Microbial, Physical, and Chemical Changes in Galveston Bay Following an Extreme Flooding Event, Hurricane Harvey

Hurricane Harvey (category four storm) made landfall along the coast of Texas (United States) and then stalled out over Texas and Louisiana, releasing 1.29 × 1011 m3 of precipitation over 5 days. This caused extensive flooding that elevated freshwater river discharge and land runoff into Galveston Bay and eventually into the Gulf of Mexico. The floodwaters delivered a significant influx of dissolved organic carbon (DOC), organic pollutants and nutrients along with terrestrial and freshwater associated microbes. Over the 24 days following the flooding event, samples were collected on five cruises across Galveston Bay from the mouth of the San Jacinto River (Houston, TX, United States) to the Gulf of Mexico. Parameters measured for this study include: water quality (temperature, salinity, pH, and dissolved oxygen), nutrients (NO3–, NO2–, NH4+, Pi, and HSiO3–), polycyclic aromatic hydrocarbons (PAHs), pharmaceuticals (cotinine, carbamazepine, carbamazepine-epoxide, and prednisone), biocide (imidacloprid), DOC, bacteria, and the eukaryotic community composition (16S and 18S rRNA genes). In the week after the flood event, bay-wide salinities decreased to 0–5 compared to the higher pre-Harvey salinities of 20–30 (recorded 5 days before the flood). Water treatment facilities and petrochemical plants were compromised due to the heavy flooding in the region. Increased concentrations of DOC, nutrients, PAHs, pharmaceuticals, and biocides were observed across Galveston Bay immediately following the storm. During the 4 weeks after Harvey, concentrations of DOC, nutrients, and organic pollutants began to decrease coinciding with rising salinities as the freshwater was flushed into the Gulf of Mexico and seawater began moving back into the Bay. Successive blooms of Chlorophyta, diatoms (Bacillariophyta), and dinoflagellates (Dinophyta) occurred similar to post-storm communities from past hurricanes that have impacted estuarine systems along the Gulf of Mexico. The bacterial community showed an increase in the abundance of bacteria associated with terrestrial soils and freshwater at the initial time point and then decreased over time and were replaced by their estuarine and marine relatives within the month following the hurricane. The eukaryotic community changed substantially following Harvey and did not recover to pre-Harvey conditions during our study period, suggesting a longer recovery time compared to the prokaryotes. Although the water quality parameters and prokaryotic community showed signs of returning to pre-Harvey conditions within the month following the flood event, long-term impacts need to be measured in the years following the flood.

Hurricane Harvey (category four storm) made landfall along the coast of Texas (United States) and then stalled out over Texas and Louisiana, releasing 1.29 × 10 11 m 3 of precipitation over 5 days. This caused extensive flooding that elevated freshwater river discharge and land runoff into Galveston Bay and eventually into the Gulf of Mexico. The floodwaters delivered a significant influx of dissolved organic carbon (DOC), organic pollutants and nutrients along with terrestrial and freshwater associated microbes. Over the 24 days following the flooding event, samples were collected on five cruises across Galveston Bay from the mouth of the San Jacinto River (Houston, TX, United States) to the Gulf of Mexico. Parameters measured for this study include: water quality (temperature, salinity, pH, and dissolved oxygen), nutrients (NO 3 − , NO 2 − , NH 4 + , P i , and HSiO 3 − ), polycyclic aromatic hydrocarbons (PAHs), pharmaceuticals (cotinine, carbamazepine, carbamazepine-epoxide, and prednisone), biocide (imidacloprid), DOC, bacteria, and the eukaryotic community composition (16S and 18S rRNA genes). In the week after the flood event, bay-wide salinities decreased to 0-5 compared to the higher pre-Harvey salinities of 20-30 (recorded 5 days before the flood). Water treatment facilities and petrochemical plants were compromised due to the heavy flooding in the region. Increased concentrations of DOC, nutrients, PAHs, pharmaceuticals, and biocides were observed across Galveston Bay immediately following the storm. During the 4 weeks after Harvey, concentrations of DOC, nutrients, and organic pollutants began to decrease coinciding with rising salinities as the freshwater was flushed into the Gulf of Mexico and seawater began moving back into the Bay. Successive blooms of Chlorophyta, diatoms (Bacillariophyta), and dinoflagellates (Dinophyta) occurred similar to post-storm communities from past hurricanes that have impacted estuarine systems along the Gulf of Mexico. The bacterial community showed an increase in the abundance of bacteria associated with terrestrial soils and freshwater at the initial time point and then decreased over time and were replaced by their estuarine and marine relatives within the month following the hurricane. The eukaryotic community changed INTRODUCTION Houston, Texas (United States) is located within the watershed of Galveston Bay, which is the second largest estuary system in the northern Gulf of Mexico. The volume of freshwater delivered to Galveston Bay during Hurricane Harvey (1.40-1.70 × 10 10 m 3 ) was similar in magnitude to the volume of freshwater inflow that is typically delivered to the Bay over the course of a year (1.86 × 10 10 m 3 ) [Du et al., 2019;Texas Water Development Board, 2019;Thyng et al., 2020 (in review)]. During the passing of Hurricane Harvey from 25-30 August 2017, more than 1.29 × 10 11 m 3 of rain fell across Texas and Louisiana (United States). It is estimated that during the flooding event, locations in Houston, TX, sank into the Earth ∼2 cm where rainfall totals reached up to 1.54 m (van Oldenborgh et al., 2017;Blake and Zelinsky, 2018;Milliner et al., 2018).
