Distributions and biogeochemistries of methylamines and ammonium in the Arabian Sea

Deep-Sea Research II 46 (1999) 593—615

Distributions and biogeochemistries of methylamines and ammonium in the Arabian Sea Stuart W. Gibb 
*, R. Fauzi C. Mantoura , Peter S. Liss
, Ray G. Barlow Plymouth Marine Laboratory (PML), Prospect Place, West Hoe, Plymouth, PL1 3DH, UK
School of Environmental Sciences, University of East Anglia (UEA), University Plain, Norwich, NR4 7TJ, UK Received 31 May 1996; received in revised form 27 September 1996; accepted 1 August 1997

Abstract The distributions of monomethylamine (MMA), dimethylamine (DMA), trimethylamine (TMA) and ammonium (NH>) were investigated in the Arabian Sea. The data set presented is  the first to describe the distribution of MAs on an oceanic scale. Throughout the region concentrations of NH> were up to two orders of magnitude greater than those of the MAs.  MMA (0—66 nM) was generally the most abundant MA, whilst TMA was only found at concentrations (4 nM. Low concentrations of MAs in open-ocean meso- and oligotrophic regions contrasted with the elevated levels recorded in the highly productive coastal upwelling waters of the NW Arabian Sea. In total the MAs contributed (1% dissolved organic nitrogen (DON). Depth maxima of MMA and DMA were generally associated with those of Chla, and in offshore regions, also with those of NH> (above the thermo-, oxy- and nitrataclines).  Maxima of TMA were recorded at the base of the thermo- and oxyclines, resolved from the other analytes. Through correlation studies, a degree of diatom specific MMA production was inferred (R"0.65, p(0.001) and microzooplankton grazing found to influence significantly all aqueous MA concentrations. Enhanced correlation of MMA concentrations with mesozooplankton abundance was attributed to their ability to graze diatoms. These observations are analogous to those made of equivalent oceanographic regimes in the Mediterranean Sea (Gibb et al., 1994) and support the idea that MA concentrations in seawater are primarily regulated by the productive aspects of their biological dynamics. We postulate that the nitrogen taken up in nutrient-rich, diatom-dominated regions of the Arabian Sea will be used both biosynthetically and anabolically. This may be accompanied by introduction of MMA and DMA into the aqueous phase through enzymatic precursor degradation, nitrogen detoxification, senescence or lysis and accelerated through grazing pressures, particularly that of mesozooplankton on diatoms. In contrast, under the more oligotrophic conditions recorded in the

*Corresponding author. Tel.: 0044 1752 220058; fax: 0044 1752 633101; e-mail: [email protected]. 0967-0645/99/$ - see front matter  1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 7 - 0 6 4 5 ( 9 8 ) 0 0 1 1 9 - 2

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remote Arabian Sea, those species of phytoplankton with a lower nitrogen demand are favoured, e.g., prymnesiophytes and dinoflagellates. Correspondingly lower MA concentrations are recorded in these regions.  1999 Elsevier Science Ltd. All rights reserved.

1. Introduction Nitrogen is a biologically essential element in the marine environment. In oxic seawater it may occur in each of its nine oxidation states, ranging from the thermodynamically most stable and fully oxidised form, nitrate (NO\, oxidation state #V)  to the reduced species ammonia (NH , oxidation state !III). The methylamines,  monomethylamine (MMA), dimethylamine (DMA), and trimethylamine (TMA) are simply the primary, secondary and tertiary methylated homologues of NH . Both  NH and MAs are widely distributed in the marine environment and contribute to the  regenerated nitrogen cycle (Carpenter and Capone, 1983; King, 1988). NH and the MAs are highly water soluble (Table 1), and undergo extensive  hydrogen bonding to form basic solutions. In aqueous solution, they partition between their dissolved gaseous (e.g. NH ) and solvated cationic forms (e.g.   NH> ), according to the ambient temperature and ionic strength. However, in    seawater the solvated cation typically accounts for '90% of the total dissolved concentration (i.e. NH> /NH> #NH> '0.9).         Since NH> is in the correct oxidation state for incorporation into organic matter, it  is often the preferred form of nitrogen by marine phytoplankton (Liss and Galloway, 1992). The importance of NH> as a nutrient is especially great in the euphotic  zone of oligotrophic regions where most of the primary production is based on regenerated nutrients. Although NH> may represent (1% of the total dissolved  nitrogen, it can account for 44—89% of total nitrogen assimilation and is capable of complete inhibition of nitrate (NO\) assimilation by phytoplankton (Wheeler  et al., 1989). Through the use of C-MMA as a non-metabolisable analogue of NH> in  mechanistic and transport studies the uptake of MAs by several classes of phytoplankton has been demonstrated, e.g., dinoflagellates and coccolithophores (Balch, 1986), diatoms (Wheeler and Hellebust, 1981) and microflagellates (Koike et al., 1983). However, MAs, as well as being taken up, may also be produced by phytoplankton (Abdul-Rashid, 1990): either through direct emission, or by degradation of quaternary amine osmolytes (QAs) such as choline and glycine betaine via Hoffman eliminationtype reactions (King, 1988). Hoffman elimination also occurs with the osmoticum dimethylsulphoniopropionate (DMSP), resulting in the formation of the reduced sulphur biogas dimethylsulphide (DMS). The transfer of NH and MA gases together  with DMS, across the air—sea interface to the atmosphere is suggested to play an important role in the regulation of rain, cloud water and aerosol pH (Quinn et al., 1988; Van Neste et al., 1987; Charlson et al., 1987). MAs are also capable of fuelling the nitrogen requirements of bacteria. Their uptake has been demonstrated with heterotrophic bacteria (Budd and Spencer, 1968),

9.25 0.0018 (298 K) 0.0420

5

NH  NH  17.03 !33.4

Ammonia

Species

2, 3 4

1 1

Source

0.0126

10.64 0.0015

MMA H CNH   31.06 !6.3

Monomethylamine

0.0021

10.77 0.0013

DMA (H C) NH   45.12 7.4

Dimethylamine

1 — Weast (1986), 2 — Bates and Pinching (1949), 3 — Smith and Martell (1975), 4 — Van Neste et al. (1987), 5 — Hidy (1984).

