Deep-sea mining discharge can disrupt midwater food webs

Domain: earth_science
Published: 2025-11-06
Editorial outcome: minor_revision

Abstract
The Clarion-Clipperton Zone contains extensive beds of polymetallic nodules on the abyssal seafloor, with vast areas (~1.5 million km2) under license for deep-sea mining. Mining companies have proposed discharging excess waste generated during nodule extraction in the lower mesopelagic and upper bathypelagic zones, which are home to a unique faunal community including zooplankton and micronekton. Here, using compound-specific isotope analysis of amino acids, we find that natural background particles larger than 6 µm form the base of the food web, but will be diluted by the same sized, nutritionally deficient mining-associated particles. Given that 53% of zooplankton taxa are particle feeders and 60% of micronekton taxa are zooplanktivores at proposed discharge depths, there is significant potential for food-web disruption. Therefore, we show that a midwater mining plume could trigger bottom-up ecosystem impacts with potentially severe consequences for the faunal community, extending beyond zooplankton and micronekton to nekton, including large marine predators.

Introduction
The Clarion-Clipperton Zone (CCZ) in the Eastern Topical Pacific is a region of great interest for proposed deep-sea mining of polymetallic nodules. The International Seabed Authority, the UN established regulatory agency1, has granted 19 exploration licenses for polymetallic nodules covering a vast area of ~1.5 million km2. Previous research has documented environmental risks of mining, including biodiversity loss in seafloor communities2,3. In addition to the seafloor, midwater communities may also be at risk. During the mining process, nodules are collected from the abyssal seafloor, along with seawater and sediments, and transferred through a pipe to a collection ship for separation of nodules from sediment waste. This seawater-containing sediment waste, as well as pulverized nodule particles, must be returned to the ocean. Although the release depth for this waste is currently unclear, some mining operators have proposed midwater mining discharge within the lower mesopelagic/upper bathypelagic zone4,5. The risks to midwater communities remain poorly understood.

A midwater mining waste plume is anticipated to negatively affect mesopelagic fauna, with impacts ranging from replacing or diluting the food of filter feeders to visual impairment of active predation and bioluminescent communication6,7. Pelagic food webs are complex, linking small primary producers in the upper ocean to top consumers at a range of depths. Diel vertical migration from deeper daytime depths to shallower nighttime foraging depths is common, vertically mixing the food web8,9. Additionally, about 5–25% of the organic matter produced in the euphotic zone is exported to deeper depths through passive sinking and active transport10,11. The material that reaches the mesopelagic undergoes aerobic microbial decomposition and transformation, resulting in a characteristic decrease in organic carbon and nitrogen content12,13. The mining plume, consisting of deep abyssal sediments from <2 µm to 63 µm in size14, is predicted to have a high concentration of low-quality particles, along with fragmented inorganic nodules. These mining waste particles could clog the respiratory and olfactory surfaces of organisms across all trophic levels and obstruct filter-feeding structures6,15.

Of importance to midwater food webs in the CCZ region is the presence of an oxygen minimum zone (OMZ), a region characterized by particularly hypoxic conditions (to <1 µM)16. Oxygen gradients influence the depth distribution of zooplankton and micronekton communities17,18, as biological adaptations to low oxygen create species-specific habitat thresholds that determine where organisms reside and feed19,20. In the mesopelagic, zooplankton biomass may exhibit a modest increase in the lower oxycline (LO) in comparison to the OMZ core18,21, however, highly mobile and migratory micronekton (small fish, cephalopods, and crustacea, ~2–20 cm in size9 often reside at these depths17,18.

Compound-specific isotope analysis of amino acids (CSIA-AA) can identify the particle size fraction that forms the base of the food web22,23, and it can characterize trophic structure of the community24,25. Understanding the contribution of different particle size fractions to the base of the food web is critical, as mining plumes will introduce particles of varying abundance, nutritional content, and quality depending on their size. This knowledge is essential for assessing the potential impacts of midwater mining plumes on the faunal community at depth.

In this study, we apply a Bayesian mixing model26 to isotopically differentiate particle sources based on size and quantify their contribution to the base of the food web. The goal of this study is to characterize the trophic sources of the faunal food web at a proposed lower mesopelagic discharge depth to evaluate how the introduction of a midwater mining waste plume could disrupt it. We examine both the composition of mining discharge plume material and the nutritional quality at the base of the food web, along with the trophic modes of the animals living there. This work provides a critical step towards understanding the potential impacts of full-scale mining on the mesopelagic community.

Results
During this study, we collected 3 types of particle samples: background, discharge, and plume samples in 3 size fractions, small (0.7–6 µm), medium (6–53 µm), and large (>53 µm) (Fig. 1). Background samples were collected at various depths (800–1500 m) during 3 cruises, either when no mining activity was occurring or at locations and depths far from mining activity, ensuring that these particles represent natural midwater particles. Discharge and plume samples were collected in Fall 2022 during a small-scale test mining operation. During this operation, a collector vehicle on the abyssal seafloor collected sediments and nodules, which were brought to a surface ship. The nodules were separated from abyssal sediments onboard, and the effluent waste was released at ~1250 m. This event represented a rare opportunity to study midwater discharge associated with deep-sea mining. Discharge particle samples consisted of shipboard effluent waste collected after nodules were removed but before midwater release. Plume particle samples were collected in situ after the mining waste was discharged, using transmissometry to measure turbidity and confirm that we were sampling within the plume.

Background particle data were obtained as a proxy for the potential base of the food web from previous cruises where particles, zooplankton, and micronekton were collected. Isotopic analysis was performed to characterize these particles for use in a mixing model to identify the base of the food web. This approach allows us to evaluate how the introduction of mining waste might affect the pelagic community in the future. Because plume generation lasted a maximum of 32 hours and no animal samples were collected during the Fall 2022 cruise, we lack direct data on the incorporation of plume material into the food web.