The Trinity and San Jacinto Rivers deliver >70% of the freshwater inflow into Galveston Bay (Guthrie et al., 2012). Highly contaminated Superfund sites which were compromised from the deluge that Hurricane Harvey produced (Chapin, 2017) are located within the lower watershed of the San Jacinto River. Spills from compromised wastewater treatment systems, industrial facilities, oil refineries, and chemical plants released > 1.17 × 10 5 m 3 of raw sewage and toxic chemicals as well as 4.53 × 10 5 kg of deadly air pollutants (Environment Texas Research and Policy Center, 2017;van Oldenborgh et al., 2017;Wolf, 2017;Kiaghadi and Rifai, 2019). Pollutants released by Texas facilities include benzene, 1,3-butadiene, hexane, hydrogen sulfide, toluene, and xylene (all known carcinogens except hexane) and >300,000 kg of sulfur dioxide, which can lead to the formation of dangerous contaminants (Wolf, 2017). Non-point sources of polycyclic aromatic hydrocarbons (PAHs) could have also included those previously found in the sealant of parking lots (Van Metre et al., 2006) and in sediments disturbed by floodwaters (Horney et al., 2018). Due to the increased industrial pollution in the region, Galveston Bay sediments and biota are traditionally key focus areas for environmental monitoring of petrogenic PAHs, and legacy pollutants such as polychlorinated biphenyls (PCBs), dioxins, and metals (Roach et al., 1993;Willett et al., 1997;Qian et al., 2001;Suarez et al., 2006;Howell et al., 2008;Lakshmanan et al., 2010;Yin et al., 2015). While attention is typically focused on monitoring such pollutants, recent concerns have grown for the environmental presence of new or "emerging" pollutants (Ankley et al., 2007;Boxall et al., 2012), such as pharmaceuticals and/or biocides (PBs). Although the occurrence and bioaccumulation potential of select PBs has been reported in surface waters and fish inhabiting river systems (Du et al., 2016), to our knowledge studies focused on the presence of PBs in the Galveston Bay watershed have not been conducted previously.
Previous studies on the water quality within Galveston Bay have been conducted which have provided a baseline of the pre-Harvey conditions (Guo et al., 1999;Örnólfsdóttir et al., 2004;Roelke et al., 2013;Dorado et al., 2015). Freshwater inflow pulses which deliver nutrients and organic matter to Galveston Bay vary seasonally, producing a dynamic and complex system (Roelke et al., 2013;Dorado et al., 2015;Pinckney et al., 2017). The microbial community responds to variations in the water quality by displaying seasonal changes in the community structure within this highly productive estuary (Lester and Gonzalez, 2011;Roelke et al., 2013;Dorado et al., 2015). Depending on the volume of freshwater delivered to Galveston Bay, phytoplankton biomass has actually been shown to decrease following these pulses due to hydraulic displacement (Roelke et al., 2013). The response of the phytoplankton community has also been shown to be dependent on the level of nutrient limitation present before the freshwater pulse occurs (Roelke et al., 2013;Dorado et al., 2015). Guo et al. (1999) observed that dissolved organic carbon (DOC) in Galveston Bay decreased non-conservatively with increasing salinity indicating a removal of the DOC with increased mixing between freshwater and seawater. At low to intermediate salinities input of DOC was observed that was attributed to primary productivity and resuspension of DOC from bottom sediments.
Hurricane Harvey caused extensive flooding of rivers and produced land runoff to the point that freshwater draining into Galveston Bay via the Houston Ship Channel pushed the leading edge of the saltwater wedge out toward the Gulf of Mexico [Du et al., 2019;Thyng et al., 2020 (in review)]. Flooding of this volume has not been recorded previously in Galveston Bay leading to questions regarding the response of the biological community. This paper summarizes the water quality parameters, PBs, DOC, PAHs, and changes in the microbial community (16S rRNA and 18S rRNA analyses) in Galveston Bay immediately following Hurricane Harvey. Collectively, these findings show a transition from an estuarine microbial community preceding the hurricane to a primarily freshwater microbial community after the flooding event, which then began the shift back to an estuarine/marine biotic community. Within the month after the passing of the hurricane, the prokaryotes showed signs of returning to a pre-flooding community while the eukaryotic community still appeared to be in transition. The impacts of the increased contaminants and the extensive period of low salinity should be monitored in years to come to determine the long-term effects on the biological community including the fisheries. This collaborative effort will allow for improved understanding of the physicochemical and biological changes following a large storm event within subtropical estuaries.

Study Area and Sampling Design
The San Jacinto and Trinity Rivers are the two major sources of freshwater inflows to Galveston Bay (Figure 1), delivering 16 and 55% of the annual riverine discharge to the northwest and northeastern reaches of the Bay, respectively (Guthrie et al., 2012). Buffalo Bayou, adjacent to the San Jacinto River, brings in an additional 12% while smaller tributaries around the Bay account for the rest of the inputs (Guthrie et al., 2012; Figure 1). Five sampling cruises T1 (09/04/2017), T2 (09/09/2017), T3 (09/16/2017), T4 (09/21/2017), and T5 (09/28/2017) were conducted during the month following Hurricane Harvey onboard the R/V Trident. Ten stations (1-10) were sampled to capture the conditions immediately following Hurricane Harvey on a transect from the San Jacinto River mouth (station 1) near the Port of Houston to the mouth of Galveston Bay near the Gulf of Mexico (station 10) (Figure 1).

