Biomonitoring in the Anthropocene: Urban estuary environmental DNA tracks marine fish, terrestrial wildlife, and human diet

1 1 Title: Biomonitoring in the Anthropocene: Urban estuary environmental DNA tracks 2 marine fish, terrestrial wildlife, and human diet 3 4 5 6 Mark Y. Stoeckle 1, Jesse H. Ausubel1 7 8 1Program for the Human Environment, The Rockefeller University, New York, New York, 9 United States of America 10 11 *Corresponding author 12 Email: [email protected] (MYS) .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 2 13 Abstract 14 Managing human impacts in urban estuaries asks for up-to-date monitoring of marine 15 life. Here we analyze vertebrate eDNA in New York City’s East River, a rocky estuary channel 16 difficult to survey with mechanical gear and subject to wastewater discharge. We collected water 17 samples weekly for one year and applied spike-in metabarcoding to quantify vertebrate eDNA. 18 Replication experiments demonstrated good reproducibility above about 10 eDNA copies/PCR. 19 We propose a fish censusing scale based on absolute eDNA abundance. Local marine fish eDNA 20 followed a classic hollow curve species abundance distribution over four orders of magnitude, 21 with abundant and common taxa comprising about 25% of species and 95% of fish eDNA. There 22 was a 10-fold increase in local marine fish eDNA in summer and seasonal differences among 23 taxa consistent with known phenology. Two fish species were newly abundant in comparison to 24 an eDNA survey at the same site in 2016. Levels of other vertebrate eDNA—domesticated 25 animal, non-fish wildlife, and dietary fish—were correlated with human eDNA levels, consistent 26 with a shared wastewater source. Wastewater eDNA identified the commonest urban mammals, 27 land birds, and household pets. Proportions of dietary animal eDNA in wastewater closely 28 approximated proportions in national consumption statistics, opening a window into human diet 29 assessment. Effort and cost for the weekly shoreline survey were modest. Vertebrate eDNA 30 metabarcoding with spike-in quantification enabled weekly monitoring of urban estuary fish 31 populations, identified overlooked newly abundant species, and reported on terrestrial wildlife 32 and human diet. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 3 33 Introduction 34 Many estuaries are sites of major cities and at the same time essential habitat for wildlife. 35 An urban estuary represents a profound transformation of the physical and living landscape 36 [1,2]. Hazards to the aquatic biosphere may arise from dredging, commercial and residential 37 wastewater discharge, maritime traffic, ballast water disposal, and engineering modifications 38 such as dikes, piers, seawalls, and filling of wetlands [3]. Assessing anthropogenic alterations 39 and mitigations needs up-to-date biological monitoring. Aquatic surveys are constrained by 40 shoreline armoring, navigation restrictions, and submerged structures including cables, tunnels, 41 bridge and pier supports, and rocky reefs. 42 Environmental DNA may offer a way to advance the science and practice of 43 biomonitoring [4]. The ease and nondestructive nature of collecting water for eDNA raises the 44 prospect of monitoring biodiversity at fine scale in space and time. For instance, there are several 45 metabarcoding primer sets targeting the mitochondrial 12S gene that amplify eDNA from most 46 bony fish species [5-8]. One liter of estuary water typically contains sufficient eDNA to assay at 47 least the more abundant taxa. Hindrances to wider adoption of metabarcoding for fish 48 assessment include persistent uncertainty about how metabarcoding reads relate to eDNA 49 abundance and how eDNA abundance relates to fish abundance [9,10]. Recent reports indicate 50 progress addressing these essential concerns. First, there is increasing evidence that relative 51 metabarcoding reads and eDNA levels are generally proportional to relative fish species 52 abundance [11-15]. Second, this correlation can be improved by weighting metabarcoding reads 53 to adjust for amplification bias, i.e., differences in PCR efficiency among species due to primer 54 mismatch or other factors [16-19]. Alternatively, adding a known quantity of a synthetic DNA .