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A Viability study on the use of enriched sediment verses

natural sediment to increase growth and survival rates of

Zostera marina.

Daniel j. Flint FdSc Marine Science

Falmouth Marine School

May 2012

Abstract

Zostera marina is a key habitat in Northern European coastal waters due to the

nursery grounds that it provides for many juvenile species. Unfortunately due to the

naturally occurring fungal disease found within Zostera marina whipping out over

90% of the naturally occurring eelgrass beds in northern European waters in the

1930’s. This naturally occurring disease which normally has no effect on the eelgrass

due to its natural defence of phenoic acid was able to take such a devastating effect

due to the very poor weather conditions in the 3 years prior to the epidemic resulted in

the eelgrass not being able to build the nitrogen reserves and therefore the phenoic

acid as a natural defence against pathogens. This study has looked into the viability of

the most effective methods of cultivating eelgrass, with the long term goal of re-

introduction in to the natural environment, either replenishing existing Zostera m.

beds or possibly seeding new Zostera m. beds. This study looks at the effect of

nutrient enriched sediment verses natural sediment of the effect of growth rate and

therefore net nitrogen gain/reserves. By creating a system in which both populations

of Zostera m. where exposed to the same environmental conditions, with only one

being in nutrient enriched sediment the sole effect of the sediments can be reliably

measured. Through statistical tests of the comparative growth rates of both

populations of Zostera m. it is proven that enriched nutrient sediments increases

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growth and survival rates of Zostera m. and therefore the viability of reintroduction

into the natural environment.

Introduction

The lower Fal and Helford intertidal

areas are classified as SSSIs (Natural

England, 2011). Within these areas

there are habitats designated as SACs.

Special Areas of Conservation (SACs)

are sites that have been adopted by the

European Commission and formally

designated by the government of each

country in whose territory the site lies

(Natural England, 2011). Zostera

marina, Eelgrass is one of four closely

related species of seagrass in European

water. Seagrasses have evolved from

different freshwater species, some

being more related than others. There

are other species of aquatic plants that

inhabit the marine areas of low to

moderate salinity however the only

group that can be classified as seagrass

are found in high salinity oceanic

waters (Borum & Greve 2004) Z.

marina inhabit from the intertidal

sones down to 5 - 15 meters depth in

northern European waters (J. Borum et

al. 2004) being able to do so has

resulted in Zostera marina or Eelgrass

is the most dominant species of

seagrass in northern temperate coastal

waters. Eelgrass (Zostera marina)

inhabits these areas due to its

preference to grow in sheltered

intertidal zones and down to 5-15

meters in northern European waters. (J.

Borum et al, 2004). With the fal having

wide areas of shallow sheltered water

produced from the tin mining further

up stream producing fine sand, mud

and silt deposits, producing anoxic

conditions under the sediment. This is

favourable for Zostera as typical

growth occurs in highly reducing

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sediments. Photosynthesis-mediated 02

supplied to below-ground tissues

sustains aerobic respiration during

photosynthetic periods. (R.D. Smith,

1998). Zostera is able to survive and

thrive in these conditions through

being able to obtain O2. Strict

anaerobic conditions inhibit plant

growth in all but a few species of plant.

The ecological success of eelgrass in

anoxic sediments must rely

predominantly on anatomical and

physiological features that prevent

below ground tissues from becoming

permanently anaerobic. (Armstrong

1978, Crawford 1978, Davies 1980).

However the physiological adaptive

features of this ecologically important

species are not well understood.

Extensive reviews by McRoy and

Mcmillan and Zieman and Witzel have

stated that there is very little

experimental data available on

photosynthetic characteristics on these

species beyond the estimates of plant

primary production. (McMillan and

McRory. 1977) and (Zieman and

Witzel. 1980) Studies have shown that

zosteras physiological responses of

temperate seagrass, Zostera marina,

ability to absorb light. These factors

have been expanded upon by the fact

that over the past 10 years 62% of all

published journals on seagrass have

purely focused on light intensity and

epiphytes while only 18% have

focused on hydrodynamics, now

thought to be one of the key factors in

eelgrass growth. 17% have focused on

sediment characteristics only major

factor contributing to eelgrass growth

that previously was thought to be a less

significant factor. Only 3 % of the

journals published have been focused

on seagrass geochemistry. (E. W. Koch

2001). Though the understanding of

eelgrass hydrodynamics it has become

clear that there are far more

understanding needed than purely the

light intensity required as even with

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high light intensity if the flow rate is

