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Development and standardization of a microalgae test for determining deathsby drowning

P.A. Dıaz-Palma a,*, A. Alucema a, G. Hayashida a, N.I. Maidana b

a Department of Chemical and Pharmaceutical Sciences, Universidad Catolica del Norte, Antofagasta, Chileb Natural and Exact Sciences Faculty, Universidad de Buenos Aires, Buenos Aires, Argentina

1. Introduction

Medicolegal investigation relied largely on laboratory-basedanalyses, and some of these, like the DNA profile of body fluid, havebecome valuable routine methods [1,2]. The so-called ‘‘diatomtest’’ for the diagnosis of death by drowning is widely practiced inNorthern Hemisphere countries like France and Japan, but is notbeing used regularly in forensic practice in Latin America.

Microalgae are of limited application in forensic sciences, butthey play an important role in certain types of research [1,2], thusemphasizing the necessity of studies on their taxonomy andecology. In fact, evidence provided by taxonomists and ecologistscan be presented by either the plaintiff or defendant to support averdict [1,2].

In forensic science, detection of diatoms in tissues maycontribute to the diagnosis of drowning, which is a very common

cause of death. The circumstances can be established by eye-witnesses’ testimony or a suicide note, but when underlying causesare unclear, the standardization of methodologies helps todetermine whether a crime was committed [1]. Death by drowningis easily recognized in a fresh cadaver, but histopathological signsare hampered by the deleterious effects of decomposition, or bystrong waves and currents crashing the body against the rocks.Therefore, in case of severe injuries, the actual cause of deathshould be ascertained [1].

The diatom test has been accepted by many authors [3–9], butthere is no consensus on whether diatoms are accurate indicatorsof drowning since they have been also found in non-drowningvictims, such as swimmers and fishermen [10,13]. These findingsled some researchers, for example Gylseth and Mowe [11],Shellman and Sperl [12] and Foged [14], to rule them out asreliable evidence.

Nevertheless, it is reasonable to assume that such cases mayrepresent false positives resulting from sample contaminationprior to the analysis.

On the other hand, Peabody [7,8] and Auer and Mottonen [15]state that diatom count can be used to discriminate betweendrowning and non-drowning cases.

Forensic Science International 184 (2009) 37–41

A R T I C L E I N F O

Article history:

Received 15 November 2007

Received in revised form 8 September 2008

Accepted 21 November 2008

Keywords:

Drowning

Microalgae

Diatoms

A B S T R A C T

The diatom test for the diagnosis of drowning is widely used in countries of the Northern Hemisphere

such as France and Japan. In Latin America, however, it has not been adopted as a routine procedure in

forensic autopsies. In aquatic ecosystems, dinoflagellates and some chlorophytes are microalgae that,

like diatoms, have cell walls and other resistant structures. As a result, they can be found in tissues from

drowning victims, which is important because diatoms may be rare under particular environmental

conditions. On this basis, we propose to extend the ‘‘diatom test’’ to include other microalgae for the

determination of death by drowning.

In this work, we developed a standardized procedure for detecting microalgae in tissues from

drowning victims, with techniques described in the international literature and designed by us. The

corpses were recovered from coastal areas in Antofagasta Region, Chile, during summer 2005. The most

effective procedure for the treatment of water and tissue samples involved the combination of enzymatic

digestion (proteinase K) and chemical digestion. The technique allowed the recovery of dinoflagellate

evidence belonging to genera Prorocentrum, Ceratium, Dinophysis and Protoperidinium; silicoflagellates of

the genus Dictyocha; an undetermined, filamentous chlorophyte; entire valves of centric diatoms and

fragments of pennate and centric diatoms. This is the first protocol using microalgae other than diatoms

for forensic cases in Latin America, and particularly in Chile.

� 2008 Elsevier Ireland Ltd. All rights reserved.

* Corresponding author at: Department of Chemical and Pharmaceutical

Sciences, Universidad Catolica del Norte, Avenida Angamos 0610, Antofagasta,

Chile. Tel.: +56 55 355624/5383; fax: +56 55 355632.

E-mail address: pdiaz@ucn.cl (P.A. Dıaz-Palma).

