Wastewater input reductions reverse historic hypereutrophication of Boston Harbor, USA.

This paper documents the changes that followed large nutrient (N and P) and organic matter input reductions to a major metropolitan marine bay, Boston Harbor (USA). Before input reduction, its N and P inputs fell in the upper range of the < 1-> 300 gN m-2 year-1 and < 0.1-> 40 gP m-2 year-1 for coastal systems. Elevated nutrient and organic matter inputs are recognized causes of coastal eutrophication. Treatment upgrades and then diversion of its wastewater discharges offshore, lowered its N, P, and organic C inputs by 80-90%. The input decreases lowered its trophic status from hypereutrophic to eutrophic-mesotrophic. With the reversal of hypereutrophication, pelagic production and phytoplankton biomass decreased, and the nitrogen limitation relative to phosphorus limitation increased. Benthic metabolism and dissolved inorganic N fluxes decreased, and benthic-pelagic coupling was altered. Bottom-water dissolved oxygen, already at healthy levels, increased, and seagrass expanded. Coastal management requires that the changes, following the nutrient and organic matter input reductions implemented to address eutrophication, be understood. Boston Harbor's recovery, because its water column was vertically well mixed and marine, was more pronounced than in many other systems.

First identification of tetrodotoxin (TTX) in the flatworm Stylochoplana sp.; a source of TTX for the sea slug Pleurobranchaea maculata.

High concentrations of the neurotoxin tetrodotoxin (TTX) were detected by liquid chromatography-mass spectrometry (LC-MS) in the Platyhelminthes Stylochoplana sp. from Pilot Bay (Tauranga, New Zealand). This is the first detection of TTX in this genus. Concentrations were monitored from March to November (2013) and found to significantly decrease from a peak in July (avg. 551 mg kg(-1)) to November (avg. 140 mg kg(-1)). Stylochoplana sp. co-occurred with TTX-containing Pleurobranchaea maculata (Opisthobranchia). A Stylochoplana sp.-specific real-time PCR assay was developed targeting the mitochondrial cytochrome c oxidase subunit I gene to determine if P. maculata consumed Stylochoplana sp. Positive Stylochoplana sp. signals were obtained for 7 of 19 P. maculata tested. Mass calculations indicate Stylochoplana sp. could supply Pilot Bay P. maculata with the TTX required to account for the concentrations reported in previous studies (ca. 1.04 mg TTX per individual) based on an ingestion rate of one individual every 2-3 days throughout their lifetime. However, due to the lack of Stylochoplana sp. in areas with dense P. maculata populations, and high concentration (ca. 1400 mg kg(-1)) of TTX detected in some individuals, it is unlikely that Stylochoplana sp. represent the sole source of TTX in P. maculata.

First detection of tetrodotoxin in the bivalve Paphies australis by liquid chromatography coupled to triple quadrupole mass spectrometry with and without precolumn reaction.

Two methods for the determination of tetrodotoxin (TTX) in marine biota have been developed and validated using ultra-performance LC coupled to triple quadrupole MS. The direct analysis of TTX is completed in one method, while the other method detects the dehydration product of TTX after reaction with base. The methods were validated in a single-laboratory trial and used to test Paphies australis (pipi) samples collected from Whangapoua, New Zealand during April 2011. Pa. australis is a commonly eaten species of bivalve that was found to contain TTX at levels up to 0.80 mg/kg in this study. The methods exhibited recoveries ranging from 94 to 120%, and the within laboratory reproducibility ranged from 6 to 27% for Pleurobranchaea maculata (grey-side gilled sea slug) and bivalve matrixes. Use of the method using a dehydration step showed no evidence of TTX analogs in any of the samples.

Investigating diet as the source of tetrodotoxin in Pleurobranchaea maculata.

The origin of tetrodotoxin (TTX) is highly debated; researchers have postulated either an endogenous or exogenous source with the host accumulating TTX symbiotically or via food chain transmission. The aim of this study was to determine whether the grey side-gilled sea slug (Pleurobranchaea maculata) could obtain TTX from a dietary source, and to attempt to identify this source through environmental surveys. Eighteen non-toxic P. maculata were maintained in aquariums and twelve were fed a TTX-containing diet. Three P. maculata were harvested after 1 h, 24 h, 17 days and 39 days and TTX concentrations in their stomach, gonad, mantle and remaining tissue/fluids determined using liquid chromatography-mass spectrometry. Tetrodotoxin was detected in all organs/tissue after 1 h with an average uptake of 32%. This decreased throughout the experiment (21%, 15% and 9%, respectively). Benthic surveys at sites with dense populations of toxic P. maculata detected very low or no TTX in other organisms. This study demonstrates that P. maculata can accumulate TTX through their diet. However, based on the absence of an identifiable TTX source in the environment, in concert with the extremely high TTX concentrations and short life spans of P. maculata, it is unlikely to be the sole TTX source for this species.

Depuration of tetrodotoxin and changes in bacterial communities in Pleurobranchea maculata adults and egg masses maintained in captivity.

Depuration of tetrodotoxin (TTX) was investigated in adult grey side-gilled sea slugs, Pleurobranchaea maculata, maintained in captivity on a TTX-free diet. Three adults were harvested every 21 days for 126 days, and TTX concentrations were measured in organs/tissues and egg masses. Automated rRNA intergenic spacer analysis (ARISA) was used to investigate bacterial community structure in selected samples. Linear modeling of adult data demonstrated a decline (P<0.001) in average total TTX concentrations over time. Temporal data obtained from a wild population showed similar depuration rates, indicating that once adults reach a certain size, or sexual maturity, TTX is no longer produced or acquired substantially. Depuration rates differed among organs, with concentrations in the heart declining the fastest. The gonads had the slowest and least significant depuration rate indicating, at most, weak depuration of this tissue. There was a strong correlation (R(2)=0.66) between TTX concentrations in the first-laid egg masses and total TTX in the corresponding adult. These data suggest that adult P. maculata transfer TTX to their offspring, and presumably that functions as a chemical defense. ARISA data showed a shift in bacterial community structure within 3 weeks of introduction to captivity. Based on the combined data, the exact origin of TTX in P. maculata is unclear, with evidence both in favor and against a dietary source, and endogenous or bacterial production.

