Sewer overflows are an environmental concern due to their potential to introduce contaminants that can adversely affect downstream aquatic ecosystems. As these overflows can occur during rainfall events, the influence of rainwater ingress from inflow and infiltration on raw untreated wastewater (influent) within the sewer is a critical factor influencing the dilution and toxicity of the contaminants. The Vineyard sewer carrier in the greater city of Sydney, Australia, was selected for an ecotoxicological investigation of a sanitary (separate from stormwater) sewerage system and a wet-weather overflow (WWO). Three influent samples were collected representing dry-weather (DW), intermediate wet-weather (IWW) and wet-weather (WW). In addition, a receiving water sample was also collected downstream in Vineyard Creek (WW-DS) coinciding with a WWO.We employed direct toxicity assessment (DTA) and toxicity identification evaluation (TIE) approaches to gain comprehensive insights into the nature and magnitude of the impact on influent from rainwater ingress into the sewer. Three standard ecotoxicological model species, a microalga, Chlorella vulgaris, the water flea, Ceriodaphnia dubia and the midge larva, Chironomus tepperi were used for both acute and chronic tests. The study revealed variable toxicity responses, with the sample of influent collected in wet-weather displaying lower toxicity compared to the dry-weather sample of influent. Ammonia, and metals, were identified in dry weather as contributors to the observed toxicity, however, this risk was alleviated through rainwater ingress in wet-weather with further dilution within the receiving water. Based on toxicity data, dilutions of influent to minimise effects on C. vulgaris and C. dubia ranged from 1 in 12 in DW to 1 in 2.8 in WW, and further diminished in the receiving water to 1 in 1.8. The successful application of ecotoxicological approaches enabled the assessment of cumulative effects of contaminants in influent, offering valuable insights into the sanitary sewer system under rainwater ingress.
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