Elsevier

Toxicon

Volume 140, 15 December 2017, Pages 45-59
Toxicon

Nodularin from benthic freshwater periphyton and implications for trophic transfer

https://doi.org/10.1016/j.toxicon.2017.10.023Get rights and content

Highlights

  • Periphyton, smallmouth juvenile bass and adult bass were collected from Pennsylvania, USA.

  • ELISA, Individual Variant Analysis (LC-MS/MS) & MMPB were used to measure Microcystins/Nodularins.

  • NOD-R was determined to be present in freshwater periphyton.

  • NOD-R was also detected in juvenile smallmouth bass and adult livers.

Abstract

In 2013 and 2015, the Pennsylvania Department of Environmental Protection conducted a survey of lotic habitats within the Susquehanna, Delaware, and Ohio River basins in Pennsylvania, USA, to screen for microcystins/nodularins (MCs/NODs) in algae communities and smallmouth bass (Micropterus dolomieu). Periphyton (68 from 41 sites), juvenile whole fish (153 from 19 sites) and adult fish liver (115 from 16 sites) samples were collected and screened using an Adda enzyme-linked immunosorbent assay (ELISA). Samples that were positive for MCs/NODs were further analyzed using LC-MS/MS, including 14 variants of microcystin and NOD-R and the MMPB technique. The ELISA was positive for 47% of the periphyton collections, with NOD-R confirmed (0.7–82.2 ng g−1 d.w.) in 20 samples. NOD-R was confirmed in 10 of 15 positive juvenile whole fish samples (0.8–16.7 ng g−1 w.w.) and in 2 of 8 liver samples (1.7 & 2.8 ng g−1 w.w.). The MMPB method resulted in total MCs/NODs measured in periphyton (2.2–1269 ng g−1 d.w.), juvenile whole fish (5.0–210 ng g−1 d.w.) and adult livers (8.5–29.5 ng g−1 d.w.). This work illustrates that NOD-R is present in freshwater benthic algae in the USA, which has broader implications for monitoring and trophic transfer.

Introduction

Cyanobacteria, both freshwater and marine, have been known to produce a variety of bioactive secondary metabolites, many that act as toxins (Chorus and Bartram, 1999; Pearson et al., 2010). Hepatotoxic classes of cyanotoxins include the heptapeptide microcystins and the pentapeptide nodularins (Rinehart and Harada, 1988). Nodularins are considered mainly marine/brackish toxins produced by Nodularia spumigena and microcystins are typically related to freshwater cyanobacteria (Chorus and Bartram, 1999, Rastogi et al., 2014, Zurawell et al., 2005). Although uncommon, nodularins have been detected in a freshwater planktonic Nodularia spumigena and a terrestrial Nostoc source (Akcaalan et al., 2009, Gehringer et al., 2012). Freshwater benthic sources of nodularin include a strain of Nodularia sphaerocarpa (PCC 7804) isolated from a thermal spring in France, in mixed assemblages of cyanobacterial mats collected from lakes in New Zealand, and from Iningainema pulvinus gen nov., sp nov. isolated from a freshwater spring in tropical, north-eastern Australia (Beattie et al., 2000, McGregor and Sendall, 2017, Wood et al., 2012a). In the United States, information on nodularin produced in freshwater is limited. In the 2007 Environmental Protection Agency National Lakes Assessment, USA, Nodularia was detected in freshwater lakes in South and North Dakota, with an Adda microcystins/nodularins ELISA used to screen samples (Loftin et al., 2016). While Adda-containing microcystins/nodularins (MCs/NODs) were detected, nodularin presence was not confirmed using a more specific technique and it is not known if microcystins or nodularins produced the positive assay response.

The detection of MCs/NODs can be achieved using multiple approaches. Nodularins and microcystins share a unique amino acid, the Adda (3-Amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) (Rinehart and Harada, 1988). The common moiety allows for the detection of free MCs/NODs with an Adda-specific enzyme-linked immunosorbent assay (ELISA), as well as the use of the MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) technique for total MCs/NODs (unbound & bound/conjugated) (Fischer et al., 2001, Sano et al., 1992, Williams et al., 1997). However, there is not a lot known about the quantitative accuracy of the Adda ELISA when measuring nodularins, which is further confounded when microcystin variants are present. The MMPB technique, when calibrated using MC-LR (M.W. 995 g mol−1) measures nodularin-R (M.W. 825 g mol−1) hypothetically at 120%, based on the stoichiometric formation. When multiple variants of microcystins and/or nodularin are present in a given sample, the error of measurements is therefore between 89 and 120% based on the molar formation of MMPB. A more accurate and specific method for microcystin and nodularin analysis is liquid chromatography coupled with mass spectrometry/mass spectrometry (LC-MS/MS). However, in order to confidently identify and quantitate specific variants in complex matrices, standards are required for instrument calibration and standard addition. Due to this limitation, individual variant analysis likely underestimates total free MCs/NODs (Foss and Aubel, 2015).

