Endocrine Disruption in Crustaceans: Mechanisms, Environmental Occurrence, and Physiological Impacts

Definitions, Classifications, and Mechanisms of Endocrine Disrupting Compounds

Definitions and Regulatory Context
- An endocrine disruptor (EDC) is defined by WHO/IPCS as an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse effects in an intact organism, or its progeny, or (sub)populations.
- A potential endocrine disruptor is an exogenous substance or mixture that possesses properties that might be expected to lead to endocrine disruption in an intact organism, or its progeny, or (sub)populations.
- The term endocrine disruptor compounds (EDCs) was introduced in $1991$ , leading to progressive restrictions in environmental legislation worldwide.
Quantitative Structure-Activity Relationship (QSAR) Drivers
- Chemical structure dictates the likelihood of a pollutant acting as an EDC.
- The presence of halogens, sulfur, and/or phosphorus within a chemical structure significantly increases the probability of endocrine-disrupting activity.
- A high degree of lipophilicity represents a primary secondary factor facilitating cellular uptake and bioaccumulation.
Cellular and Molecular Modes of Action
- Hormone Synthesis in Endocrine Cells: EDCs alter the transcription of DNA to mRNA (including epigenetic modifications), the translation of mRNA to proteins (disrupting peptide hormones or key enzymes involved in steroidogenesis and hormonal synthesis), post-translational protein processing, and/or exocytotic hormone secretion.
- Hormone Distribution and Clearance: EDCs alter systemic transport by interfering with binding proteins in blood or hemolymph, or by modifying the enzymatic rates of hormonal degradation and clearance.
- Signal Transduction in Target Cells: EDCs interact directly with nuclear or cell-surface receptors as agonists or antagonists (e.g., xenoestrogens acting as agonist ligands in vertebrates). EDCs also disrupt signal transduction by down-regulating or up-regulating receptor expression levels or inhibiting intracellular second-messenger cascades.
- Cellular Proliferation: EDCs disrupt normal mitotic activity and tissue turnover in both endocrine glands and target organs.

Possible modes of action of EDCs in cellular pathways

Major Classes of Endocrine Disruptors
- Phenol-Derived Compounds: Includes alkylphenols and bisphenol A (BPA). BPA is widespread in plastics, food-storage materials, and thermal receipt paper, operating as a potent xenoestrogen.
- Pesticides and Polychlorinated Biphenyls (PCBs): Includes organochlorines such as endosulfan, DDT, and DDT metabolites. Triazine herbicides, such as atrazine, stimulate estrogen production by inhibiting the aromatase enzyme.
- Flame Retardants: Brominated and organophosphorus flame retardants reduce circulating thyroid hormone levels in aquatic species.
- Pharmaceuticals and Personal Care Products (PPCPs): Non-steroidal anti-inflammatory drugs (NSAIDs) disrupt cyclooxygenase activity and prostaglandin synthesis. Synthetic estrogens like $17\beta\text{-estradiol}$ and $17\times10^0\text{-ethinylestradiol}$ ( $17\text{-ethinylestradiol}$ or EE2) act as direct xenoestrogens. Ultraviolet (UV) filters used in sunscreens also exert xenoestrogenic effects.
- Organometallic Compounds: Tributyltin (TBT) disrupts nuclear receptor signaling, ecdysteroid pathways, and neuroendocrine regulation.
- Insect Growth Regulators (IGRs): Juvenile hormone mimics (e.g., pyriproxyfen, fenoxycarb, methoprene) designed to prevent insect maturation cross-react with crustacean juvenoid receptors due to ancestral structural conservation.