Contents lists available at ScienceDirect

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0379-0738/$ – see front matter � 2008 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.forsciint.2008.11.015

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Taking into account the small number of diatom valves that canbe recovered from cadaveric tissue [15], a test using microalgaeother than diatoms (e.g. dinoflagellates and chlorophytes) mayprovide a more reliable diagnosis.

It is worthwhile to mention that both the ‘‘diatom test’’ and the‘‘microalgae test’’ proposed by us do not constitute by themselves aconclusive proof of death by drowning, from a legal point of view.Instead, they represent tools that, together with other analyses(toxicology testing, determination of chloride and strontium levelsin blood and presence of hemoglobinemia, etc.), help the PublicProsecutors to reach a final decision.

Diatoms and other algae that can be of use in forensicinvestigation are identified by the morphology of the frustule,theca, endoskeleton, cell wall, etc. The observation of thesestructures requires the removal of organic matter represented bymicroalgae content and tissue debris. The three most commonmethods used for preparing samples for diatom analysis involvetreatment with a strong acid, tissue solubilizers and/or enzymaticdigestion [16]. However, some of these methods fail when used formarine diatoms [16] and consequently there is an increasing needfor developing methodologies that can be extrapolated to differentaquatic systems.

In the present work we present a standardized procedure fordiagnosing death by drowning. In addition, we compare differentdigestion techniques to determine which provides the maximumamount of forensic evidence (microalgae) for taxonomicalidentification. The techniques were performed using bone marrowand water samples, and were designed by us or obtained fromliterature.

2. Methods

All plasticware used was carefully washed with free-phosphorus detergent and

soaked in 20% HCl for 24 h, rinsed with distilled water and finally rinsed with

diatom-free distilled water (DFDW), obtained by filtration through a 0.45-mm-

pore-size nitrocellulose membrane.

Tissue samples were obtained from autopsies performed in the Medicolegal

Service of Antofagasta City (Chile). The samples came from three individuals drown

during summer 2005; one neonate, who died of sudden death that same year,

served as control. A sample of sternal bone marrow was taken from each individual,

and a lung sample from one of them. Each sample was washed with DFDW before

cooling at 4 8C.

Almost simultaneously with the finding of the victims, seawater samples were

collected along the coast of Antofagasta Bay, close to the sites where bodies were

recovered. Samples were taken in triplicate with a 45-mm mesh plankton net, fixed

in 2% formalin and stored at 4 8C until processing.

Prior to any treatment, seawater samples were examined under the microscope

to estimate qualitatively the density of microalgae, and they were concentrated by

evaporation and centrifugation when the number of microalgae was small.

We chose as the best digestion method the one yielding the cleanest sample, the

largest number of evidences (complete valves, thecae and endoskeletons, or

identifiable microalgal fragments), and the optimum visualization of the characters

for species identification. To determine the most appropriate procedure, we

modified methods widely reported in the literature by varying digestion time and

type and concentration of reagents. We also tested an alternative method

(Treatment 4) applied by one of the authors to another type of diatom analysis [17].

Bone marrow and water samples were treated as follows (see Table 1 for

details)—Treatments 1 and 2, digestion with strong acids HCl and H2SO4 [5]: acid

concentration were 5 and 100%, and digestion times were 2 and 24 h, respectively.

Treatment 3 (enzymatic): digestion using a solution of 0.5 U/ml proteinase K and

0.01 M Tris–HCl, pH 7.5, with 2% sodium dodecyl sulfate (SDS), neutralized with

concentrated HCl [6].

Treatment 4: 1–2 ml of concentrated hydrogen peroxide (H2O2) were added to

each sample. The samples were put in a double boiler and heated in the microwave

at 80% power (around 650 W) for 3 min. After cooling, 1 to 2 ml of 20% HCl were

added. Following Treatments 1, 2 and 4, samples were neutralized by successive

washes with DFDW and centrifugations.

The treated water samples were observed in permanent preparations mounted

on a hydrophobic adhesive medium (Entellan or Eukitt), whereas tissue samples

were observed in temporary preparations. Samples were examined under a

binocular microscope Zeiss Axiostar equipped with a digital camera System

Olympus DP70.

On the basis that bone marrow samples were taken from subjects known to have

died by drowning, we verified the efficiency of the procedure proposed in this work,

by comparing the number of complete and incomplete diatoms obtained from

tissues with reference values given by Ludes and Coste [1].