Water Quality Sampling
While the average water depth in the bay is ∼2.4 m, sampling close to the ship channel allowed us to evaluate a deeper profile. A calibrated Hydrolab MS5 datasonde was used to collect water quality parameters from surface down to 4 m at each station including temperature ( • C), salinity, dissolved oxygen (mg L −1 and %), and pH. Secchi depth (m) was collected simultaneously. Unfiltered surface water samples (≥100 mL) were collected for total nitrogen (TN) and total phosphorus (TP). Surface water (≥100 mL) was also vacuumfiltered through a pre-rinsed, 47 mm glass fiber filter (GF/F), and the filtrate was used to measure the dissolved nutrients including: nitrate (NO 3 − ), nitrite (NO 2 − ), ammonium (NH 4 + ), phosphate (P i ), and silicate (HSiO 3 − ). Filtrate was placed in acid-washed (10% HCl) 250-mL polyethylene Nalgene bottles rinsed three times with filtrate from the sample and then frozen until analysis. All water samples collected for total and dissolved nutrient analysis were shipped to the Geochemical and Environmental Research Group at Texas A&M University for analysis on the Lachat QuikChem AE autoanalyzer (American Public Health Association [APHA], 1985). The sum of NO 3 − , NO 2 − , and NH 4 + was used to calculate dissolved inorganic nitrogen (DIN). The resulting DIN value was then divided by P i to calculate the ratio of inorganic nitrogen to phosphate (DIN:P i ). Samples for DOC were filtered through 0.2 µm Whatman-Nucleopore Q-TEC filters (Filtration Solutions) on board. Concentrations of DOC were determined using a high temperature-combustion method and a Shimadzu TOC-V analyzer (Benner and Strom, 1993). Total suspended sediments (TSS) were collected from the surface water samples during all time points at all stations; 150 mL of surface water was filtered through a pre-weighed and combusted 47 mm GF/F. After filtration the sample was placed in a drying oven at

16S rRNA and 18S rRNA Gene Community Analyses
For the 16S and 18S ribosomal RNA (rRNA) gene analysis samples were collected at every station during each time point. The water samples were filtered on 0.2 µm polyethylsulfone (PES) membrane filters immediately after returning to shore. Two pre-Harvey samples (named pre-Harvey controls) were collected off the Texas A&M University at Galveston Boat Basin (coordinates) on 07/31/2017 and 08/22/2017. Each sample was filtered through a GF/F (with a 0.7 µm poresize), followed by a 0.22 µm pore-size polyvinylidene fluoride (PVDF) filter. Filters were stored in -80 • C freezer until total nucleic acid was extracted, using the MO Bio PowerSoil DNA Isolation Kit (cat. no. 128888-50). DNA could not be extracted from the pre-Harvey controls GF/F filters, therefore all further PCR amplifications were performed from the PVDF DNA extractions. PCR amplification using Promega GoTaq Flexi DNA Polymerase was performed following the 16S/18S rRNA gene Illumina amplicon protocol from the Earth Microbiome project. 1 Each sample was amplified in triplicate 25 µL reactions with the following cycling parameters: 95 • C for 3 min, 30 cycles of 95 • C for 45 s, 50 • C for 60 s, and 72 • C for 90 s, and a final elongation step at 72 • C for 10 min. For the prokaryotes, amplifications were performed using the 515F-806R primer pair (10 µM each) modified to include recently published revisions that reduce bias against the Crenarchaeota and Thaumarchaeota lineages as well as the SAR11 bacterial clade (Apprill et al., 2015;Parada et al., 2016). The primer pair was additionally modified to include Golay barcodes and adapters for Illumina MiSeq sequencing. Final primer sequences are detailed in Walters et al. (2016). The primer pair utilized for the Eukaryote analysis was V8f-1510r (Bradley et al., 2016). For eukaryotes the V8V9 hypervariable region on the 18S rRNA and for prokaryotes the V4 hypervariable region on the 16S rRNA were used for amplification (Caporaso et al., 2011;Bradley et al., 2016). Following amplification, the triplicate products were combined together and run on a 1.5% agarose gel to assess amplification success and relative band intensity. Amplicons were then quantified with the QuantiFluor dsDNA System (Promega), pooled at equimolar concentrations, and purified with an UltraClean PCR Clean-Up Kit (MoBio Laboratories; Carlsbad, CA, United States). The purified library, along with aliquots of the three sequencing primers, were sent to the Georgia Genomics Facility (Athens, GA, United States) for MiSeq sequencing (v2 chemistry, 2 × 250 bp). Sequence reads for both the 16S rRNA and 18S rRNA were processed separately using mothur v.1.39.5 following the MiSeq SOP https://www.mothur.org/wiki/MiSeq_SOP (Schloss et al., 2009;Kozich et al., 2013).
Samples (1 L) for PBs analysis were collected at each station during every time-point. Collectively, these can be used as proxies for human activities and in the case of this study, for excess flows, over flows, and other flood impacts on waste water treatment facilities that would release them into the environment. PBs were quantified using an Agilent 1260 UHPLC system with triple-quad 6420 mass detector using an Agilent jet-stream electrospray source. PBs from Galveston Bay water samples (500 mL) were spiked with an internal standard (deuterated carbamazepine or d10-carbamazepine) and extracted using Oasis HLB solid phase cartridges (500 mg, Waters Cat#186000115). The mass spectrometer settings included a capillary voltage of 3.5 kV and nebulizer gas temperature of 350 • C and gas flow of 12 L/min. All PBs were quantified using multiple-reaction-monitoring (using previously optimized precursor > product ions) in positive electrospray ionization (ESI +) mode. These included: 177.1 > 80.2 (cotinine), 237.1 > 194 (carbamazepine), 253.1 > 180 (carbamazepine epoxide), 359.2 > 341.1 (prednisone), and 256.1 > 175 (imidacloprid). Deuterated carbamazepine (d10-carbamazepine, 247.2 > 204.1) was used as internal standard and all compounds were chromatographically separated on an Agilent poroshell EC-C18 column (3 × 50 mm, 2.7 µm). The mobile phase comprised of LC-MS grade Milli-Q water and methanol, with each containing 5 mM ammonium formate. The mobile phase gradient transitioned from 30%, increasing linearly to 70% in 3 min, and then to 95% over 6 min. The gradient was subsequently decreased from 95 to 70% over 3 min and from 70 to 30% (initial condition) in 3 min with a constant flow rate of 0.4 mL/min. The total chromatographic runtime was 25 min.