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 4 55 template as a standard to each PCR assay (“spike-in”) helps quantify absolute eDNA levels [20- 56 22]. A related approach uses qPCR with metabarcoding primers to quantify total fish eDNA 57 [23]. Quantification offers significant benefits. Absolute eDNA concentrations can be directly 58 compared within and among studies. Spike-in quantification reveals limits to reproducible 59 detection and adjusts for amplification of nontarget DNA [24]. Additional informative work 60 regarding eDNA levels and fish abundance includes mesocosm testing of eDNA shedding and 61 decay, effects of temperature and sunlight exposure on DNA degradation, modeling ocean 62 dispersal including tidal effects, and accounting for allometric scaling [25-29]. Particularly in 63 urban settings, further concerns arise regarding interference from wastewater DNA [30]. eDNA 64 has inherent limitations as a biomonitoring tool, including absent information on life stage, age, 65 health, weight, sex. 66 In this report we applied spike-in metabarcoding with Riaz 12S gene primers to quantify 67 vertebrate eDNA in New York City’s East River, a tidal strait in the lower Hudson River estuary 68 [31]. The Hudson estuary has a storied history [32,33]. Environmental restoration efforts dating 69 from the Clean Water Act of 1972 have begun to reverse centuries of neglect [34,35]. The East 70 River study site challenges gear-based fish surveys because of the channel’s armored shoreline, 71 irregular rocky bottom topography, and rapid tidal currents [36]. Wastewater permeates the 72 estuary. Like many municipalities, New York City is served by a combined sewer system that 73 conducts household sewage and stormwater from street runoff to underground reservoirs and 74 then to treatment plants [37]. When conduits are overloaded, as frequently occurs in New York 75 City after even modest rainfall, the effluent empties at combined sewer overflow (CSO) outfalls 76 into waterways. CSO outfalls in New York City currently discharge about 18 billion gallons of 77 untreated wastewater into the estuary annually. Although significant, this represents an 80% .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 5 78 reduction in CSO discharges since 1985, thanks to more than $40 billion in infrastructure 79 improvements [38]. We test two hypotheses: first, that estuary eDNA quantifies local marine fish 80 populations without interference from wastewater DNA and second, that wastewater DNA 81 usefully reports on other aspects of urban environment. 82 Materials and Methods 83 Replicate 1 L water samples were collected weekly at an East River shoreline site, used 84 in prior studies, beginning May 2, 2024 through May 1, 2025 (Fig 1) [39]. As the shoreline is 85 armored and elevated about 2 meters above water level, paired samples were obtained with 86 separate throws of a bucket on a rope and transferred at site to laboratory bottles. Given tidal 87 current up to 3 m/sec, estimated average water surface distance between samples was about 100 88 m (range 0 m – 400 m) [36]. Samples were brought to the laboratory within 15 min and each 89 liter was filtered separately using a 4.5 cm, 0.45  M pore size nitrocellulose membrane 90 (Millipore) and wall suction. Once complete, filters were folded to protect retained material and 91 stored in 15 mL centrifuge tubes at -20C. Between uses, collection and filtration equipment 92 were washed extensively with tap water and air dried. DNA was extracted from filters within 93 three months using Qiagen DNeasy PowerSoil Pro Kit and eluted in 100 l of Buffer 6. DNA 94 yield was measured with Qubit and samples were stored at -20C. Negative field controls 95 consisted of 1 L samples of laboratory tap water filtered and extracted for DNA using the same 96 equipment as for field samples. 97 98 Fig 1. Survey design. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 6 99 Upper left, Collection location in New York City’s East River, a tidal channel between Long 100 Island Sound and New York Harbor. Asterisk marks collection site. Right, map of lower Hudson 101 River estuary and surrounds. Lower left, collection and analysis strategy. Map generated in 102 Photoshop using USGS templates (https://apps.nationalmap.gov/viewer/). Photo credit Mark 103 Stoeckle. 104 105 PCR was done as previously described except that the spike-in standard was a 768 bp 106 synthetic gene block (IDT), based on native ostrich 12S DNA amplicon standard in prior study 107 (S1 Fig) [40]. Unlike the native ostrich 12S sequence, the synthetic gene block has M13 tails and 108 three bases modified to match the MiFish-U forward primer [6]. Primary PCR assays were 109 carried out with TaKaRa Titanium Taq hot start High-Yield EcoDry premix in 25 L volume 110 with 200 M tailed Riaz primers, 250 copies gene block standard, and 5 L DNA or 5  L 111 reagent-grade H2O, the latter as PCR negative control. Unlike other commonly used primer sets 112 for fish metabarcoding, Riaz 12S primers are effective for most vertebrates, as 12S binding sites 113 are conserved not only in bony fish but also in mammals and birds [5]. DNA samples were 114 amplified in replicate and negative field and PCR controls were included in each amplification 115 set. Primer sequences and thermal cycling conditions are in S1 Table. After amplification, 5  L 116 of reaction mix were run on 2% agarose gel with SyberSafe to assess products and the remaining 117 20 l were diluted 1:20 in Buffer EB and stored at -20C. Following procedure described in 118 prior studies, 5 L of diluted primary PCR product were indexed with Nextera XT Index Kit v2 119 Set A and Cytiva PuReTaq Ready-to-Go PCR beads in 25  l volume. To assess products, 5 L 120 of index reaction mix were run on a 2% agarose gel, and the remaining 20 L were pooled, .