Environmental Occurrence, Bioaccumulation, and Ecological Exposure

Ecotoxicological Evidence in Wildlife
- Aquatic organisms experience widespread reproductive failure, including imposex in gastropod mollusks exposed to TBT antifouling paints and elevated intersexuality in marine copepods exposed to municipal sewage and industrial effluents.
- Wild invertebrate populations exhibit feminization, altered sex ratios, structural malformations, and reduced population fecundity.
Quantified Contamination in Edible and Wild Crustaceans
- Seafood Monitoring: Edible crabs and shrimps contain accumulated UV-filters ranging from $1.3\text{ to }103\,\text{ng/g}$ dry weight. Canned king crab and fresh grey shrimp exhibit brominated phenol concentrations up to $22.2\,\text{ng/g}$ wet weight, surpassing safety thresholds for human and ecosystem health.
- Surface Water Steroid Burden: Natural and synthetic steroids occur globally at $\text{ng/L}$ concentrations in surface waters, driving altered sex ratios and abnormal sexual differentiation in wild copepods, Gammarus pulex, and Carcinus maenas.
- Wastewater Treatment Plant (WWTP) Effluents: Chemical and receptor-based bioassays across $14$ European countries confirm high concentrations of complex EDC mixtures entering river basins.
- Zooplankton Bioaccumulation: Early life stages of copepods (Acartia spp., Temora longicornis, and Pseudocalanus sp.) in the Baltic Sea bioaccumulate bisphenol A, octylphenol, and nonylphenol. Bisphenol A concentrations reach maximum values of $769.2\,\text{ng/g}$ dry weight.
- Estuarine and Coastal Environments: In the Tagus estuary (Portugal), UV filters accumulate in bivalves and crustaceans, with a mean concentration of $3.93\,\mu\text{g/kg}$ wet weight in Carcinus maenas. In the Adriatic Sea, nonylphenol contaminates $100\%$ of sampled crustacean species, with tissue burdens between $118\text{ and }399\,\text{ng/g}$ in Squilla mantis and Nephrops norvegicus.
- Polar Environments: Untreated wastewater outfalls along the Antarctic coast release personal care products, pharmaceuticals, antibiotics, and alkylphenols at $\mu\text{g/L}$ concentrations, posing significant physiological risks to native polar crustaceans.