Polycyclic Aromatic Hydrocarbons (PAHs)
The 16 PAHs included in this study are naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene, dibenzo(a,h)anthracene, and benzo(ghi)perylene. Water samples were collected at each station during each time point throughout the sample period. The PAHs from the water samples were extracted using liquid-liquid extraction based on Wade et al. (2011) with few modifications. Briefly, the water sample was placed in a 2-L separatory funnel, acidified by sulfuric acid, and extracted three times with 30 mL of dichloromethane. Prior to extraction, a mixture of deuterated standards containing naphthalene-d 8 , phenanthrene-d 10 , and perylene-d 12 was added (Kamalanathan et al., 2019). The extract was filtered through a chromatography column containing anhydrous sodium sulfate and concentrated by rotary evaporator. The samples were then analyzed by gas chromatography (HP-6890 Series) interfaced with an Agilent 5973 inert mass selective detector (MSD), and operated in a selective ion-monitoring (SIM) mode (Bacosa et al., 2018). The PAHs were quantified using a calibration curve of PAHs standards. The final concentration was corrected based on the recovery of the standards.

Multivariate Statistical Analysis
Statistical analyses were conducted using PRIMER v6 + PERMANOVA add-on package software (Plymouth Routines in Multivariate Ecological Research; Clarke and Warwick, 1994). Environmental variables including water quality parameters, nutrients, DOC, PAHs, and PBs were square root transformed, normalized to place all parameters on the same unit less scale, and then used to build a similarity matrix based on the calculated Euclidean distances. The 16S rRNA gene and 18S rRNA gene community analyses were conducted on the operational taxonomic unit (OTU) abundance data which were square root transformed and then used to calculate a Bray-Curtis similarity index matrix. This index best represents the similarities (or dissimilarities) within the biotic community due to the fact these data are not normally distributed and have unequal variance distribution characteristics (Clarke and Gorley, 2006).
The relationship between the distributions of the biological and environmental variables was investigated using the distancebased linear model (DISTLM) in the PRIMER v6 software package (Clarke and Warwick, 2001). The DISTLM calculates the correlations between each of the environmental variables and the biological dataset. The DISTLM was run on the Bray-Curtis similarity matrix of the 16S rRNA and 18S rRNA OTU abundances. The DISTLM routine was carried out to provide a method of matching the microbial community from the 16S rRNA and 18S rRNA results with the environmental variables. A permutational analysis of variance (PERMANOVA) was run to determine significant differences between time points and stations for both the environmental and biological data. PERMANOVA was run on the Euclidean distance matrix of the transformed and normalized environmental data and the Bray-Curtis similarity matrix built from the square root transformed biological data. The biological data points were displayed in an nMDS to visualize the distribution of the biotic data in ordination space. The environmental vectors were overlain on the nMDS to show the Pearson correlations in relation to the biotic community changes temporally and spatially.

Water Quality
The elevated precipitation and freshwater inflows entering the Bay from the San Jacinto and Trinity Rivers resulted in decreased salinity and increased sediment load (Figures 1, 2, Supplementary Figures S1, S2, and Table 1). During the T1 cruise (1 week after Hurricane Harvey passed over the Houston metro region) salinities across the Bay were low (0.02-6.57 at surface and 0.04-13.90 at 4 m depth). Near the mouth of the San Jacinto River (station 1) salinities were 0.02 at the surface and 0.36 at 4 m depth (Figure 2). Four weeks after hurricane passed the salinities began to increase ranging from 7.82 at the surface closest to the San Jacinto River up to 21.14 (4 m depth) at the Gulf of Mexico location (station 10) (Figure 2). Temperatures were lowest during the T2 sampling and then increased over time through T5 ( Figure 3A and Table 1). The pH was lowest during the T1 sampling event and then increased during T2 and T3 and began to plateau during T4 and T5 ( Figure 3B and Table 1).
Dissolved organic carbon concentrations were highest (308-566 µM) during the first cruise at stations 1-8 ( Figures 3C, 4 and Table 1). As the salinity began to increase during the last three cruises, DOC concentrations declined and ranged from 267 to 401 µM across the bay (Figures 2, 4). The DOC displays an inverse relationship with Secchi depth with increasing time after the flooding event (Figures 3C, I and Table 1). Highest TSS concentrations were recorded during T1, T2, and T4 ( Figure 3F and Table 1). The highest TSS concentration was observed during the T4 cruise at 122 mg L −1 at station 10 while the lowest value (20 mg L −1 ) was observed during the T1 cruise ( Figure 3F and Table 1).