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 7 121 cleaned with AMpure beads at 1:1, and resuspended in Buffer EB. Pooled libraries were 122 sequenced at AZENTA using MiSeq, 2 x 150 bp, 10% PhiX, and 7.5 GB depth. Each library 123 represented a single PCR run on a single DNA sample. The 192 field libraries plus 21 controls 124 (11 field, 10 PCR) were analyzed in four sequencing runs together with unrelated libraries. To 125 assess potential primer bias, a set of DNA samples (64 field samples plus negative controls) 126 were amplified with MiFish-U-F/R2 primers and ostrich g-block spike-in as described above. 127 MiFish-U-F/R2 primers have an extra 3’ base in reverse primer to reduce amplification of 128 bacterial 16S DNA [40]. Primer sequences and thermal cycling parameters are in S1 Table. The 129 protocol was otherwise the same as for Riaz primer amplifications. Pooled libraries were cleaned 130 with AMpure and sequenced at AZENTA on a MiSeq, 2 x 250 bp with 10% PhiX. 131 Bioinformatics used a DADA2 pipeline as previously described [24]. DADA2 output 132 files were transferred to Excel. Amplicon sequence variants (ASVs) were filtered to exclude 133 detections representing less than 0.1% of total reads for a given ASV. ASVs were identified by 134 100% match to a local library of reference sequences representing local marine fish, local 135 freshwater fish, nonlocal fish, non-fish wildlife, domesticated animal, and human (S2 Table). 136 Unmatched sequences were manually submitted to GenBank using BLAST; additional matches 137 were added to the reference library. For each library, reads per copy of ostrich standard were 138 calculated. This proportion was then applied to convert reads to eDNA copies for all ASVs in 139 that library (reads per ASV ÷ reads/copy standard = copies per ASV). As previously reported 140 some local marine fish species shared Riaz segment 12S sequences, and some species were 141 represented by more than one ASV (S2 Table). Results are expressed as eDNA copies per PCR 142 assay or as per liter water sample, the latter obtained by multiplying copies per PCR by 20 to 143 account for the proportion of DNA extract used for each PCR. For MiSeq fastq files, the pipeline .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 8 144 was adjusted to accommodate the longer amplicon. Statistical tests were performed in GraphPad 145 Prism 10.5.0. 146 Results 147 Vertebrate eDNA by category 148 A total of 96 samples were obtained on 48 collecting days. eDNA was successfully 149 extracted and amplified from all water samples and negative controls, generating 192 field 150 sample and 21 negative control NGS libraries. ASVs in DADA2 output files were identified to 151 species and assigned to vertebrate categories as described in Materials and Methods. Sequencing 152 depth appeared sufficient to detect single copy eDNA. Average sequencing depth was 116,983 153 vertebrate reads per library (range 21,045 – 275,753); average reads per eDNA copy gene block 154 standard were 88 (range 4 – 326) (S3 Table). Local marine fish eDNA was detected on all days 155 (average copies/L, 12,507; range 1,161 – 58,738) but accounted for less than half of total 156 vertebrate eDNA (Fig 2, S6 Table). The majority was human eDNA (average copies/L, 27,629; 157 range, 1,295 – 279,273). Domesticated animal and other categories of vertebrate eDNA were 158 commonly present. Negative control libraries yielded low levels of human (average copies/L, 24; 159 range 0 – 173) and domesticated animal (average copies/L, 15; range 0 – 143) eDNA. (Fig 2, S4 160 Table). Other categories of vertebrate eDNA were not detected in negative controls. 161 162 Fig 2. Vertebrate eDNA abundance in field samples and negative controls. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 9 163 Values represent daily averages (left) or individual negative controls (right). Note scale differs 164 between graphs. Box indicates 25th-75th percentile, whiskers, 1.5x interquartile range, and 165 outliers are shown as points; x marks average and line is median. Graph at left does not display 166 outliers in human eDNA; plot otherwise represents values for complete dataset (S4 Table). 