Month: August 2018

APL003 mussels

 

 

 

 

 

Sources of organic detritus under mussel farms in the Ría de Ares-Betanzos

By Joeri Kaal and CSIC-PROINSA Mussel Lab Team

The Galician coastline is enormously productive of high-quality seafood due to the vast supply of nutrients, oxygen and plankton in this unique coastal upwelling system¹. One of the characteristics of the Rías is the abundance of raft farms, “bateas”: floating wooden structures with 500 ropes hanging into the water column and on which mussels grow (suspended mussel farming). Galician mussel farms represent the highest mussel (Mytilus galloprovincialis) producing area in Europe (>250 Million kg per year)². These farms are known to both boost biodiversity in the water column, providing refuge to fauna and anchoring for macroalgae, but also to cause alteration of benthic communities due to the massive production of “biodeposits” (mussel fall-out and faeces), sometimes resulting into anoxia at the seabed^3,4. The Marine Research Institute (IIM) of the Spanish Research Council (CSIC) has been studying the ecology, chemistry and sustainability of these suspended bivalve systems for many years^2,5. Now, an effort has been made to identify the potential of analytical pyrolysis techniques to provide information on biodeposit composition by means of analysis of particulate organic matter (POM) from sediment traps deployed underneath the farms.

 

 

MATERIALS AND METHODS

The samples were obtained from a mussel culture polygon in the vicinities of Lorbé in the Ría de Ares-Betanzos (Sada, A Coruña province, Galicia, NW Spain), consisting of 107 rafts and an annual production of 10,000 tons of mussels. The particulate organic matter (POM) samples were obtained from multitrap collectors that were deployed beneath raft #46 (further from coast, 16 m water depth) and raft #14 (closer to coast and fish cages, 14 m depth) (Figure 1) on the 2^nd and 3^rd of May, 2011 (Spring bloom oceanographic scenario⁵). Samplers were deployed at the bow (or proa, i.e. water entry) and stern (popa, water exit) sides of the rafts. The traps contained four collectors which were open for no more than 12 h to mitigate the influence of microbial decomposition in the traps. Two of the 4 collectors within each trap were analyzed, T1 and T2 (these correspond to adjacent PVC cylinders within the multitrap). No preservatives were used (for details, see Zúñiga et al. 2014 ⁵). Hence, a total of 8 samples were considered (2 rafts x 2 positions x 2 replicates) as a first approach to determine biodeposit sources and identify mechanisms that control variability in POM’s molecular composition. After retrieving the suspensions from the traps their content was filtered through Whatman GF/F filters (0.7 μm pore size). Due to the low inorganic matter content of these samples (<10 %), no sample treatment other than drying at 100 ºC, was performed (it would be a different scenario for the samples from Winter, which may have inorganic contents exceeding 75 %)⁵.

The Py-GC-MS analyses were performed by inserting solid POM (that was scraped directly from the filters) into quartz tubes with quartz wool on both ends. Pyrolysis was performed for 10 s at 650 °C, with a Pyroprobe 5000. The pyrolyzer was connected to a 6890 GC and 5975 MSD from Agilent Technologies. For chromatographic temperature profiles and MSD parameters, see Kaal et al. ⁶. Major peaks were quantified on the basis of characteristic m/z fragments. Data evaluation was based on i) pyrolysis compound percentages, analysis of variance (one-way ANOVA) and linear correlation analysis.

Table 1 presents the list of samples analyzed and parameters that are used for comparison with the Py-GC-MS data: fluxes of nitrogen (N_total), carbon (C_total) and organic C (C_org), which is modified data after Zúñiga et al. (2014)⁵.

