# 2003 Kluwer Academic Publishers. Printed in the Netherlands.

The use of ultra-sound in the preparation of carbonate and clay sedimentsfor chironomid analysis

Barbara Lang1,*, Alan P. Bedford1, Nigel Richardson1 and Stephen J. Brooks2

1Department of Natural, Geographical and Applied Sciences, Edge Hill, St. Helens Road, Ormskirk,

Lancashire L39 4QP, UK; 2Department of Entomology, Natural History Museum, Cromwell Road,

London SW7 5BD, UK; *Author for correspondence (e-mail: langb@edgehill.ac.uk)

Received 11 April 2001; resubmitted 30 October 2002; accepted 1 June 2003

Key words: Carbonate sediment, Chironomidae, Clay sediment, Hawes Water, Paleolimnology,

Ultra-sound

Abstract

Initial investigations of Holocene carbonate sediment from Hawes Water, Northwest England, yielded lower

numbers of chironomid head capsules than anticipated. Standard techniques used to prepare sediment for

chironomid analysis were ineffective in breaking up the coarse crystalline sediment structure sufficiently.

This led to large amounts of sediment being retained and increased sample processing times. The low yield of

head capsules also meant that more sediment was needed to produce adequate numbers of head capsules for

analysis. The use of ultra-sound as part of the sediment processing was investigated. This technique reduced

the amount of sediment left for sorting and yielded significantly more head capsules which were of equivalentstructural condition and cleaner than those produced by conventional methods. The technique was extended

to clay samples where similar results were obtained although shorter treatment times are recommended. The

proportion of Tanytarsini and Tanypodinae heads increased significantly in carbonate and clay samples,

respectively; both sediment types showed a significant decline in the proportion of Chironomini. The results

indicate that ultra-sonic preparation of samples will yield a more accurate representation of chironomid

assemblages in sediments leading to greater sensitivity and reliability in analysing past environmental

conditions.

Introduction

The use of sub-fossil Chironomidae as indicators

of past environmental change is becoming increas-

ingly popular. The value of Chironomidae in this

field is due to several attributes of the family. They

are present and abundant in most freshwater

environments. The larval head capsules preserve

well in sediment and are identifiable at least to

generic level. Many are stenotopic. The rapid gen-eration time plus the fact that the adult midge is

able to disperse rapidly means that they are quick

to respond to environmental change. These

factors suggest that the chironomid assemblage of

a water body is in approximate equilibrium with

the environmental conditions of the time and itis, therefore, possible to derive environmental

inferences from fossil assemblages.

Lindegaard (1995) describes the versatility of

Chironomidae as aquatic bio-indicators. They

have been used to establish a lake classification

system, grouping lakes according to their trophic

level (Brundin 1949; Sæther 1979), to indicate and

assess the extent of freshwater acidification(Wiederholm and Erikson 1977; Henrikson et al.

1982; Walker et al. 1985; Brodin 1990; Johnson

et al. 1990; Schnell and Willassen 1996), to investi-

gate anthropogenic eutrophication and oxygen

451Journal of Paleolimnology 451–460, 2003.30:

levels (Clerk et al. 2000; Francis 2001; Little and

Smol 2001; Merilainen et al. 2001), to examine

variations in lake salinity (Paterson and Walker

1974; Clair and Paterson 1976; Walker et al. 1995;

Heinrichs et al. 1997; Verschuren et al. 2000) and toassess the impact of heavy metal contamination on

freshwater ecosystems (Armitage and Blackburn

1985; Kansanen 1985; Yasuno et al. 1985;

Ilyashuk and Ilyashuk 2001). Chironomidae have

also been used to infer temperatures for the Late

Glacial and early Holocene by means of transfer

functions (Walker et al. 1991; Olander et al. 1997;

Brooks and Birks 2000a, b; Larocque et al. 2001;Korhola et al. 2002). The use and significance of

Chironomidae in the fields of paleoecology have

been reviewed in detail by Hofmann (1986, 1988)

and Walker (1987, 1995, 2001).

