Environ. Sci. Technol. 2009, 43, 2092–2098
Exposure to Treated Sewage Effluent Disrupts Reproduction and Development in the Seasonally Breeding Ramshorn Snail (Subclass: Pulmonata, Planorbarius corneus) N E I L C L A R K E , ,† E D W I N J . R O U T L E D G E , † ANTHONY GARNER,† DAIRE CASEY,‡ RACHEL BENSTEAD,‡ DAVID WALKER,§ BURKARD WATERMANN,| K GNASS,| A T H O M S E N , | A N D S U S A N J O B L I N G * ,† Brunel University, Uxbridge, Middlesex, UB8 3PH, Waterlooville Laboratory, National Laboratory Service, Environment Agency, 4 The Meadows, Waterbury Drive, Waterlooville, Hampshire, PO7 7XX, Hanningfield Treatment Works, Essex and Suffolk Water, South Hanningfield, Chelmsford, Essex, CM3 5HS, LimnoMar, Bei der Neuen Muenze 11, D-22145 Hamburg, Germany
Received August 18, 2008. Revised manuscript received December 29, 2008. Accepted January 6, 2009.
Experiments were conducted to assess the impacts of exposure to sewage treatment works (STW) effluent upon the growth, reproductive function, and sexual development of the European mollusc, Planorbarius corneus under seasonally varying temperatures and photoperiodic conditions. In river water, a clear seasonal change in the number and weight of egg masses (during both 2003 and 2004), and in the number of eggs produced, was found, providing evidence for profound effects of both changing temperature and photoperiod on reproduction. Exposure to STW effluent caused disturbances in this seasonal reproductive cycle at all concentrations tested. The effects included significant dose-dependent increases in fecundity and in the overall length of the reproductive cycle in adult snails exposed to both 50% and 100% effluent relative to river water for a period of up to 14 weeks. Disturbances in the development of both the male and female gametes of the both the adult snails and their developmentally exposed offspring were also seen. These effects were more evident in the offspring than in the adults.
Introduction Many freshwater mollusc species have become threatened or extinct over the last 400 years (1). Suggested explanations for this include eutrophication, changed farming practices, and pollution, although none of these have been well described. Discharges from sewage treatment works (STWs) are a major factor influencing the chemical content of fresh waters all over the world. In small densely populated countries * Corresponding author e-mail:
[email protected]. † Brunel University. ‡ Waterlooville Laboratory. § Essex and Suffolk Water. | LimnoMar. 2092
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 6, 2009
such as the UK, STW effluent discharges can often comprise 50% of the flow of some rivers, a figure that can routinely rise to 90% or more in dry conditions. Effluents contain tens of thousands of chemicals in everyday use, including natural hormones produced by animals, synthetic steroids found in contraceptives, pesticides, surfactants, and plasticizers. Although these chemicals are not overtly toxic, it has been hypothesized that in combination, they are capable of producing a variety of adverse effects including cancers, reproductive and fertility disorders, and immune and thyroid dysfunction in vertebrates. In particular, feminized responses, have been reported in both caged and wild fish as a result of exposure to low concentrations of estrogens present in effluents from wastewater treatment works (WWTW). Studies on wild populations of fish in UK rivers have shown that intersex fish (feminized males) are compromised in their reproductive capacity which may, in turn, have populationlevel consequences (2). Little is known about whether similar effects might occur in invertebrates, and there is general disagreement among scientists regarding whether or not their endocrine systems share sufficient similarities with vertebrates to suspect that they would be similarly affected. Only a few studies report the effects of STW effluent on reproduction in freshwater molluscs (3, 4), and no published reports are available that directly associate STW effluents with population declines in molluscs. Some reports suggest that reproduction and development in mollusc species may be influenced by exposure to chemicals also known to influence reproduction in vertebrates (e.g., ref (5)), while others strongly refute these claims (6). One study (5) clearly suggests that there are temperature-driven differences in sensitivity of molluscs to endocrine active chemicals. This may have particular relevance for seasonally breeding species, where reproduction is strongly influenced by temperature. Links between chemical exposure, biological effects and changes in physical environmental factors are not yet well developed in the literature. The aim of this study was to investigate these interactions by exposing the egg laying pulmonate gastropod Planorbarius corneus to sewage effluents in outdoor mesocosms, where temperature and photoperiod change seasonally. We provide evidence that exposure to sewage effluents greatly influences seasonal reproduction and sexual development in this freshwater mollusc.