too high (above 150 cm/s there will be

reduced average growth rates due to

self shading from neighbouring

eelgrass. (E. W. Koch 2001) however a

flow rate lower than 18 cm/s will result

in a increased DBL thickness in turn

reducing the CO2 absorption resulting

in reduced photosynthesis, over

prolonged periods can be the cause of

death in the species. (Jones et al.

2000). Having said these studies still

have shown that light intensity is a

major contributing factor, as light

shading studies indicated a massive

reduction in standing stock of Zostera

marina due to light irradiance,

(Dennison 1979). Though studies

carried out on light intensity and

nutrient enrichment it was clear that

only in light saturated conditions were

nutrients an additional factor in

increased seagrass production. The

results of the experiment suggested

that the available light is the principle

factor governing seagrass production

in moderately nutrient enriched

environments. (K. A. Moore & R. L.

Wetzel). As is apparent there is still

much debate between the effects and

causes of Zostera standing stock

diminishment. These factors have to be

further understood as to provide

adequate conservation. The first step in

conservation is understanding the

biological makeup and physical

identification of Zostera marina.

Zostera marina has leaf bundles with

terminal shoots on horizontal

rhizomes. Branching of the rhizomes

occurs during the growth season

forming the new terminal shoots. (J.

Borum and T. M. Greve. 2004). Each

new leaf formed produces a new

rhizome segment (internode) and two

bundles of roots develop from the

nodes between the segments. The roots

themselves are thin (0.2-1mm),

covered in fine hairs that may be up to

20cm long. The rhizomes segments on

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which the roots develop are generally

2-6mm in thickness and vary from 5-

40mmin length. Begin as a white/green

colour and change to dark brown in the

older segments. (J. Borum and T. M.

Greve. 2004). Male and female flowers

are found on the same individual. Male

and female flowers of Zostera marina

are small, greenish and hidden in

pockets within leaf sheaths. Zostera

marina may produce thousands of

seeds per square meter and flower

regularly under optimum conditions

predominantly flowering from early

spring till autumn. During this period

the Zostera begins to change

morphology to produce more leaf

bundles separated by long thin stem

segments. (J. Borum and T. M. Greve.

2004). Being an angiosperm Zostera

marina has the same nutrient

requirements as terrestrial plants, but

obviously with a few key differences.

The major nutrients required for

Zostera growth are Carbon dioxide,

Nitrogen and phosphate. The Redfield

ratio of C:N at 106:16 of carbon to

nitrogen (Redfeild et al. 1963) being a

marine primary producer one might

expect that eelgrass would contain the

same ratio of carbon to nitrogen

however it considerably lower than in

other marine species such as

phytoplankton. (Atkinson & Smith.

1984, Duarte 1990). Although this is

the case overall nitrogen requirement

for maximum eelgrass production per

m2 is still 3 time higher than that of

phytoplankton production. (M. F.

Pedersen. J. Borum. 1992). Eelgrass

unlike most angiosperms not only

absorb nutrients from the sediment

they also uptake nutrients from the

water column. (Iizumi & Hattori, 1982,

Thursby & Harlin, 1982, Short &

McRoy, 1984). Sediments where

Zostera can be located have generally a

much high concentration of nitrogen

than in the water column. (Lizumi &

Hattori, 1982, Boon 1986, Dennison et

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al. 1987) although this is the case

Zostera marina leaves have a much

higher uptake affinity than root

rhizomes (Short & McRoy 1984)

suggesting that the sediment is not the

primary source of nutrients. however

later studies have shown that nutrient

uptake can be accredited to both the

leaves and roots-rhizomes as both

contribute significantly to overall

nitrogen uptake in eelgrass (Iizumi &

Hattori, 1982, Short & McRoy 1984,

Zimmerman et al, 1987).