The method used to prepare chironomid samples

for analysis as described by Brooks and Birks

(2000a), is now fairly standard, although slight

variations to this method do exist in terms of theconcentrations of chemicals used, lengths of time

spent in these chemicals, temperatures at which sedi-

ments are deflocculated and microscope magnifica-

tion. It should be noted that most chironomid

workers use wet sediment for analysis and this is

recommendedbyWalker (2001); someworkershow-

ever have dried the sediment prior to deflocculation.

A study of sediments from Hawes Water,England, aims to establish a fine resolution multi-

proxy paleoclimatic reconstruction for Northwest

England. The study uses a wide variety of indepen-

dent biological and chemical indicators including

both stable isotope analysis (18O/16O and 13C/12C)

and chironomid analysis. Chironomid data fit well

into such a study both complementing and enhanc-

ing interpretation of results from other indicatorgroups. Due to the inclusion of stable isotope ana-

lysis in the project the choice of site for the study

was limited to lakes which had produced carbo-

nates in the past. The sedimentary record at

Hawes Water extends from the onset of the

Devensian Late Glacial up to the present day, thus

giving a complete Holocene record. Carbonate

deposition appears to have taken place from thebeginning of the Late Glacial period up until

around 4500 years BP (Mann, unpublished data).

The carbonate from Hawes Water is primarily a

shell rich, fine-grained calcitic micrite consisting

mainly of CaCO3 precipitate with a small amount

of organic detritus and little or no clastic mineral

content. Carbon analysis (Mann, unpublished

data) shows carbonate percentages of the sediment

to be well in excess of 97%. Little work has been

done on carbonate lakes. Bryce (1962) looked atchironomids from Malham Tarn, Yorkshire,

England, and recently Lotter et al. (1997) and

Brooks (2000) have worked on Swiss carbonate

lakes. None of the sediment from the Swiss lakes,

however, was as pure as that derived from Hawes

Water. Initial chironomid analysis of the Holocene

carbonate from Hawes Water prepared using the

standard method (Brooks 2000), yielded very fewheads. Six tests were run on samples weighing

around 10 g each (dry weight). Only one sample

produced more than 70 heads and the average head

count per sample was 56.5. A total of 61.08 g (dry

weight) of sediment produced only 339 heads, an

average of 5.5 heads g�1. Although the low yield of

chironomid heads may have been due to a rapid

carbonate precipitation rate or poor head preserva-tion within the carbonate we felt that it most

likely arose from inefficient extraction techni-

ques. Recent publications (Heiri and Lotter 2001;

Larocque 2001; Quinlan and Smol 2001) suggest

that the number of heads extracted from some of

the samples may have been adequate for statistical

inferences to be made, however 56.6 heads g–1 was

the average and some of the individual samples hadvery low counts. The process of picking the chiro-

nomid head capsules from the sediment, which is

often noted by chironomid workers as being

tedious was prolonged further because of the nat-

ure of the micrite. The crystalline structure and the

low density of the carbonate meant that to extract

the number of head capsules necessary for analysis

more than 6 cm3 of sediment would have to bepicked through. This would leave little sediment

for analysis of other proxies from the same

core. In addition, the chironomid head capsules

extracted from the sediments were often crumpled

and contorted. They were covered in a fine carbo-

nate dust and many had large amounts of sediment

within the head capsule, which made identification

difficult.We investigated a number of different approaches

to destroy or break down the carbonates without

destroying the chironomid heads. This paper

proposes an alternative preparatory method for

chironomid analysis of carbonate sediments using

452

ultra-sound treatment. Such sediments have often

been classified as ‘marl’. However, it must be noted

that marl is defined as having a clay content in

excess of 10% (Reijers and Hs€uu 1986) and with a

carbonate content in excess of 97%, classificationof the Hawes Water sediment as marl would there-

fore be inaccurate. This point is important as sedi-

ments with a clay content react differently to the

ultra-sonic treatment. Adaptations to the pro-

posed method are included for samples with a

clay content.