Reproductive Endocrine Disruption and Transgenerational Toxicity

Ovarian Development, Vitellogenesis, and Steroidogenesis
- Transcriptomic and Proteomic Disruption:
  - Exposure of copepods (Eurytemora affinis) to pyriproxyfen ( $10\,\mu\text{g/L}$ for $48\,\text{h}$ ) or chlordecone causes dysregulation of over $30$ functional genes controlling gametogenesis, vitellogenin ( $\text{Vg}$ ), and FMRF-like amides.
  - Macrobrachium rosenbergii exposed to chlordecone ( $0.2\,\mu\text{g/L}$ for $30\,\text{d}$ ) exhibits severe proteomic down-regulation of proteins responsible for reproductive and developmental control.
  - Macrobrachium nipponense exposed to polystyrene nanoplastics ( $<1\,\mu\text{m}$ at $5\,\text{mg/L}$ for $28\,\text{d}$ ) shows dose-dependent transcription anomalies: low doses stimulate vertebrate-like sex steroid titers and gene expression, while high doses suppress transcription, causing complete reproductive arrest.
- Vitellogenin ( $\text{Vg}$ ) mRNA Expression Dynamics:
  - Crustaceans lack functional, classic estrogen receptors; therefore, using $\text{Vg}$ as an exclusive biomarker for xenoestrogen exposure is scientifically controversial.
  - In Daphnia magna, 4-nonylphenol ( $0.2\,\text{mg/L}$ for $72\,\text{h}$ ) and cadmium ( $20\,\mu\text{g/L}$ for $72\,\text{h}$ ) increase $\text{Vg}$ expression through anti-ecdysteroidal pathways (since ecdysteroids normally suppress $\text{Vg}$ in cladocerans).
  - Cyproterone acetate ( $2.1\,\text{mg/L}$ for $72\,\text{h}$ ) decreases $\text{Vg}$ expression in D. magna via ecdysteroidal agonist action.
  - In male amphipods (Gammarus fossarum), methoxyfenozide ( $0.001\,\mu\text{g/L}$ for $21\,\text{d}$ ), propiconazole ( $0.1\,\mu\text{g/L}$ for $21\,\text{d}$ ), and cyproterone ( $0.1\,\mu\text{g/L}$ for $21\,\text{d}$ ) induce ectopic $\text{Vg}$ accumulation.
  - Chlordane ( $1\,\text{ng/L}$ for $72\,\text{h}$ ) and lindane ( $0.1\,\mu\text{g/L}$ for $24\,\text{h}$ ) elevate $\text{Vg}$ protein levels in female Neocaridina denticulata.
  - Nonylphenol ( $0.01\,\mu\text{g/L}$ for $96\,\text{h}$ ) elevates $\text{Vg}$ in Neomysis integer.
- Biphasic Effects of Herbicide Formulations vs. Active Ingredients:
  - Chronic exposure ( $3\text{ months}$ ) of the estuarine crab Neohelice granulata to the commercial formulation Roundup Ultramax® suppresses ovarian growth, promotes mature oocyte reabsorption, and depletes $\text{Vg}$ content.
  - In contrast, pure glyphosate ( $2.5\,\text{mg/L}$ for $32\,\text{d}$ in vivo or $0.2\,\text{mg/L}$ for $24\,\text{h}$ in vitro) accelerates ovarian growth and acts synergistically with exogenous progesterone to hyper-stimulate oocyte maturation and $\text{Vg}$ synthesis.
- Ecdysteroid-Driven Ovarian Maturation:
  - In caridean shrimps and peracarids, elevated premolt ecdysteroid titers stimulate ovarian and extraovarian $\text{Vg}$ synthesis.
  - para-Phenylphenol ( $0.2\,\text{mg/L}$ for $6\,\text{h}$ ) down-regulates ecdysone receptor and $\text{Vg}$ genes in D. magna, delaying first brood release and lowering overall fecundity.
  - The fungicide fenarimol blocks $20\text{-hydroxyecdysone}$ synthesis, disrupting both ecdysis and vitellogenesis in amphipods.
  - Ovarian tissue secretes ecdysteroids directly to soften vulvar cuticle hinges in N. granulata, facilitating copulation during hard-shell intermolt periods.
Interference with Methyl Farnesoate (MF) Signaling
- Methyl farnesoate (the unepoxidated form of insect juvenile hormone III) regulates crustacean gonad maturation and mediates environmental sex determination.
- Pyriproxyfen ( $2\,\text{kg/ha}$ equivalent field dose for $65\,\text{d}$ ) forces precocious ovarian maturation and structural damage in the land crab Gecarcoidea natalis.
- Juvenoid mimic potency for inducing male offspring in D. magna follows the hierarchy: Pyriproxyfen ( $\text{EC}_{50} = 0.11\,\mu\text{g/L}$ across $7\text{--}10\,\text{d}$ ) = Fenoxycarb ( $\text{EC}_{50} = 0.1\,\mu\text{g/L}$ across $7\text{--}10\,\text{d}$ , inducing $100\%$ male broods at $1\,\mu\text{g/L}$ ) > Methoprene ( $\text{EC}_{50} = 0.49\,\text{mg/L}$ , inducing weaker male production, delayed maturity, and reduced fecundity at $0.2\,\text{nM}$ over $7\,\text{d}$ ).
- Atrazine ( $2.5\,\text{mg/L}$ ) and endosulfan sulfate ( $0.23\,\text{mg/L}$ ) act as weak MF agonists, antagonizing the stronger juvenoid activity of fenoxycarb in D. magna.
- NMDA receptor agonists/antagonists and serotoninergic modulators (desipramine at $1.1\,\mu\text{M}$ ; MK-801, fluoxetine, and citalopram at $1\,\mu\text{M}$ ) alter upstream neurotransmission, disturbing MF synthesis and triggering male offspring production in Daphnia pulex.
Disruption of Neuroendocrine Axis and Neurohormones
- Bisphenol A ( $2\,\mu\text{g/L}$ for $14\,\text{d}$ ) and polystyrene microspheres ( $0.69\,\text{mg/L}$ for $14\,\text{d}$ ) upregulate gene expression of Gonad Inhibiting Hormone (GIH) in Litopenaeus vannamei, suppressing gonadal development.
- Atrazine suppresses ovarian growth in N. granulata ( $3\,\text{mg/L}$ for $3\text{ months}$ in vivo; $0.3\,\text{mg/L}$ for $24\,\text{h}$ in vitro) and Procambarus clarkii ( $1\text{ and }5\,\text{mg/L}$ for $1\text{ month}$ ) by hyper-stimulating GIH neurosecretion from the X organ-sinus gland complex.
- Cadmium ( $0.5\,\text{mg/L}$ for $24\,\text{h}$ in vitro) stimulates GIH release from Uca pugilator eyestalks, whereas cadmium ( $0.5\,\text{mg/L}$ ) and copper ( $0.1\,\text{mg/L}$ ) inhibit GIH secretion in N. granulata.
- Fluoxetine ( $0.5\,\mu\text{M}$ for $12\,\text{h}$ ) suppresses Crustacean Hyperglycemic Hormone (CHH) gene expression in Carcinus maenas.
- Xenoestrogens disrupt serotonin ( $5\text{-HT}$ ) receptor expression and monoamine oxidase degradation, indirectly suppressing $5\text{-HT}$ -stimulated Gonad Stimulating Hormone (GSH) release.
Male Reproductive Functions, Sexual Differentiation, and Offspring Development
- $17\alpha\text{-Ethinylestradiol}$ ( $0.1\,\mu\text{g/L}$ for $245\,\text{d}$ ) disrupts spermatogenesis and decreases male gnathopod size in second-generation Hyalella azteca.
- 4-Nonylphenol ( $10\,\mu\text{g/L}$ for $120\,\text{d}$ ) alters male secondary sexual characteristics (producing abnormally long second antennae) in Corophium volutator.
- Atrazine ( $2.5\,\text{mg/L}$ for $28\,\text{d}$ ) skews sexual differentiation toward a higher female-to-male ratio in juvenile Cherax quadricarinatus.
- Cadmium, methoxyfenozide, and pyriproxyfen alter over $800$ male gonadal proteins in G. fossarum, causing severe sperm count depletion.
- Glyphosate ( $1\,\text{mg/L}$ for $30\,\text{d}$ in vivo; $18\,\text{h}$ in vitro) reduces sperm count and sperm viability in N. granulata, blocking androgenic gland (AG) regulation of spermatophore synthesis.
- Prostaglandin inhibition by NSAIDs (naproxen at $3.3\,\text{mg/L}$ for $21\,\text{d}$ in Moina macrocopa; diclofenac at $1\,\text{mg/L}$ for $63\,\text{d}$ in Neocaridina davidi) impairs ovarian maturation, lowering total offspring production.
- Exogenous testosterone ( $0.31\,\text{mg/L}$ chronic or $8\,\mu\text{M}$ for $21\,\text{d}$ ) induces egg abortion, embryo malformations, and reduced fecundity in D. magna via anti-ecdysteroidal action.
Transgenerational Toxicity and Epigenetic Inheritance
- Zinc oxide nanoparticles ( $0.3\,\text{mg/L}$ for $21\,\text{d}$ ) down-regulate ovarian steroidogenic genes in D. magna, delaying first pregnancy and decreasing brood sizes; these effects persist until recovering in the $\text{F}_3$ generation.
- Simvastatin ( $0.32\,\mu\text{g/L}$ for $55\text{--}65\,\text{d}$ across generations $\text{F}_0\text{ to }\text{F}_3$ ) alters gene transcription across ecdysteroid, catecholaminergic, GABAergic, and cholinergic pathways in Gammarus locusta, driving persistent transgenerational reproductive failure via inherited DNA methylation and histone modifications.
- Pyriproxyfen ( $14.02\,\mu\text{g/L}$ for $42\,\text{d}$ ) causes multi-generational reproductive impairments in $\text{F}_0\text{ and }\text{F}_1$ lineages of D. magna.