We found that mining-associated particles were nutritionally poor. Background particle amino acid concentrations were 4.7 ± 2.7, 41.1 ± 25.3, and 46.3 ± 34.7 ng N/µg PN for small, medium, and large particles, respectively. In contrast, plume and discharge particle amino acid concentrations were substantially lower at 3.8 ± 4.4, 1.7 ± 1.5, and 4.2 ± 4.7 ng N/µg PN (Fig. 1 and Table S1). There was no significant difference in amino acid concentration of small particles between background and plume/discharge samples (p=0.663, one-way ANOVA, Table S2). However, medium and large background particles had significantly higher amino acid concentrations compared to plume/discharge particles (p = 0.028, p = 0.035, one-way ANOVA, Fig. 1 and Table S2). These findings indicate that the medium and large particles in the plume were significantly depleted in amino acid content relative to background material.

Background particle concentrations were much lower than plume particle concentrations. During the Fall 2022 plume sampling, we used a LISST (Laser In Situ Scattering and Transmissometry, LISST-DEEP, Sequoia Scientific) mounted on the CTD rosette to measure in situ particle concentration and size in 32 logarithmically spaced classes centered between 1.36 and 230.14 µm. As the medium and large plume and discharge particle pools were significantly different in amino acid content compared to background particles, samples were pooled into small (0.7–6 µm) and large (>6 µm, medium + large) size fractions for this analysis. Background particle concentrations were 0.08 and 0.23 µL/L for small and large size fractions, respectively, while plume particle concentrations were 9.80 and 2.18 µL/L for the same size fractions (Fig. 2 and Table S3). Background particle counts were 0.014 and 0.003 × 108 particles/L for small and large size fractions, respectively, compared to plume particle counts of 11.15 and 0.12 × 108 particles/L for small and large size fractions (Table S4).

Previous analyses of CCZ abyssal seafloor sediments suggest that sedimentary particles range from <2 µm to 63 µm in size14. The plume particle size distribution showed maxima between 2.6 and 4.3 µm (Fig. S1). Visual analysis of plume samples corroborates this pattern, showing an increase of small (0.7–6 µm) particles in the plume compared to the background. These particles were likely sedimentary in nature due to its light brown color, while dark pulverized nodule particles were observed in the 6–53 µm size range (Fig. 1). It should be noted that the “plume” is not a well-defined entity but rather an area where particle concentrations form a gradient exceeding background levels (see Supplementary Note 1). Some parts of this plume may be highly opaque, while others are only slightly turbid. Consequently, our particle concentrations represent a point along the continuum of the plume. The maximum particle concentration and count reflect the extent of our sampling but may not necessarily represent the true maximum within the plume itself.

To assess the potential impact of a plume on the faunal community, it is first necessary to understand the current contribution of particles as trophic sources to the base of the food web. Using δ15N values of source and δ13C values of essential amino acids (see Supplementary Note 2, Tables S5 and S6, and Fig. S2), we constructed a Bayesian mixing model to estimate plausible ranges to consumers. While diel vertical migration (DVM) is a common phenomenon and occurs at this site, an epipelagic source was not included in the mixing model, a decision based on several considerations. First, the consumer samples analyzed were collected during nighttime tows, meaning that most individuals sampled were likely non-migratory residents. Second, only ~20% of the zooplankton biomass present below the OMZ in the deep mesopelagic migrates to the upper ocean at night. Finally, the micronekton taxa included in this analysis are non-migratory, so any influence from an epipelagic DVM source from DVM should be minimal.

We found that particles >6 µm make up a significant proportion of the base of the food web in lower mesopelagic and upper bathypelagic depths below the OMZ (Fig. 3). Of the 46 animal samples collected below the OMZ (700 to 1500 m), these large particles accounted for at least 50% of the food web trophic base in 30 samples. Specifically, at the discharge depth (1000–1500 m), 16 of the 26 animal samples had large particles making up at least 50% of the food web base (Fig. 3, Source Data 3). Overall, on an individual animal basis, 65% of consumers relied on particles >6 µm for more than half of their food web base. When grouped by zooplankton size fraction/micronekton taxa, 5 of the 8 groups showed a mean contribution of particles >6 µm exceeding 50% (Fig. 3 and Table S7).

In sum, our mixing model indicates that large particles make a substantial contribution to the base of the food web below the OMZ (Fig. 3), a region expected to be impacted by mining waste plumes. Furthermore, we found that large plume and discharge particles are significantly lower in amino acid content (Fig. 1) and significantly higher in concentration compared to background particles (Fig. 2). This material would serve as the main food source for fauna, and the reduced amino acid concentration of these particles is a key indicator of their reduced nutritional value, leading to lower protein intake for the same foraging effort. Together, these findings show that a mining plume would drastically dilute the native particle supply for suspension feeders, replacing it with low nutritional quality seafloor sediments and inorganic fragmented nodules, which could inhibit the ability of organisms to meet their metabolic demands by diluting the relatively organic-rich native particles27,28. This alteration of the in situ particle field has serious implications for the faunal community at the depth of the proposed mining plume.

The breadth of impact of the midwater plume on pelagic food webs is likely to be high based on our knowledge of the diversity and trophic ecology of zooplankton and micronekton below the OMZ. The deep (700–1500 m) zooplankton community contained 186 amplicon sequence variants (ASVs) that were classified into 79 distinct taxa (e.g., genus, family) based on whole-community metabarcoding (18S rDNA), with 53% classified as particle feeders (proportions were nearly identical for just the discharge depth from 1000 to 1500 m; Supplementary Data 1). These taxa, including calanoid copepods, ostracods, and oncaeid copepods rely on suspended particulate organic matter as a primary food source29–32 and may be particularly vulnerable to resuspended sediments from mining plumes. Euphausiids and midwater shrimp may also be impacted as they are known to opportunistically consume detrital aggregates at depth33,34. In addition, gelatinous taxa such as siphonophores, tunicates, and scyphozoans accounted for 20% of the taxa in this deep midwater community, increasing in relative read abundance with depth. These organisms may be particularly susceptible to plume-related impacts, as suspended particles can adhere to their gelatinous bodies, clog feeding structures, disrupt buoyancy, and increase mucous production and energy demands6,7 (zooplankton community analysis detailed further in Supplementary Note 3: Functional Ecology Analysis, Zooplankton Functional Group Results). Given the importance of particle-feeding and gelatinous taxa at midwater depths, mining-induced plumes could significantly disrupt zooplankton communities and trophic interactions, with cascading effects on micronekton and higher trophic level predators.