Methods

The carbonate sediment used was from an 8.5-m

marginal core taken from the south side of Hawes

Water. One-centimetre core slices were taken at

intervals throughout the cores. The standard pre-

paration for extracting chironomid head capsules

from these core slices involved deflocculation for

15 min in 10% KOH heated to 75 �C and then

sieving on 212- and 90-�m meshes (Brooks 2000).The sediments were transferred to a sorting tray

and examined under a binocular microscope (�40

magnification). Chironomid head capsules were

extracted using fine forceps, dehydrated in 100%

ethanol and then mounted in Euparal1. Identi-

fication of chironomid head capsules was made

using a number of reference sources including

Hofmann (1971), Cranston (1982), Wiederholm(1983), Oliver and Roussel (1983) and Rieradevall

and Brooks (2000).

The use of wet samples is normally recom-

mended (Walker 2001). Drying of sediments in

many cases causes samples to become hard and

concrete like and therefore difficult to process.

These problems however are not found with the

carbonate sediments from Hawes Water. The crys-talline nature of the sediment is not changed by air-

drying or oven-drying at a low temperature. Due to

the alkaline nature of the sediment standard pro-

cedures (i.e., KOH deflocculation) fail to break

down the sediment and the use of even weak con-

centrations of HCl to remove the carbonate and

clean the heads is unsatisfactory because of the

purity of the carbonate sediment. The reactionscreate gas bubbles in the heads, are too violent and

destroy or badly damage the heads. The sediment

can only be broken down by a gentle mechanical

process, which in itself can damage the heads. The

use of oven-dried (e.g., 35 �C) sediments has the

advantage that accurate comparisons between sub-samples can be made and the number of heads per

gram of sediment can be calculated.

The impact of drying on extraction efficiency

was investigated using oven dried and fresh (wet)

sediment samples from the same horizon. The two

sub-samples had the same sedimentary history and

should, therefore, contain a similar chironomid

assemblage. We examined a total of five pairs ofsamples from different horizons. Following stan-

dard preparation, head concentrations (numbers

per gram dry weight) were compared using a

matched-pairs t-test. The head concentrations for

the wet sediments were calculated using a dry

weight calibration figure derived from the corre-

sponding dry sample. No significant difference was

found (Table 1).Ultrasound treatment was investigated as an

alternative method for extracting head capsules.

Individual samples were weighed, placed into a

250-ml beaker in 100 ml of warm water (40 �C)

and gently broken up with a glass rod. They were

then left to stand for 10 min and stirred intermit-

tently. This treatment was found to be as effective

as the traditional methods in initially breaking upthe core material. The beakers containing the

sediment were then placed into the sonic bath

(Branson, 200 ultra-sonic cleaner, 40 kHz) for

varying trial times. After ultra-sonic treatment the

samples were sieved on 212- and 90-�m meshes and

Table 1. Comparison of head densities in wet and oven dried

carbonate samples using a matched-pairs t-test (ns – not

significant).

No. heads g�1

Sample Dried sediment Wet sediment

1 9.75 6.46

2 8.99 9.05

3 13.35 15.90

4 6.57 14.41

5 15.46 7.33

Mean 10.82 10.63

t 0.07 ns

The head concentrations for the wet sediments were calculated

using a dry weight calibration figure derived from the

corresponding dry sample. The samples were prepared using

standard preparation techniques (Brooks 2000).

453

the residues examined. Ultrasound treatment

appeared to yield more heads, which were cleaner.

Far less sediment was retained on the sieves reduc-

ing considerably the overall extraction time.

Prolonged ultra-sound treatment however causednoticeable damage. On the basis of a number of

these trials, the following protocol was developed.

– The samples are broken down in warm water

(40 �C) and then sieved on 212- and 90-�m

meshes. (Tests had shown that the larger heads

were more susceptible to damage if they remained

in the ultra sound once freed from the sediment.