Fig 1. Depiction of natural geographical distribution of

Zostera marina.

However due to a disease in the 1930’s

sweeping across Europe depleting the

population by 90% (Koch 2001) only

those in brackish waters survived.

Zostera marina is under huge amounts

of pressure still due to human activity,

sediment disturbance is a massive

influence to the growth and well being

of Z.marina if the sediment is

disturbed, the turbidity increases, this

reduces light intensity to the Z.marina

leaves this as we know from Zosteras

high light intensity requirements very

detrimental to growth rates. For these

reasons, this study was carried out to in

a bid to asses the viability of culturing

Zostera marina in the lab, with the

idea to reintroduce Zostera m. back

into the natural environment either

reseeding existing Zostera m. beds or

potentially seeding new Zostera m.

beds.

Method and materials

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A 130 litre tank was divided into two

sections by the use of a purpose built

Perspex divider see fig. 2. This

allowed the separation of the two

sediment types, natural sediment. Both

sections where then filed with a base

layer of coral sand this was placed on

the bottom of both sections to enable

water flow to prevent excess anoxic

conditions. This can be seen on the left

of Fig. 2.

Fig. 2 Cultivation tank set up. (D.Flint 2011)

Once the base layer was in place and

levelled out, the sediments where then

put into place one side containing the

natural sediment a mud/slit mixture

that was taken from Deveron mudflats,

see fig. 3 for details of specific area.

The sediment was taken from this area

as this is consistent with the natural

sediment of Zostera m. beds in this that

area.

Fig. 3 Google maps, location for source of natural

sediment.

The second section was filled with a

nutrient enriched sediment, (Aquatic

fertiliser). On top of these sediments a

second layer of fine coral gravel and

sand was placed this was to prevent the

water movement causing the sediments

to be stirred into the water column

causing a increase in turbidity and

therefore effecting light intensity and

therefore growth rates. Also the use of

coral gravel provides a natural

buffering of the water to help maintain

the natural pH of the water maintaining

a level between 7.4 and 8.0 recreating

the natural conditions (J. Borum and T.

M. Greve. 2004). The lighting required

to recreate the natural light conditions,

a Halide lighting system with a 150

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watt 5000k bulb alongside two 24watt

T5 bulbs. These were put on a

electronic timer which was set for 16

hours a day for recreate the natural

light duration at the time of the start of

the study. The filtration system used

was a canister filter to provide good

water quality and decent water flow

without reducing aquarium space or

causing shading. To increase water

flow as recommended by (J. Borum

and T. M. Greve. 2004) two 1600lph

power heads were placed on opposite

ends of the tank on a waver maker

system this allowed not only increased

water flow but intermittent water flow

causing a wave motion in the tank

preventing the build up of macroalgaes

and filamentous algae which would

reduced growth of Zostera m. as stated

by (J. Borum. 2004) Zostera m.

requires a wave period of 0.4-0.7

seconds. Once set up the Zostera m.

may be planted placed into the

sediment only up to the crown under

low light intensity to establish the

growth of the root systems. Once

established and any non viable

specimens removed form the tank and

the study may begin.

Measurements were taken on a weekly

basis measurement to be taken from

the crown to the tip of the tall frond.

Each specimen was numbered by a

small cable tie being numbered and

placed around the base of the

specimen. The number of fronds is to

be recorded this together with the

labelling system will allow reliable

record to be kept.

Results.

From the data collected from the

cultivation tanks, the overall growth

has been established and placed on to

the graph in Fig. 4 taking into account

the loss of the samples in the natural

sediment as opposed to no losses in the

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enriched sediment once the trial had

begun. As can been seen form the 2.