Molecular weight, MW Boiling point, b.p. (°C) Thermodynamic Stability constant, pK(298 K, ionic strength, I"0) Henry’s law constant (293 K) Solubility coefficient, a (298 K, I"0)

Abbreviation Formula

Variable (units)

Table 1 Structural formulae and properties of NH and methylamines (MAs) 

0.0015

9.80 0.0023

TMA (H C) N   59.11 2.9

Trimethylamine

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non-methylotrophs (Bicknell and Owens, 1980), cyanobacteria (Gunnersen et al., 1988), methylotrophs (Budd and Spencer, 1968) and methanogens (Winfrey and Ward, 1983). Zooplankton and higher trophic levels are proposed to contribute to MA cycling through grazing and excretion processes (King, 1988). The oceanic distribution and biogeochemical cycling of NH> and occurrence of  MAs in marine biota have been studied over many years. However, until recently the ability to reliably measure NH> and MAs in seawaters outwith coastal and shelf seas  fell beyond the capabilities of conventional analytical techniques. The introduction of highly sensitive techniques by Brzezinski (1987), Jones (1991), Hall and Aller (1992) and Harabin and Van Den Berg (1993) for the nanomolar determination of NH>, and  by Van Neste et al. (1987), Abdul-Rashid et al. (1991), Yang et al. (1993), and Gibb et al. (1995a, b) for a range of alkylamines, including the MAs, now permits their oceanic distributions to be studied. However, to date there have been no ocean-scale studies of MAs. The objective of this study was to characterise the spatial distributions of NH> and MAs in the  contrasting oceanic and biogeochemical regimes of the Arabian Sea and to interpret these distributions in relation to the gradients in phytoplankton abundance, oxygen levels and the monsoonal upwelling characteristic of the region. Since the chemical oceanography of NH> in the Arabian Sea is considered together with other micronut rients elsewhere in this volume (Woodward et al., 1999), this report will primarily focus upon the distributions and biogeochemistries of the MAs.

2. Methods Studies of MAs and NH> were carried out as part of the ºK ‘ARABESQUE’  programme; a contribution to the Joint Global Ocean Flux Study (JGOFS) in the Arabian Sea. Samples were collected and analysed on-board research cruises 210 and 212 of R.R.S. Discovery, 27/8/94—4/10/94 (ARABESQUE 1) and 16/11/94—19/12/94 (ARABESQUE 2) respectively (Fig. 1). Seawater samples were collected either using a 12-bottle CTD rosette system equipped with depth, salinity and temperature probes, oxygen electrode, transmissometer, underwater light meter and fluorimeter, or from the ship’s non-toxic supply (in-take depth &3 m). CTD bottles were sub-sampled into modified gas-tight polythene bottles (Gibb, 1994), stored in a cool-box and analysed onboard unfiltered within 6 h of collection. Concentrations of analytes in seawater were determined simultaneously using automated Flow Injection Gas Diffusion coupled to Ion Chromatography (FIGD-IC) (Gibb et al., 1995a, b). Briefly, NH> and MAs are deprotonated by addition of base  (NaOH to pH'12), and selectively transferred via diffusion across a gas-permeable Goretex membrane into an dynamic, acidic acceptor stream (40 mM methane sulphonic acid, MSA), in which they were resolvated and enriched (20—40 min). Ethylenediaminetetraacetic acid (EDTA) was added on-line to prevent precipitation of alkali earth metals under the pH conditions employed. The enriched acceptor

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597

Fig. 1. Locations of stations in the Arabian Sea occupied during Cruises 210 (27/8/94—4/10/94) and 212 (16/11/94—19/12/94) of R.R.S. Discovery. Filled station markers correspond to stations discussed in text.

stream was transferred to a Dionex DX-100 ion chromatograph (IC) in which NH>  and the MAs were separated isocratically in a MSA eluent (40 mM, at 1 ml/min) on 2;Dionex CG-10 columns within 15 min. Analytes were quantified by chemically suppressed conductimetry, using cyclo-propylamine (c-PA) and sec-butylamine (s-BA) as internal standards. IC background conductivity was maintained (0.6 lS and column back-pressure 1000—1200 psi. An eluent head pressure of N (5 psi) was applied to the eluent reservoir.

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Table 2 Operational performance data for the FIGD-IC system during ARABESQUE Species

NH> 

MMA

DMA

TMA

c-PA*

Limit of detection (nM) Linearity (R2, n"5) 0—100 nM 0—1000 nM
Reproducibility (RSD%) #20 nM #100 nM

42

2.3

0.81

0.21

n/a

n/a 0.988

0.997 0.992

0.991 0.992

0.992 0.990

0.998 0.996

15.2 8.0

6.8 4.3

6.1 2.8

4.5 2.1

2.53 3.83

All data Arabian Sea, 1994, 20 min diffusion time unless otherwise stated. *c-PA"cyclo-propylamine, internal standard. Limit of detection"3 pn (blank);
Determined by Mediterranean (1993) n/a — not applicable. Based on triplicate analyses of '2000 m oligotrophic seawater with 20 and 100 nM standard additions.