167 168 Local marine fish 169 eDNA copy number reproducibility 170 Species detection and eDNA copy number were largely reproducible in single PCR 171 replicates, particularly for more abundant eDNA (S5 Table, S2 Fig). For species with more than 172 10 copies/PCR, most (414/443; 94%) were detected in replicate library, whereas for those with 173 fewer than 10 copies/PCR, only about half (124/278; 45%) were present. Excluding 174 nonreplicated detections, the average absolute fold-difference in copies per species was modest 175 (average, 2.0; standard deviation (SD),  2.5). As expected, there were larger differences 176 between paired field collections (S5 Table, S2 Fig). About half of field sample detections above 177 and below 10 copies/PCR were present in replicate library (225/443 (51%) and 125/278 (45%), 178 respectively). Excluding non-detections, the average absolute fold-difference in copies per 179 species was 3.1, SD,  8.2. Pooled PCR and field replicate datasets were reproducible over a 180 wide concentration range (Fig 3). The average absolute fold-difference in copies per species was 181 1.3 (SD  0.4) and 1.5 (SD  1.3), respectively (S6 Table). Amplification with MiFish-U-F/R2 182 primer set yielded copy numbers per species similar to that with Riaz primers (Fig 3; S7,S8 183 Tables; S3 Fig). One exception was cunner (Tautogolabrus adspersus), which amplified weakly .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 10 184 with MiFish-U-F/R2 primers; this discrepancy was predictable based on primer mismatch (S4 185 Fig). 186 187 Fig 3. Reproducibility of pooled PCR and field replicates. 188 Each point represents one local marine fish species in pooled sets of A) PCR or B) field 189 replicates (96 PCRs/set). Sets are named as in Fig 1. Values are copies per pool. C) Fold- 190 difference copies/species for pooled datasets generated with MiFish-U-F/R2 as compared to Riaz 191 primers. Shading covers  4-fold difference. Asterisk indicates cunner (T. adspersus). Data 192 sources in text. 193 194 Species abundance distribution (SAD) 195 Seventy-one local marine fish species were detected (S3 Table). Species yearly average 196 eDNA abundance followed a hollow curve distribution ranging over four orders of magnitude 197 (Fig 4). We propose an abundance scale based on order of magnitude differences in absolute 198 eDNA concentration as shown in Fig 4. Abundant species were commonly detected and when 199 detected, present in many copies (S5 Fig). Conversely, rare species were rarely detected and 200 when detected, present in few copies. These two properties generated the hollow curve SAD. 201 Abundant and common species (n = 16; 23% of species) were mostly reef-associated, consistent 202 with the rocky nature of the East River site and accounted for the great majority (95%) of fish 203 eDNA (Table 1). 204 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 11 205 Fig 4. Local marine fish SAD and proposed abundance categories. 206 Each column represents one species. Species are rank ordered by average yearly abundance and 207 shown in linear and log scale. 208 209 Table 1. Abundant, common local marine fish. 210 Species are ranked by decreasing abundance. 211 eDNA copies/L % eDNA Reef Tautog Tautoga onitis 4298 34.4 Y Cunner Tautogolabrus adspersus 1198 9.6 Y Menhaden, river herrings Brevoortia tyrannus, Alosa sp 1101 8.8 Skilletfish Gobiesox strumosus 1068 8.5 Y Feather blenny Hypsoblennius hentz 531 4.2 Y Striped bass Morone saxatilis 506 4.0 Y Bay anchovy Anchoa mitchilli 458 3.7 Oyster Toadfish Opsanus tau 439 3.5 Y Seaboard goby Gobiosoma ginsburgi 419 3.3 Y Spot Leiostomus xanthurus 401 3.2 Y Black sea bass Centropristis striatus 362 2.9 Y Atlantic silverside Menidia menidia 299 2.4 American eel Anguilla rostrata 251 2.0 Scup Stenotomus chrysops 144 1.1 Y .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 12 Atlantic herring Clupea harengus 132 1.1 Tomcod Microgadus tomcod 108 0.9 Bluefish Pomatomus saltatrix 104 0.8 SUM 11819 94.5 212 213 214 Phenology 215 There was 10-fold seasonal variation in total marine fish eDNA which roughly paralleled 216 seasonal variation in New York Harbor water temperature (Fig 5). There was a similar seasonal 217 pattern in daily species richness, such that no species were abundant in winter and few were 218 common. Daily species richness for uncommon and rare species did not show a clear seasonal 219 trend. Individual species differed in seasonality (Fig 6). 220 221 Fig 5. Local marine fish eDNA seasonal abundance and diversity. 