The two fractions of were therefore subjected toseparate periods of ultra-sound with the coarse

fraction ultra-sonic time being shorter).

– The coarse fraction is placed into a 250-ml

beaker in 100 ml water and sonic bathed for

2 min. (The sediment must be stirred whilst in

the sonic bath to ensure that all the sediment re-

mains in suspension in the water column while

being subjected to the ultra-sonic treatment).– This fraction is then re-sieved on 212- and 90-�m

meshes.

– The fine fraction is placed into 250-ml beakers

in 100 ml water and sonic bathed and stirred

for 3 min.

– This fine fraction is then re-sieved on a 90-�m

mesh.

– The residues are then sorted and the Chironomidheads extracted, mounted and identified using

standard techniques.

In order to ascertain if any heads were being

broken down to such an extent that they would

pass through the sieves, the sonic bathed sediment

from four samples was passed through an extra

sieve (63 �m). The sediment from the 63-�m sieve

was then examined for head fragments.Having established a working protocol, the

efficiency of this method compared to standard

procedures was examined. Fifteen samples from

different core slices were each split into two sub-

samples. These two sub-samples were then pre-

pared using the differing methodologies and the

results compared.

Late Glacial clay samples

The Hawes Water cores, whilst primarily micrite,

did include a band of Late Glacial clay. Even when

working with this fresh, wet clay material, defloc-

culation with KOH fails to break the sediment

down to particle size (clays and silts <65 �m).The use of ultra-sound to prepare and further

break down this sediment was investigated. A com-

parison of head extraction efficiency between fresh

(wet) and pre-dried material found no significant

difference (Table 2). To enable direct and accurate

comparison of head numbers the ultra-sound tests

were therefore run on dried samples. The clay sedi-

ments were deflocculated in KOH and then sub-jected to ultra-sonic treatment as described above.

After treatment the clay sediments were totally

broken down and passed through the sieves leaving

only a small amount of organic material to be

sorted. As with the carbonate samples, this short-

ened the overall extraction time. The samples

yielded far more heads although these heads had

suffered considerable damage. Further sampleswere prepared using shorter periods of ultra-

sound. The aim was to establish the point at

which the sediment was broken down and examine

the condition of the head capsules at this point. On

the basis of these trials, the following successful

protocol was developed.

– The samples are deflocculated for 15 min in 10%

KOH heated to 75 �C and then sieved on 212-and 90-�m meshes.

– The coarse fraction is placed into a 250-ml

beaker in 100 ml water and sonic bathed and

stirred for 10 s.

– This fraction is then re-sieved on 212- and 90-�m

meshes.

Table 2. Comparison of head densities in wet and oven dried

clay samples using a matched-pairs t-test (ns – not significant).

No. heads g�1

Sample Dried sediment Wet sediment

1 11.30 4.66

2 35.45 54.00

3 24.57 12.75

4 20.92 21.49

5 60.40 78.00

Mean 30.5 34

t 0.59 ns

The head concentrations for the wet sediments were calculated

using a dry weight calibration figure derived from the

corresponding dry sample. The samples were prepared using

standard preparation techniques (Brooks 2000).

454

– The fine fraction is placed into 250-ml beakers

in 100 ml water and sonic bathed and stirred

for 15 s.– This fine fraction is then re-sieved on a 90-�m

mesh.

– The residues are then sorted and the chironomid

heads extracted, mounted and identified using

standard techniques.

As with the carbonate method the efficiency of

this new treatment was compared to standard pro-

cedures. Fifteen samples from different core sliceswere each divided into two sub-samples. These two

sub-samples were then prepared using the differing

methodologies and the results compared.