A custom-built PC-interfaced control unit and data capture unit (DCU, Phillips) were employed in series to direct switching of a solenoid valve in the flow injection system, to control IC operation, and to collect data (Gibb et al., 1995b). Operational sensitivity, reproducibility and linearity of response of the automated FIGD-IC system during the Arabian Sea studies are given in Table 2. FIGD-IC gives a measure of the total dissolved concentrations of analytes, i.e. dissolved gas plus solvated cationic forms (e.g. NH #NH> ). All data and      subsequent discussion thus refer to total dissolved concentrations. The term NH> will  be used to refer to (NH #NH> ) whilst analogously MA will be used to express      (MA #MA.H> ). Representative chromatograms for analysis of spiked and un    spiked seawater samples are shown in Fig. 2. 2.1. Other measurements Chemotaxonomic pigments were determined by reverse-phase HPLC with absorbance and fluorimetric detection (Barlow et al., 1998). Nutrients were determined by Technicon autoanalyser, and NH> using Jones’ (1991) fluorescence technique  (Woodward et al., 1999). Dissolved organic nitrogen (DON) was measured by high-temperature catalytic oxidation with chemiluminescence detection (HTCOCLD, Miller, pers. comm.). Mesozooplankton and microzooplankton data were provided by Edwards and Stelfox (pers. comm.) and bacterial counts by Pomeroy (pers. comm.).

3. Results NH> and MA data from the ARABESQUE programme are summarised in  Table 3, together with previously reported data. From Table 3 and Figs. 3 and 4,

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599

Fig. 2. Representative FIGD-IC chromatograms for 100 nM spiked abyssal seawater (upper) and unspiked surface seawater (lower). (c-PA"cyclo-propylamine, internal standard spiked to sample; s-BA" sec-butylamine, internal standard added to acceptor stream).

it may be seen that NH>  and MAs were ubiquitous in the photic zone throughout the Arabian Sea. NH> was, without exception, the dominant analyte, with concentrations  of up to two orders of magnitude greater than those of the MAs in both oligotrophic and coastal waters. Of the MAs, MMA was most abundant, whilst TMA was present only at low concentrations ((4 nM). Ethylamine (EA) also was detected occasionally but largely unquantifiable due to its low concentration and partial co-elution with DMA. Concentrations of MMA and DMA were highest in the shallow, productive, coastal waters along the Arabian Peninsula (Figs. 4A, B and 5). However, mean MMA concentrations showed greater contrast between coastal and oligotrophic regions than did those of DMA (Table 3). Consequently, the contribution of DMA to & MAs was greater offshore under more nutrient deficient conditions. TMA concentrations meanwhile were low in both coastal upwelling and offshore regions.

Arabian Sea — ‘AS series’ Aug—Oct 1994 (30) Nov—Dec 1994 (27) Arabian Sea — ‘A series’ Aug—Oct 1994 (82) Nov—Dec 1994 (44) Gulf of Oman — ‘GOM series’ Aug—Oct 1994 (40) Nov—Dec 1994

Pacific—Hawaii coastal (9) Atlantic—Massachusetts coastal (3) (3)

Irish Sea

Flax Pond, New York Mediterranean a. offshore b. coastal (Gulf of Lions)

This work

Van Neste et al. (1987)

Abdul-Rashid (1990)

Yang et al. (1993, 1994) Gibb et al., (1994)

11$9 (32) nd

113$102 (397) nd

5—60 7.5$5.5 18$10.0

33$9.6 252$506

0—619

nd

nd

52$20 200$58 32$5

6$7 (31) 12$7 (34)

91$91 (480) 112$(76)

nd nd

12$20 (66) 22$13 (55)

MMA mean$p, (max)

139$135 (538) 206$271 (778)

NH>  mean$p, (max)

Concentration (nM)

4.6$3.0 12$11.4

15—180

0—100

1.5$2.0 8.9$4.4 8.9$1.1

2.8$3.1 (11) nd

2.9$2.8 (12) 2.9$1.6 (7)

3.0$4.1 (14) 4.2$2.8 (14)

DMA mean$p, (max)

1.4$1.6 10$6.9

(3—80

0 —4

12$3.0 41$27 10$13

0.19$0.42 (1.9) nd

0.05$0.21 (1.8) 0.13$0.24 (0.8)

0.10$0.37 (2.0) 0.45$0.81 (4.0)

TMA mean$p, (max)

Techniques — Van Neste et al. (1987), GC-CLD; Abdul-Rashid (1990), micro-diffusion#GC-NPD; Yang et al. (1993, 1994), cell diffusion#GC-NPD; Gibb et al. (1994), FIGD-IC.(lod"below limit of detection, nd"not determined.

Location (Number of samples)

Authors (Yr)

Table 3 Summary of MA and NH> concentrations determined during the ARABESQUE programme (September—December 1994), collated with previously reported  oceanic MA data

600 S.W. Gibb et al. / Deep-Sea Research II 46 (1999) 593—615

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601

Fig. 3. Vertical profiles showing the intermediate and deep water distribution of MAs and NH> in relation  to in situ temperature (°C) and nitrate (lM), nitrite (nM/10) and dissolved oxygen concentrations at A. station A1 (24/11/94) and B. station A9 (25—26/94). Left column: MMA (nM) (—䉬—), DMA (nM) (—䊐—), TMA (nM) (—䢇—) and NH> (nM) ( - - - - ). (Nutrient data—Woodward pers. comm.). 