Tetrodotoxin concentrations in Pleurobranchaea maculata: temporal, spatial and individual variability from New Zealand populations.

Tetrodotoxin (TTX) is a potent neurotoxin that has been identified in a range of phylogenetically unrelated marine and terrestrial organisms. Tetrodotoxin was recently detected in New Zealand in Pleurobranchaea maculata (the grey side-gilled sea slug). From June 2010 to June 2011 wild specimens were collected from 10 locations around New Zealand. At one site (Narrow Neck Beach, Auckland) up to 10 individuals were collected monthly for 6 months. Attempts were also made to rear P. maculata in captivity. Tetrodotoxin was detected in samples from eight of the ten sites. The highest average (368.7 mg kg⁻¹) and maximum (1414.0 mg kg⁻¹) concentrations were measured in samples from Illiomama Rock (Auckland). Of the toxic populations tested there was significant variability in TTX concentrations among individuals, with the highest difference (62 fold) measured at Illiomama Rock. Tetrodotoxin concentrations in samples from Narrow Neck Beach varied temporally, ranging from an average of 184 mg kg⁻¹ in June 2010 to 17.5 mg kg⁻¹ by December 2010. There was no correlation between TTX levels and mass. The highest levels correspond with the egg laying season (June-August) and this, in concert with the detection of high levels of TTX in eggs and early larval stages, suggests that TTX may have a defensive function in P. maculata. Only one larva was successfully reared to full maturation and no TTX was detected.

Detection of tetrodotoxin from the grey side-gilled sea slug - Pleurobranchaea maculata, and associated dog neurotoxicosis on beaches adjacent to the Hauraki Gulf, Auckland, New Zealand.

Investigations into a series of dog poisonings on beaches in Auckland, North Island, New Zealand, resulted in the identification of tetrodotoxin (TTX) in the grey side-gilled sea slug, Pleurobranchaea maculata. The levels of TTX in P. maculata, assayed by liquid chromatography-mass spectrometry (LC-MS) ranged from 91 to 850 mg kg(-1) with a median level of 365 mg kg(-1) (n = 12). In two of the dog poisoning cases, vomit and gastrointestinal contents were found to contain TTX. Adult P. maculata were maintained in aquaria for several weeks. Levels of TTX decreased only slightly with time. While in the aquaria, P. maculata spawned, with each individual producing 2-4 egg masses. The egg masses and 2-week old larvae also contained TTX. Tests for other marine toxins were negative and no other organisms from the area contained TTX. This is the first time TTX has been identified in New Zealand and the first detection of TTX in an opisthobranch.

Algal populations controlled by fish herbivory across a wave exposure gradient on southern temperate shores.

Consumers that forage across habitats can affect communities by altering the abundance and distribution of key species. In marine communities, studies of trophic interactions have generally focused on the effects of herbivorous and predatory invertebrates on benthic algae and mussel populations. However, large mobile consumers that move across habitats, such as fishes, can strongly affect community dynamics through consumption of habitat-dominating species, but their effects often vary over environmental gradients. On temperate rocky shores, herbivorous fishes are generally a small part of the fish fauna compared to the tropics, and there is sparse evidence that they play a major direct role in algal community dynamics, particularly of large brown algae that dominate many reefs. In New Zealand, however, a wide-ranging herbivorous fish, Odax pullus, feeds exclusively on macroalgae, including Durvillaea antarctica, a large low-intertidal fucoid reaching 10 m in length and 70 kg in mass. In four experiments we tested the extent of fish herbivory and how it was affected by algal canopy structure across a gradient of wave exposure at multiple sites. Exclusion experiments showed that fish impacts greatly reduced the cover and biomass of Durvillaea and that these effects decreased with increasing wave stress and algal canopy cover, effectively restricting the alga to exposed conditions. Almost all plants were entirely removed by fish where there was a sparse algal canopy in sheltered and semi-exposed sites, but there was significantly less grazing in exposed sites. Recruit Durvillaea beneath canopies were less affected by fish grazing, but they grew slowly. Successful natural recruitment, therefore, occurred almost exclusively on exposed shores outside canopies where many plants escaped severe grazing, and growth to maturity was far greater than elsewhere. Such large and direct impacts on the local and regional distribution of large brown algal populations by mobile vertebrate consumers are rare and were mediated by an environmental gradient and plant density, both of which interact with algal demographics. The study highlights that, even though herbivorous fish diversity may be low, the impacts of particular species may still be high, even in cool temperate waters where fish herbivory is usually considered to be minimal.

The Boston Harbor Project, and large decreases in loadings of eutrophication-related materials to Boston Harbor.

Boston Harbor, a bay-estuary in the north-east USA, has recently been the site of one of the largest wastewater infrastructure projects conducted in the USA, the Boston Harbor Project (BHP). The BHP, which was conducted from 1991 to 2000, ended over a century of direct wastewater treatment facility discharges to the harbor. The BHP caused the loadings of total nitrogen (TN), total phosphorus (TP), total suspended solids (TSS) and particulate organic carbon (POC) to the harbor, to decrease by between 80% and 90%. Approximately one-third of the decreases in TSS and POC loadings occurred between 1991 and 1992; the remaining two-thirds, between 1995 and 2000. For TN and TP, the bulk of the decreases occurred between 1997 or 1998, and 2000.