Results

Carbonate sediments

Visual comparisons of the amounts of sediment

remaining after treatment showed that much less

sediment had been retained in the samples sub-

jected to ultra-sound than in conventionally pre-

pared samples. This was true for both the fine and

the coarse fractions. As a result extraction time was

considerably shorter (approximately 50%).The sediments subjected to ultra-sound were

much cleaner than conventionally prepared sam-

ples. This eased identification as the chironomid

heads were free from micritic particles and other

carbonate fragments. Examination showed that all

of the heads from the ultra-sonic samples were in asimilar condition structurally to heads from the

conventionally prepared sediment; no damage

had occurred to the heads whilst in the ultra-

sound. This was confirmed by examining the four

sediment samples retrieved from the 63-�m sieve.

From this sediment only one very small

Tanytarsini head and one mandible were found

and extracted.Head densities for standard and ultra-sonic

treatments were compared using a matched-pairs

t-test (Table 3). Significantly more heads were

extracted from the samples subjected to ultra-

sound treatment than from the conventionally pre-

pared samples. This was true for both size fractions

but the most substantial increase was seen in the

numbers of heads extracted from the fine fraction.Whilst all of the subgroups increased significantly,

Tantytarsini showed the biggest increase where on

average, nearly three times as many heads were

found. As chironomid analysis in climate recon-

struction work uses percentages rather than head

densities the percentage contribution of the main

groups to the total sample were compared using a

t-test (arcsine transformed data). Ultra-sonic treat-ment leads to a significant increase in the propor-

tion of Tanytarsini but a significant decrease in the

proportion of Chironomini.

Table 3. Comparison of head extraction efficiency between standard methods and ultra-sound treatment for oven-dried carbonate

sediments.

Head densities (heads g�1) Mean proportion of sample (%)

Mean

density,

standard

prep.

Mean

density,

ultrasonic

prep. N

Mean %

increase

of heads in

ultrasonic

samples t Significance Standard Ultrasonic t Significance

Total number of heads 9.59 15.13 15 61 7.58 ***

Heads retained on fine sieve 5.48 10.07 15 98 7.32 ***

Heads retained on coarse sieve 4.11 5.03 15 26 3.36 **

Tanypodinae 1.1 1.89 15 102 3.32 ** 13.42 13.03 0.11 ns

Orthocladiae 0.65 1.34 15 111 3.79 ** 6.5 9.1 1.81 ns

Chironominae 7.67 11.62 15 57 5.57 *** 79.01 75.83 1.65 ns

Chironomini 5.38 6.78 15 30 3.2 ** 56.3 44.29 4.65 ***

Tanytarsini 2.27 4.86 15 186 6.8 *** 22.6 31.83 4.73 ***

Head density data compared using a matched-pairs t-test. Proportion data (%) compared using a t-test following arcsine transformation.

Mean percentage increase derived from individual sample increases. A total of 4096 heads extracted. ns p > 0.05, *0.01 < p < 0.05,

**0.001 < p < 0.01, ***p < 0.001.

455

Examination of the conventionally prepared

samples (whether oven dried or fresh) identifieddistinct ‘clumps’ of organic material coated with a

fine dust of micritic particles in the fine fraction.

Similar clumps were not present in the ultra-sonic

samples. Manual attempts to break up these

clumps by prodding with a mounted needle did

not reveal any more heads.

Late Glacial clay sediments

As with the carbonate samples more heads wereextracted from the ultra-sound treated sediments

(Table 4) with the most substantial increase being

seen in the fine fraction. The mean percentage incr-

easesareconsiderably larger thanwiththecarbonate

samples with the greatest increase occurring with

the Tanypodinae where four times as many heads

were recovered. The percentage contribution of the

main groups to the total sample were again com-pared using a t-test (arcsine transformed data).

Ultra-sonic treatment leads to a significant increase

in the proportion of Tanypodinae and, as with

the carbonate samples, a significant decrease in the

proportion of Chironomini. Examination of the

sediment from the conventionally prepared sam-

ples shows that some of the sediment remains as

small balls of clay which soaking in KOH had failedto break up. The ultra-sonic treatment described

above breaks down clay sediments to particle size.

These sediments then passed through the sieves

leaving little or no inorganic sediment to be sorted.