Indeed, we found 60% of 80 unique micronekton taxa from 700 to 1500 m were zooplanktivores, 37.5% were pelagic micronektonivores (feeding on smaller zooplanktivorous micronekton and occasionally zooplankton), 1.25% were gelatinous zooplanktivores, and 1.25% were pelagic generalists (proportions were nearly identical for just the discharge depth from 1000 to 1500 m; Supplementary Data 2). Furthermore, 85% of the density of micronekton within the discharge depth is zooplanktivores (micronekton community analysis detailed further in Supplementary Note 4). Specifically, among the micronekton sampled for CSIA-AA, Cyclothone spp., Eucopia spp., and Japatella spp. are zooplanktivores35,36, whereas Acanthephyra brevicarinata shrimps are classified as pelagic micronektonivores34,37. Overall, the results suggest that the potential impacts on the zooplankton community due to mining plumes could significantly diminish the primary food source for micronekton, potentially leading to starvation or, for more mobile micronekton, migration from the area.

Accurately identifying the scale of these food web effects is challenging but essential to consider. Factors such as particle flow rate, particle size, and concentration thresholds all significantly influence the vertical and horizontal dispersion, and consequently, the volume of water affected by the sediment discharge38,39. Depending on the exact metrics applied, the scale of effect of the impact is estimated to be on the order of 1000s km3/yr38,39. Given the potential for multiple simultaneous operations in the eastern CCZ, this represents considerable environmental risk to these communities. They provide essential ecosystem services, playing crucial roles in the biological pump8,40, provisioning commercially harvested fishes6,9,41, and maintaining a healthy, functioning ecosystem (Fig. 4).

There are potential options for mitigating the effects of the plume, though each comes with its own risks. Discharging at a shallower depth than the proposed discharge depth could result in similar or worse impacts, because it would affect regions of the water column with significantly higher biomass and important top predators18,21. Releasing the mining plume in the oxyclines above and below the OMZ core, or within the OMZ core itself, could also be especially harmful. Some zooplankton species are highly sensitive to small oxygen variations and adjust their vertical position to remain within their preferred oxygen levels17,19,21. In low oxygen conditions (<10 µM), changes in zooplankton abundance have been linked to oxygen concentration shifts of only a few micromolars21. This finely tuned behavior could be disrupted by introducing an oxygenated midwater mining waste plume into the oxyclines or within the OMZ core (see Supplementary Note 1, Table S8 and Fig. S3). A number of zooplankton and micronekton also vertically migrate within the top 1000 m, potentially expanding the zone of a plume’s influence18,19. Some larger nekton also dive into these depths to forage42,43, although it should be noted that this may be limited by the strong OMZ in this region. Discharging below the current depth, into the deeper bathypelagic, remains largely unstudied. Limited data suggest that while the bathypelagic is an area of low abundance, it harbors high biodiversity, including species that are distinct from the mesopelagic44,45. Furthermore, this community may depend on large particles as the base of its food web, like the processes observed from 700 to 1500 m in this study. Introducing a mining plume into this zone could therefore have comparable adverse effects.

A final potential option is to return the sediment waste to the seafloor. In the case of nodule mining, a seafloor sediment plume will already be generated by the collector vehicle46. A few studies of mining tests suggest that this plume may behave as a turbidity flow, staying within 10’s of meters of the seabed46,47. Introduction of the mining waste material near the seabed could alter the dynamics of this plume, requiring further study and modelling to evaluate its effects on dispersion. However, this approach may reduce the overall impact footprint by releasing one mining plume instead of two. Given our results showing that a midwater release will disrupt whole food webs, including those linked to seafood sources, bringing the discharge plume back down to the seafloor may minimize environmental risks, and we urge that it be considered.

In conclusion, this study provides empirical evidence that discharging mining waste in midwaters is likely to disrupt midwater food webs. The base of the zooplankton and micronekton food webs at this site, located below the OMZ, rely heavily on large particles (65% of consumer samples). Approximately half of zooplankton taxa in this community are particle feeders, employing suspension feeding or detritivory, and their diets would be severely impacted by the introduction of a midwater mining waste plume composed of similarly-sized particles but of lower nutritional quality. As more than half of the micronekton taxa in this community are zooplanktivores, the micronekton community would also face severe disruption due to the reduction or loss of their zooplankton food source. There are confirmed stress responses to sediment exposure in gelatinous plankton7, and other risks such as suffocation and toxic metal exposure are also possible. This unique and diverse faunal community is at significant risk if full-scale mining proceeds (Fig. 4). Mitigation is possible through careful selection of discharge depths by mining companies, combined with regulation by national and international agencies.

Methods
Ethical guidelines
Vessels employed procedures for collection and combustion of trash as standard to at-sea science research vessels to minimize dumping of trash and disturbance to the ecosystem. Waste disposal at sea followed the internationally recognized standards included in the MARPOL Annex IV and V regulations. All possible efforts were taken to minimize disturbance to the environment.
All animal handling and sampling were state-of-the-art and were in accordance with the American Society of Ichthyologists and Herpetologists “Guidelines for Use of Fishes in Field Research.” To minimize pain and stress in the captured animals, they were euthanized as soon as possible after capture if they were still alive. Any that were alive but not needed returned to the ocean as recommended by the ASIH guidelines. The smallest number of animals was used to achieve statistical validity of isotopic analyses for food web analysis. All fishes were collected under UH IACUC permit 14-1934-9. UH IACUC approved this study protocol.