NO 3 − + NO 2 − (NO x ) concentrations increased with time at the stations nearest the river mouth with highest concentrations observed during the last time point (Figure 3G and Table 1). NH 4 + was observed in the highest concentrations at stations 1-8 during the first cruise (T1) ranging from 4.77 to 14.16 µM ( Figure 3H and Table 1). The concentrations of NH 4 + remained elevated at the stations 1 and 2 nearest the San Jacinto River mouth during the first three cruises (T1-T3) ranging from 14.17 to 14.97 µM ( Figure 3H and Table 1). Over time and with increasing distance from the mouth of the river, the NH 4 + concentrations decreased to concentrations < 6 µM ( Figure 3H and Table 1). The TN ranged from 23.26 to 114.98 µM with lowest concentrations occurring near the Gulf of Mexico and higher concentrations present near the river mouth during T3 and T4 ( Figure 3I and Table 1). The orthophosphate (P i ) concentrations were lowest immediately following the flooding event at all stations ranging from 1.11 to 3.74 µM ( Figure 3J and Table 1). The highest concentrations of P i were observed at the northernmost stations during T2-T4 and also at mid bay stations during the latest time point (Figure 3J and Table 1). TP exhibited two peaks along the cruise transect at stations 1 and 2 and stations 8 and 9 during the latter time points (T3-T5; Figure 3K and Table 1). The DIN:P i was below 16 during the entire cruise period indicative of nitrogen limitation across the bay system ( Table 1). The highest concentrations of silicate were observed in the samples collected during T2 ranging from 68.01 µM at the northernmost site (station 1) to 49.86 µM at the furthest point from the river mouth (station 10; Table 1). At the last time point, the HSiO 3 − remained high at station 1 (63.37 µM) but decreased to 30.69 µM at station 10 nearest the Gulf of Mexico ( Figure 3L and Table 1).

16S and 18S rRNA Gene Community Analysis
Prokaryotic community analysis (16S rRNA) revealed the generally dominant members of the marine bacteria coastal community pre-Harvey (07/31/2017 and 08/22/2017) including Acidimicrobiia, Cyanobacteria, Betaproteobacteria, SAR11, Gammaproteobacteria, and Oceanospirillales (Figures 5, 6). These estuarine/marine bacteria were replaced by microorganisms of terrestrial origin including bacteria within the phyla Actinobacteria and Verrucomicrobia (class Opitutae), sedimentary classes with the phylum Actinobacteria, and freshwater origin phyla Betaproteobacteria, Burkholderiales, and Sphingobacteria (associated with treated wastewater) following Harvey (Figures 5, 6 and Supplementary Figure  S4). Spartobacteria began to increase in relative abundance during time points T3 and T5 (Figure 5). Marine Cyanobacteria, dominated by Synechococcus sp., normally represent ∼20-30% of the total abundance of all OTUs on average in Galveston Bay. After Hurricane Harvey, <5% of all OTUs were Cyanobacteria and then increased back to normal levels within 6 weeks following Harvey. Betaproteobacteria followed an opposite trend and decreased in abundance from early to late time-points over time (Figures 5, 6 and Supplementary Figure S4). Although the relative abundance of the phyla Proteobacteria did not change greatly over the sample period, the classes within this group of bacteria did fluctuate with the variability in salinity and other environmental parameters (Figure 6). The Alphaproteobacteria, Deltaproteobacteria, and Gammaproteobaceteria increased in OTU relative abundance with increasing salinities post-Harvey (Figure 6). The Betaproteobacteria groups made up a majority of the relative abundance immediately following the flooding event   and then decreased with time as the salinities increased across the Bay (Figure 6).
The eukaryotic community (18S rRNA gene) analysis shows that the dominant members of the community were Cryptophyta, Oligotrichea (ciliates), and Ochrophyta (mostly comprised of species within Bacillariophyta hereafter referred to as diatoms). Immediately following Hurricane Harvey (T1), the relative abundance of both Cryptophyta, Oligotrichea, and diatoms reflected that of Pre-Harvey conditions (Figure 7). At the next time point (T2), the highest relative abundance of freshwater chlorophytes were observed at transect stations closer to the San Jacinto River mouth (Figure 7). The relative abundance of diatoms dominated the community at all stations during T2 and even more so during T3 cruise (Figure 7). During the T3 cruise, the chlorophytes were replaced by Ochrophyta (predominantly diatoms) and Myzozoa (mostly Dinophyceae). In contrast, the relative abundance of Dinophyceae (dinoflagellates) was lower during these two cruises in and was highest at stations closest to the Gulf of Mexico during T4 and T5 cruises (Figure 7).
Water sampling in Galveston Bay during all cruises (T1-T5) showed the presence of PBs in the weeks following Hurricane Harvey varying on both temporal and spatial scales ( Figure 8A, Table 2, and Supplementary Figure S5). Temporally the concentrations of cotinine, carbamazepine, carbamazepineepoxide, and imidacloprid, behaved similarly to each other, while prednisone did not ( Figure 8A and Table 2). Levels for cotinine (0.005-0.013 ng L −1 ), imidacloprid (0.026-0.074 ng L −1 ), carbamazepine (0.017-0.036 ng L −1 ), and carbamazepineepoxide (0.004-0.011 ng L −1 ) were all lower immediately following Hurricane Harvey during T1, and then steadily increased over successive weeks ( Figure 8A and Table 2). However, prednisone levels were highest immediately following the storm (21.07 ng L −1 ) at station 1 and then decreased with increasing distance from the mouth of the San Jacinto River to station 10 (7.29 ng L −1 ). After T1, prednisone was only detected at station 4 during T2 (0.154 ng L −1 ) and then during T3 at stations 1, 2, 4, and 6 in low concentrations (0.291-1.23 ng L −1 ; Figure 8A and Table 2). PAHs in the water column were relatively higher during T1 at stations 1, 2, and 6 and then declined with increasing distance from the mouth of the San Jacinto River (Figure 8B). At stations 1, 2, and 6, the concentration of PAHs was 102, 167, and 103 ng L −1 , respectively. The concentrations of PAHs decreased to 24 ng L −1 at station 10 closest to the Gulf of Mexico. For the time points T2-T5, the concentrations of PAHs ranged from a minimum of 19 ng L −1 at station 7 during the T4 cruise to a maximum of 69 ng L −1 at station 2 during the T2 cruise. Overall, PAHs levels declined with increasing time after the hurricane passed.