To date, there are 10 naturally occurring nodularins described (Table 1) and two dihydrostereoisomers ([D-MeAbu5]nodularin & [L-MeAbu5 ]nodularin) derived artificially in the lab (Namikoshi et al., 1993). Nodularin (NOD-R) was first purified from Nodularia spumigena following livestock losses in New Zealand and found to be structurally similar to microcystins (Rinehart and Harada, 1988). Motuporin (NOD-V) was then isolated from a Papua New Guinea sponge, Theonella swinhoei, and is thought to have originally derived from a microbial source (Silva et al., 1992). Three additional Nodularia spumigena derived variants were elucidated by Namikoshi et al. (1994), including a non-toxic [(6Z)Adda3]NOD-R, and two desmethylated variants, [DMAdda3]NOD-R and [DAsp1]NOD-R. The [Glu4(OMe)]NOD-R, originally described by Rinehart et al. (1994) and reported to be non-toxic, was also detected in Nodularia from the Baltic Sea (Mazur-Marzec et al., 2006). Other nodularins described by Mazur-Marzec et al. (2006) included the desmethylated [DHb5]NOD-R and a nodularin with an additional methyl group on the Adda moiety, [MeAdda3]NOD-R. Another nodularin with homoarginine instead of arginine (NOD-Har) has been found to be the dominant variant in Nodularia sphaerocarpa (PCC 7804) (Beattie et al., 2000, Saito et al., 2001).

The trophic transfer of nodularin from cyanobacteria to both invertebrates and vertebrates has been illustrated, with the majority of current research conducted relating to saline or brackish systems. However, one freshwater study demonstrated that NOD produced by benthic freshwater cyanobacteria mats accumulated in freshwater crayfish (Paranephrops planifrons) through direct and potentially secondary consumption (Wood et al., 2012b). Nodularia blooms in the Baltic Sea have contributed to the bulk of the research conducted to date relating to nodularin exposure and trophic transfer, including primary and secondary prey items. The copepod Eurytemora affinis has been shown to accumulate NOD from multiple exposure routes, including the direct ingestion of N. spumigena, exposure to dissolved fractions and through the microbial food web (Sopanen et al., 2009). Zooplankton field collections containing Eurytemora affinis and the cladoceran Pleopsis polyphemoides have also been shown to accumulate nodularin up to 2360 ng g−1 dry weight (d.w.) (Karjalainen et al., 2008, Karjalainen et al., 2006, Karjalainen et al., 2005). Small marine fish, such as herring (Clupea harengus membras), sprat (Sprattus sprattus) and three-spined stickleback (Gasterosteus aculeatus) also accumulate NOD, with levels reported up to 800 ng g−1 d.w. (Karjalainen et al., 2008). The species of herring studied is described as a facultative zooplanktivorus filter-feeder (Blaxter, 1990), the sprat feeds on planktonic crustaceans (Flintegård, 1987), and the three-spined stickleback, which is generally confined to coastal areas when found at sea, is considered omnivorous (Scott and Crossman, 1973).

The transfer of NOD to higher trophic levels has been investigated as well. NOD from three-spined stickleback (2.8–700 ng g−1 d.w.) and herring (0–90 ng g−1 d.w.) to salmon (Salmo salar) in the northern Baltic Sea was investigated, with only a single low level (10 ng g−1 d.w.) detected in the salmon liver (Sipiä et al., 2006). Adult European flounder (Platichthys flesus) livers collected from the Baltic Sea have been found to accumulate levels of NOD, sometimes > 2000 ng g−1 d.w. (Kankaanpää et al., 2005, Mazur-Marzec et al., 2007, Persson et al., 2009, Sipiä et al., 2002a, Sipiä et al., 2002b). High levels of NOD (>2000 ng g−1) in the blue mussel (Mytilus edulis), a flounder prey item, further supports higher level trophic transfer of NOD in the Baltic Sea (Sipiä et al., 2001). In a brackish New Zealand lake, NOD-R accumulation reached 147 ng g−1 in shortfin eels (Anguilla australis), a potential food source to the indigenous Māori people (Dolamore et al., 2016). In southeast Queensland, Australia, a fish mortality event led to the detection of high NOD-R concentrations in sea mullet (Mugil cephalus) livers (median 43,600 ng g−1 d.w.) (Stewart et al., 2012). Extrapolating results of field studies conducted in saline/brackish waters to freshwater systems is not fully representative, but may still be considered informative since freshwater nodularin studies are limited. Additionally, species-specific effects and biotransformation coupled with temporal limitations in sample collection highlight the difficulty in drawing conclusions with regard to specific toxic effects from environmental exposure to NOD.