Low concentrations of MMA, DMA and TMA in deep waters (e.g. (10, (5 and (2 nM, respectively, Fig. 3) indicate that dissolved MAs are unlikely to contribute a significant flux of fixed nitrogen to the deep ocean, but are instead more intimately involved in euphotic zone processes.

602

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Fig. 4. Vertical profiles of MAs and NH> in the upper water column in relation to a series of chlorophyll  and carotenoid chemotaxonomic pigments at A. station AS5 (9/9/94), B. station AS5 (27/11/95) and C. station A7 (10/12/94) Left column: MMA (nM) (—䉬—), DMA (nM) (— 䊐—), TMA (nM) (—䢇—) and NH> (nM) ( - - - - ). Right column: Chlorophyll c3 (Chlc3), a chemotaxonomic marker of prymnesiophytes  and pelagiophytes; Chlorophyll c1c2 (Chlc1c2), diatoms; peridinin (PER), dinoflagellates; fucoxanthin (FUC), diatoms; 19-hexanoyloxyfucoxanthin (HEX), prymnesiophytes; zeaxanthin (ZEA), cyanobacteria/prochlorophytes; Chlorophyll b (Chlb), chlorophytes and Chlorophyll a (Chla), total algal biomass.

S.W. Gibb et al. / Deep-Sea Research II 46 (1999) 593—615

603

Fig. 5. Contoured spatial distributions of [MMA], [DMA], and [TMA] (nM) along the trans-shelf transect AS5—AS1 in the region of Masira Bay in the N.W. Arabian Sea (9—10 September 1994).

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Concentrations of DON, determined by HTCO-CLD, were in the range of 4—6 lM (Station A1, Miller, pers comm.). Total & MAs accounted for (1% DON, with a maximum contribution of &0.8% in the region of the sub-surface Chla maximum. Whilst a poor relationship was observed between NH>  concentrations and phytoplankton biomass in the coastal upwelling region (station AS5, Fig. 4A and B), offshore the vertical maxima of NH>  and biomass often coincided (e.g., Fig. 4C Chla profile). Meanwhile MMA, and DMA vertical concentration maxima generally coincided with those of the Chla in both shallow coastal waters (e.g., AS5; surface Chla maxima, Fig. 4A and B) and offshore waters (e.g., A7; sub-surface Chla maximum). However, the spatial association of MMA and DMA depth maxima with that of Chla maxima was not always observed; e.g., in Fig. 4C the vertical profile of DMA shows a minimum at the Chla maximum, indicating removal of DMA, whilst in other incidences no relationship is apparent. Distinct spatial profiles and relationships are less evident for TMA, largely due to its low concentrations. However, maximum TMA concentrations do appear to occur lower in the water column than those of MMA and DMA, at the base of the temperature and oxygen gradients and below the Chla maxima. The vertical distribution of TMA would therefore appear to be uncoupled from the other MAs. 3.1. Seasonal variations Between ARABESQUE 1 (Aug.—Oct.) and ARABESQUE 2 (Nov.—Dec.) mean concentrations of NH>, MMA, DMA and TMA recorded in the stations on the ‘AS  series’ increased by 48, 83, 43 and 350%, respectively, whilst those of the ‘A series’ increased by 23, 100, 0 and 160%, respectively. Nevertheless, throughout, the order of abundance of [NH>]'[MMA]'[DMA]'[TMA] was conserved. In addition,  considerable changes were observed in the structure of vertical profiles of NH> and  MAs (Fig. 4A and B).

4. Discussion 4.1. Chemical oceanography Abundances of MMA and DMA recorded in the stations along the ‘AS series’ concur with the coastal measurements made by Van Neste et al. (1987; Table 3), whilst those of MMA in the ‘A series’ stations (Fig. 1, Table 3) are comparable to those measured in the open ocean by Mopper (1—5 nM, see Van Neste et al., 1987). Data are also consistent with measurements made in the contrasting regions of the oligotrophic N.W. Mediterranean Sea and the eutrophic Gulf of Lions (Table 3). Offshore oligotrophic waters of the Mediterranean were characterised by some of the lowest reported MA and NH> concentrations. Although moderate increases occurred in  association with the rising bathymetry of the coastal shelf, only in the shallow eutrophic coastal waters of the Gulf of Lions were concentrations greatly enhanced (i.e. MAs, 3—58 nM; NH> 20—1440 nM). 

