of the two ponds at each of the three dosing levels was placed in (i) Undosed control vessels to assess the effect of pond-dosed selenium on gametogenesis alone. (ii) Well water continuously dosed to the nominal concentration of selenium existing in the pond of origin. This was done to examine the effect of waterborne selenium on the gametogenesis and early life stages of perch.
M. Crane et al,
442
Table 1. Experimental design for laboratory perch early life stage test (each treatment duplicated) Nominal Se concentration in ponds (Ixg/L)
Pond numbers
Nominal Se concentration in laboratory (Ixg/L)
0 0 0 0 2 2 10 10 25 25
l&6 l&6 l&6 l &6 3&8 3&8 4&7 4&7 2& 5 2&5
0 2 10 25 0 2 0 10 0 25
nominal concentrations although there was considerable variability about these means (measured mean total selenium concentrations = < 1 . 0 Ixg/L, 2.0 Ixg/L (STD = 0.8), 8.0 Ixg/L (STD = 5.6) and 24.4 Ixg/L, (STD = 14.9)). Concentrations of selenium remained constant for several weeks after the initial dose, with levels in the 25 Ixg Se/L nominal concentration ponds generally measuring between 30 and 40 Ixg Se/L (Figure 2). Total selenium concentrations in the other ponds were nearer their nominal concentrations during this period. During the second half of the dosing period, however, selenium disappeared from the water column of the ponds more rapidly than before, and more frequent dosing was required to maintain mean nominal concentrations in the water column. This led to fluctuations in selenium concentrations around mean values.
Plants 2. Egg ropes from controlponds: Subsamples of eggs from each of the two ponds were placed in: (i) Undosed control vessels to assess hatching success. (ii) Well water continuously dosed to the nominal concentrations in the treated ponds (2, 10, and 25 Ixg Se/L). This was done to isolate the effects of waterborne selenium on perch early life stage development alone. Glass flow-through aquaria were set to contain 10 litres of test medium with a flow rate of 100 ml/min via single-axis siphon dosers. The egg subsamples were exposed in 500-ml Pyrex vessels with 500-1xm mesh collars, designed to allow the flow of medium over the eggs, but prevent the escape of hatching larvae. Two of these vessels were placed in each aquarium, each containing eggs from one of the duplicate pond treatments. Each laboratory treatment was also duplicated in a randomised block design, so that eggs from two ropes from each pond could be used, if available (Table 1). If only one rope was produced in a pond, then eggs from that rope were placed in all the vessels available for that pond. Water samples were taken from each test aquarium for total selenium analysis one week before and immediately after the addition of the first egg rope subsamples, and then at weekly intervals until the end of the experiment. Temperature, dissolved oxygen, pH and hardness were measured daily in each aquarium for the duration of the experiment. The eggs were observed daily, and the vesselsremoved when the larvae had successfully hatched. The number of live and dead larvae, deformities and unhatched eggs were then recorded.
Results Water Chemistry Dissolved oxygen, pH, temperature, total hardness, total ammonia, nitrite, nitrate, and soluble reactive phosphorus levels are presented as the mean of all eight ponds on each sampling date (Figure 1). There were few unusual features about these measurements, except that pH remained high for much of the study, apparently due to the photosynthetic activity of pondweed. There were also a number of peaks in the concentration of total ammonia measured in the ponds, but these were at levels below those likely to cause damage to freshwater systems (Seager et al. 1988). Mean measured selenium levels in the ponds averaged over the course of the experiment were close to
The pondweed Elodea canadensis rapidly colonized the experimental systems, forming dense mats in every pond. There were no apparent differences in percentage cover between any of the ponds. No other macrophytes established populations over the course of the experiment. Chlorophyll a levels oscillated independently of each selenium concentration during the experiment, reflecting the seasonally-related abundance of phytoplankton species (Figure 3). Although chlorophyll levels dropped to zero in both of the ponds dosed at 25 Ixg Se/L immediately after dosing began, levels had risen by the next sampling date, and thereafter remained similar to those at the other concentrations.
