Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

Population survey of Holothuria (Halodeima) grisea

(Aspidochirotida: Holothuriidae) at its limit of geographic distribution

in the Western South Atlantic

Guilherme Sabino Rupp1*; https://orcid.org/0000-0002-5476-9689

Adriano Weidner Cacciatori Marenzi2; https://orcid.org/0000-0002-8154-5867

Robson Ventura de Souza1; https://orcid.org/0000-0003-0588-0038

Rafael Schroeder2,3; https://orcid.org/0000-0001-7340-0214

1. Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina (EPAGRI), Centro de Desenvolvimento em

Aquicultura e Pesca, Florianópolis, SC, Brazil; rupp@epagri.sc.gov.br (*Correspondence),

robsonsouza@epagri.sc.gov.br

2. Universidade do Vale do Itajaí (UNIVALI), Escola Politécnica, Rua Uruguai 458, Centro 88302-901, Itajaí, Brazil;

marenzi@univali.br, schroederichthys@gmail.com

3. Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Terminal de Cruzeiros do Porto de

Leixões, Avenida General Norton de Matos S/N, 4550-208 Matosinhos, Portugal.

Received 23-I-2023. Corrected 14-IX-2023. Accepted 27-IX-2023.

ABSTRACT

Introduction: The sea cucumber Holothuria (Halodeima) grisea Selenka, 1867 is a common echinoderm in inter-

tidal regions along the Brazilian coast, which recently became the focus of unreported and unregulated fisheries.

This study was carried out in sandy-rocky substrates at Armação do Itapocoroy, Penha, Santa Catarina (26o47’ S;

48o36’ W), near its southern limit of geographic distribution.

Objective: To determine the densities (individuals*m-2) of Holothuria (H.) grisea within a spatial-temporal per-

spective as well as to determine biometric and growth characteristics of the population.

Methods: Two-meter wide transects perpendicular to the coastline were carried out in winter and spring 2019

and in summer and spring 2020, in periods of spring low-tides. In each sampling occasion the total number of

specimens of H. grisea were determined, and a group of 90 organisms was submitted to in situ biometrics (weight,

length and width), and immediately returned alive to their habitat.

Results: The densities of H. (H.) grisea were significantly higher in the subtidal sector and lower in the upper

intertidal sector with no indication of significant differences among sampling campaigns. Depth was the primary

factor explaining the observed density patterns and rugosity of the substrate was secondary but also important.

The body length ranged from 5.2 to 22.5 cm, whereas the weight varied from 6.0 to 230 g. The mean and modal

lengths were 12.54 and 13 cm, respectively. Approximately 75 % of the population sampled was between 10 and

14 cm and the average weight was 60 g. Estimates from von Bertalanffy growth function indicate that the young-

est sea cucumber was one year-old, and the oldest had approximately two and a half years.

Conclusions: This is the first study to determine biometric parameters for H. (H.) grisea in southern Brazil and

the first one to estimate growth and age estimates for a wild population of this species. The densities recorded

in the present study were lower than those previously reported for this region, suggesting anthropic influence.

Key words: Holothuria (Halodeima) grisea; Santa Catarina; densities; spatial distribution; population.

https://doi.org/10.15517/rev.biol.trop..v72iS1.58623

SUPPLEMENT

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

INTRODUCTION

Sea cucumbers play an important role in

marine ecosystems (Purcell et al., 2016), and

present outstanding commercial value through-

out the world (Purcell et al., 2018), however

little is known about their populations in Brazil

(Mendes et al., 2006; Ponte & Feitosa, 2019;

Souza Jr. et al., 2017; Ventura et al., 2013). The

greed for these high priced-organisms has led

to intense capture in more than 70 Countries,

threatening the populations of various species

around the planet (Purcell et al., 2013; Purcell

et al., 2023), including Latin America (Conand,

2018; Sonnenholzner, 2021).

Although a wide diversity of sea cucumbers

is described for shallow waters of the Brazilian

coast (Martins, 2012; Martins & Souto, 2020;

Martins & Tavares, 2021; Martins et al., 2022),

only three species are described for south-

ern Brazil (Rupp et al., 2023). Among them,

Holothuria Halodeima grisea Selenka, 1867 is

considered the most common, being recorded

in the intertidal region at the base of the rocks,

usually in contact with the bottom sand (Bueno

et al., 2015; Martins, 2012; Mendes et al., 2006;

Tiago & Ditadi, 2001; Tommasi, 1969). Nev-

ertheless this species is already threatened by

unreported and unregulated capture in Brazil

(Ponte & Feitosa, 2019; Rupp & Marenzi, 2021;

Souza Jr. et al., 2017). The geographic distribu-

tion of H. (H.) grisea is predominantly tropical,

and includes the west coast of Africa, Florida,

Gulf of Mexico, Panamá, Caribbean islands,

Colombia, Venezuela, and Brazil up to Santa

Catarina (Martins, 2012; Pawson et al., 2010).

In latter location Rupp et al. (2023) recorded

the occurrence of H. (H.) grisea in nine out of

the eleven intertidal sampled sites, with higher

densities (> 1.7 ind.m-2) recorded in the central

portion of the state, and none found further

south than the locality of Garopaba.