Study site
Samples were collected in the NORI-D mining claim in the Clarion-Clipperton Zone (CCZ) during 3 cruises: DG5B in Spring (March–April) 2021, DG5C in Fall (October) 2021, and DG7B in Fall (October–November) 2022. In 2021, samples were collected at two sites: the Preservation Reference Zone (PRZ) and the Collector Test Area (CTA). In 2022, sampling was conducted only at the CTA. This study includes particle samples collected during all 3 cruises at both sites, zooplankton samples were collected in Spring and Fall 2021 from the CTA site, and micronekton were collected in Fall 2021 at the CTA.

Environmental data
Conductivity-temperature-depth (CTD) casts were conducted during all cruises to measure temperature, salinity, transmissometry, and dissolved oxygen concentrations. Oxygen sensors were calibrated using Winkler oxygen titrations on seawater samples collected in Niskin and GoFlo bottles. In situ particle abundance and size distributions were characterized using a LISST (LISST-DEEP, 650 nm, Sequoia Scientific) for 32 logarithmically spaced classes centered between 1.36 and 230.14 µm, with bandwidths ranging from 0.22 to 38 µm. Data were binned to 1 db depth intervals and calibrated to the particle minima using the deepest 10 m of the cast.

Particle collection
Particulate matter was collected using in situ Large Volume Water Transfer Systems (McLane WTS-LV) pumps equipped with tiered filters on top pre-cleaned 0.7 µm glass fiber filters, and 6 µm and 53 µm Nylon or polyester screens to separate particles into 3 size fractions: small (0.7–6 µm), medium (6–53 µm), and large (>53 µm). Filters were subsampled at sea immediately after the sampling cast for bulk and compound-specific isotope analysis, wrapped individually in baked (500°C, 5 h) aluminum foil, and frozen at −80°C prior to analysis in the lab (detailed further in Supplementary Note 5).
Background particle samples were collected in the lower mesopelagic (above the proposed discharge depth and below the OMZ core; 800–1000 m) and in the upper bathypelagic zone (1000–1500 m). Thirty one particle samples were collected from below the OMZ core across all 3 cruises; however, only 21 samples (7 small, 7 medium, 7 large) possessed the full suite of amino acids and were used as separate sources in the model. Samples collected when no mining activity was occurring or at locations and depths far from mining activity are referred to as “background samples” in the text. During the Fall 2022 cruise, a small-scale test mining operation was conducted5, generating a midwater mine waste plume for up to 32 h of sample collection. Pumps were mounted on a rosette equipped with a CTD package (as described above) and a LISST-DEEP to record real-time turbidity and particle size distributions, aiding in plume identification. Tow-Yo casts were performed while the pumps were active to maintain sampling within the plume, approximately 300–1000 m from the point of discharge (detailed further in the Supplementary Notes 5 and 6). These are referred to as “plume samples” in the text. “Discharge samples” were collected aboard the test mining vessel prior to mine waste being discharged at depth. The three same size-fractions of particles were collected.

Zooplankton collection
Zooplankton were collected using a 1 m2 Multiple Opening/Closing Nets and Environmental Sensing System (MOCNESS) from 1500 m to the sea surface. Samples were collected from 9 depth-stratified nets, targeting depths below the OMZ core in the LO and suboxycline (SO) of 1500–1250 m, 1250–1000 m, 1000–800 m, and 800–700 m. Bulk zooplankton was split quantitatively at sea using a Folsom plankton splitter and fractions preserved for isotope and metabarcoding analyses. A ¼ fraction was separated into 5 size fractions, 0.2–0.5 mm, 0.5–1.0 mm, 1.0–2.0 mm, 2.0–5.0 mm, and >5 mm, and stored at −80°C. These samples were weighed, lyophilized, ground, and stored at −20°C until stable isotope analysis. In total, 24 zooplankton samples, collected from below the OMZ core in Spring and Fall 2021, were used for isotope analysis. A ¼-⅛ fraction was preserved in RNAlater, stored at −80°C, and analyzed by community metabarcoding to assess zooplankton diversity and community composition. A ~365-bp fragment of the V1-V2 region of nuclear 18S rRNA was PCR amplified for these analyses, with reads classified using a Naïve-Bayes Classifier in Qiime248 using multiple referenced databases (detailed further in Supplementary Note 3: DNA Extraction, Amplification, and Sequencing, Bioinformatics; Supplementary Data 3).

Micronekton collection
Micronekton were collected using a 10 m2 MOCNESS equipped with 3 mm mesh nets, sampling from 1500 m to the sea surface. Samples were collected from 5 depth-stratified nets, targeting depths below the OMZ core in the LO and SO of 1500–1000 m and 1000–700 m. In the field, micronekton were individually sorted to family level, then transported to the lab for further identification to the lowest taxonomic unit possible, often to species level, prior to analysis. Specific micronekton taxa analyzed included Acanthephyra brevicarinata, caridean shrimp, Cyclothone spp. fish, Japatella spp. octopod, and Eucopia spp. mysid shrimp. These taxa were selected as abundant representatives collected in both the LO and SO. In total, 21 micronekton samples from below the OMZ core in Fall 2021 were analyzed in this study.