Extraction time was therefore greatly reduced.The structural condition of the heads after

10–15 s of ultra-sound was the same as the condi-

tion of the heads in the conventional samples. Heads

from the ultra-sonic sample were cleaner and there-

fore identification was easier and more complete.

The cleaning effect of ultra-sound treatment for

both sediment types was particularly beneficial

when identifying the Tanypodinae as the cephalicsetae were much more visible. As a result the ultra-

sonic samples contained far fewer ‘unidentified

Tanypodinae’ than the conventional samples.

Discussion

Ultra-sonic treatment was introduced as an alter-

native method for processing carbonate samples as

a result of the poor yield of chironomid head cap-sules. In order to allow for statistical comparisons

of head densities, we used dried sediment. Most

workers now work with wet or freeze-dried

material and Walker (2001) specifically recom-

mends that samples should not be oven- or air-

dried. Comparison of extraction efficiency in this

study between dried and fresh samples processed

using standardprocedures (Brooks and Birks 2000a)found no significant difference. Considering the

crystalline nature of carbonate sediments this is not

surprising and corresponds with our observations.

Table 4. Comparison of head extraction efficiency between standard methods and ultra-sound treatment for oven-dried clay sediments.

Head densities (heads g�1) Mean proportion of sample (%)

Mean

density,

standard

prep.

Mean

density,

ultrasonic

prep. N

Mean %

increase of

heads in

ultrasonic

samples t Significance Standard Ultrasonic t Significance

Total number of heads 22.40 52.84 15 145 8.85 ***

Heads retained on fine sieve 5.78 18.73 10 255 3.56 **

Heads retained on coarse sieve 16.22 34.20 10 166 4.53 **

Tanypodinae 5.40 17.01 15 152 6.83 *** 22.69 30.33 2.43 *

Orthocladinae 5.70 11.20 15 128 3.88 ** 18.33 16.75 0.68 ns

Chironominae 14.33 28.83 15 87 7.97 *** 58.98 52.92 1.88 ns

Chironomini 10.14 18.14 15 87 5.41 *** 41.78 32.02 4.76 ***

Tanytarsini 4.18 10.68 15 199 6.07 *** 17.19 20.9 1.49 ns

Head density data compared using a matched-pairs t-test. Proportion data (%) compared using a t-test following arcsine transformation.

Mean percentage increase derived from individual sample increases. A total of 4125 heads extracted. ns p > 0.05, *0.01 < p < 0.05,

**0.001 < p < 0.01, ***p < 0.001.

456

With clay sediments, however, this difference is

more surprising. Together the results demonstrate

that drying sediments does not interfere with head

extraction and means that chironomid analysis is a

possibility with archived sediments that have par-tially or fully dried out. These sediments might

previously have been considered inappropriate for

this technique.

The initial trials demonstrated clearly that the

use of ultra-sonic treatment would break up

carbonate-rich material leading to considerably

less sediment being retained following sieving. As

a result, processing times were reduced consider-ably (by approximately 50%). Excessive ultra-sonic

treatment can result in damage to head capsules

and a reduction in overall yield. Using appropriate

time periods, however, prevents this damage and

significantly increases yields. Evidence for lack of

head damage is provided by the fact that virtually

no fragments or heads were retained in the 63-�m

sieve used in the final stages of the method in fourof the samples.

In our first trials on clay samples we found that

damage to the chironomid heads would occur with

excessive treatment times. Tests showed that the

clays were broken down within the first few seconds

of ultra-sonic treatment. We suggest that the

damage seen arises from the sheet-like structure

of clays. Once broken down into individual parti-cles, the small platy clay minerals become agitated

and are more abrasive than the more even carbo-

nate particles. The clay content of sediments may

vary and chironomid workers using any sediment

containing clays should be aware that continued

exposure in the ultra-sonic bath after the clays

have broken down will damage the heads. It is

advisable, when starting work on an unfamiliarsediment to sieve the sample after just 5 s of ultra-

sound, to see if the clays have been broken down

and to examine the condition of the heads.