Table 1. Chi Squared Statistical test of overall growth rates

of the two populations of Zostera marina

graph there is not the same amount of

data for each sediment type, this is due

to in the settlement period there was

considerably more loss of Zostera m

samples within the natural sediment

that in the enriched sediment. However

sediment type was not the sole

contributor to this as the time that the

study was carried out coincided with

the time of year Zostera m begins

to stop growth, as well as the fact that

Zostera is a protected species could

not be harvested for this study due to

Zostera m being a Protected species

within the Fal under SSSI and SAC

legislation. The Zostera m. had to be

Growth (mm)

Nutrient Enriched Natural

9 5

10 9

10 XXX

3 12

14 XXX

10 9

10 12

13 10

11 9

8 XXX

7

12

10

11

10

10

Averages 9.875 4.125

O E O-E (O-E)2 (O-E)2/E

Nutrient 9.875 7 2.875 8.265625 1.180803571

Natural 4.125 7 -2.875 8.265625 1.180803571

2.361607143

Critical value 3.841

Statistical value 2.362

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collected form specimens that had been

washed ashore is rough weather

therefore prior damage to the Zostera

may have occurred that may not have

been apparent, degradation of the

Zostera may have already begun. This

being the case the Zostera samples in

the nitrogen rich, nutrient enriched

sediment did show a marked

improvement in the survival of the

specimens, as show in table 1, this may

be due to the nitrogen deficit normally

occurred in nitrogen recycling by the

Zostera (J. Borum. 2004). Carrying out

a chi squared statistical test on the

samples as shown in table 2. shows a

significant difference in the growth

rates of the two populations favouring

the enriched sediment see table 2 for

details. The statistical value is lower

than that of the Critical value therefore

significantly different. Due to the

limited amount of data that could be

collect from this study it would appear

that enriched sediment does have a

huge bearing on the growth and

survival rates of Zostera which is

reinforces the hypothesis of (J. Bourm

2004) who stated that the only effect of

higher nitrogen levels within the

sediment will only replace the nitrogen

lost from nitrogen recycling by the

Zostera as the species is very effective

at recapturing any nitrogen lost from

decaying leaves and roots. To back up

these results a much more

comprehensive study on a larger

scales would be needed to qualify

these results.

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Overall Growth measurements.

0

2

4

6

8

10

12

14

16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Specimen number

Gro

wth

in

mm

Nutrient enriched

sediment

Natural sediment

Fig. 4 A graph to show the Overall growth rates of the Two populations of Zostera marina.

The three troughs in the graph of the

natural sediment show the number of

species that did not survive in the

cultivation tank. This may be due to

the bridging of the nitrogen deficit of

nitrogen recycling (J. Bourm 2004).

However this may also be due to the

several contributing factor that affected

the results that were collected, firstly

one of the power heads designed to

created irregular water flow preventing

shelf shading (Koch, 2001) fell of its

placement into the sediment on the

natural sediment section. This caused

massive disturbance of the sediment

and resulted in several specimens

being uprooted. These specimens had

to be replanted this would have set

back growths rate and may very well

have resulted in the loss for specimens

in this section. Problems were also

experienced with the lighting system, a

malfunction in the electronic timers

resulted in the constant output of high

intensity light. This resulted in excess

algae within the tank, lots of macro

and filamentous algae was produced

which has a hugely negative effect on

the growth of Zostera marina. (J.

Bourm, 2004).

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Discussion

Through this study it is apparent that

the theory proposed by (J. Bourm,

2004) that increased levels of nitrogen

in the sediment will provide the small

percentage of nitrogen that is lost to

the environment from nitrogen

recycling by Zostera marina. Enabling

Zostera not only to survive stressful

conditions i.e. uprooting and replanting

but to also show a growth pattern

larger than that of natural sediment.

Having said that it may also the case

that the contributing factors in this

study such. The contributing factors in

this study being the power head

disturbance and the faulty lighting

timer may have had adverse effects on

the natural sediment populations

causing a less pronounced growth and

survival rate seen in the enriched

sediment population. If this study were

to be carried out again, suggestions

would be to carry out the study on a

larger scale using more specimens in

each sediment sample. Also it would

be advisable to collect Zoster m. in

early spring to summer as this is in

Zostera m primary growth period

therefore being healthier, stronger and

have larger nitrogen and phenoic acid

reserves (Koch 2001). It would also be

advisable to use larger power heads to

create a larger intermittent flow

reducing shelf shading and a more

adequate wave period of 0.4-0.7

seconds (J. Bourm, 2004). This would

result in more accurate and reliable

results to more accurately identify the

importance of using a nutrient enrich

sediment to increase growth and

survival rates. As (Borum et al, 1989)

