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Phosphatase activities of mosses, algae and cyanobacteria as biomonitoring tools for upland water nutrient status.

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Presentation on theme: "Phosphatase activities of mosses, algae and cyanobacteria as biomonitoring tools for upland water nutrient status."— Presentation transcript:

1 Phosphatase activities of mosses, algae and cyanobacteria as biomonitoring tools for upland water nutrient status

2 The WFD derived from gaps in previous legislation to protect Europe’s water. Two key requirements: To classify ecological status and, where necessary, restore the structure and function of aquatic ecosystems. Introduction of a river basin management planning system. Biological classification systems have been intercalibrated at EU level for comparability between member states. Present biomonitoring methods can contribute to ecological status classification and the restoration but mainly for lowland rivers with high inorganic nutrient loads. Planned management measures (basic measures) have been set out to tackle pollution problems In upland areas however, ‘basic measures’ are mostly not applicable and so ‘supplementary measures’ need developing. Classification using standard monitoring methods of water chemistry can be misleading. The implementation of the diatom methodology for the WFD is suited for more downstream sites (useful for inorganic nutrients, less environment variation). Need to develop a new biomonitoring system for uplands Water Framework Directive and uplands

3 The large surface area of upland catchments and the associated meteorological conditions and exposure to climate warming and atmospheric N deposition all result in a highly vairable and shifting stream water quality. Another confounding feature of upland areas is that much of the filtrable nutrient fractions/species in flowing waters can be organic, deriving from organic rich soils (e.g. peat, forested soils). At present the ‘basic measures’ of biological classification under the WFD does not take organic P into consideration. Ideal organisms for this purpose should be widespread and tolerant of a range of nutrient levels, as opposed to species with narrow ecological ranges. Organisms should "integrate" the environment over much longer period, such as long- lived algal colonies and, ideally, also aquatic bryophytes. Biological monitoring and upland streams

4 Most, if not all, phototrophs can utilize inorganic phosphate in their environment, and it is commonly assumed that the ability to utilize organic phosphate is more restricted. Yet there is a very large and increasing body of evidence proving that many organisms can use organic compounds as a P source. And it has also been shown that organisms can utilize organic phosphates as their sole phosphorus source, much of the evidence has come indirectly from studies on phosphatases and phosphatase activity…. ….and research has shown that many organisms from upland regions have high surface phosphatase activity and, where tested, can grow on organic P sources, so organic P is clearly important. Surveys of species distributions and water chemistry have shown that some organisms are actually favoured by organic P. Bioavailability of organic phosphorus

5 Growth of the aquatic moss Warnstorfia fluitans (Hedw.) Loeske from an acidic stream in North-East England in media containing inorganic P (  ), PME (G-1-P;  ) and PDE (DNA; ) over 31 days. 0.25 mg L -1 P0.5 mg L -1 P1 mg L -1 P Biofilms consisting of Chlorococcum sp., Phormidium sp. and Synechocystis sp. medium containing KH 2 PO 4 at 3.68 mg L -1 P ( ) and 0.368 mg L -1 P and organic P only (glucose-6-phosphate) at 0.368 mg L -1 P ( □ ). Growth of organisms using organic phosphorus Organic P typeNos of strains growing para-nitrophenyl phosphate50 β-glycerophosphate 50 bis-para-nitrophenyl phosphate 47 DNA49 ATP40 phytic acid35 A survey of 50 cyanobacterial strains cultured in media containing organic P (Whitton et al. 1991).

6 mosses are non-motile, evergreen, relatively tolerant to various pollutants and easy to find and identify. Assessment of atmospheric N deposition studied using the internal N content and phosphatase activities of terrestrial mosses. Mosses growing at terrestrial and aquatic sites display ‘surface’ phosphatase activity, which varies in response to the concentrations of N and P in the moss tissue. This in turn reflects the relative availability of these nutrients in the ambient environment. Mosses possess the ability to use a wide range of organic phosphate esters present in the ambient environment. Whole catchment monitoring – as broadly the same methods can be used for terrestrial and aquatic mosses, and similar sets of samples can be used for monitoring phosphorus status and heavy metal contamination. Bryophytes and nutrient status of upland areas

7 Phosphatases: background information Phosphatases are enzymes that hydrolyse organic P compounds: –Phosphomonoesterases (PMEase) hydrolyse phosphomonoesters e.g. sugar phosphates, inositol hexaphosphates –Phosphodiesterases (PDEase) hydrolyse phosphodiesters e.g. DNA, RNA, phospholipids Activity can often be demonstrated simply by staining (BCIP- NBT, ELF ® 97).