222 Each column represents one collection day. Collection months, colored by quarter, are indicated; 223 overlay shows New York Harbor water temperature. Left, total copies/L local marine fish. Right, 224 number of species, colorized by abundance rank on collection day as in Fig 4 (source data S3 225 Table). 226 227 Fig 6. Seasonal eDNA abundance individual species. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 13 228 Selected species with differing phenology are shown. Each column represents one collection 229 day; scale is copies/L. Scale differs between graphs. Color bar indicates months as in Fig 5. 230 Scientific names, source data in S3 Table. 231 232 Other vertebrate eDNA 233 Human, domesticated animal, non-fish wildlife, and nonlocal fish eDNA was commonly 234 detected in estuary samples (Fig 2). Unlike local marine fish eDNA, daily abundances of other 235 vertebrate eDNA categories were directly correlated to that for human eDNA, consistent with a 236 shared wastewater source (Fig 7). Estuary eDNA correctly identified the commonest urban 237 mammals, land birds, and household pets (Table 2) [41,42]. Dietary animal eDNA proportions 238 closely tracked proportions in national statistics on meat and fish consumption (Fig 8) [43-46]. 239 240 Fig 7. Other vertebrate eDNA abundance by category in relation to human eDNA 241 abundance. 242 Each point represents one collection day. Scale is log10 copies/L. 243 244 Table 2. Top species other vertebrate categories. 245 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 14 Copies/L Detections Non-fish wildlife (62) Mammals (22) Rat 684 40 Gray squirrel 23 20 Raccoon 19 14 Beaver 13 14 House mouse 12 14 White-tailed deer 11 10 Deer mouse 11 10 Land birds (19) Rock pigeon 254 36 Starling 19 11 House sparrow 11 8 Waterbirds (19) Canada goose 219 33 Ring-billed gull 89 26 Herring gull, other Larus sp 74 30 Mallard, other Anas sp 59 33 DC cormorant 42 31 Greater scaup, other Athya sp 11 8 Domesticated animal (16) Dietary (11) Chicken 1601 47 Cow 867 46 Pig 716 43 Turkey 57 29 Sheep 56 26 Goat 26 14 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 15 Pets (5) Dog 252 40 Cat 104 22 Nonlocal fish (55) Atlantic salmon 80 30 European sea bass 43 16 Tilapia 24 12 Rainbow trout, other Oncorhynchus sp 20 13 Red snapper, other Lutjanus sp 19 7 Human 27629 192 246 247 Average copies/L and days detected (total collection days n = 48) are shown. Taxa are ranked by 248 decreasing abundance within each category; rare species (<10 copies/L) not shown. Total 249 detected in parentheses. Scientific names, complete dataset in S3 Table. 250 251 Fig 8. Proportions dietary animal eDNA compared with proportions US consumption per 252 capita. 253 Data sources in text. 254 255 Discussion 256 In this investigation we employed spike-in metabarcoding to quantify vertebrate eDNA 257 weekly for one year in an urban estuary. There were two main findings. First, results supported 258 hypothesis that eDNA indexes local marine fish abundance. eDNA followed expected fish 259 survey characteristics including a classic hollow-curve species abundance distribution, reef .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 16 260 specialists as top species, summer peak in total fish abundance, and species-specific seasonal 261 patterns consistent with life histories [47-51]. Human and other wastewater eDNA was plentiful 262 but total vertebrate eDNA levels were below the threshold expected to suppress fish assessment. 263 Second, wastewater eDNA tracked terrestrial wildlife abundance and human consumption of 264 meat and fish. 265 We propose a fish abundance scale based on order of magnitude differences in absolute 266 eDNA concentration (Fig 2). An order of magnitude scale has been applied to rank numerical 267 abundance of fish species [52]. Standardized numerical categories of eDNA abundance could 268 help communicate survey findings to general as well as scientific audiences. eDNA rarity was 269 the main limit to eDNA detection and quantification, a recognized constraint in fish eDNA 270 surveys [53-55]. The protocol employed a PCR input of 1/20th of the DNA obtained from 1 liter 271 of water, a similar proportion as that in other studies. Fish eDNA copies per PCR aliquot were 272 surprisingly low (average, 625; range, 21 – 3271). This was typically sufficient to detect a dozen 273 or so species (average, 15; range, 4 – 26). Across the one-year survey, most of 71 local marine 274 fish species were present in fewer than 10% of PCRs (S3 Table). At the other end of the 275 distribution, skilletfish (G. strumosus) and feather blenny (H. hentz) were newly abundant (Table 276 1). These taxa were rare in an eDNA survey at this site in 2016 and increased in 2022 (Fig 9) 277 [39,56]. Both species were rare or absent in regional surveys up to 2020 [52,57-59]. Hudson 278 River Park Fish Survey (HRPFS) corroborates recent increases [60]. Skilletfish and feather 279 blenny were first recorded in HRPFS traps in 2020 and subsequently commonly collected. Both 280 species favor oyster reef habitat; the current plenitude might be consequent to restoration of 281 oyster beds in New York Harbor that began in 2015 [61]. 