In addition to reducing processing time, ultra-

sound treatment actually eases identification as

heads are much cleaner. Chironomid head capsules

retrieved from carbonate sediments can often be

very contorted. This is probably due to recrystal-lisation of calcium carbonate within the sediment

after it has been deposited. As a result, many of

the heads contain large amounts of sediment within

the head capsule, hindering identification. Very

little sediment remained within the heads after

sonic bathing and the heads were generally less

crumpled. The heads from the clay samples were

also noticeably cleaner than the un-bathed

material. For both types of sediment, the cleaner

heads led to greater accuracy in identification andreduced the number of head capsules which could

not be assigned to a particular genus. Producing

cleaner heads is particularly important where fine

detail such as cephalic setae (Rieradevall and

Brooks 2000) or premandible structure is used in

identification.

The increases in yield following ultra-sound

treatment of both carbonate and clay sedimentssuggest that head extraction using standard pre-

paration techniques was not complete and that

heads were being overlooked. Within the carbonate

sediments, organic ‘clumps’ were present in all

samples prepared using standard methods

(whether fresh or oven dried) and absent in ultra-

sonic samples. Clay sediments contained small

balls following conventional processing, againindependent of whether the sediment samples had

been pre-dried. Both clay and carbonate lumps

were also resistant to manual attempts at disrup-

tion. The lumps did not break up sufficiently to

release heads and, if this treatment had been suc-

cessful, it would certainly have caused severe head

damage. With the carbonate sediments, the organic

debris released on breaking up these clumps wascovered with a very fine carbonate dust and uni-

dentifiable. The absence of these organic clumps

and clay balls within the corresponding ultra-sonic

samples suggests that the ultra-sound had broken

them down. Any heads bound within these clumps

are therefore released into the water column for

ultra-sonic cleaning. From these observations it

seems that the increase in the number of chirono-mid heads seen in carbonate ultra-sonic samples is

due to the break up of these organic clumps and

further break up of the carbonate itself, which held

them together. In the clay sediments the increase in

head numbers is due mainly to the release of heads

when the sediment is completely broken down.

The significant overall increase in head numbers

recorded means that less sediment is needed in orderto produce a representative sample. This is a vitally

important fact as it would facilitate the use of chir-

onomid analysis when only small amounts of sedi-

ments are available. This may be particularly useful

when chironomid studies are part of a multi-proxy

457

investigation where several analyses have to be

performed on a limited number of cores. In both

carbonate and clay sediments, the major contribu-

tion to the increased yield in head capsules is made

by Tanytarsini and the Tanypodinae. Tanytarsinihave relatively small heads and would tend to be

trapped in clumps or be difficult to recognise within

the sediment. Tanypodinae have large heads but are

very thin and weakly chitinised so do not stand out

immediately from the many cladoceran carapaces

that are often found. Within the carbonate sedi-

ments, they may well be trapped within the organic

clumps. These tanypod heads are more easily recog-nised following the cleaning effects of the ultra-

sound treatment. Chironomini show the smallest

percentage increases. These are relatively large,

heavily chitinised heads which are the easiest to

recognise in all samples. The effectiveness of the

ultra-sound technique is demonstrated by the fact

that even these heads show a significant increase.

In most palaeoenvironmental studies usingchironomid analysis percentage abundance rather

than head densities are used. Examination of the

proportional data for subfamilies and tribes shows

that with carbonate and clay sediments, there is a

significant decline in the percentage of Chirono-

mini following ultra-sound treatment. Considering

that more of these heads were actually extracted,

this decline must be due to proportionately largerincreases in the other groups. With the carbonate

sediments there is a significant increase in the pro-

portion of Tanytarsini heads whilst in Late Glacial

clays Tanypodinae increased significantly.

One of the principles upon which any climatic

reconstruction is based is that the fossil evidence is

representative of the living assemblage of the past

environment. The accuracy of a reconstruction isdependent on the accuracy of the initial fossil data.

