DOI: 10.6084/m9.figshare.28212716.v1
title: Data and R-Code used for Zooplankton Publication

description: Combined sewer systems are common throughout much of the world, allowing the conveyance and of both storm- and waste- water within one system. Many of these systems were built with overflows (termed “CSO”) leading to local waterways so that large rain events do not overwhelm sewer treatment plants nor cause urban flooding. Chicago, IL USA has the addition of pumping stations which, during extreme wet weather events, actively pump combined storm- and waste- water into the Chicago River. In 2023 12.3 billion liters of untreated storm- and waste-water was pumped into the Chicago River, 9.1 B l of which was discharged into Bubbly Creek. Increased conductivity was noted In Bubbly Creek following the CSO event and dissolved oxygen levels remained ≤ 5.0 mg l for several weeks following the event. We show that populations of Chydoridae were lost and Moinidae populations reached high (&gt;100 individuals l<sup>-1</sup>) abundances within Bubbly Creek following the CSO event whereas populations of zooplankton taxa at locations elsewhere in the system remained relatively unchanged. We posit that nutrients provided by the sewage fueled a phytoplankton bloom, while low oxygen levels remove predators allowing densities of zooplankton (primarily Moinidae) to exceed 100 l<sup>-1</sup> in Bubbly Creek. We offer some of the first <i>in-situ</i> evidence that releases of untreated combined storm- and waste-water (i.e., CSOs) alter zooplankton communities of the receiving waterbody.Between June 25 and July 15 2023 27.7 cm rain fell, including ~10 cm within 24 hrs on July 2 2023 (as recorded at Midway Airport), overwhelming much of the Chicago’s storm and sewer system. To alleviate pressure in the sewage system and avoid flooding communities and homes, 3.4 billion liters of untreated waste and storm water was pumped into Bubbly Creek (aka, South Fork of the South Branch of the Chicago River) over the course of 67 hours between July 2nd and 5th and 10 CSO outfalls activated. At the same time, 2.2 bn l were pumped into the North Branch over 28.4 hrs and 21 CSO outfalls activated. A second event occurred July 12-15 2023 where 2.5 bn l was pumped into Bubbly Creek via the Racine Avenue pumping station, no CSO outfalls registered as being active during this time. MWRD maintains 7 continuous dissolved oxygen monitoring stations (Eureka Manta2™ or Manta+™ Probes of Austin, Texas) within the Chicago River and North Shore Channel which are situated 1 m below the water surface and probes rotated monthly (Minarik et al. 2024). North Branch main channel locations (Station names: Church Street, Addison Avenue, Division Avenue) exhibited low oxygen (&lt; 5.0 mg l-1) for a period of ≤ 12 hours following the early July 2023 CSO event. The station within the South Branch of the Chicago River (Station name: Loomis Avenue), exhibited low oxygen for 28 hrs July 3-4 and 21 hrs July 6-7. Downstream of our locations included in this study (Station name: Cicero Avenue), low oxygen occurred for 6 consecutive days starting July 3rd 2pm and for nearly 88% of the hourly recordings between July 13- 23rd remained &lt; 5.0 mg l-1. Over the course of 2023, ≤ 6 % of recordings of at this downstream location exhibited oxygen levels &lt; 5.0 mg l-1, whereas ≤ 3 % of recordings during 2023 at others mentioned so far were &lt; 5.0 mg l-1. Within Bubbly Creek, anoxic and hypoxic conditions existed from July 2nd until July 21st, and hypoxia occurred for 76.5% of hourly recordings between July 22nd and Aug 2nd, and then 56.4% of recordings through Aug 12th (Station names: I-55 and 36th Street). Over the course of 2023, 20 and 24 % of recordings of the two Bubbly Creek monitoring stations exhibited oxygen levels &lt; 5.0 mg l-1. For comparison, in 2022 no CSO events occurred within Bubbly Creek and ≤ 5 % of recordings across the year exhibited oxygen levels &lt; 5.0 mg l-1 (Minarik et al. 2023).Seven locations were chosen to represent the North and South Branches of the Chicago River (Fig 1.). These included three locations on the North Shore Channel and North Branch (referred to as “North”), two locations in the South Branch main channel, and two locations to represent the main treatment location, Bubbly Creek. All sites were accessible via a pier or wading, allowing sampling of water from a depth 0.6 m.All locations were sampled mid-day, between 09:30 and 15:00, typically in order from North to South, with some variation in the order of the Southern locations which is not thought to influence results. A 5 l horizontal Niskin Bottle (General Oceanics) and a 19 l bucket were used to collect 20 l of sub-surface (top 0.6 m) water which was filtered through a 53 micron mesh sieve. Collected organisms were then washed into 20 ml scintillation vials and preserved onsite with 90 % Ethanol for later enumeration. A handheld YSI meter was used to collect data on temperature, conductivity, and dissolved oxygen during each sampling event. Samples were taken as close to the center of the waterway as possible. Sites were visited approximately weekly between June 8<sup>th</sup> and September 12<sup>th</sup> 2023, for a total of 15 sample dates. Due to visible clouds of zooplankton on July 27<sup>th</sup> in Bubbly Creek, 2 20 l samples were taken via bucket. We sampled using both the Niskin bottle and bucket concomitantly on August 1<sup>st</sup>, 8<sup>th</sup>, and 15<sup>th</sup> to facilitate analysis of any differences in the communities or abundances they captured.Zooplankton were enumerated under a dissection microscope using a gridded square petri dish. Samples with large numbers of zooplankton were diluted to a known volume (i.e., 300 ml) and at minimum 4 subsamples were taken using a 10 ml Hensen-Stempel sampler following agitation. Subsample counts were adjusted to reflect the full 20 l sample volume, and averaged to obtain 1 value for that date, location, and sampling method combination. During lab work, the Bubbly Creek sample from August 22<sup>nd</sup> was lost after one 10 ml subsample was counted (beaker broke), we maintained this sample in the analysis despite lower confidence in community composition (78 individuals counted compared to mean of 124, median of 54, and standard deviation of 176 across 146 samples). Zooplankton were classified into the following groups: Bosminidae, Calanoida, Chironomid larvae, Cyclopoida, Chydoridae, other Daphniidae (including <i>Scapholeberis</i> spp., <i>Daphnia lumholtzi</i>, among others), Diaphanosoma (Sididae), Moinidae, Naididae, Ostracoda, <i>Polyphemus pediculus </i>(Polyphemidae), and the presence of nauplii and rotifers were noted but not counted.<br>