 

Table 1. List of samples analyzed and selected parameters

Raft	Date	Sampler	Sample	Ntotal	Ctotal	Corg
	m/d/yr	–	–	mg/m2d	mg/m2d	mg/m2d
#46 bow	5/2/2011	T1	17	405	2503	1603
	5/2/2011	T2	18	304	1866	2092
#46 stern	5/2/2011	T1	19	813	5017	5101
	5/2/2011	T2	20	619	4340	2833
#14 bow	5/3/2011	T1	27	186	1283	1045
	5/3/2011	T2	28	210	1409	1321
#14 stern	5/3/2011	T1	29	447	3157	1958
	5/3/2011	T2	30	488	3576	1930

 

 RESULTS AND DISCUSSION

A large array of carbohydrate- and especially protein-derived pyrolysis products were detected (pyrroles, pyridines, diketodipyrrole, indoles) (Table 2). They include markers of N-acetylglucosamine polymers (acetamidosugars), which can be found in chitin from arthropod/crustacean/mollusk exoskeleta (and squid beaks), bacterial cell wall material, and the mucus of mollusks that cover faeces and pseudofaeces (Figure 2). The majority, if not all, of the other carbohydrate and protein products probably originate from mussel derivatives or marine biota such as phytoplankton.

 

Table 2. List of pyrolysis products and source allocation (CARB=carbohydrate, LIG=lignin, MAH=monocyclic aromatic hydrocarbon, MCC=metylene chain compound, NCOMP=N-compound, PAH=polycyclic aromatic hydrocarbon, PHEN=phenol)

#	RT (min)	compound	m/z	group
1	1.075	MeBr	94+96	other
2	1.371	benzene	78	MAH
3	1.569	pyridine	79+52	NCOMP
4	1.601	pyrrole	67	NCOMP
5	1.704	toluene	91+92	MAH
6	1.78	acetamide	59	NCOMP
7	1.897	3/2-furaldehyde	95+96	CARB
8	2.037	C1-pyrrole	80+81	NCOMP
9	2.146	C1-pyridine	93+66	NCOMP
10	2.396	styrene	104+78	MAH
11	2.786	5-methyl-2-furaldehyde	110+109	CARB
12	2.942	benzonitrile	103+76	NCOMP
13	3.082	phenol	94+66	PHEN
14	3.701	C1-phenol	107+108	PHEN
15	3.888	C1-phenol	107+108	PHEN
16	4.075	carbohydrate compound	126	CARB
17	4.569	C2-phenol	107+122	PHEN
18	4.845	naphthalene	128	PAH
19	5.115	propylcyanobenzene	91+131	NCOMP
20	5.193	4-vinylphenol	91+120	LIG
21	5.266	C3-phenol	121+136	PHEN
22	5.739	indole	117+90	NCOMP
23	5.843	3-acetamido-5-methylfuran	97+(69+)139	NCOMP
24	5.858	C1-naphthalene	142+141(+115)	PAH
25	5.864	p-tert-butylphenol	135+107(+150)	cont
26	5.931	C3:1 phenol (isopropenyl?)	134+119	cont
27	5.994	C1-naphthalene	142+141(+115)	PAH
28	6.139	3-acetamido-2/4-pyrone	111+(82+)153	NCOMP
29	6.55	C1-indole	130+131	NCOMP
30	6.566	biphenyl	154	PAH
31	8.214	fluorene	166+165	PAH
32	8.458	5-bromoindole	197+195(+116+89)	NCOMP
33	8.521	levoglucosan	60+73	CARB
34	8.988	diketodipyrrole	186+93	NCOMP
35	9.623	phenanthrene/anthracene	178	PAH
36	9.68	C14-fatty acid	60+73	MCC
37	10.117	alkane/alkene pair	55+57	MCC
38	10.158	phenylnaphthalene	204+203	PAH
39	10.283	phytadiene 1	68+95(278)	MCC
40	10.496	diketopiperazine	70+154+194	NCOMP
41	10.507	C16-alkylnitrile	97	NCOMP
42	10.579	phytadiene 2	81+82(278)	MCC
43	11.006	C16-fatty acid	60+73	MCC
44	12.191	C16-alkylamide	59+72	NCOMP
45	12.243	p,p‘-isopropylidenebisphenol	213+228	cont
46	12.243	C18-fatty acid	60+73	MCC
47	14.229	unknown compound	(91+)129+207	other
48	16.013	triterpenoid compound	368+353	other
49	16.663	triterpenoid compound	69+269(+298)	other