S.W. Gibb et al. / Deep-Sea Research II 46 (1999) 593—615

605

4.1.1. Dissolved organic nitrogen In the oceanic and coastal waters an average of 25—42% of the dissolved inorganic nitrogen taken up by phytoplankton is released as DON (Bronk et al., 1994). Whilst chemical speciation studies of seawater DON have concentrated on the more abundant species such as proteins and amino acids, some minor constituents such as purines, pyrimidines and nucleotides have also been characterised. Nevertheless, a significant proportion of this DON pool remains uncharacterised, including the contribution of MAs. During the ARABESQºE programme we estimate the contribution of & MAs to seawater DON was (1%. Bronk et al. (1994) estimated turnover times of 10 days for oceanic DON and conjected that resultant flux rates were of the same order of magnitude as the inorganic fluxes traditionally considered. Since a large proportion of the Arabian Sea DON was thought be refractory in nature (&50%; Miller pers. comm.), such short turnover times imply that the labile DON component is even more rapidly turned over. Since MAs are biologically labile species, and they have been shown to be a preferred nitrogen source to NO\ and NO\ by certain species of phytoplankton   (Wheeler and Hellebust, 1981; Pelley and Bannister, 1979), it may be hypothesised that MAs are rapidly recycled within the euphotic zone and that their influence on oceanic nitrogen cycling may be disproportionate to their dissolved concentrations. Clearly, process studies are necessary to examine this matter further. 4.1.2. Oxygen, nitrate and nitrite The oxygen minimum zone (OMZ), extending from &150 to 1000 m, is a characteristic feature of the northern Arabian Sea and is established as one of the largest oceanic reducing sites, accounting for ca 10—30% of global water column denitrification (Naqvi, 1987; Law and Owens, 1990). The vertical coincidence of maximum NH>  concentrations with the primary nitrite maximum has previously been reported in Saragasso Sea and Gulf Stream oligotrophic waters (Brzezinski, 1988). In the oligotrophic provinces studied in this work NH> maxima were also observed  near the primary nitrite (NO\) maxima (just above the thermocline and oxycline and  well resolved from the nitratacline; Fig. 3). Depth maxima of MMA and DMA were coincident with those of the primary nitrite maximum throughout the Arabian Sea and, in remote oligotrophic waters, also with those of NH> (e.g., Fig. 3). However, concentra tions MAs did not appear to be greatly influenced by the reducing conditions of the OMZ water, even in the region of maximum denitrification (station A9, Fig. 3). 4.2. Biological oceanography 4.2.1. Phytoplankton In depth profiles of oligotrophic Arabian Sea waters, the maxima of NH> and  biomass were found to coincide vertically (Fig. 4C). Due to the fundamental lack of sensitivity of classical oceanographic analytical techniques this observation has only been made recently. Brzezinski (1988) first recorded NH> maxima (70—100 m) near  the deep Chla maximum in Saragasso Sea and Gulf Stream oligotrophic waters employing a highly sensitive extraction technique.

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Although vertical profiles also indicated a general coincidence in the maxima of MAs and Chla, overall correlation studies revealed only a weak numerical relationship (Table 4). Analogous studies on oceanic populations and laboratory cultures of phytoplankton have shown poor correlation of DMS with total biomass. A more robust relationship is observed instead with the carotenoid biomarkers peridinin (PER) and 19’Hexanoyloxyfucoxanthin (HEX), indicating dinoflagellates and prymnesiophytes to be significant DMS producers (Turner et al., 1988, 1989). Investigations of chlorophyll and carotenoid pigments have proved invaluable in providing chemotaxonomic information about the abundance, composition and distribution of phytoplankton in the ocean. Each class of marine microalgae possesses a distinct pigment signature that may be used to characterise its occurrence (Barlow et al., 1993). Thus, through correlation of MA concentrations with concurrently determined pigment abundances, it is possible to determine whether any specific algal taxonomic class exerts a particular influence on the distribution of MAs. Indications of taxon specific MA production are given by coefficients in excess of those obtained from correlation with the universal biomass marker Chla (Table 4). Strongest evidence of algal taxon specific MA production was inferred from correlation of the diatom marker, fucoxanthin (FUC), and its complimentary pigment Chlorophyll c1c2 (CHLc1c2) with MMA and DMA concentrations (Table 4). A significant correlation was also observed between PER, and chlorophyll c3 (Chlc3) with MMA, indicating a potential production bias by dinoflagellates and either prymnesiophytes or chrysophytes, respectively. Furthermore, it is suggested that phytogenic MA production is weighted toward primary amines since all pigment concentrations show more significant correlation to MMA alone than to & MAs. The relationship between diatom abundance and MMA, and to a certain extent DMA, is illustrated in the seasonal shifts in the respective MA and pigment vertical profiles at station AS5 (Fig. 4), and also along the transect AS5 to AS1 (Fig. 5). At the oceanic extreme of the transect, AS1, the phytoplankton community was jointly dominated by diatoms and prymnesiophytes, as inferred through respective FUC and HEX abundances (Barlow et al., 1999). However, whilst HEX concentrations remain relatively stable with the shoreward progression to AS5, there was a four-fold increase in FUC concentrations. It is within this region that the highest levels of MMA and DMA were measured. Analogous correlation studies of a comparable Mediterranean data set also indicated that MMA was related more closely to diatoms than to total biomass (MMA vs. FUC, R"0.65, n"24; MMA vs. Chla, R"0.42, n"24, Gibb et al., 1994). However, in the absence of more extensive oceanic data complimented by data for mono-cultures, it is unrealistic to imply that MA production is confined to a single taxa. It is more plausible that oceanic MA distribution is subject to an intermediate regime in which phytogenic production is dominated by a range of ecologically important groups e.g. dinoflagellates also appear to be significant MMA producers (Table 4). Concentrations of TMA were found to correlate with neither the total biomass abundance nor any of its composite algal classes (Table 4). This may be due, in part, to an inability to measure changes in ambient TMA concentrations since these were

'2000 lm 1000—2000 lm 500—1000 lm 200—500 lm

All Dinoflagellates Diatoms Diatoms Prymnesiophytes/ pelagiophytes

115 115 115 115

9 9

7 7 7 7

84 (0.588) (0.033) (0.069) (0.124)

0.35 (0.000) /

0.59 (0.096) 0.66 (0.060)

0.25 0.79 0.72 0.64

!0.05 (0.676)

0.41 (0.000)

115

(0.000) (0.000) (0.000) (0.000)

0.35 0.53 0.65 0.56

MMA

(0.018) (0.006) (0.000) (0.000)