We compared the species composition of microalgae in seawater and bone

marrow samples with the Jaccard index of similarity [18], using the BioDiversity

PRO program (version 2).

3. Results

The results of the chemical digestion techniques are shown inTable 1. We selected Treatment 4 for water samples because itachieved the best performance.

The enzymatic Treatment 3 resulted in partial digestion of bonemarrow and lung samples and therefore microalgae observationwas hindered by the large proportion of organic matter (Fig. 1).However, results improved when Treatment 4 was applied afterTreatment 3. The combination of the enzymatic and chemicaldigestions increased the number of the specimens recovered, withmost of the microalgal structures (frustules, thecae and endoske-letons) being complete. This allowed the identification of a highdiversity of taxa including diatoms, dinoflagellates, silicoflagellatesand chlorophytes, thus increasing the accuracy of the forensicdiagnosis.

Table 1Results obtained with the different digestion techniques.

Procedure no. Concentration Digestion time (h) Results

1 (HCl) 5% 2 and 24 Partial digestiona, there is still a large amount of organic matter.

10% 2 and 24

20% 2 and 24

50% 2 Aggressive digestionb, there is a decrease in the amount of organic

matter but the number and diversity of species recovered are low.80% 2

Concentrated 2

2 (H2SO4) 5% 2 and 24 Partial digestion, there is still a large amount of organic matter.

10% 2 and 24

50% 2 Aggressive digestion, there is a decrease in the amount of organic matter,

but the number and diversity of species recovered are low.80% 2

Concentrated 2

3 (H2O2) 20% 2 Partial digestion, there is still large amounts of organic matter.

Concentrated 2 and 24

4 (combined) conc. H2O2, Ø and HCl 20% 0.05 + Ø Optimum digestionc, there is a low amount of organic matter and

the number of species recovered is low.

a The amount of organic matter is higher enough to hinder the visualization of microalgal structures.b Destruction of microalgae frustules and thecae.c The removal of a larger amount of organic matter from the sample allows the clear visualization of the microalgal structures.

P.A. Dıaz-Palma et al. / Forensic Science International 184 (2009) 37–4138

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The control sample was negative for microalgae, and showed fatcells some blood cells like megakaryocytes and bone structures likehaversian canals.

In the sample from subject 1 (Table 2), we identified taxa ofdiatoms, dinoflagellates and silicoflagellates. The number ofdinoflagellates and silicoflagellates was comparable to that ofdiatoms. With regard to diatoms, the proportion of completevalves was higher than that of incomplete ones for most of the taxa(Table 2). No marine microalgae were recovered from the lungsample of subject 2.

Table 2 also shows the taxonomic diversity found in the sampleof subject 3. The sample contained different taxa, includingdiatoms, dinoflagellates and silicoflagellates. Microalgal structureswere less abundant than in the sample from subject 1 and alsothere were fewer structures for each taxon (Table 2). Incompletevalves, thecaes and endoskeletons outnumbered complete ones. Asin the sample from subject 1, the silicoflagellates, representeduniquely by Prorocentrum micans, were the only ones presentingcomplete thecae.

Table 3 shows that the counts of diatom valves in the samplesfrom subjects 1 and 3, expressed per unit of tissue, were higherthan those established by Ludes and Costes [1]; this wasparticularly the case for the sample from subject 3, who wasfound in the warm waters of a small semi-closed bay withmicroalgae blooms occurring almost all-year-round at intervalsonly depending upon the tidal regime (Gob. Marıtima deAntofagasta, personal communication).

Table 4 shows the microalgae identified in water samples takenat the coasts of beaches El Trocadero, Los Pinares and Juan Lopez.The water samples from El Trocadero and Los Pinares wereanalyzed together with the tissue sample of subject 1 because thecorpse was found in the midway between the two beaches. Weidentified seven and six different taxa at El Trocadero and LosPinares, respectively, with diatoms, dinoflagellates and silico-flagellates represented at both sites.

The microalgae from the water sample taken at the coast of thelocality Juan Lopez (Table 4); was compared with the tissue sampleof subject 3, who was recovered at this same site. We identified 15taxa including diatoms, dinoflagellates and silicoflagellates.Dinoflagellates showed the highest abundance of microalgalstructures and species richness in both water and tissues samples;in particular, the one responsible of the red tides occurring in theregion during summer [19].