282 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 17 Fig 9. Newly abundant species. Data are from this study and previously reported East River eDNA surveys (see text for references). 283 284 Wastewater DNA offered insights. Daily levels of non-fish vertebrate eDNA correlated 285 with daily levels of human DNA, consistent with a shared wastewater source. The highest levels 286 of human and other vertebrate eDNA were obtained after significant rainfall, evidence that peak 287 concentrations resulted from CSO discharge of untreated waste (S6 Fig). Whether processed 288 wastewater contributes human and other vertebrate eDNA to the Hudson estuary is unknown. It 289 is recently reported that dietary fish eDNA persists even after quaternary treatment of sewage 290 [62]. Future work could examine eDNA content of untreated sewage and street runoff at CSO 291 outfalls, at different points in the processing system, and elsewhere in the estuary. Human and 292 domesticated animal DNA may serve as markers of human environmental impact [63]. 293 Wastewater is often considered a nuisance in eDNA surveys because of possible suppression of 294 fish reads by human DNA and misleading detection of dietary species [30,64]. Some studies use 295 human blocking primers, which have unknown effects on amplification of target taxa. Present 296 results suggest this precaution may be unnecessary, the more so if primers are selective for fish 297 vs. other vertebrates. In this report the apparent copy number of human DNA as assayed with 298 MiFish-U-F/R2 primers, which have multiple binding site mismatches in mammals, was about 299 1/1000th of that obtained with Riaz primers (S8 Table). 300 Rats are the most abundant wild mammal in New York City [65,66]. Monitoring rat 301 eDNA in the estuary may help assess pest control programs. Standardizing against other .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 18 302 terrestrial wildlife eDNA such as pigeon might compensate for the variable content of street 303 runoff in estuary samples, or street runoff could be directly sampled. Routine estuary eDNA 304 testing could greatly inform urban wildlife management. More than 60 non-fish wildlife species 305 were identified including fauna rarely seen (S3 Table). Nearly all were taxa known to be resident 306 within city limits, although transport from distant sites or local zoos cannot be excluded [41,42]. 307 Proportions of livestock and dietary fish eDNA in wastewater closely approximated proportions 308 in national consumption statistics. To our knowledge this is the first demonstration that 309 wastewater eDNA reports on human diet, a finding of potential value to public health and 310 commercial interests. The proportions of goat and sheep in East River eDNA were greater than 311 in national data, which might reflect increased consumption by ethnic populations in New York 312 City. This hypothesis could be explored with local sales data. 313 Limitations 314 This report has several limitations. A concurrent gear-based survey is not available for 315 benchmarking eDNA. Assessment of eDNA performance for fish censusing rests on features 316 outlined above. Sampling was conducted at a single location, so findings incompletely depict 317 lower Hudson River estuary fish populations. Nonetheless, fish eDNA diversity broadly 318 overlapped with traditional surveys conducted in the estuary over the past 35 years [52,57-59]. 319 Potential tidal effects were not directly assessed. Given semidiurnal tides and weekly water 320 sample collection, consecutive samples were drawn on approximately opposite tides. Inspection 321 did not show consistent week-to-week variation in eDNA profiles, and exploratory plots of 322 eDNA copies/species vs. tide timetable were similarly unrevealing. The apparent absence of 323 differences may reflect limited dispersion, as tidal excursion in the East River is less than the .