(0.346) (0.536) (0.749) (0.546)

0.33 (0.000) 0.65 (0.000) /

0.11 (0.772) 0.48 (0.187)

0.42 0.29 0.15 0.28

!0.04 (0.733)

0.28 (0.003)

0.22 0.26 0.43 0.37

DMA

Pearson correlation coefficient, R (‘p’)

115 115 115 115

n

(0.692) (0.447) (0.799) (0.975)

(0.243) (0.165) (0.109) (0.137)

0.089 (0.350) 0.11 (0.232) 0.22 (0.022) /

!0.32 (0.408) !0.22 (0.569)

0.52 !0.59 !0.66 !0.62

!0.07 (0.528)

!0.00 (0.963)

0.04 !0.07 0.02 !0.00

TMA

(0.000) (0.000) (0.000) (0.000)

(0.490) (0.073) (0.141) (0.171)

0.36 0.99 0.76 0.16

(0.000) (0.000) (0.000) (0.088)

0.52 (0.156) 0.69 (0.041)

0.32 0.72 0.62 0.58

0.05 (0.645)

0.40 (0.000)

0.34 0.50 0.63 0.55

& MAs

Note: Bacteria data courtesy of Pomeroy. Mesozooplankton courtesy of Edwards, Microzooplankton courtesy of Stelfox, both correlations based on depthintegrated samples.

Amine (nM) NH>  MMA DMA TMA

Microzooplankton Biomass (mgC m\ Grazing (lg Chla d\)

Zooplankton (mgC m\)

Bacteria Abundance (cell counts)

Algal Pigment (ng/l) Chlorophyll a (Chla) Peridin (PER) Fucoxanthin (FUC) Chlorophyll c1c2 (Chlc1c2) Chlorophyll c3 (Chlc3)

Class

Table 4 Correlation (Pearson linear) and significance of MA concentrations with microbiological parameters. (Pigment data only includes coefficients exceeding those with Chlorophyll a)

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typically around the detection limit of the analytical method. However, given this limitation, it may be speculated that phytogenic inputs play a minor role in the regulation of seawater TMA concentrations relative to those of MMA and DMA. Whilst the natural production of MAs by phytoplankton is proposed to occur via Hoffman elimination of intracellular QAs (King, 1988), relatively little else is known of their extracellular production. However, Abdul-Rashid (1990) studied MA production in bacteriostatic and non-bacteriostatic culture media populated by Chlorophyceae (Dunaliela minuta, Dunaliella tertiolecta and Chlorella salina), Chrysophyceae (Pavlova lutheri) and diatoms (Phaeodactylum tricorutum). Abdul-Rashid observed MA production per cell to increase with NO\ concentration in the growth media. This is  consistent with our observation of highest recorded MA concentrations occurring in the nutrient-rich coastal upwelling region of the Arabian Sea. In all culture studies Abdul-Rashid (1990) found MMA to be the most abundant MA, accounting for 86—98% of total molar MA production, followed by DMA (1.7—12%) and then TMA (0.2—3%). This also corresponds to the relative abundances of MAs found throughout Arabian, Mediterranean and Irish Sea waters (Table 3), and hence emphasises the importance of phytoplankton to the distribution of MMA and DMA, but not TMA. Although the distribution of MAs in Arabian Sea, can be partially explained through consideration of their algal production, paradoxically the uptake of MAs by phytoplankton is also reported in the literature. Pelley and Bannister (1979) showed green algae (Chlorella pyrenoidosa) capable of MMA uptake and Wheeler and Hellebust (1981) and Wright and Syrett (1983) the diatoms Cyclotella cryptica and Phaeodactylum tricorutum i.e. the same species Abdul-Rashid (1990) observed to produce MMA. Proposed to occur by an NH> transport mechanism, the uptake of MMA at  elevated concentrations was found to inhibit NO\ and NO\ utilisation via a mecha  nism thought to involve the suppression of nitrate reductase activity and synthesis. No effect was observed on utilisation of NH> (Gunnersen et al., 1988). Furthermore it  has been shown that MAs represent a non-metabolisable nitrogen source to phytoplankton and hence are not used in growth, but instead are retained in intracellular solution (Wheeler and Hellebust, 1981). Thus it is conceivable that in the presence of an abundance of NO\ or NH>, MMA will not be taken up, but will instead be   released either as a consequence of either osmolyte breakdown or nitrogen detoxification. Only under nitrogen-deficient conditions where the NH> assimilation mecha nism is under-utilised will MMA be taken up. Why energy-rich metabolites such QAs and MAs are retained in intracellular solution to fulfil osmoregulatory functions rather than more readily available inorganic ions may appear incongruous (Warren and Pierce, 1982). However, MAs and QAs are more compatible with macromolecular structure and functionality, and also reduce the need for modifying proteins in the concentrated intracellular fluids (Yancey et al., 1982). They are also implicated in the regulation of intracellular nitrogen toxicity: methylation being a common biological detoxification mechanism, e.g., urea, an important by-product of nitrogen metabolism, is accumulated by some species as an intracellular osmolyte, yet it exerts a stongly perturbing effect on macromolecules