Multivariate Analysis
The results of the DISTLM analysis showed salinity, pH, DIN:P i , secchi, temperature, dissolved oxygen, HSiO 3 − , and DIN were all strongly significantly correlated (p < 0.001) with the changes in the biotic community ( Table 3). The P i and TN were also significantly correlated (p < 0.05) with the changes in the biotic community ( Table 3). The nMDS shows the distribution of the biotic community measured at each time point and station with a stress level of 0.10 which corresponds to an acceptable ordination that represents the distribution of data points accurately (Figure 9). Increased salinity, pH, dissolved oxygen, and temperature were all strongly correlated (ρ > 0.70) with biotic community that was present during the later time points (T4 and T5) and stations that were closest to the Gulf of Mexico (stations 6-10; Figure 9). Increased nutrients (DIN:P i and HSiO 3 − ) were correlated (ρ > 0.4) with the biotic community that was present during the early time points (T1, T2 and select stations during T3) and stations located closer to the mouth of the San Jacinto river stations 1-5; Figure 9). The results of the PERMANOVA confirm that there was a significant difference in the biological community when comparing T1 to T2 (p < 0.05) and T3-T5 (p < 0.001; Table 4 and Supplementary  Table S1). The biotic variables during T2 were also significantly different from T3-T4 (p < 0.01) and T5 (p < 0.001; Table 4). T3 biological variability was significantly different from T4-T5 (p < 0.001) and T4 was significantly different from T5 (p < 0.01; Table 4). Regarding the environmental variability across time points, T1 and T2 were significantly different from each other (p < 0.001) and were also significantly different from all other time points (p < 0.001; Table 4). T3, T4, and T5 were also significantly different from each other in terms of environmental variability (p < 0.01; Table 4). When considering biological community variability between stations looking at all the time points combined stations 2-4 were all significantly different from station 10 (p < 0.05) and station 5 was significantly different from stations 8 and 9 (p < 0.05; Table 4). While the environmental variability between station 1 and station 2 was not different they were both significantly different from stations 3-8 (p < 0.05) and from stations 9-10 (p < 0.01; Table 4).

DISCUSSION
The total volume of freshwater that entered Galveston Bay by rainfall and runoff during Hurricane Harvey was approximately three to six times the bay volume [Du et al., 2019;Thyng et al., 2020 (in review)]. Historically salinities at the same locations and time of year would range from 20 to 30 (Roelke et al., 2013;Dorado et al., 2015;Pinckney et al., 2017) which is similar to what was observed in during the pre-Harvey sample collections. This flooding event pushed the saltwater out of the bay via hydraulic flushing creating freshwater conditions for over 2 weeks. Although the salinities did begin to increase in the weeks following the flood event, pre-flood salinities were not reached during this study period reflective of the large displacement of bay waters associated with this storm (Figure 2 and Supplementary  Figures S1, S2). This large pulse of freshwater via river discharge, runoff, and direct precipitation delivered 105 ± 10 Gg  of terrigenous DOC to the bay within one week following the hurricane (Yan et al., this issue). With this initial pulse of freshwater, elevated concentrations of ammonium were measured in the bay from and were believed to be from a variety of sources associated with Harvey including agricultural runoff, waste water, and storm water. The  ammonium concentration was elevated during the first two cruises and then declined as it was potentially taken up by the bacterioplankton and phytoplankton. The TN also increased in concentration during the first cruise and showed a decrease over time potentially indicating that the microbial community was remineralizing this source of organic nitrogen to ammonium and also converting the ammonium to NO x . Flooding of the bay impacted microbial communities such that the generally dominant members of the marine coastal bacteria community (Acidomicrobia, Alphaproteobacteria, Cyanobacteria, Betaproteobacteria SAR11, and Gammaproteobacteria Oceanospirillales), were replaced by microorganisms of terrestrial (Actinobacteria, OBP45 Soil Group, Opitutae, OBP35 soil group), sedimentary (Actinobacteria), agricultural (OM190 soil group), and freshwater (Betaproteobacteria: Burkholderiales and Methylophilales) origin (Figures 5-7, 9; Chistoserdova, 2011a,b;Beck et al., 2013). During the first cruise, the bacterial communities at all stations were very homogenous, consistent with the parallel trends observed in the water quality values. Sphingobacteria, more commonly found in activated sludge from wastewater treatment plants (Herlemann et al., 2013), had higher relative abundance during the first two cruises and at the freshwater sites for the later time points. Microbes associated with surface sediments (species within the class Opitutae) were observed in the earlier cruises, which could explain times when terrestrial soils may have been present in the floodwaters. Four weeks after the hurricane, a shift in the members of the phylum Verrucomicrobia occurred from the soil-based Opitutae class to the aquatic Spartobacteria class indicating that the estuarine/marine conditions were returning to the system (Herlemann et al., 2013). There was no increase in the relative abundance of gut-associated bacteria (i.e., Enterobacteria and Firmicutes), suggesting there was minimal human influence on the bacterial community in earlier time points compared (Zhang et al., 2015). van Oldenborgh et al. (2017) calculated that there was a pulse (1.14 × 10 5 m 3 ) of untreated wastewater and raw sewage into the bay during the flooding event. For this study, the first cruise took place approximately 1 week after the flooding event, thus the gut-associated signal may have been missed if it was present during and immediately following the storm, or that our detection limit is too high to pick up the least abundant microorganisms. Members of the microbial community typically present in the bay and normally associated with coastal marine environments, such as Oceanospirillales, SAR11, and Marine Group A, were barely detectable after Hurricane Harvey and slowly increase with time after the storm. Marine Cyanobacteria dominated by Synechococcus sp. typically represent ∼20-30% of the total abundance of all OTUs within Galveston Bay. After Hurricane Harvey, Cyanobacteria represented <5% of all OTUs and gradually came back to pre-hurricane levels within the 6 weeks following the flood. The observed decrease in marine Cyanobacteria could be due to the lower salinity and lower water temperatures, a seasonal trend that has been observed along the coast of Texas (Suttle and Chan, 1994) and other environments (Xia et al., 2015;Kim et al., 2018).