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 19 324 length of the channel [67]. Many fish species spawn in the estuary [35]. Possible contributions of 325 spawning, larval, or juvenile fish to eDNA levels are unknown. Water temperature might alter 326 eDNA shedding or decay rates [26]. However, such across-the-board factors insufficiently 327 account for species-specific phenologies obtained with eDNA. eDNA persistence beyond weekly 328 sampling intervals might blunt reporting on current fish abundance. Individual species 329 demonstrated strong seasonal patterns consistent with known phenology, consistent with 330 inference that the assay indexes current or recent fish density (Fig 6). In addition, peak values of 331 human and domesticated animal eDNA associated with recent rainfall did not persist into the 332 following week (S6 Fig). Assignment of species to categories carries uncertainties. Some local 333 marine species are also consumed by humans—striped bass (M. saxatilis), for example. eRNA 334 assays may enable distinguishing nucleic acid signals due to resident fish from those introduced 335 by waste [68]. Nonlocal fish eDNA attributed to wastewater may have originated from 336 extralimital strays. 337 Reproducibility and accuracy are desirable attributes in surveying marine biodiversity. 338 These considerations apply to both questions raised in the Introduction—whether metabarcoding 339 reports on eDNA levels and whether eDNA levels report on fish abundance. Replication 340 experiments demonstrated good reproducibility in measuring eDNA levels, particularly for more 341 abundant eDNAs. Accuracy might be distorted by differences among species in amplification 342 efficiency. Quantitatively similar results for most species with MiFish-U-F/R2 as compared with 343 Riaz primers suggest this is not a major factor for bony fish (Fig 3, S3 Fig). Cunner (T. 344 adspersus) was an exception. The apparent concentration of cunner eDNA was about 70-fold 345 lower with MiFish-U-F/R2 than with Riaz primers, a deficit predicted by binding site mismatch .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 20 346 (S4 Fig). Dietary animal eDNA added evidence that the Riaz assay accurately reported relative 347 eDNA levels. 348 There are limits to knowledge about abundance of marine biodiversity [69]. Censusing is 349 inherently imprecise for fish and other nekton [70]. Given that fish SADs typically extend over 350 four or five orders of magnitude, a reproducible protocol that indexes eDNA or fish abundance 351 within, say, half an order of magnitude (about threefold) of the reference value would have wide 352 use. There is no certain gold standard in benchmarking eDNA for marine fish assessment—all 353 methods have catchability biases [71,72]. Estuary eDNA detected 22 freshwater fish species, all 354 uncommon or rare (S3 Table); these findings may be of interest for future work. Waterbird 355 eDNA was grouped with that of other non-fish wildlife but probably originated from birds in the 356 estuary rather than from wastewater; this could be tested directly. The survey required about 357 25% effort and direct costs of about $15,000 (S9 Table). About half of effort was devoted to 358 bioinformatics; this might be reduced by automating some procedures performed in Excel. 359 Conclusion 360 Vertebrate eDNA metabarcoding with spike-in quantification offers a practical approach 361 to biomonitoring in urban estuaries. This methodology holds promise as an aid to estuary fish 362 and wildlife management and opens a window into human diet. Regular reporting of such 363 findings would likely be of interest to both government and non-government entities [73]. 364 Absolute quantification of eDNA levels could yield heretofore unprecedented ability to map fish 365 abundance across diverse sites and habitats. 366 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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Environ DNA. 2024;6: e432. 560 561 Supporting Information .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint 31 562 S1 Table. PCR primers, protocols. 563 S2 Table. Reference sequences. 564 S3 Table. eDNA copies per ASV by PCR, day. 565 S4 Table. eDNA copies per day by category for field samples, negative controls. 566 S5 Table. Reproducibility individual PCR and field replicates. 567 S6 Table. Reproducibility pooled PCR and field replicates. 568 S7 Table. MiFish-U-F/R2 copies/ASV. 569 S8 Table. MiFish-U-F/R2 vs Riaz copies/ASV. 570 S9 Table. Effort, costs. 571 S1 Fig. Gene block spike-in standard. 572 S2 Fig. Reproducibility of individual PCR and field replicates. 573 S3 Fig. MiFish-U-F/R2 vs Riaz pooled copies/L. 574 S4 Fig. Primer binding sites for local marine fish species detected in this study. 575 S5 Fig. Frequency of detection, copies per detection vs eDNA overall abundance. 576 S6 Fig. Human eDNA abundance and recent rainfall. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 8, 2025. ; https://doi.org/10.1101/2025.09.04.674353doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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