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(Yancey et al., 1982). MAs (and QAs) have been shown to be potent counteractants of protein perturbation by urea (Yancey and Somero, 1978). However, it was also demonstrated that use of amines alone to deal with nitrogen toxicity was equally deleterious since they could induce protein rigidity. It is noteworthy that correlation coefficients of MMA with FUC (Table 4) for the Arabian Sea are numerically comparable to those of DMS with coccolithophores in natural populations of marine phytoplankton reported by Turner et al. (1988): R"0.67 (n"12) in blooms in which they represented '20% algal biomass. Equivalent correlations are recorded in dinoflagellate dominated blooms ('40%; DMS to Gyrodinium aereolum R"0.79, n"25; DMS to other dinoflagellates R"0.46, n"21). In contrast, Turner et al. (1988) observed poor correlation between diatoms and DMS concentrations (R"0.19, n"19), comparable to those observed between MMA and DMA concentrations with HEX abundance in this work (R"0.1, p"0.30 and R"0.03, p"0.77 respectively). As noted earlier, the relative importance of diatoms and prymnesiophytes to MA concentrations may be seen in Fig. 4 by relating dominant pigment abundances to MMA and DMA concentrations at the contrasting stations AS5 (diatom dominated, maximum MA concentrations) and A7 (prymnesiophyte dominated, low MA concentrations). In the coastal Arabian Sea, high seasonal primary production is observed with coastal upwelling as a major force in supplying nutrients to the euphotic zone (Mantoura et al., 1993). This upwelling, a consequence of Eckman transport in response to winds blowing parallel to the coast, generally starts in May and peaks in late July—August (Swallow, 1984). The southern Omani coastal region (Fig. 1), is one of the most productive areas of the world’s oceans, with NO\ fuelling new production  ( f-ratio"0.9) (Owens et al., 1993). Thus, it may be hypothesised that in this productive coastal region, subject to a greater upwelled nutrient supply, some of the NH>  and NO\ taken up by phytoplankton will be used in support of growth, whilst  a proportion will be anabolically utilised in the synthesis of osmotica such as QAs and MAs. Under such conditions diatoms, being adapted to higher nutrient supplies than other algae, will flourish. This may be accompanied by greater MA introduction into the aqueous phase through enzymatic degradation of QAs and direct extracellular losses consequent on nitrogen detoxification. Offshore, under more oligotrophic conditions, those species of phytoplankton better adapted to nitrogen limitation will be favoured, e.g., those requiring nitrogen for growth only, rather than for osmotic function. Hence, taxa capable of osmotica synthesis utilising sulphur, never a limiting nutrient, may be favoured (e.g., dinoflagellates, prymnesiophytes). Under such nitrogen limitation (and consequently reduced need for cellular nitrogen detoxification), lower MA (and possibly higher DMS) concentrations are found. Relative MA and DMS abundances may thus be indicative of algal class predominance and succession. Furthermore, gradients in nitrogen limitation may also account for the decrease observed in the mean MMA/DMA concentration ratio between coastal and open ocean regimes. In addition to taxonomic phytoplankton shifts, changes in MA (and QA) concentrations may result from changes in nutrient abundance and consequent osmoregulatory adaptation within individual classes of phytoplankton; e.g., Andreae (1986)

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suggests that under nitrogen limitation phytoplankton may switch to DMSP production. Turner et al. (1988) supported this idea via culture studies that showed that Emiliania huxleyi produced less DMSP in a NO\ supplemented medium.  4.2.2. Zooplankton Despite being constrained to a limited number of concurrent observations, correlations of MMA concentrations with zooplankton and microzooplankton were the strongest to emerge from the Arabian Sea data set (with the exception of the '2000 lm zooplankton fraction; Table 4). It may also be noted that coefficients resulting from MMA correlation with mesozooplankton abundances are augmented with respect to those with microzooplankton. This may be a consequence of grazing habits, since diatoms, inferred earlier to be the principle producers of MMA, are grazed by mesorather than micro-zooplankton. DMA concentrations meanwhile exhibited a poorer (yet still positive) correlation with both mesozooplankton and microzooplankton abundances than did MMA (Table 4). Whilst zooplankton herbivory may contribute directly to the aqueous budget of MMA and DMA, microzooplankton bacteriovy may also exert a complimentary, and indirect impact on MA concentrations through regulation of the principle consumers dissolved MAs. However, in the course of these studies it was not possible to differentiate between these mechanisms. Zooplankton (Artemia sp.) grazing activity on bacteriostatic cultures (Chlorophyceae, Chrysophyceae and diatoms) has been shown to result in exponential increases in aqueous MA concentrations over the first 1—2 days, with levels reaching 5—6 times those of ungrazed cultures after 7 days by Abdul-Rashid (1990). In these studies MMA was the most abundantly produced MA, followed by DMA, while TMA represented (1% of & MAs. This is again entirely consistent with our field observations (Table 3). The influence of zooplankton on aqueous MMA and DMA concentrations is analogous to their influence on DMS concentrations: Dacey and Wakeham (1986) observed that ambient DMS release increased 24 fold when dinoflagellates (inferred as ecologically important DMS producers) were subjected to copepod grazing. However, it was not known whether this was a consequence of cell damage during ingestion, of digestive precursor decomposition or of bacterial activity in faecal material. In contrast to MMA and DMA, concentrations of TMA related inversely to all zooplankton biomass and grazing parameters (again excluding the '2000 lm fraction; Table 4). These, the only significant correlations recorded for TMA, are indicative of its removal from the aqueous phase. Such removal of TMA from the aqueous phase by zooplankton, has been reported by Strom (1979, 1980) who demonstrated that live copepods oxidised C-TMA from seawater using an NADPH-monooxygenase and accumulated it as TMAO. Since TMA is reported to occur in many marine algae (Steiner and Hartmann, 1968) and enzymatic oxidation is the only known mechanism for TMAO formation in animals (Strom, 1979), this mechanism may help account for the low TMA concentrations recorded in the Arabian Sea. However, in contradiction to the above, limited incubation studies on natural populations netted from the Mediterranean Sea demonstrated that zooplankton did