Stable isotope analysis
Samples for stable isotope analysis were processed using routine procedures25,49. Bulk isotope samples were packed into tin capsules and processed using a Costech ECS 2010 Elemental Combustion System coupled to a Thermo Finnigan Delta Plus XP or Thermo Scientific Delta V Advantage via a Thermo Scientific Conflo IV. Reference material (characterized glycine standard and characterized in-house tuna white muscle tissue) was analyzed every 6–12 samples, and the corresponding response factor (ratio of nitrogen peak area, in volt-seconds, to nitrogen reference size, Vs [µg PN]-1) of the reference material was used to determine the particulate nitrogen (PN) concentration (µg N-PN) of samples.
For CSIA-AA, samples were hydrolyzed using 6N hydrochloric acid and purified using cation exchange chromatography. Samples were esterified with isopropanol and trifluoracetic acid (TFAA) and then further purified with liquid-liquid extraction using a phosphate buffer:chloroform mixture. Samples were stored in a dichlormethane-TFAA solution at −20°C and transferred into ethyl acetate before analysis.
Nitrogen isotopic composition was measured using a Thermo Scientific Delta V Plus IRMS interfaced to a Trace gas chromatograph. Internal references L-2-aminoadipic acid (AAA) and L-(+)-norleucine (NOR) with known δ15N values were co-injected with each sample, and a reference suite of 14 pure amino acids with known δ15N values was co-injected with NOR and AAA every 2–4 sample runs. The corresponding response factors (ratio of AA peak area, in volt-seconds, to AA reference size, Vs [nmol AA]-1) of the reference suite were used to determine AA concentration (ng N-AA) of samples. Samples were measured in triplicate when enough material was available.
Total amino acid concentration (as presented in Fig. 1) was calculated by summing the individual amino acid concentrations from CSIA-AA divided by total nitrogen concentration from bulk analysis. A known subsample of the total filter was split for bulk and CSIA-AA, the response factor of the reference material (in either method) was then used to calculate the concentration of the sample analyzed. The subsample percentage (e.g. ½ for bulk analysis, ½ for CSIA-AA) was used to calculate total carbon and nitrogen contents within the sample. Total amino acid content (CSIA-AA, ng N-AA) was normalized to total nitrogen content (Bulk, µg N-PN) in order to compare results between samples.
Carbon isotopic composition of AA was measured using a MAT 253 IRMS interfaced with a Trace GC Ultra. Internal isotopic references AAA, NOR, and ann-C alkanes were co-injected with each sample, and a reference suite of 14 pure amino acids with known δ13C values was co-injected with NOR and AA every 2-4 samples.

