Selection and qualification of standard test organisms for BWMS type-approval testing

Seasonal and regional variability of natural plankton assemblages can constrain biological validity in BWMS land-based type-approval testing, particularly when challenge-water concentrations fall below regulatory requirements. This study proposes an evidence-based workflow to select and qualify standard test organisms (STOs) for the ≥10 μm to <50 μm and ≥50 μm size classes. Survivor records from full-scale type-approval tests were first used to prioritize taxa that persist through treatment and subsequent handling. Four candidates were finalized: two centric diatoms for the ≥10 μm to <50 μm class (Stephanocyclus meneghinianus and Actinocyclus sp.) and two zooplankton for the ≥50 μm class (Moina macrocopa and Tigriopus sp.). Taxonomic assignments were supported by DNA barcoding, and regulatory compliance was evaluated using minimum-dimension measurements under practical microscopy views. All candidates consistently satisfied the relevant size thresholds. Operational feasibility was assessed through routine maintenance, showing stable culture growth for diatoms (OD750) and maintained or increasing total abundance for zooplankton. Control-holding performance under test-relevant conditions was examined during short-term dark holding across freshwater, brackish, and marine waters over the protocol temperature range (5-35 °C) for up to 5 days. The ≥10 μm to <50 μm candidates remained above the minimum required concentration throughout the holding period, whereas the ≥50 μm candidates were more sensitive under extreme conditions but remained viable under typical holding durations and non-extreme temperatures. Collectively, the finalized candidates provide practical STO options that combine size compliance, routine maintainability, and control-holding validity for reproducible challenge-water preparation in land-based type-approval testing.