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produce TMA, and at concentrations to rival those of MMA and DMA. However, animals collected from below the [Chla] maximum (&50 m) produced 8—30 times the TMA of those collected above this depth. This feature, attributed to zooplankton diurnal migrational feeding patterns in the Mediterranean, and not observed for MMA or DMA, is consistent with observation of TMA concentration maxima at 50—120 m in some Arabian Sea vertical profiles. 4.2.3. Bacteria Whilst the microbial loop envisages that bacteria utilise dissolved organic matter (DOM), and dominate the regenerative aspects of the nitrogen cycle, poor correlations were observed between MA concentrations and bacterial cell counts (Table 4). However, since the bacterial population as a whole may be involved in both the production and consumption of MAs, unless one mechanism strongly prevails then no discernible relationship will be observed between MA concentrations and bacterial abundance. Furthermore, those classes of bacteria involved in MA turnover are likely to represent such a minor fraction of the total bacterial community that any correlation is numerically swamped. In the bacteriostatic algal cultures studied by Abdul-Rashid (1990), levels of MMA, DMA and TMA reached 92—136, 5—37 and 0.7—4 times those, respectively, measured under non-bacteriostatic conditions. This indicates effective removal of MAs from the medium by heterotrophic bacteria and preferential utilisation of MMA over DMA which is in turn preferred to TMA. Budd and Spencer (1968) similarly reported decreasing Micrococcus growth rates with increasing methyl substitution of the substrate. All MA uptake has been shown to be inhibited by the addition of NH> in  cultures of nitrogen fixing bacteria (Mazzucco and Benson, 1984) and nitrifying bacteria (Glover, 1982). Given this preference for lower molecular weight nitrogen compounds, the bacterial utilisation of MAs may be influenced by the availability of NH> and hence also upon  the relative abundance and activity of methanogens and methylotrophs within the bacterial community. The preferential utilisation of MMA over DMA may also help to account for decreasing MMA/DMA in offshore oligotrophic waters (Table 3). However, the fact that MMA was found to be the most abundant MA followed by DMA then TMA, in both this and Abdul-Rashid’s studies, despite bacterial preference for the lower molecular weight compounds, suggests that regulation of aqueous MA concentrations in the euphotic zone of oceanic waters is dominated by microbial production rather than consumption.

5. Summary and conclusions Concentrations of MAs were up to two orders of magnitude lower than those of NH> and accounted for (1% [DON]. MMA was generally the most abundant MA,  whilst TMA was only found at concentrations (4 nM. Concentrations of MA were insensitive to the vertical oxygen gradients characteristic of the region, compared to gradients in biological activity. Poor correlation of TMA with both MMA and DMA

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concentrations, suggests that the individual MAs are subject to different regulatory mechanisms and that the dynamics of TMA are decoupled from those of MMA and DMA. Data presented, and its comparison to previous laboratory studies, supports the idea that the levels of MAs in seawater are predominantly regulated by producers (phytoplankton and zooplankton) rather than by consumers (bacteria). Overall a degree of diatom specific production of MMA, and to a lesser extent DMA was inferred via chemotaxonomic pigment associations. Microzooplankton appear to promote significantly aqueous MMA and DMA concentrations, through their grazing, ingestion, digestion and excretion habits. Enhanced MMA and DMA correlations with mesozooplankton may be a consequence of their ability to graze diatoms. Low concentrations of MAs in offshore mesotrophic—oligotrophic regions (A7—A9) contrasted with the elevated levels recorded in the productive upwelled waters of the coastal NW Arabian Sea. It is postulated that in this nutrient rich region, dominated by diatoms, some of assimilated nitrogen will be used for biosynthesis whilst a proportion will be anabolically utilised in osmotica synthesis. This may be accompanied by introduction of MMA and DMA into the aqueous phase through enzymatic QA degradation, nitrogen detoxification, senescence or lysis and accelerated through grazing pressures, particularly those of mesozooplankton predation of diatoms. Offshore, under more oligotrophic regimes chemotaxonomic shifts, accompanied by osmotic adaptation, will favour those species of phytoplankton with a lower nitrogen demand. Concentrations of MAs will be suppressed through reduced osmolyte turnover and nitrogen detoxification, and possibly complimented by non-metabolised MMA uptake. It is evident that many aspects of the biogeochemical cycling of MAs parallel those of their sulphur cycle analogue, DMS. Since knowledge of the cycling of DMS is relatively advanced compared to that of the MAs, this understanding may be used as a conceptual template for interpreting the oceanic distribution and biogeochemical cycling of MAs in future studies.

Acknowledgements The authors would like to both acknowledge and thank the following people for analysis of samples or for supplying data for use in interpreting the distribution and biogeochemical cycling of MAs: Denise Cummings (chlorophyll and carotenoid pigments); Malcolm Woodward, Andrew Rees and John Stephens (nutrients); Axel Miller (dissolved organic nitrogen); Alan Pomeroy (bacterial counts); Claire Stelfox (microzooplankton) and Elaine Edwards (Zooplankton and grazing). Thanks also to the full complement of Officers, Crew and Scientists onboard R.R.S. Discovery during ARABESQUE cruises 210 and 212. This work was carried with the financial support of NERC Research Grant, Ref no. GR3/08715 and MOD/DRA Joint Grant TQ/10/3/2. This manuscript has benefited from the constructive comments of two anonymous referees.

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