Materials and Methods Snails. Adult P. corneus were obtained from Blades Biological (Cowden, Edenbridge, Kent, TN8 7DX. England.) in early (experiment 1) and late (experiment 2) spring of 2003 and 2004, respectively. The supplier informed us that the snails were collected from a lowland ditch near Worthing (514704E, 103103N) Sussex, U.K., which has no STWs inputs. Treated Sewage Effluent. The treated sewage effluent was derived from Chelmsford STW, Chelmsford, Essex, U.K. The diluent river water was drawn from the River Blackwater, 12km away from any major inputs of STW effluent. Experimental design. All snails were acclimated to the diluent river water, then weighed and measured prior to deployment into each of four large 685 L capacity exposure tanks where they were acclimated to river water for 2 weeks. They were then exposed to 25, 50, or 100% STWs effluent mixed with river water or to river water alone. Replication was provided by suspending six groups of 11 (experiment 1) or nine (experiment 2) adult P. corneus in enclosures at different positions within the tank to try and mimic different 10.1021/es8020167 CCC: $40.75
2009 American Chemical Society
Published on Web 02/06/2009
positions within a river, by repeating the experiment over two consecutive summers (experiment 1 from April to July 2003 and experiment 2 from May to September 2004) and by performing the sampling every two weeks. Replication applied in this way is a reasonable way to test hypotheses referring to large-scale systems, where replication of the river itself is not possible or not practical (7). Both studies were conducted in an aerated flow through system for periods of 12 (experiment 1) and 14 (experiment 2) weeks. The flow rates of river and/or treated sewage effluent to individual tanks were recorded daily, and adjusted to nominal if necessary. Water temperature was monitored and recorded daily and oxygen levels were checked weekly. Snails were fed organic “little gem” lettuce ad libitum. In experiment 2, the diet was also supplemented with tetramin flake fish food once per week (3.6 g per enclosure). The F1 P. corneus originating from egg masses that had been laid by adults throughout experiment 2 were exposed further to the same effluent for a period of up to 3 months and then allowed to grow on in river water until they were at least 6 months old when they were removed and counted. Mortality. Mortalities were counted every two weeks, and the lengths of dead snail shells were recorded to determine size at time of death. Growth. Thirty adult snails were sampled prior to exposure in each experiment to gain initial measurements of total weight and length (from the far tip of the operculum to the farthest part of the shell), for comparison with the exposure period. These measurements were also taken at the end of week 6 (in experiment 1 only) and at the end of both experiments. Biochemical Parameters. In order to obtain a measure of the energy content of the snails and to ensure that any differences in reproductive output of the snails were not due to differences in the available energy stores between the exposure groups, the protein content and glycogen phosphorylase activity of the mantle tissue in snail samples from the reference (river water) and 100% effluent tanks were measured at both the 6 and 12 week sampling points (experiment 1 only; see Supporting Information (SI) and refs 8, 9). Reproductive Output. Every two weeks, the numbers and weights of all egg masses per group of replicate snails were counted. From week 4 until the end of each experiment, the number of eggs per mass and weight of individual masses were recorded. Where there were more than 40 intact masses in a particular group of snails, the numbers of eggs of only the first 40 intact masses were counted. A small proportion of the egg masses were damaged by the scalpel blade as they were removed. The eggs from these damaged masses were not counted. A small proportion of the egg masses (