The response of the microbial community (including bacteria and phytoplankton) after a flooding event can be dependent on the nutrient concentrations present in the estuary before the storm waters were introduced to the system (Roelke et al., 2013;Dorado et al., 2015). Initially DIN:P i observed in Galveston Bay following Harvey was relatively high (∼10), particularly adjacent to the river mouth, and then returned closer to ∼1 by the end of the month, indicating the phytoplankton populations were nitrogen limited as has been observed previously (Roelke et al., 2013;Dorado et al., 2015). The organic phosphorus continued to increase with time as the flood waters continued to move out of the bay toward the Gulf of Mexico. The P i also increased with time after the initial flooding event supporting the idea that the bacterial community was remineralizing the organic phosphorus throughout the bay. In previous studies, members of the phytoplankton community (diatoms) have responded by increasing in biomass if nutrients were limiting prior to a flooding event (Paerl et al., 2001;Roelke et al., 2013;Dorado et al., 2015). When pulses of nutrients are introduced into a system that is nutrient replete, the phytoplankton biomass may not increase in response to additional nutrients (Roelke et al., 2013;Dorado et al., 2015). Inversely, the Dinophyceae have shown a negative correlation with freshwater inflows or added nutrients (Roelke et al., 2013;Dorado et al., 2015). Other factors that may contribute to changes in the phytoplankton abundance depend on the volume of floodwater and how the residence time of the system is affected. Phytoplankton biomass has been shown to decrease following large flooding events due to the hydraulic flushing physically pushing the bay waters out of the system (Paerl et al., 2001;Pinckney et al., 2009;Roelke et al., 2013). Immediately following Harvey, a small phytoplankton bloom occurred with a dominance of diatoms, cryptophytes, and chlorophytes, in response to the freshwater inflow and pulse of nutrients from Hurricane Harvey suggesting nutrients may have been limiting prior to this flooding event (Piehler et al., 2004). Blooms of both diatoms and dinoflagellates have previously been seen after flooding events in an Australian lagoon (Cook et al., 2010;Hallegraeff, 2010) and in North Carolina's Neuse River Estuary after Hurricane Isabel (Wetz and Paerl, 2008) and in Lake Ponchetrain, LA (United States) following Hurricanes Katrina and Rita (Pinckney et al., 2009).
Alongside changes in phytoplankton communities are corresponding changes in eukaryotic grazers. Ciliate grazers, including select freshwater Oligotrichea, preferentially consume cryptophytes (Müller and Schlegel, 1999;Posch et al., 2015). Before Harvey, Oligotrichea (a ciliate grazer) was dominant alongside its prey the cryptophytes. Similar to what Posch et al. (2015) had observed following the hurricane, diatoms were in high in relative abundance along with the ciliate grazers Listomatae. During the third cruise, the ciliate grazer Spirotrichae increased in abundance compared to earlier time points, after which Oligotrichea had the highest abundance of the ciliates which is a pattern that had previously been documented during Hurricanes Katrina and Rita and in freshwater lakes (Amaral-Zettler et al., 2008;Posch et al., 2015). Notably, the ciliate grazer sequences which were discovered in this study were similar to that observed after Hurricanes Katrina and Rita by Posch et al. (2015). After Hurricane Harvey, the eukaryotic community changed drastically, and did not recover to pre-Harvey conditions within the month of sampling, suggesting a longer recovery period compared to the prokaryotes. While much is known about commercial and recreational fisheries (Thronson and Quigg, 2008;Lester and Gonzalez, 2011;Tolan, 2013;Steichen and Quigg, 2018;Oakley and Guillen, 2019) less is known about the lower eukaryotic trophic levels and how they respond to extreme events.
The Galveston Bay watershed is a sink for runoff, major industrial, agricultural, municipal effluent discharges, and also serves as a high-intensity transportation hub for regional oil refineries and industries with intensive shipping traffic visiting its three major ports (Steichen et al., 2012). In addition to these anthropogenic pressures, spills from tankers and barges have released a variety of chemicals (including petroleum products) into Galveston Bay (Williams et al., 2017). As a result of these various contamination sources, Galveston Bay waters, sediments, and biota (microbes, invertebrates, finfish, etc.) are traditionally key focus areas for environmental monitoring studies observing legacy pollutants, such as PAHs, PCBs, dioxins, and metals (Roach et al., 1993;Willett et al., 1997;Qian et al., 2001;Suarez et al., 2006;Howell et al., 2008;Lakshmanan et al., 2010;Yin et al., 2015). Flooding from large storm events has been shown to affect estuarine systems by decreasing residence times, delivering increased concentrations of nutrients, organic matter, and potentially pollutants (Paerl et al., 2001;Peierls et al., 2003;Pinckney et al., 2009).