Data analysis
The suite of AA’s used as tracers in this mixing model was selected by iterative statistical tests. First, AA’s missing from at least 4 samples per size fraction were excluded. Second, AA’s that showed no significant difference between particle size fraction as determined by one-way ANOVA were also excluded. This process resulted in the selection of two nitrogen source amino acids (phenylalanine [Phe] and lysine [Lys]) and one carbon essential amino acid (EAA, leucine [Leu]). Within a particle size fraction, there was no difference between site, cruise, or depth as determined by way of Pairwise PERMANOVA.
The base of the food web was analyzed using a Bayesian isotope mixing model using the package runjags (version 2.2.2-4)26 in R (version 4.2.2)50 with dirichletReg (0.7-1)51. Three sampling chains were run, each containing 100,000 steps after a 50,000-step adaptation, 40,000-step burn-in, and thinning factor of 50, using an “uninformative”/generalist prior. This mixing model uses δ15N Phe, δ15N Lys, and δ13C Leu values as tracers. Since these selected amino acids serve as source and essential amino acids, they can be considered non-fractionating during trophic transfer52,53. The model assesses the 3 particle size fractions as potential sources that best matched the consumer isotope profile (Source Data 5). Animal samples were run through the model individually by size fraction/taxa (Supplementary Data 4). Given that the amino acid concentrations of the medium and large size fraction plume samples were distinct from the background material, these size fractions were pooled post-modeling to evaluate their mean contribution to the base of the food web (Tables S1 and S2).
Statistical analyses were conducted in R (version 4.2.2)50, using MASS (version 7.3-58.1)54, pairwise Adonis (version 0.4.1)55, and vegan (version 2.6-4)56. Plots were generated with ggplot2 (version 3.5.1)57.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The goal of this study was to characterize the trophic sources of the faunal food\nweb at the CCZ pelagic community at the lower mesopelagic/upper bathyal depth and to understand if a mining plume will\naffect the nutritional quality of the trophic sources and if the food web could be disrupted. These goals will help to\nunderstand the mining plume impact.\nThe results are interesting showing that several functional groups will be affected due to a decrease of nutritional quality of\nthe particles, mainly due to dilution with the mining plume particles.\nThis work is very relevant and timely since it shows clearly evidence of the impact of the mining\nplume on an ecosystem compartment that will affect upper and lower bathymetric layers since the present organisms are food source for pelagic and\nbathyal organisms, and some of them are even the connecting link between ecosystems due their daily migrations.\nThe paper is well written, the methods are appropriated, results are sounding and the authors present good discussion.\nHere in some suggestion to clarify and improve the manuscript:\nMethods:\n-Please specify the type of filters used, how the filters were preserved (some of the information is on the supplementary\nmaterial, but would help the reader here)\n-Also, it would help the reader to specify here how did you measure the amino acids concentrations. You present results as\nN/PN, but you do not explain what these units are. Is amino acid concentration measure as ratio between Nitrogen and\nParticulate Nitrogen?\n- Bayesian model, you should specify the trophic enrichment factors. You measure bulk stable isotope and Compound\nspecific. Which did you use for the Bayesian model? It is not clear if the bulk or the Compound specific. Maybe a table with\nthe values of source and TEF would be helpful for the reader to reproduce the model.\nResults and Discussion:\nIn the supplementary material you explain the analyses of the functional groups, which are key for the mixing model\nanalyses. Somehow you should include the results and discussion of the functional groups in the main text.\nFigure 3- In the caption explain that the graph is the results of the mixing model\nLine 171- How did you measure the organic? Which organic are you referring? please explain and rephrase.\nline 332- Please rephrase for clearness the sentence Reference material was analysed every 10 samples, and the\ncorresponding response factor of the reference material was used to determine the\nparticulate nitrogen (PN) concentration ( g N-PN). What do you mean with response factor? Please explain\nSupplementary material\nS8- please define SD on the table caption\nS10, please explain why just these taxa were used.", "reviewer_2": "The authors evaluated the potential impact of deep sea mining discharge on midwater food webs via comparison of natural\n& mining-associated particles in the context of zooplankton & micronekton communities. The paper is well written, thorough,\nand highly readable. This study clearly has important implications for future deep sea mining activities and mitigation of their\necological impacts. I have some minor concerns and suggestions for improvement of this manuscript, details below.\nAbstract:\nLine 29: suggest change to “…community including zooplankton and micronekton” for clarity. Initial wording suggests there\naren’t any other fauna in the midwater. I anticipate this abstract will be read by non-biologists interested in mining impacts\nwho may not be familiar with mesopelagic communities.\nLine 37: “entire community” – as with previous comment, it would be beneficial to (briefly) outline the members of this\ncommunity. I.e., that the community extends beyond zooplankton and micronekton to large marine predators, etc.\nIntroduction:\nLine 65-66: it would be helpful to give an approximate size range of the particles & fragmented nodules at this point in the\npaper for reader visualization.\nLine 71-74: I don’t believe there’s a definition of “micronekton” in the ms. Suggest including the size class definition here to\nincrease ms readability across disciplines\nResults & Discussion:\nLine 119: why are plume and discharge particles combined here?\nLine 124: This is the only mention of “phytodetritus” in the paper. This is a useful description of the medium/large particles.\nSuggest either incorporating this description throughout or remove the single mention.\nLine 154 paragraph and associated methods: Regarding the Bayesian mixing model, please expand on why the only source\ninputs are the 3 particle size fractions collected at depth & the implications of only using those source inputs. Many\nmesopelagic micronekton undergo DVM to surface waters, where they would have access to alternate basal food web\nsources. I understand that using only the particles as inputs makes sense in the context of this study’s sample collection and\nam not suggesting a change to the methods. However, I would like to see the potential inputs of other basal sources (e.g.,\nepipelagic phytoplankton) to this food web touched on in the discussion.\nRelated comment: throughout the ms, the only mention of DVM is in Figure 4’s caption. This is an important aspect of the\ncommunity to touch on at least briefly in the context of mesopelagic food web dynamics & basal sources.\nLine 168: repeated use of “large” is a bit awkward for readability.\nLine 171: please expand on why the concentration of AAs matters biologically to increase ms readability across disciplines\nLine 205-206: suggest removing the ## values inside the parenthetical – they are nearly identical to the primary values and it\nis hard to read so many numbers in one (long) sentence. Leave the parenthetical ##s to the supplemental\nLine 213: horizontal or vertical migration away from the impacted area?\nLine 227: note that many top predators travel to the sampled mesopelagic/bathypelagic depths as well\nLine 253-261: suggest reducing the number of numeric values in this concluding paragraph for readability as they are\ndistracting from the larger themes. E.g., remove the “(85%...)” parenthetical in line 260\nLine 262-263: need more than one citation in this sentence if it refers to “other studies” plural\nLine 266: suggest change “and/or” to “alongside”. Surely both careful decision making by mining companies AND some\nlevel of regulation by agencies are needed\nLine 288 (and 97): it would be helpful to have the background particle collection depths here (without needing to go\nsearching in the SI), as they are important to interpretation of the study results.