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9 Phosphatases: background information Phosphatases are represented by a "whole bunch of enzymes" (Boavida, 1990), characterized by different half-saturation constants, temperature and pH optima and substrate specificity. SubstrateProductEnzyme para-nitrophenyl phosphate (pNPP) para-nitrophenolPMEase 4-methyumbelliferyl phosphate (MUP)methylumbelliferonePMEase bis-para-nitrophenyl phosphate (bis-pNPP) para-nitrophenolPDEase bis-4-methylumbelliferyl phosphate (bis-MUP)methylumbelliferonePDEase ELF ® 97 phosphate ELF ® 97 alcoholPMEase

10 Phosphatase activity: characterisation (  mol pNP mg -1 d.wt h -1 ) PMEase PDEase

11 Phosphatase activity: characterisation V max KmKm R2R2 PMEase0.461000.98  PDEase0.331390.93 PMEase and PDEase ctivities of phytobenthos dominated by Cladophora glomerata in Lake Albano, Rome, Italy

12 Cranecleugh Burn (CB) N. T. W. Ellwood, S. M. Haile, B. A.Whitton 2008. Aquatic plant nutrients, moss phosphatase activities and tissue composition in four upland streams in northern England. Journal of Hydrology. 350, 246– 260

13 Yet Burn (YB) Cranecleugh Burn (CB)

14 Stonesdale Beck (SD)

15 Stony Gill (SG) Stonesdale Beck

16 Moss species

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19 Filtrable Reactive Phosphorus and Filtrable Organic Phosphorus concentrations of the four sample streams (n = 3) Seasonal streamwater phosphorus concentrations

20 vv Water quality variability of two upland streams

21 Seasonal coefficients of variation of streamwater P status, and moss tissue nutrient status and surface PMEase and PDEase activities (%) Seasonal variation of environmental and biological variables

22 Relationships between cellular and ambient water nitrogen and phosphorus

23 Relationship between phosphatase activity and tissue nutrient status MUP (  ) pNPP (  ) bis-pNPP( ) pNPP (  ) MUP (  )

24 Christmas, M & Whitton, B.A. (1998b) Phosphorus and aquatic bryophytes in the Swale - Ouse river system, north- east England. 2. Phosphomonoesterase and phosphodiesterase activities of Fontinalis antipyretica. Science of the Total Environment 210/211: 401-409. River transect study

25 Didymosphenia geminata an invasive species or and bioindicator of global warming or catchment disturbance?

26 River Coquet Northumberland Changes in FOP as % FTP from Jan to Aug 2000. Stony Gill North Yorkshire

27 Global distribution of Didymosphenia geminata

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29 Nutrients fractions in upland environments can be dominated by organic forms Increasing evidence indicates that organisms can obtain nutrition from organic compounds Basic biological measures do not take this into consideration Phosphatases are ubiquitously produced and simple assays give much information of nutrient status of environment (PMEase and PDEase) Relationships between activity and tissue and environment nutrient status Indication of organic nutrient dynamics Surveys using longer living organisms that tolerate a wide range of nutrient conditions, i.e. with a wide distribution, are particularly useful for both spatial and temporal monitoring Changes in activities integrate large ambient variation and better indicate trends. Relative concentrations of organic P compared to inorganic P offers competative advantages to certain species. A simple measure, avoiding lengthy taxonomic identification Similar methodology for aquatic and terrestrial mosses for whole catchment surveys Conclusions

30 Thank you !

31 * P < 0.05 and ** P < 0.01 Surface phosphatase activity, cellular and ambient nitrogen and phosphorus

32 B.L. TURNER, R. BAXTER, N.T.W. ELLWOOD and B. A. WHITTON (2003) Seasonal phosphatase activities of mosses from Upper Teesdale, northern England. Journal of Bryology 25: 189–200 Relationships between PMEase activity and tissue N, tissue P and tissue N: P ratio of four species sampled from contrasting environments (terrestrial and semi-aquatic) from Nov 1999 to Oct 2000 from Widdybank Fell. Terrestrial and semi-aquatic

33 Phosphatase, tissue N and P and environment realtionships The interrelations between tissue N and P concentrations, tissue N : P ratio, phosphatase activities and aqueous variables showed : 1) Significant +ve relationship between tissue N and aqueous NO 3 -N in some populations, but not between tissue P and aqueous P concentration; 2) Significant +ve relationships between phosphatase activities and aqueous organic N, but none with aqueous organic P; 3) Significant +ve relationships between phosphodiesterase : phosphomonoesterase activities and aqueous organic N; 4) Significant -ve relationships between phosphatase activities and tissue P concentration; 5) Significant +ve relationships between phosphatase activities and tissue N : P. Both types of biological measurement are valuable for monitoring ambient nutrients in upland streams. Neither method is clearly better than the other, so both should be included in surveys.

34 Natural sources of phosphorus that supply freshwaters derive from the weathering of the catchment parent rock and soil, and from atmospheric input. Particulate P can be composed of many mineral, amorphous precipitates and sorbed reaction products. Dissolved P is comprised of orthophosphate, inorganic polyphosphates and organic P. Dissolved organic P generally occurs as phosphomonoesters (PME) and phosphodiesters (PDE).............and can be the dominant P form in Upland water courses (> 90%). Phosphorus in freshwater Changes in FOP as % FTP from Jan to Aug 2000. Stony Gill, North Yorkshire, UK

35 Phosphatase activity and pH

36 River transect study Christmas, M & Whitton, B.A. (1998a) Phosphorus and aquatic bryophytes in the Swale - Ouse river system, north-east England. 1. Relationship between ambient phosphorus, internal N:P ratio and surface phosphatase activity. Science of the Total Environment 210/211: 389-399.


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