 The most abundant group of products are the N-containing compounds, which account for 27.4 ± 3.1 % of TQPA (percentage of total quantified peak area), followed by monocyclic aromatic hydrocarbons (MAHs; 23.1 ± 3.7 %) and phenols (22.8 ± 5.1 %). This set of compounds is a clear indication that the POM originates from predominantly N-rich sources. The abundance of not only many protein markers (such as indoles from tryptophan or diketopiperazines from dimerization reactions of peptide chains during pyrolysis) but also products N-acetylglucosamine biopolymers suggests that mussel detritus, whether it be chitin from shells or mucus in faeces droppings, is a significant, if not dominant source of POM. Note that theoretically psuedofaeces (droppings of mucus-entangled inorganic matter filtered by the animal but which did not pass its digestive system) may also provide mucus to the biodeposits but due to the low seston concentration in the clear waters of NW Spain in general, production of pseudofaeces is unlikely (seston below threshold of 4 mg/L)⁵.

 

The proteins may originate from a combination of the mussel’s faeces, and alternative (marine) sources such as macroalgae and plankton. According to the high chlorphyll a content of these samples⁵, detritus from primary production is expected to be significant. The phenols may largely originate from aromatic amino acids in proteins such as tyrosine moieties, and the same can be argued for toluene among the MAH which is associated with phenylalanine⁷. Another component of the POM is of pyrogenic origin: products of e.g. charcoal from wildfires or soot from incomplete fuel combustion. This pyrogenic POM is reflected by PAHs (1.6 ± 0.4 % naphthalene, alkylnaphthalenes, fluorene, biphenyl, phenylnaphthalene, etc.), but also benzene (MAH) and benzonitrile⁵ (N-containing pyrogenic POM). This reaffirms previous indications⁵ of the presence of a minor fraction of allochthonous POM from runoff, with a high C/N ratio, and which is concentrated in the littoral areas in the studied ría ⁸. One sample produced a significant amount of a 5-methylbromoindole (sample 20, Table 1), whereas the pyrolyzates of the other samples contained trace amounts of this molecule. This may be a natural organobromine compound from marine biota⁹, or a secondary pyrolysis reaction between inorganic bromine and protein-derived indole, and has not been detected previously identified in POM pyrolyzates. Methyl-bromide (MeBr) is also abundant.

The long-chain aliphatic products (8.9 ± 1.9 %), or methylene chain compounds (MCC), also have various potential sources. Two phytadienes were detected among the MCC, which act as probably products of chlorophyll, from phytoplankton and/or macroalgae. The remaining products, notably fatty acids, may originate from any source but especially algae and mussel faeces were expected to be prolific of these compounds. Carbohydrate products account for 8.7 ± 2.3 %. They may be of any biological source mentioned above, but the high abundance of N-acetylglucosamine polysaccharide products among the N-compounds suggests that the carbohydrates may originate predominantly from chitin and/or mucus. Contamination indicators (4.9 ± 4.7 %) are tert-phenols and a bisphenol product. They probably both originate from bisphenol-type anthropogenic contamination. The C_3:1-phenol compound shows similar differences in proportion among samples, and is therefore likely to originate from such contamination as well. Finally, lignin from terrestrial plants could not be unequivocally identified but the presence of a compound with m/z 120 and 91 at the expected retention time of 4-vinylphenol might reflect traces of land plant debris (if so, probably sedges or grasses including seagrasses).

For the ANOVA, we used all continuous variables as input attributes and the discrete parameters raft number (raft #46 vs. raft #14), trap position (bow vs. stern), and sampler (T1 vs. T2) as target variables.

As expected, none of the variables is affected by sampler number (T1/T2) located at the same spot (different cylinders in the same trap). For trap position, some significant (P<0.05) differences were observed. Firstly, the fluxes of C_total and N_total higher in the trap deployed at the stern position (popa). This suggests that the waters that flow through the rafts and the mussel cords gain in POM content along this path through the raft and illustrate biodeposition intensity differences. In other words, the effects of the rafts on the deposition of faeces can be observed not only from mass fluxes but also from relative contributions to POM. Among the Py-GC-MS products, stern samples are enriched in 3-acetamidopyrone (mussel chitin/mucus) and levoglucosan (chitin and perhaps other polysaccharides). Bow samples are enriched in MeBr. All the other >60 variables did not show significant differences.