\nFigure 1: This may be a problem with my computer, but this figure appears slightly blurry. Suggest increasing text size in the\nlegend box\nFigure 2: suggest moving legend into white space of panel B for space efficiency, especially as the figure text is very small\nFigure 4: the smiley faces on the copepods are charming but perhaps distracting/emotionally biasing from the main purpose\nof the figure.\nLine 559: “vertical migration that provisions commercially harvested fishes” text is confusing. Which fisheries are you\nreferring to? Suggest ending the sentence after “migration”\nOverall a well-designed study with interesting and important results."}, "author_response": "We thank the reviewer for their kind words and for her/his careful reading of our manuscript and\nproviding useful comments and critiques.\nHere in some suggestion to clarify and improve the manuscript:\nMethods:\n-Please specify the type of filters used, how the filters were preserved (some of the information\nis on the supplementary material, but would help the reader here)\nWe used 0.7 m glass fiber filters, 6 and 53 m Nylon and polyester screens. The Nylon and\npolyester screens were cleaned using sequential rinses with HCl, DI and MeOH, while the glass\nfiber filters were combusted at 500C for 5 hours. Filters were subsampled at sea immediately\nafter the sampling cast for bulk and compound-specific analysis, wrapped individually in baked\n(500C 5 hours) aluminum foil, and frozen at -80C prior to analysis in the lab. These details\nhave been added to the “Particle Collection” section of the Methods (line 376-381). We also\nclarified in this section that the full methodology is described in the Supplementary Materials.\n-Also, it would help the reader to specify here how did you measure the amino acids\nconcentrations. You present results as N/PN, but you do not explain what these units are. Is\namino acid concentration measure as ratio between Nitrogen and Particulate Nitrogen?\nWe added a paragraph in the “Stable Isotope Analysis” section, positioned after the CSIA-AA\nNitrogen protocol and before the CSIA-AA carbon protocol, to explain how we measured the\nconcentration of amino acids (line 458-466). Briefly, we calculated the concentration of each\namino acid from the combined peak area for all masses. The peak area was calibrated using\nreference compounds (in nanograms). The total AA content from in a sample was divided by the\ntotal N content (in micrograms) from bulk analysis in the same sample. This ratio is used to\nfacilitate comparisons between samples.\n- Bayesian model, you should specify the trophic enrichment factors. You measure bulk stable\nisotope and Compound specific. Which did you use for the Bayesian model? It is not clear if the\nbulk or the Compound specific. Maybe a table with the values of source and TEF would be\nhelpful for the reader to reproduce the model.\nThe input to the Bayesian mixing model were 15N , 15N , and 13C , values all collected\nPhe Lys Leu\nthrough CSIA-AA; no bulk isotope data were used. We selected these amino acids because\nthey are source and essential amino acids, which have been shown in the literature to be non-\nfractionating during trophic transfers. Consequently, no trophic discrimination factors need be\napplied. We added text in the paragraph discussing the mixing model parameters to clarify this\n(line 485-488).\nResults and Discussion:\nIn the supplementary material you explain the analyses of the functional groups, which are key\nfor the mixing model analyses. Somehow you should include the results and discussion of the\nfunctional groups in the main text.\nWe respectfully disagree with the reviewer's suggestion. Including additional detailed results\nand discussion of the functional groups in the main text would unnecessarily lengthen the\nmanuscript and divert attention from its primary focus. Furthermore, separate manuscripts that\nspecifically address the ecology of zooplankton and micronekton are currently in preparation\nand will be submitted to more specialized journals.\nFigure 3- In the caption explain that the graph is the results of the mixing model\nWe added text that this plot shows the mixing model mean contribution of particles in the figure\ntitle and caption.\nLine 171- How did you measure the organic? Which organic are you referring? please explain\nand rephrase.\nWe apologize for the confusion. This refers to the total AA content in the particles and should\nhave stated “significantly lower in amino acid content”. We have removed the word “organic” for\nclarity and added the relevant figure number to those sentences (line 211-215). The following\nsentence, which essentially repeated the information about higher particle concentrations within\nthe plume compared to the background, has been replaced with content aimed at improving\nreadability for reader from other disciplines, as requested by Reviewer #2.\nline 332- Please rephrase for clearness the sentence Reference material was analyzed every\n10 samples, and the corresponding response factor of the reference material was used to\ndetermine the particulate nitrogen (PN) concentration (g N-PN). What do you mean with\nresponse factor? Please explain\nThe response factor is based on the peak area (measured in volt-seconds, Vs) and known\nconcentration of the reference materials, and is used to determine the concentration of\nindividual amino acids in the sample. The same procedure is used to determine the\nconcentration of particulate carbon and nitrogen content using Elemental Analysis-Isotope ratio\nmass spectrometry. We added text to clarify this, including the appropriate units: (Vs [ug PN]-1)\nfor bulk particulate nitrogen (line 438-439) and (Vs [nmol AA]-1) for CSIA-AA (line 451-452).\nSupplementary material\nS8- please define SD on the table caption\nWe added a sentence to the Table S8 caption clarifying SD is the standard deviation of the\ngrouped results.\nS10, please explain why just these taxa were used.\nTaxa were selected as they were abundant representatives of different functional groups\ncollected in both the Lower Oxycline and Suboxycline, as described in the Methods Section. We\nadded a sentence to Table S10 caption clarifying this.\nWe thank the reviewer for their thoughtful comments. We have re-written parts of the manuscript\nto make it more understandable to a general audience. Responses to specific comments follow.\nAbstract:\nLine 29: suggest change to “…community including zooplankton and micronekton” for clarity.\nInitial wording suggests there aren’t any other fauna in the midwater. I anticipate this abstract\nwill be read by non-biologists interested in mining impacts who may not be familiar with\nmesopelagic communities.\nExcellent suggestion. We added “including” as suggested (line 31).\nLine 37: “entire community” – as with previous comment, it would be beneficial to (briefly)\noutline the members of this community. I.e., that the community extends beyond zooplankton\nand micronekton to large marine predators, etc.\nWe added “extending beyond zooplankton and micronekton to nekton, including large marine\npredators” (line 37-39).\nIntroduction:\nLine 65-66: it would be helpful to give an approximate size range of the particles & fragmented\nnodules at this point in the paper for reader visualization.\nWe added the typical size range of <2 – 63 um to this sentence, and include an appropriate\ncitation (line 78).\nLine 71-74: I don’t believe there’s a definition of “micronekton” in the ms. Suggest including the\nsize class definition here to increase ms readability across disciplines\nWe added the clarification “small fish, cephalopods, and crustacea, ~2-20 cm in size” in the last\nsentence, with citation, to increase readability (line 96).\nResults & Discussion:\nLine 119: why are plume and discharge particles combined here?\nWe were allocated limited time on the mining test cruise to collect plume and discharge particle\nsamples. Of those particles collected, we found no difference between plume and discharge\nparticles. Therefore, these sample types were combined, providing a direct comparison between\nbackground particle samples and mining-associated particles. We changed “plume particles” in\nthe first sentence of this paragraph to “mining-associated particles” to avoid ambiguity (line\n148).