Specific to this work, wild populations of smallmouth bass (Micropterus dolomieu) in Pennsylvania, just as with those species from saline and brackish systems, are likely exposed to nodularin via trophic transfer. However, ontogenetic diet shifts alter the specific pathways of exposure as the bass matures. The first foods consumed by juvenile smallmouth bass include chironomid larva, rotifers and micro-crustaceans, such as copepods (Easton and Orth, 1992). In general, as smallmouth bass mouth gape increases and their swimming prowess improves due to fin development, they feed increasingly on larger invertebrates (e.g Ephemeroptera, Gammarus and caddis fly larva) and other fish (Brewer and Orth, 2014, Easton and Orth, 1992, George and Hadley, 1979). However, fish never became a dominant prey choice with the juvenile smallmouth bass in the New River, perhaps due to the appreciable availability of invertebrates at that location (Easton and Orth, 1992). Thus, it would seem likely that ontogenetic shifts in diet, though generally predictable, should be expected to vary from site to site based upon prey availability, thereby changing potential sources of NOD exposure.

Adult smallmouth bass are considered opportunistic top carnivores with feeding behavior more benthically oriented since they typically forage lower in the water column and near the stream bottom (Brewer and Orth, 2014). In a Wisconsin lake, adult smallmouth bass diet comprised largely of crayfish throughout the summer, with seasonal Ephemeroptera and Oodonate consumption being the next most abundant food source (Frey et al., 2003). As noted in the Wood et al. (2012b) study, freshwater crayfish have the potential to accumulate NOD from benthic cyanobacteria. In this Wisconsin lake, fish, such as yellow perch and burbot, always comprised a smaller portion of the adult smallmouth bass diet compared to that of crayfish. Based on these studies, the trophic transfer pathway of cyanotoxin to juvenile and adult smallmouth bass is likely through secondary consumption of prey rather than by direct consumption of cyanobacteria.

Adult smallmouth bass will occupy both run and pool habitats in streams (Brewer and Orth, 2014). During the spawn, males establish territories near the shoreline and also provide care during the egg larval development (Brewer and Orth, 2014). In general, juvenile microhabitat selection is said to be “plastic” and is related to ontogeny (Brewer and Orth, 2014). Juvenile smallmouth bass, much like adults, prefer cover but find it in lower velocity areas (Brewer and Orth, 2014), which may increase the chances of immersion exposure to NOD. If absorption of NOD by immersion occurs in a fashion similar to that of microcystin, then it may be expected that nodularin would absorb through the dermal surface and the gills (Pavagadhi and Balasubramanian, 2013).

Background: In 2013, the Pennsylvania Department of Environmental Protection (PA DEP) conducted a pilot survey of lotic habitats from the same study areas reported in this work. A total of 17 periphyton samples were analyzed using the Adda ELISA, individual variant analysis (LC-MS/MS) and MMPB. The results (unpublished data) showed nine positive for MCs/NODs by ELISA (15–923 ng g−1 d.w.), of which, four had NOD-R (5–302 ng g−1 d.w.) and one [DAsp3]MC-LR (12 ng g−1 d.w.). MMPB values were found to be between the individual variant analysis and ELISA values (5–187 ng g−1 d.w.). This investigation led to an expansion of the original collection efforts to include more collection sites and smallmouth bass (Micropterus dolomieu) juvenile whole fish and adult livers. The methods used in this work included the Adda ELISA for screening samples, followed with confirmatory analyses for positive samples. Confirmatory techniques included LC-MS/MS to quantitate specific variants of microcystins/nodularins (MCsNODs) with commercially available standards (15 analytes) and the MMPB technique to address quantitative disparities and as further confirmation of microcystins/nodularins presence.

Section snippets

Site description

Periphyton collection sites were chosen in this survey to characterize microcystins/nodularins (MCs/NODs) in the Susquehanna, Ohio, and Delaware River basins within Pennsylvania. Juvenile and adult smallmouth bass were also harvested from these same river basins. The decline of the smallmouth bass sport fishery, primarily in the middle Susquehanna River Mainstem (Susquehanna River from Selinsgrove to York Haven) and the need to realize any possible cyanotoxin involvement in that decline, led to

ELISA and MMPB response to NOD-R

Nodularin-R resulted in approximately 200% (199–242%) cross-reactivity with the microcystins/nodularins Adda ELISA, when compared to MC-LR (certified reference standards). The curves were generated using 4-parameter logistic regression (Fig. 2). The ELISA data reported for the periphyton and tissue samples were integrated from an MC-LR curve and are likely overestimations of total toxin levels when NOD-R is present.