For raft location, #14 was enriched in benzene, benzonitrile and fluorene (black-carbon derived) whereas #46 is enriched in some aliphatic products (an alkane/alkene pair and C₁₆-alkylamide). This suggests that the site that is closer to the coastline is enriched in terrestrial black carbon materials, possibly indicative of fluxes of charcoal particles from rivers and streams that discharge in the area.

The linear correlations between the C and N  fluxes showed some interesting relationships. Strong positive correlations between C_total and 3-acetamidopyrone (r=0.92!, P<0.005), C₁-indole, diketopiperazines, levoglucosan and C₁₆-fatty acid probably show the effect of POM sedimentation rate and proportions of mucus (delivering the N-acetylglucosamine polymer) and phytoplanikton (delivering protein). Negative correlations between C_total and MeBr, styrene and bisphenol products probably represents a dilution effect (less biodeposits, more background signal). The same patterns are observed for C_org and N_total. The C/N ratio is not correlated to any pyrolysis product.

 

Conclusions

The molecular properties of the POM could be largely traced back to nitrogen-containing biopolymers, in particular protein and mucus/chitin. Mucus and chitin cannot be distinguished on the basis of the available data yet, as we have not analyzed references materials for mussel mucus. The chitin/mucus originates predominantly from the mussel particles that trickle down from the rafts into the traps, possibly representing fall-off (although unlikely) and, most importantly, faeces. The protein originates from mussel debris (mucus/chitin) and plankton, and the presence of phytadienes provide strong evidence for algae-derived aliphatic POM (chlorophyll). The relative proportions of mucus/chitin and phytoplanktonic protein and aliphatic POM cannot be addressed: pyrolytic reactions are complex and chitin is entangled in protein which is difficult to distinguish from non-entangled protein in phytoplankton. However, from the ANOVA it was concluded that mussel debris was more abundant in the exit (stern) than entry (bow) of the mussel farms, which may reflect the background signal of POM from sources that are not directly associated to the bivalve cultures such as plankton. Other materials that were identified were traces of bisphenol contamination of unknown origin and fire residues (charcoal, black carbon), the latter of which had a stronger signal in the pyrolysis chromatograms from raft that was closest to the coastline (#14). In this context, it is worthy to mention that the region is subjected to a very intense fire regime. There is a clear potential of molecular characterization of particulate debris underneath mussel rafts to understand organic matter cycling and seafloor ecology.

 

ACKNOWLEDGEMENTS

The efforts of Uxío Labarta, María-José Fernández-Reiriz, Diana Zúñiga and Carmen González Castro, among other members of the CSIC-PROINSA Mussel Farm Team, allowed for the materialization of this collaboration.

 

References

1. Fraga, F., 1981. Upwelling off the Galician Coast, Northwest Spain. Book Series: Coastal and Estuarine Sciences, Volume 1: 176-182. https://doi.org/10.1029/CO001p0176.
2. Labarta, U., Fernández-Reiriz, M.J., Pérez-Camacho, A., Pérez-Corbacho, E., 2004. El mejillón, un paradigma bioeconómico. In: Fundación Caixa Galicia (Ed.), Bateeiros, mar, mejillón. Una perspectiva bioeconómica: Centro de Investigación Económica y
   Financiera. Editorial Galaxia, Vigo, pp. 19–479
3. Otero, X.L., Calvo de Anta, R.M., Macías, F., 2008. Iron geochemistry under mussel rafts in the Galician ria system (Galicia-NW Spain). Estuarine and Coastal Shelf Science 81, 83–93.
4. Chamberlain, J., Fernandes, T.F., Read, P., Nickell. T.D., Davies, I.M., 2001. Impacts of biodeposits from suspended mussel (Mytilus edulis L.) culture on the surrounding surficial sediments. ICES Journal of Marine Science, 58: 411–416, doi:10.1006/jmsc.2000.1037.
5. Zúniga, D., Castro, C.G., Aguiar, E., Labarta, U., Figueiras, F.G., Fernández-Reiriz, M.J., 2014. Biodeposit contribution to natural sedimentation in a suspended Mytilus galloprovincialis Lmk mussel farm in a Galician Ría (NW Iberian Peninsula). Aquaculture 432, 311–320.
6. Kaal, J., Martínez Cortizas, A., Nierop, K.G.J., Buurman, P., 2008. A detailed pyrolysis-GC/MS analysis of a black carbon-rich acidic colluvial soil (Atlantic ranker) from NW Spain. Applied Geochemistry 23, 2395-2405.
7. Tsuge, S., Matsubara, H., 1985. High-resolution pyrolysis-gac chromatography if proteins and related materials. Journal of Analytical and Applied Pyrolysis 8: 49-64.
8. Sánchez-Mata, A., Glémarec, M., Mora, J., 1999. Physicochemical structure of the benthic environment of a Galician ría (Ría de Ares–Betanzos, north-west Spain). Journal of the Marine Biological Association of the United Kingdom 79, 1–21.
9. Andreotti, A., Bonaduce, I., Colombini, M.P., Ribechini, E., 2004. Characterisation of natural indigo and shellfish purple by mass spectrometric techniques. Rapid Communications in Mass Spectrometry 18: 1213–1220.