In general, the low volatility and polar nature of PBs ensure widespread distributions through primarily aqueous transport routes (mainly surface waters) or through foodchain (trophic) transfer (Daughton and Ternes, 1999). Major sources of pharmaceuticals in the environment include effluent outfalls from wastewater treatment plants and runoff from agriculture or from landfill leachates (Boxall et al., 2012;Gaw et al., 2014). In this study, the PBs (cotinine, imidacloprid, carbamazepine, and carbamazepine-epoxide) increased in concentration with time indicating a dilution effect by the freshwater pulse following this flooding event. Prednisone, a commonly prescribed steroidal (glucocorticoid) antiinflammatory drug in North America (Cdc Md STARnet, 2019), had been measured in Galveston Bay previously by Rashid and Duffett (2016). The level of prednisone following Harvey presents an exception to the trends observed for the remainder PBs showing an increase from pre-Harvey conditions, compared to the undetectable levels at the subsequent time points. This result is difficult to explain as it is counter to the expectation of PB dilution following the six orders of magnitude of rainfall relative to sewage overflow. It may be likely that a point-source release of prednisone due to the flooding caused by Harvey is responsible for the observed trends (i.e., high levels immediately following Harvey, with levels decreasing thereafter). Overall, our results indicate that there is likely to be a low level of PB contamination (∼0.01-0.1 ng L −1 ) in Galveston Bay, with elevated levels more characteristic of the upper reaches of the Bay and around the entrance to the Houston Ship Channel. It is likely that these compounds constitute commonly occurring PBs that represent an "anthropogenic footprint" in the Houston/Galveston Bay watershed but further work is necessary to provide confirmation.
In what is argued to be an extremely rare event, the 1000year flooding associated with Hurricane Harvey occurred in a region already showing positive trends in terms of intensity and frequency of extreme precipitation events (van Oldenborgh et al., 2017). In a modeling study, Emanuel (2017) estimated that the annual probability of 500 mm of area-integrated rainfall was about 1% in the period 1981-2000 but increased to 18% in the period 2081-2100. Both studies make it clear that extreme rainfall events along the Texas Gulf Coast are on the rise. Recent events along the northern Gulf of Mexico coast, including two 500-year floods in the years immediately prior to Hurricane Harvey (May 2015 and April 2016) as well as several other severe storm events which directly impacted the Houston area this century (Tropical Storm Allison, 2001 andHurricane Ike, 2008), indicate these models are tracking observations. Extreme rainfall events should be part of the discussion of environmental management and mitigation and future improvements to flood protection systems in major cities around the Gulf and globally. Although the water quality and microbial community showed signs of returning to pre-Harvey conditions within the month following the flood event, longterm impacts need to be measured in the years following the flood. While the nekton community in estuarine systems is resilient to short-term disturbances, there may be more lasting effects with the predicted rise in the frequency and magnitude of these large rain events (Waide, 1991;Paperno et al., 2006;Oakley and Guillen, 2019). With an increase in frequency and intensity of these coastal disturbances and extreme flooding events, there may be a shift in the amount of time needed for a system to recover (Paerl et al., 2001). Rapid declines in the salinity of estuarine systems such as what was observed in Galveston Bay have been shown to impact the resident nekton community by decreasing the availability of suitable habitat for these macrofauna (Lenihan and Peterson, 1998;Eby et al., 2000;Paerl et al., 2001;McFarlin et al., 2015). Future work should be done on the effects this freshening had on the organisms in the bay following a disruption at the base of the food web. The impacts of the increased contaminants and the extensive period of low salinity created due to this flooding event warrant continued monitoring of the water quality conditions and microbial community of this system in years to come to determine the long-term effects on the biological community from the base of the food web upward within Galveston Bay.
Collectively, these findings showed that over the month following the storm, a transition occurred from an estuarine biotic community before the hurricane to a primarily freshwater biotic community after the flooding event and then began the shift back to an estuarine/marine biotic community within about a month after the flood event. There was a large pulse of organic matter that provided substrate for microbial degradation. The bacterial community showed an increase in the abundance of bacteria associated with terrestrial soils and freshwater at the initial time point and then decreased over time and were replaced by their estuarine and marine relatives within the month following the hurricane. The microbial eukaryotic community (phytoplankton and grazers) displayed a rapid transition from an estuarine community to a freshwater community following the storm with a slower recovery compared to the 16S rRNA community. This collaborative effort will allow for improved understanding of the physicochemical and biological changes following a large storm event within subtropical estuaries. While this work captured the immediate impact of a hurricane on an estuarine system, future work is recommended to enhance our understanding pertaining to the long-term effects of a storm of this magnitude on various levels of the food web.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the BioProject #PRJNA558756. This research was made possible by a grant from The Gulf of Mexico Research Initiative to support consortium research entitled ADDOMEx (Aggregation and Degradation of Dispersants and Oil by Microbial Exopolymers) Consortium. Data are publicly available through the Gulf of Mexico Research Initiative Information and Data Cooperative (GRIIDC) at http://data.gulfresearchinitative.org (doi: 10.7266/ PGC99C7D).

ACKNOWLEDGMENTS
We would like to thank all members of the Phytoplankton Dynamics lab at Texas A&M University at Galveston (TAMUG) that assisted with cruise plans and organization, sample collection, and processing. Many thanks to the crew of the TAMUG R/V Trident and to everyone from TAMUG that came along (with very short notice) to assist in sample collection, sample processing, and data analysis.
TABLE S1 | Non-significant results of the PERMANOVA analysis that was run on biological data matrix with the environmental data as the predictor variables. There were 9999 permutations ran to achieve the results.