suggested that nitrogen conservation

i.e. reclamation could potentially be an

important mechanism for eelgrass

nitrogen nutrition, as studies have

shown that this is the case, even with

deciduous terrestrial, evergreen plants

living in nutrient poor environment

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therefore providing the nitrogen to the

eelgrass that would normally have

been lost though this almost

completely effective process can

explain the reason for the population of

Zostera marina in the nutrient enriched

sediment to be healthier than the

population in the natural sediment.

Acknowledgements

I gratefully acknowledge the help and support provided by Craig Baldwin, Falmouth

Marine School for help, advice and concerns with aquaculture as well as use of lab

and equipment with funding from Southampton University, Falmouth Harbour

commission and Falmouth Marine School. I would also like to thank Luke Marsh,

Falmouth Marine School, for his help taking measurements and helping with

maintenance of the study tank.

References

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Koch, E.W., 1996. Hydrodynamics of shallow Thalassia testudinum beds in Florida, USA. In: Kuo, J., Phillips, R.C., Walker, D.I., Kirkman, H. (Eds.), Seagrass Biology: Proceedings of an International Workshop, Rottnest Island, Western Australia, 25±29 January 1996. Sciences UW, Nedlands, Western Australia, pp. 105±110.

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Muehlstein, L.K., Porter, D. and Short, F.T., 1991. Labyrinthula zosterae sp. nov., the causative agent of wasting disease of eelgrass, Zostera marina. Mycologia, 83: 180-191. Patriquin, D. G. (1972). The origin of nitrogen and phosphorus for growth of the marine angiosperm Thalassia testudinum. Mar. Biol. 15: 35-46 Pedersen, M. F., Borum, J. (1992). Nitrogen dynamics of eelgrass Zostera marina during a late summer period of high growth and low nutrient availability. Mar. Ecol. Prog. Ser. 80: 65-73 Phillips RC, McRoy CP (1990) Seagrass research methods. Monographs on oceanographic methodology. UNESCO, Paris. 210 pp Rasmussen, E., 1977. The wasting disease of eelgrass (Zostera marina) and its effects on environmental factors and fauna. In: C.P. McRoy and C. Helfferich (Editors), Seagrass Ecosystems. A Scientific Perspective. Marcel Dekker, New York/Basel, pp. 1-52 Renn, C.E., 1936. The wasting disease of Zostera marina: I. A phytological investigation of the diseased plant. Biol. Bull., 70: 148-158.

Short, F.T., Mathieson, A.C. and Nelson, J.I., 1986. Recurrence of the eelgrass wasting disease at the border of New Hampshire and Maine, U.S.A. Mar. Ecol. Prog. Set. 29: 88--92.ng, E.L., 1938. Labyrinthula on Pacific coast eelgrass. Can. J. Res., 16:115-117. Thayer, G.W., Engel, D.W., LaCroix, M.W., 1977. Seasonal distribution and changes in the nutritional quality of living, dead, and detrital fractions of Zostera marina L. J. Exp. Mar. Biol. Ecol. 30, 109–127 Tutin, T.G., 1938. The autecology of Zostera marina in relation to its wasting disease. New Phytol., 37: 50-71. Vergeer, L.H.T. and den Hartog, C., 1991. Occurrence of wasting disease in Zostera noltii. Aquat. Bot., 40: 155- 163. Vergeer, L.H.T. and den Hartog, C., 1994. Omnipresence of Labyrinthulaceae in seagrasses. Aquat. Bot., 48: 1- 20.

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Fig. 1. Natural Geographical Distribution of Zostera marina, D. Flint 2012, map sourced from

google maps.

Fig. 2 Cultivation tank set up sediment set up through to filling. D. Flint 2011.

Fig 3. Google maps Deveron mudflats [Online] available at:

http://maps.google.co.uk/maps?hl=en&tab=wl (Accessed on 29th

April).

Fig. 4. A graph to show the Overall growth rates of the Two populations of Zostera

marina