The values of the Jaccard index of similarity obtained for waterand bone marrow samples are shown in Table 5. Hurlimann et al.[15] stated that algae from water and tissue samples belong to thesame community when the similarity index is higher than 60%.According to this, there was a low similarity between bone marrow

Fig. 1. Map of the Bahıa de Antofagasta, Chile, showing the location of the sites

where victims of drowning were recovered and, where seawater samples were

collected.

Table 2Results obtained with the bone narrow sample from individuals: no. of complete

frustules, thecae or endoskeletons/no. of incomplete frustules, thecae or

endoskeletons/total of algal remains.

Class Individual 1 Individual 3

Diatoms (Bacillariophyceae) 2/0/2 –

1/3/4 –

0/3/3 0/3/3

– 1/0/1

Dinoflagellates (Dinophyceae) 183/0/183 2/0/2

1/1/2 –

8/10/18 –

Silicoflagellates (Dictyochophyceae) 22/21/43 0/4/4

Green algae (Chlorophyceae) 1/0/1 –

Total 217/38/256 3/7/10

Table 3Number of expected valves per mass unit (*) and observed valves for samples 2 and

4. (*) According to Ludes and Coste [12].

Sample No. Sample weight (g) Expected valves Observed valves

1 3.52 1.76 ffi 2* 9

3 5.68 2.84 ffi 3* 4

Table 4Algal remains recovered in the samples from different localities. FTC, number of complete frustules, thecae or endoskeleton; FTI, number of incomplete frustules, thecae or

endoskeleton; R, replicate; x, mean values; S.D., standard deviations. Localities ET: El Trocadero; LP: Los Pinares and JL: Juan Lopez.

Algal remains Locality R1 R2 R3 x S.D.

FTC FTI Total FTC FTI Total FTC FTI Total

Diatoms ET 5 14 19 14 24 38 0 0 0 28.5 26.1

LP 4 4 8 11 0 11 5 10 15 11.4 4.7

JL 42 33 75 141 32 173 47 19 66 104.5 78.4

Dinoflagellates ET 101 22 123 254 1 255 0 0 0 189 117.4

LP 35 1 36 29 3 32 13 2 15 27.6 16.1

JL 30 96 126 24 0 24 16 7 23 57.7 62.3

Total locality ET 106 36 142 268 25 293 0 0 0 217.5 143.6

Total locality LP 39 5 44 40 3 43 18 12 30 39 20.7

Total locality JL 72 122 201 165 32 197 63 26 89 162.2 140.6

P.A. Dıaz-Palma et al. / Forensic Science International 184 (2009) 37–41 39

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samples and seawater samples taken from the site where bodieswere found because indices ranged between 29 and 55%.

4. Discussion

The enzymatic digestion treatment was more effective toremove the organic matter from human tissues than frommicroalgae. On the other hand, the addition of sodium dodecylsulfate detergent (SDS) was found to be effective in causingmembrane lysis and protein degradation, as well as in reducinglipids.

In the chemical digestion treatment, the addition of hydrogenperoxide at high temperature was effective in removing organicmaterial. Hydrogen peroxide is a very powerful oxidant, which isdecomposed into products at different oxidation states under hightemperature conditions, since it oxidizes and reduces itself.

In most cases, the digestion using strong acids was lessaggressive due to the low concentration of chloride acid (20% HCl)and shortening of the process. This was reflected in theexperiments performed to determine the most appropriatechemical digestion procedure. Thus, at a concentration of 20%HCl, the number of taxa was smaller at longer digestion times.Likewise, a similar result was obtained at concentrations above20%.

Finally, the combination of enzymatic and chemical digestionsin tissue samples resulted in a synergism that substantiallyincreased the diversity of diatom, dinoflagellate and chlorophytespecies. Consequently, we conclude that this combination yields abetter performance than other procedures reported in literature[1,20,21].

Silica is scarcer in marine than in freshwater environments,thus representing a limiting factor for seawater diatoms. Thesecompete with other species that also incorporate this element, andtheir valves contain less silica than freshwater diatoms [22]. As aresult, seawater diatoms are vulnerable to aggressive chemicaltreatments, and this accounts for the efficiency of the combineddigestion method used here. This interpretation also applies tosilicoflagellates because of their siliceous endoskeletons [23].Dinoflagellate thecae, which are composed of glucan [23], wereresistant to the combined chemical and enzymatic treatment. Thefinding of a chlorophyte in bone marrow samples (Table 2) wassurprising because its external structures are usually vulnerable tostrong acids, and supports our conclusion that this technique issuccessful in recovering a large number of taxa.