\nLine 124: This is the only mention of “phytodetritus” in the paper. This is a useful description of\nthe medium/large particles. Suggest either incorporating this description throughout or remove\nthe single mention.\nThank you for this observation. We removed the term “phytodetritus” since medium/large\nparticles could also be composed of fecal material or aggregations, and this paper does not\nexplicitly explore the origins and composition of the particles, although this will be a subject of a\nsubsequent manuscript.\nLine 154 paragraph and associated methods: Regarding the Bayesian mixing model, please\nexpand on why the only source inputs are the 3 particle size fractions collected at depth & the\nimplications of only using those source inputs. Many\nmesopelagic micronekton undergo DVM to surface waters, where they would have access to alternate basal food web\nsources. I understand that using only the particles as inputs makes sense in the context of this study’s sample collection and\nam not suggesting a change to the methods. However, I would like to see the potential inputs of other basal sources (e.g.,\nepipelagic phytoplankton) to this food web touched on in the discussion.\nWe agree with this comment and have added text to the manuscript addressing this point.\nAcknowledging the presence of DVM, we had already considered its potential influence and\ntook steps to minimize its impact. As described in the revised text, all samples were selected\nfrom nighttime tows to preferentially target non-migratory resident species. While this\nmanuscript only briefly mentions it, ongoing studies (manuscripts in preparation) will explore this\ntopic in greater detail with additional data. Current evidence indicates that only ~20% of the\nzooplankton biomass present below the OMZ, in the deep mesopelagic, migrates to the upper\nocean at night. Lastly, the micronekton taxa analyzed were specifically selected for their non-\nmigratory behavior, further limiting potential contributions from epipelagic or DVM-influenced\ntrophic sources (line 193-200).\nRelated comment: throughout the ms, the only mention of DVM is in Figure 4’s caption. This is\nan important aspect of the community to touch on at least briefly in the context of mesopelagic food web dynamics & basal sources.\nWe agree with this suggestion and have now added text to the Introduction (lines 72-74) with\nappropriate citations that acknowledges that DVM vertically mixes food webs. We have also\nadded clarity at lines 193-200 regarding the rationale for a limited influence of DVM on our\nmeso- and bathypelagic material. Lastly, we have added text (lines 298-300) that describes how\nDVM may expand the zone of influence of the mine waste plume.\nLine 168: repeated use of “large” is a bit awkward for readability.\nThank you. We changed this sentence to: “In sum, our mixing model indicates that large\nparticles make a substantial contribution to the base of the food web below the OMZ (Fig 3), a\nregion expected to be impacted by mining waste plumes.” (Line 211-213)\nLine 171: please expand on why the concentration of AAs matters biologically to increase ms\nreadability across disciplines\nWe added a sentence explaining that reduced AA concentration is a key indicator of the reduced\nnutritional value of particles, leading to less protein intake for the same foraging effort. (Line\n215-218).\nLine 205-206: suggest removing the ## values inside the parenthetical – they are nearly\nidentical to the primary values and it is hard to read so many numbers in one (long) sentence.\nLeave the parenthetical ##s to the supplemental\nWe removed ##’s inside parenthetical as suggested to improve readability. They are also\npresent in supplemental materials.\nLine 213: horizontal or vertical migration away from the impacted area?\nThis is true for the more mobile micronekton, although we suspect that less mobile animals may\nnot be able to easily swim away from the plume, and we now say this on line 270. Zooplankton\nwould only be capable of vertical repositioning relative to the plume.\nLine 227: note that many top predators travel to the sampled mesopelagic/bathypelagic depths\nas well\nThis is a good point, although the depths of migration for a number of taxa are likely shallower in\nthis region due to the presence of the pronounced OMZ. There isn’t space to go into the details,\nbut we agree with the reviewer on the general point and have added a statement and citations\non line 300-302.\nLine 253-261: suggest reducing the number of numeric values in this concluding paragraph for\nreadability as they are distracting from the larger themes. E.g., remove the “(85%...)”\nparenthetical in line 260\nThank you for the suggestion. We removed (85%), changed 53% of zooplankton taxa to\n“Approximately half” and 60% of micronekton taxa to “more than half”. We kept 65% of\nconsumer samples as this is based on the isotopic mixing model and is the main focus of the\npaper. (line 325-329)\nLine 262-263: need more than one citation in this sentence if it refers to “other studies” plural\nWe changed “other studies have also confirmed…” to “there are confirmed…”. (line 331)\nLine 266: suggest change “and/or” to “alongside”. Surely both careful decision making by mining companies AND some level of regulation by agencies are needed\nWe changed this sentence to read: “Mitigation is possible through careful selection of discharge\ndepths by mining companies, combined with regulation by national and international agencies.”\n(line 334-336).\nLine 288 (and 97): it would be helpful to have the background particle collection depths here\n(without needing to go searching in the SI), as they are important to interpretation of the study\nresults.\nWe added depth ranges of 800-1500m on line 127-128, and clarifying lower mesopelagic below\nthe OMZ core 800-1000m and upper bathypelagic 1000-1500m on line 382-384.\nFigure 1: This may be a problem with my computer, but this figure appears slightly blurry.\nSuggest increasing text size in the legend box\nWe changed the figure so that the legend text size is increased to be the same size as the axis\ntitle and particle size label. We slightly increased the width of the figure to accommodate the\nincrease in legend text size.\nFigure 2: suggest moving legend into white space of panel B for space efficiency, especially as\nthe figure text is very small\nWe moved the legend as recommended, and increased the text size to improve readability.\nFigure 4: the smiley faces on the copepods are charming but perhaps distracting/emotionally\nbiasing from the main purpose of the figure.\nWe aim to convey that the health of zooplankton affected by the mining plume may be\ncompromised. We regretfully agree with Reviewer #2 that, while the smile and frown on the\ncopepods are charming, they may be distracting and introduce emotional bias. Live copepods\nhold their antennules perpendicular to their body; however, after death, the antennules typically\ndroop and lie more parallel to the body—a characteristic \"dead pose.\" We have included this\nvisual cue to indicate \"unhappy\" copepods in the plume-influenced food web, but recognize that\nit may be too subtle for those unfamiliar with zooplankton. We note that X-shaped eyes are a\nwidely recognized cartoon convention for depicting dead or dying animals. Therefore, we use\nboth droopy antennules and X-eyes to convey comprised zooplankton heath in this figure.\nLine 559: “vertical migration that provisions commercially harvested fishes” text is confusing.\nWhich fisheries are you referring to? Suggest ending the sentence after “migration”\nWe ended sentence after “migration” as suggested.\nOverall a well-designed study with interesting and important results."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I am happy with the way the authors improved the manuscript with my suggestions and reviewer #2.\nThe paper is well-written, comprehensive, and highly accessible. It offers valuable insights with clear implications for future\ndeep-sea mining operations and the mitigation of their ecological impacts.\nI recommend the publication of the manuscript NGS-2013-03-00534.", "reviewer_2": "The authors have nicely revised the manuscript. I recommend it for publication"}, "author_response": ""}]

Decision letter