Invertebrate Abundance There were no major differences over the course of the study in the benthic macroinvertebrate communities found in ponds dosed at different selenium concentrations. The species richness was similar in all the ponds, with 20 to 24 different taxa present in each. The abundance of the most common species at each selenium concentration are plotted in Figure 4. These results suggest that seasonal factors were a more important determinant of benthic invertebrate abundance than the concentration of selenium within each pond. Table 2 shows the mean abundance of commonly occurring invertebrates (i.e., those taxa found in more than 50% of the samples taken from control ponds) in the benthic samples taken from ponds at each concentration at the end of the experiment. These data indicate the patchy occurrence of pond invertebrates: many samples did not include representatives from even these four most common taxa, and variation among samples was high. Chironomid larvae were the most abundant organisms in this set of samples, and significant differences were apparent among ponds dosed at different selenium concentrations. A Newman-Keuls test (Zar 1984) showed that fewer chironomids were found in ponds dosed at 10 Ixg Se/L compared with those dosed at 0 or 2 Ixg Se/L (q = 6.8 and 6.3, respectively, p < 0.001), but there were no significant differences between low dose ponds and those dosed at 25 Ixg Se/L. This suggests that the difference in chironomid abundance was not directly related to selenium toxicity.
SeleniumToxicityin FreshwaterPonds
443 Dlssolved Oxygen
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4.66, p < 0.01) and Simocephalus expinosus was more abundant in the control ponds compared with those dosed at 10 and 25 Ixg Se/L (q = 4.04 and 3.85, p < 0.025 and p < 0.05, respectively). This apparent response to selenium was not evident in populations of Eurycereus lamellatus and Acroperus harpae. The former showed a
Selenium Toxicity in Freshwater Ponds
445
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M. Crane et al.
446
Table 2. Abundance (number per sample) of common benthic invertebrate taxa in samples taken from ponds on 3 April 1991, Day 513 (n = 10) Abundance Taxon
Concentration (pog Se/L)
Median
Range
Kruskal-Wallis H
Probability
Chironomids
0 2 10 25 0 2 10 25 0 2 10 25 0 2
33.5 31.0 4.5 13.5 1.5 1.0 3.5 0.5 2.5 3.5 1.0 4.0 1.5 2.0
11-62 0-85 0-14 0-77 0-9 0-5 0-10 0-8 0-39 0-13 0-11 1-17 0-9 0-7 0-5 0-21
15.02
0.05). The concentrations of selenium found in perch muscle and gonads at the end of the experiment are also shown in Table 4. Levels of selenium were similar in the two types of tissue and increased significantly with environmental concentrations up to approximately ten times control values. (ANOVA on log-transformed data: muscle, F = 49.73, P < 0.001; gonad F = 85.38, P < 0.001). Tukey's Least Significant Difference test (Zar 1984) showed that gonad selenium levels were significantly different at p < 0.025 between all treatments. Muscle selenium levels were significantly different at p < 0.05 between all treatments, with the exception of the difference be-
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tween the 10 Ixg Se/L and 25 Ixg Se/L ponds which was not statistically significant. Two grass carp (the initial number added) were recovered from all of the ponds dosed below 25 txg Se/L. One dead carp was recovered from one of the 25 ixg Se/L ponds (pond 2 on 7 January 1991, Day 427), and the remaining three carp from these ponds were missing at the end of the experiment. The concentrations of selenium found in grass carp muscle at the end of the experiment are shown in Table 5. Concentrations increased ten-fold compared with controls in those carp exposed to 10 p~g Se/L. Only two sticklebacks were recovered from the entire set of ponds at the end of the experiment. Both sticklebacks were recovered dead from pond 2, on 25 February 1991 (Day 476) and 4 March 1991 (Day 483), respectively.
Perch Spawning and Hatching Success and Larval Survival The perch began to spawn on the trays placed in the ponds on 13 April 1991 and continued until 3 May 1991 (Table 6). Egg ropes were found in all of the ponds, with the exception of pond 2 (25 Ixg Se/L). Fertilization success was greater than 90% of eggs in subsamples taken from all of the ropes, except for the rope recovered from pond 3 (2/xg Se/L) on 13 April 1991 (Day 523), in which there was no evidence of fertilization success. Water quality during the perch early life stage test was maintained at 15°C -+ 1.3°C, pH 7.86 + 0.15 and 95-110% ASV. Water hardness was 300 mg/L as CaCO 3. Selenium concentrations were maintained near their nominal values (range of measured values (ixg S e / L ) =