Despite the fact that both chloride and sulfuric acids areclassified as strong acids, the former was less aggressive than thelatter based on the larger number of evidences obtained from eachsample.

An appropriate digestion of the organic matter from intra- andextra-cellular components is needed to improve the visualizationof structures used for microalgal identification.

We obtained a higher diversity of taxa (diatoms, dinoflagellates,silicoflagellates and chlorophytes) from tissue samples than thatreported in other studies [14,24]. This result indicates that,conversely to the traditional methods, the combined procedureallows the recovery of a larger number of evidence and does not

depend exclusively on habitat features conditioning the avail-ability of diatoms.

The lung is expected to show the highest diversity of taxabecause it is one of the first organs to come into contact with waterduring the drowning process, and from which water enters thebloodstream to reach the different tissues. This discordance withour results is likely to be due to the small sample volume, andreflects the importance of using samples from different sources(bone marrow, lung, blood) of the same subject to validate theprocedure.

A small number of non-marine diatom valves were recoveredfrom the lung sample; these are frequently found in localcontinental environments [17]. Their presence can be attributedto contamination occurring while the victim was alive because thevalves, which can be easily transported by the wind, may haveentered into the lung.

In addition, contamination could have taken place duringcorpse manipulation, autopsy, laboratory analysis, or in anyother stage of the process. The efficacy of the method used in thepresent work is supported by the large number of valves andfragments recovered from tissue samples of subjects 1, 2 and 3,which even exceeded the minimum number suggested by Ludesand Coste [1]. With regard to algae other than diatoms that canbe recovered by this technique, we propose to standardize theminimum number of structures required to consider the test aspositive.

According to the Jaccard similarity indices, algae found in bonemarrow and water samples were not likely to belong to the samecommunity. This result is probably due to environmental factorsnot considered in our analysis on the basis that water sampleswere not taken simultaneously with the finding of the body.

The probability of microalgae to reach tissues during submer-sion may depend on their morphology. Thus, the maximum lengthsof several taxa exclusively found at the site of drowning werehigher than the average length of algae from the tissue, such asSynedrosphenia sp. and Fragilaria fasciculata. In this respect, studieson the use of different morphological features as forensic evidenceare needed to validate the Jaccard index of similarity of 60%proposed by Hurlimann et al. [15].

5. Conclusion

We developed a reproducible protocol for both water and tissuesamples. The digestion procedure used in the present studyincreased the recovery of evidences (microalgae taxa) in compar-ison with various methods usually referred to in literature. Inaddition this novel technique facilitates the visualization ofrepresentative microalgal structures and provides a number ofundamaged cell coverings large enough to allow an accuratetaxonomic identification. The importance of this result lies in thefact that traditional techniques are very aggressive and cause notonly the digestion of tissues, but also the fracture of most of thealgal material.

We established that microalgae found in one of the analyzedtissue samples did not enter during drowning based on thedetection of foreign material, which emphasizes the importance ofidentifying local microalgal flora.

A standardized microalgal test for the diagnosis of death bydrowning provides valuable evidence for the interpretation offorensic cases. However, this evidence must be contrasted withthat obtained from other sources because of the numerous factorsinvolved in the accident, such as the type of environment wheredrowning took place (marine or freshwater ecosystems), the lifehistory of victims, presence of eyewitnesses, and routineprocedures performed by qualified forensic professionals (e.g.toxicologists).

Table 5Jaccard indices of similarity (JIS%) between bone marrow and water samples.

Locality Bone marrow samples

Sample 2 Sample 4

Los Pinares 55.55

El Trocadero 50.00

Juan Lopez 28.57

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Our results contribute to enhance the current knowledge offorensic biology, and the microalgal test proposed here appears asa valuable, reliable and sensitive diagnostic tool to be used inroutine forensic procedures.

Acknowledgments

We thank Drs. Carlos Contreras O. and Jaime Llanos S. for criticalreading of the manuscript. Other acknowledgements to MissKarina Penaloza for the analysis of diatoms samples.

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