 

 

 

APL002 moss

 

 

 

Itla-okla (Tillandsia usneoides) fibre temper in Pre-Columbian ceramics

By Joeri Kaal (Pyrolyscience) and Zackary Gilmore (Department of Anthropology, Rollins College, USA)

Itla-okla, which means “tree hair”, was the Indian name of the epiphyte Tillandsia usneoides, also known as “Spanish beard” or “Spanish moss”. Pre-Columbian societies of the Late Archaic period (Orange and Stallings traditions; 3000-1800 B.C.) used these epiphytes as an organic temper in the production of pottery. In collaboration with Zackary Gilmore (Rollins College, Florida, USA), Pyrolyscience studied archaeological sherds of this fiber-tempered (FT) ware in order to identify (other) organic materials used for their fabrication and assess the production conditions of this extraordinary pottery (Gilmore, 2015).

Materials and Methods

The materials analyzed are (1) ceramics, ball-milled to powder and treated with HF to eliminate oxides, (2) isolated charred fiber materials, scraped from the archaeological sherds and (3) Spanish moss, both uncharred and charred in a temperature series between 300 and 600 ºC. The analysis of charred “moss” (it is neither a moss nor a lichen, but an angiosperm from the Bromeliaceae family) was thought to enable the estimation of the firing temperature (in terms of muffle furnace equivalent temperature, T_MFE) of the isolated fibres and whole ceramic fragments. HF treatment was performed to eliminate reactive minerals. Pyrolysis-GC-MS was performed at 750 ºC to stimulate fragmentation of thermoresistant components such as charred organic matter (Black Carbon) (Kaal et al., 2009). Extant moss was double-wrapped in aluminum foil to create oxygen-limited conditions and charred between 300 and 600 ºC (e.g. Turney et al., 2006).

Results and Discussion

First we’ll discuss the molecular chemistry of the plant and it’s experimentally created charred equivalents. The loss of weight of the sample of fresh Itla-okla as induced by charring in the muffle furnace shows a typical increase from low to high temperatures: 50 % at T_MFE=300 ºC towards 85 % at T_MFE=600 ºC (Figure 1). This is known to reflect dehydration and condensation reaction as the temperature rises.

With Py-GC-MS, the uncharred sample (feedstock) is prolific of many polysaccharide products (acetic acid, furans, cyclopentenones, pyranones), lignin products (4-vinylphenol, guaiacols, syringol) and some aliphatic products such as phytadienes (from chlorophyll) and fatty acids. With THM-GC-MS, the main peaks are fatty acid methyl esters (C₁₆, C₁₈, C₂₄-C₂₈), mid-chain methoxylated C₁₆ fatty acid methyl esters (from cutin in cuticula) and the lignin products P18 (p-coumaric acid methyl ester), G18 (ferulic acid methyl ester). These results represent the first molecular screening with Py-GC-MS and THM-GC-MS of Tillandsia usneoides feedstock.