Endocrine Disruption of Molting, Somatic Growth, and Intermediate Metabolism

Ecdysteroid Receptor (EcR) Cascades and Molting Inhibition
- The ecdysteroid signaling pathway requires the ecdysteroid receptor (EcR) to form a functional heterodimer with the Retinoid X Receptor (RxR / Ultraspiracle), binding to ecdysone response elements (EcRE) to initiate gene transcription.
- Tributyltin ( $3.8\,\text{nM}$ TBT combined with $1\,\text{nM}$ $20\text{-hydroxyecdysone}$ ) hyperactivates the RxR:EcR reporter complex in Drosophila S2 cells and causes non-physiological overexpression of the HR3 ecdysteroid-inducible gene in D. magna, arresting ecdysis and causing lethal molting failure.
- Heptachlor ( $0.33\text{ and }1\,\text{mg/L}$ for $24\,\text{h}$ ) alters circulating ecdysteroid titers and delays ecdysis in Homarus americanus larvae.
- Endosulfan ( $0.092\,\mu\text{g/L}$ for $21\,\text{d}$ ) delays molting in D. magna via competitive EcR antagonism.
- Diacyl hydrazine pesticides ( $10\,\mu\text{M}$ for $24\,\text{h}$ ) bind the crustacean EcR, functioning as weak agonists.
- Chlordecone ( $0.2\,\mu\text{g/L}$ for $30\,\text{d}$ ) depletes systemic $20\text{-hydroxyecdysone}$ titers and inhibits chitobiase activity (the $20\text{-hydroxyecdysone}$ -induced enzyme that degrades inner exoskeleton layers) in M. rosenbergii.
- Nonylphenol ( $10\text{ and }30\,\mu\text{g/L}$ for $14\,\text{d}$ ) lowers total body $20\text{-hydroxyecdysone}$ levels and reduces molting frequency in Americamysis bahia.
- Bisphenol A ( $10.66\,\text{mg/L}$ for $21\,\text{d}$ ) inhibits ecdysis in D. magna.
- Carbamazepine ( $10\,\mu\text{g/L}$ for $4\,\text{d}$ ) inhibits molting in Eriocheir sinensis by upregulating Molt-Inhibiting Hormone (MIH) gene expression in eyestalks while suppressing EcR and RxR transcription in the hepatopancreas.
- UV filters ( $75\,\mu\text{g/L}$ for $24\,\text{h}$ ) disrupt ecdysteroid-responsive transcript expression in D. magna.
- Heavy metals directly block ecdysis: Cadmium ( $0.5\,\text{mg/L}$ for $42\,\text{d}$ ) competes with calcium entering Y-organ epithelia via calcium channels during mid-premolt, suppressing the ecdysone surge required for molting in N. granulata. Conversely, in vitro Cd exposure ( $3\,\mu\text{g/L}$ for $48\,\text{h}$ ) of isolated Y-organs post-surge elevates ecdysteroid release in G. fossarum.
Endocrine Regulators of Glycemia and Energy Metabolism
- Crustacean Hyperglycemic Hormone (CHH) secreted from the X organ-sinus gland complex regulates hemolymph glucose levels during stress.
- Cadmium ( $0.5\,\text{mg/L}$ for $14\,\text{d}$ ) and copper ( $0.1\,\text{mg/L}$ for $14\,\text{d}$ ) induce severe hypoglycemia in N. granulata by blocking endogenous CHH secretion from the sinus gland; normal glycemia is fully restored by exogenous CHH injection.
- TBT suppresses CHH neurosecretion and target tissue signaling in Oziotelphusa senex and M. rosenbergii.
- Zinc oxide nanoparticles ( $0.3\,\text{mg/L}$ for $21\,\text{d}$ ) down-regulate insulin-like peptide transcripts in D. magna, reducing total body length.
- Persistent Organic Pollutants (POPs, such as PCBs and benzo[a]pyrene) induce cytochrome P450 monooxygenases (CYP45-P450 pathways), accelerating metabolic degradation and elimination of circulating ecdysteroids and juvenile hormones.