The use of standard chironomid preparation

techniques may lead to misrepresentations of the

chironomid assemblages retained within the sedi-

ment. With carbonate sediments, standard methods

fail to release the smaller Tanytarsini heads for

extraction. If these smaller heads were simply earlier

instars of the species already represented this maynot affect analysis. However, the bias towards

larger heads means that the smaller taxa are being

under represented. Chironomini and Tanypodinae

typically characterise temperate periods and certain

Tanytarsini typically characterise cool periods

(Brooks and Birks 2000a). The result of using

standard preparation techniques may be a mis-

representation of the fauna and errors in climate

inference perhaps leading to an overall warmer

prediction. The significant increase in the propor-tion of Tanypodinae in the Late Glacial clay sam-

ples is balanced by a decline in the Chironomini.

The impact of these differences on climate inter-

pretation is probably small. However, taxa with

both cold and warm preferences do exist within

all of the main groupings and differences may be

seen in climate reconstruction following ultra-sonic

treatment.As ultra-sound treatment releases more heads

from the clay and carbonate sediments and there is

no evidence that any are being destroyed, we suggest

that the assemblages recorded are more accurate

representations of the actual fossil assemblage

found in the sediments. These results would suggest

that ultra-sonic preparation would give a more

complete representation of fossil assemblages andtherefore ultimately enhance the accuracy of the

final climate analysis.

Conclusions

Oven drying sediment samples has no significant

impact on head capsule yield. Drying sediments

enables accurate head density values to be deter-

mined for statistical analysis. This observation alsomeans that existing dry core samples, previously

considered to be unsuitable, may be used for

chironomid analysis.

Controlled ultra-sonic treatment breaks down

sediment without damaging the chironomid heads

contained within the sediment. More sediment

passes through the sieves and therefore less time is

spent sorting and extracting heads. More chirono-mid heads are extracted largely due to the break up

by ultra-sound of organic clumps or clay balls

within the sediment. The chironomid heads extrac-

ted from the ultra-sonic samples were cleaner and

those extracted from the carbonate samples were

less crumpled. This eases identification particularly

with regard to Tanypodinae. Evidence suggests that

the larger heads particularly Tanypodinae may bedamaged if subjected to excessive periods of ultra-

sonic treatment. It is therefore preferable to treat

the coarser fraction of sediment, which contains

458

these heads separately and to subject this fraction to

a shorter period of ultra-sound. Risk of damage to

the heads will be increased when clays are involved

because of the rapid break down of clays during

treatment. In order to avoid such damage it is there-fore necessary when working with a sediment con-

taining any clay to run an initial check to establish at

what point the clays are broken down.

Although the ultra-sonic method yielded more

head capsules for most taxonomic groups, this

increase is most apparent for Tanytarsini (carbo-

nate samples) and Tanypodinae (clay samples).

The increases seen within the ultra-sonic samplesmeans that taxonomic representation differ from

those seen in the conventionally prepared materi-

als. This difference may cause variations to climatic

reconstructions performed using these data. As a

much larger number of heads are being released

after ultra-sound we suggest that the assemblages

seen in the ultra-sonic samples are more represen-

tative of the sediment assemblages. We thereforesuggest that analysis of the ultra-sonic data would

give a more accurate reconstruction.

The increases seen indicate that the use of the

ultra-sound considerably decreases the amount of

sediment needed, as adequate numbers of heads

can be extracted from smaller amounts of sedi-

ment. This is a vitally important fact as it facilitates

the use of chironomid analysis when only smallamounts of sediments are available. This fact may

be particularly useful when chironomid studies are

taking place as part of a multi-proxy investigation

where several analyses have to be performed on a

limited number of cores.

Acknowledgements

Thanks are due to Gareth Thompson for his helpduring the establishment of the methodology and

Richard Jones and two anonymous reviewers for

their helpful comments on the manuscript. The

research was completed while BL was in receipt of

an Edge Hill research studentship.

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