With increasing T_MFE, the peak intensities (Py-GC-MS only) of these polysaccharide and lignin products decrease and those of monocyclic and polycyclic aromatic hydrocarbons (MAHs/PAHs) increase, which is a feature that has been observed in many kinds of feedstock materials of which experimental “thermosequences” were analyzed by Py-GC-MS. There is an intermediate range of thermal modification as recognized from Py-GC-MS fingerprints between 300 and 400 ºC with abundance of aliphatic products (alkanes, alkenes) and phenols (degraded lignin). The abundance of these phenols and aliphatic products (n-alkanes and n-alkenes from aliphatic biopolymers such as cutin) is high in the samples produced at 300 and 400 ºC_MFE but not at 500 ºC and higher (here, the dominance of MAHs and PAHs is very strong) (Figure 2). These results are in agreement with previous studies of plant-derived chars.

 

 

The relative abundances of benzene and toluene (B/T ratio) and naphthalene and methylnaphthalens (N/C₁N ratio) has been used as an indicator of charring intensity.  The graph below shows how these parameters increase with T_MFE in the experimental graph, especially between 400 and 550 ºC. These trends can be used to compare with the fibres and whole sherds from the FT ceramics. The figure to the right shows the B/T –  N/C₁N plot of the laboratory chars and the isolated fibers, and shows that three of the fibers plot in the low-thermal impact range (<500 ºC_MFE) whereas the other two plot in the high temperature range (450–600 ºC_MFE). For the whole sherds, we could not establish B/T due to the existence of double peaks (implying a combination of volatiles and polymeric sources) and the methylnaphthalenes could not be reliably quantified due to low peak intensities.

 

 

This information can now be used to interpret the Py-GC-MS fingerprints of the fibers extracted from sherds and whole sherd samples (Figure 4). From the chromatograms of the isolated fibers (charred elements in sherds), there are clear differences in thermal impact, which were also observed by tracking the B/T ratio (Figure 3, right graph). Some samples are dominated by MAHs and PAHs, and benzofuran, such as sample FT3 in Figure 4. Others have higher proportions of aliphatic compounds, such as FT1 and FT4, indicative of lower thermal impact.

 

 

The chromatograms of whole sherds are more surprising. Sample sherd2, for example, shows a pyrolysis fingerprint that is strongly dominated by alkanes and alkenes, and with a chain length pattern that is clearly distinct as what was observed for the experimental chars or the isolated fibers from the archaeological sherds. This may be indicative of other organic ingredients (bitumen, dung or plant materials, for example) that were added to the ware before firing and were absorbed to it during its use. Sherd4 has a fingerprint that can be largely ascribed to Itla-okla remains.

Conclusions

Clearly, there are differences in firing intensity as observed from the balance between aliphatic products and phenols, vs. MAHs and PAHs. This became evident by comparing the Py-GC-MS fingerprints of the experimental thermosequences and the archaeological ware (isolated fibers). The range of temperatures are <500 and 500-600 ºC_MFE. Note that these differences do not necessarily imply that there are large differences in the maximum temperature during firing: other factors such as the thickness of the material, and the duration of the process can explain the results as well. For example, relatively short firing or the use of thick ware may cause incomplete burn-off in the center of the ware where the organic matter may be least affected by the thermal alteration: such features can be recognized as sandwich morphologies of sherds (see figure below: dark core, lighter interior and exterior surfaces). The results of the whole sherd analysis may be indicative of other organic constituents and future studies will focus on the characterization of those materials.

 

References

Gilmore, Z.I., 2015. Direct radiocarbon dating of Spanish moss (Tillandsia usneoides) from early fiber-tempered pottery in the southeastern U.S. Journal of Archaeological Science 58, 1–8.

Kaal, J., Nierop, K.G.J., Martínez Cortizas, A., 2009. Characterisation of aged charcoal using a coil probe pyrolysis-GC/MS method optimised for Black Carbon. Journal of Analytical and Applied Pyrolysis 85, 408-416.

Kaal, J., Lantes Suárez, O., Martínez Cortizas, A, Prieto, B., Prieto Martínez, M.P., 2014. How useful is pyrolysis-GC-MS for the assessment of molecular properties of organic matter in archaeological pottery matrix? An exploratory case study from NW Spain. Archaeometry 56, 187-207.

Turney, C.S.M., Wheeler, D., Chivas, A.R., 2006. Carbon isotope fractionation in wood during carbonization. Geochimica et Cosmochimica Acta 70, 960–964.