Experimental Approaches, Research Gaps, and Environmental Remediation

Adverse Outcome Pathway (AOP) Framework
- Regulatory toxicity testing integrates Adverse Outcome Pathways (AOPs) to link Molecular Initiating Events (MIEs) (e.g., receptor binding or enzyme inhibition) to organismal and population-level adverse outcomes.
- Assay methodologies combine in vitro screening (transfected cell lines, enzymatic biosensors, structural QSAR models) with chronic in vivo life-cycle bioassays.
Anatomical Distribution of Endocrine Organs and Scientific Coverage
- Research coverage is disproportionately heavy for non-nervous peripheral endocrine glands compared to neuroendocrine structures.
- Brain (B): Secretes neurohormones controlling growth and reproduction; relative proportion of studies is low to intermediate.
- X Organ-Sinus Gland Complex (SG): Located in the eyestalks; secretes neurohormones (GIH, MIH, CHH); relative proportion of studies is intermediate to high.
- Mandibular Organ (MO): Secretes methyl farnesoate; relative proportion of studies is intermediate.
- Y Organ (YO): Secretes ecdysone and ecdysteroids; relative proportion of studies is high.
- Thoracic Ganglion (TG): Secretes neurohormones (GSH, FmIHH); relative proportion of studies is low to intermediate.
- Ovary (OV): Synthesizes vitellogenin and putatively secretes steroids; relative proportion of studies is high.
- Androgenic Gland (AG): Secretes Insulin-like Androgenic Gland Hormone (IAG), directing male sexual differentiation; relative proportion of studies is intermediate.

Endocrine and neuroendocrine organs in crustaceans and study proportions

Critical Knowledge Gaps in Crustacean Endocrinology
- The precise chemical structures and signal transduction pathways of several key crustacean hormones remain uncharacterized (e.g., the exact structure of GSH).
- Emerging neurohormonal targets requiring ecotoxicological evaluation include:
  - The FmIHH gene product expressed in the thoracic ganglion, identified as a primary candidate for GSH.
  - Crustacean Female Sex Hormone (CFSH) expressed in the eyestalk, which drives phenotypic feminization and is regulated via feedback loops with IAG in hermaphroditic species.
Environmental Remediation and EDC Neutralization Technologies
- Photocatalytic Degradation: Hybrid polymer-based photocatalytic materials degrade complex organic EDCs in aqueous media.
- Fungal and Bacterial Bioremediation: Non-ligninolytic fungi (such as Umbelopsis isabellina) and specialized bacterial strains metabolize alkylphenols (nonylphenol, 4-tert-octylphenol, 4-cumylphenol), eliminating toxicity.
- Polymer Nanocomposites: Biodegradable polymers and nanocomposite filters remove pharmaceutical and industrial EDCs from wastewater stream effluents.
- Genetic Engineering: Genetically engineered microbes, optimized using in silico structural predictive modeling, degrade target EDCs in contaminated aquatic environments.