The experimental oxidation and MMPB analysis of certified reference standards

Discussion

Three different analysis techniques were employed for the analysis of periphyton, juvenile whole fish and adult liver samples. Considerations regarding the quantitative data achieved from each analysis approach used in this work are outlined in Table 3. While analysis techniques are evolving to be more specific and comprehensive, each approach has its own set of caveats, as illustrated in this work.

The total MCs/NODs concentrations achieved from MMPB analysis of periphyton samples illustrated

Conclusions

The results of this research showed that nodularin is present in freshwater benthic habitats in North America, and the analysis approach is imperative to making conclusions based on MC/NOD measurements. For instance, ELISA is frequently used as the primary method for MC/NOD analysis, with positive responses not further investigated, as in the National Lake Assessment (Loftin et al., 2016). Therefore, it is currently unknown to what extent that samples sourced from freshwater contain nodularin,

Conflicts of interest

The authors declare no conflict of interest.

Ethical statement

The authors affirm that the submitted manuscript is an original work, has not been published before, and is not submitted for publication elsewhere. It does not contain unlawful statements, and it does not infringe on the rights of others. The authors have no relationship with any manufactures or distributors of products used in this manuscript. This paper reflects our own research and analysis and does so in an accurate and unbiased manner. All authors have contributed significantly to the

Acknowledgments

Funding for this work was provided under contract# 35-131571 through the PA DEP. The authors recognize the following PA DEP staff for their valuable assistance: Gary Gocek, Charlie McGarrell, Travis Stoe, William Brown, Michael Lookenbill, Dustin Shull, Erika Bendick, Mark Hoger, Justin Lorson, and Mark Brickner.

References (54)

  • V.O. Sipiä et al.

    Bioaccumulation and detoxication of nodularin in tissues of flounder (Platichthys flesus), mussels (Mytilus edulis, Dreissena polymorpha), and clams (Macoma balthica) from the northern Baltic Sea

    Ecotoxicol. Environ. Saf.

    (2002)
  • S.A. Wood et al.

    Consumption of benthic cyanobacterial mats and nodularin-R accumulation in freshwater crayfish (Paranephrops planifrons) in Lake Tikitapu (Rotorua, New Zealand)

    Harmful Algae

    (2012)
  • J.H.S. Blaxter

    The herring

    Biology

    (1990)
  • S. Brewer et al.

    Smallmouth bass Micropterus dolomieu lacepede, 1802

  • F.M. Buratti et al.

    Human glutathione transferases catalyzing the conjugation of the hepatoxin microcystin-LR

    Chem. Res. Toxicol.

    (2011)
  • I. Chorus et al.

    Toxic Cyanobacteria in Water: a Guide to Their Public Health Consequences, Monitoring and Management

    (1999)
  • B. Dolamore et al.

    Accumulation of nodularin in New Zealand shortfin eel (Anguilla australis): potential consequences for human consumption

    New Zeal. J. Mar. Freshw. Res.

    (2016)
  • R.S. Easton et al.

    Ontogenetic diet shifts of age-0 smallmouth bass (Micropterus dolomieu Lacépède) in the New River, West Virginia, USA

    Ecol. Freshw. Fish.

    (1992)
  • W.J. Fischer et al.

    Congener-independent immunoassay for microcystins and nodularins

    Environ. Sci. Technol.

    (2001)
  • H. Flintegård

    Fishes in the north sea Museum's aquaria

    (1987)
  • A.P. Frey et al.

    Diet overlap and predation between smallmouth bass and walleye in a north temperate lake

    J. Freshw. Ecol.

    (2003)
  • M.M. Gehringer et al.

    Nodularin, a cyanobacterial toxin, is synthesized in planta by symbiotic Nostoc sp

    ISME J.

    (2012)
  • E.L. George et al.

    Food and habitat partitioning between rock bass (Ambloplites rupestris) and smallmouth bass (Micropterus dolomieui) young of the year

    Trans. Am. Fish. Soc.

    (1979)
  • M. Karjalainen et al.

    Nodularin accumulation during cyanobacterial blooms and experimental depuration in zooplankton

    Mar. Biol.

    (2006)
  • M. Karjalainen et al.

    Nodularin concentrations in Baltic Sea zooplankton and fish during a cyanobacterial bloom

    Mar. Biol.

    (2008)
  • M. Karjalainen et al.

    Trophic transfer of cyanobacterial toxins from zooplankton to planktivores: consequences for pike larvae and mysid shrimps

    Environ. Toxicol.

    (2005)
  • F. Kondo et al.

    Formation, characterization, and toxicity of the glutathione and cysteine conjugates of toxic heptapeptide microcystins

    Chem. Res. Toxicol.

    (1992)
  • Cited by (0)

    View full text