The objective of the present study was

to evaluate the abundance and densities

RESUMEN

Estudio poblacional de Holothuria (Halodeima) grisea (Aspidochirotida: Holothuriidae)

en su límite de distribución geográfica en el Atlántico Sur Occidental

Introducción: El pepino de mar Holothuria (Halodeima) grisea Selenka, 1867 es un equinodermo común en

las regiones intermareales a lo largo de la costa brasileña, que recientemente se convirtió en foco de pesquerías

no declaradas y no reguladas. Este estudio se realizó en sustratos arenosos-rocosos en Armação do Itapocoroy,

Penha, Santa Catarina (26o47’ S; 48o36’ W), cerca del límite sur de su distribución geográfica.

Objetivo: Determinar las densidades (individuos*m-2) de Holothuria (H.) grisea dentro de una perspectiva

espacio-temporal así como determinar las características biométricas y de crecimiento de la población.

Métodos: Se realizaron transectos de dos metros de ancho perpendiculares a la línea de costa en invierno y pri-

mavera de 2019 y en verano y primavera de 2020, en periodos de bajamar sicigia. En cada ocasión de muestreo se

determinó el número total de especímenes de H. (H.) grisea, y se sometió un grupo de 90 organismos a biometría

in situ (peso, longitud y ancho), e inmediatamente se los devolvieron vivos a su hábitat.

Resultados: Las densidades de H. (H.) grisea fueron significativamente más altas en el sector submareal y más

bajas en el sector intermareal superior sin indicios de diferencias significativas entre las campañas de muestreo.

La profundidad fue el factor principal que explica los patrones de densidad observados y la rugosidad del sustrato

fue secundaria pero también importante. La longitud del cuerpo varió de 5.2 a 22.5 cm, mientras que el peso varió

de 6.0 a 230 g. Las longitudes media y modal fueron 12.54 y 13 cm, respectivamente. Aproximadamente el 75 %

de la población muestreada midió entre 10 y 14 cm y el peso promedio fue de 60 g. Estimados de la función de

crecimiento de von Bertalanffy indican que el ejemplar más joven presentaba un año de edad, mientras el más

viejo presentaba cerca de dos años y medio.

Conclusiones: Este es el primer estudio que determina parámetros biométricos para una población de H. (H.)

grisea en el sur de Brasil y el primero en estimar el crecimiento y edades para una población salvaje de esta especie.

Las densidades registradas en el presente estudio fueron inferiores a las reportadas previamente para esta región

sugiriendo la ocurrencia de influencia antrópica.

Palabras clave: Holothuria (Halodeima) grisea; Santa Catarina; densidades; distribución espacial; población.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

(individuals.m-2) of H. (H.) grisea within a

spatial-temporal perspective, at a locality where

a previous study has been conducted (Mendes

et al., 2006) nearly 16 years earlier. Addition-

ally, biometric parameters of the population

as such length and weight distributions were

determined, as well as growth parameters. Such

basic information could be useful for future

conservation practices as well as for fishery

management and aquaculture of this species.

MATERIALS AND METHODS

Field methods: The study site is in the lit-

toral zone of Armação Itapocoroy cove, Penha,

Santa Catarina (26º 47’S; 48º36’W) (Fig. 1),

which is a low-energy wave action area. The

site has a slight slope and rocky-sandy sub-

strate consisting of coarse grain-sized sedi-

ments (Mendes et al., 2006). This site is located

approximately 150 km from the southernmost

point where the Holothuria (H.) grisea have

been recorded (Rupp et al., 2023). Four Holo-

thuria (H.) grisea sampling campaigns were

carried out between August 2019 and Octo-

ber 2020 during low spring tides. Restrictions

imposed by the Covid-19 pandemic prevented

sampling from being carried out from autumn

to winter 2020. On each occasion, three two-

meter wide transects (T1, T2 and T3) distant

15 m from each other (Fig. 2) were deployed

perpendicularly to the shore, with lengths rang-

ing from 33 to 50 m (Table 1), depending on the

tidal level. The transect areas were surveyed by

visual and tactile inspections and the numbers

of H. (H.) grisea in each one-meter division

of the transects were recorded. The samplings

always started at T2, then proceeding to T3 and

finally to T1. In three opportunities it was not

possible to finalize the sampling at T1 and T3

due to tidal surge. The rocks at the sampling

area were not revolved in order to avoid habitat

disturbance. The length of the transects was

divided in three sectors based on tidal expo-

sure: A – upper intertidal (length 0 to 16 m),

B – lower intertidal (length 17 to 33 m) and C –

subtidal (length 34 to 50 m). Their depths were

determined in the middle of each sector. To do

that, tidal tables from the Brazil’s Navy Hydrog-

raphy and Navigation Center (Centro de Hidro-

grafia e Navegação [CHN], n.d.) and from a

nearby tide gauge from the Santa Catarina State

Institution for Agricultural Research and Rural

Extension (Empresa de Pesquisa Agropecuária

e Extensão Rural de Santa Catarina, n.d.) were

used as references. The rugosity of each sector

was estimated using the chain method (Luck-

hurst & Luckhurst, 1978). A metal chain with

small links was laid along the central portion of

the transects, so that it followed the contours of

Fig. 1. Location of the study site at Armação do Itapocoroy, Penha (SC).

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

the substratum as closely as possible. The ratio

between the contour length of the chain and

the sector actual length (Rg) was used to infer

rugosity index:

Ri = 1-(1/Rg)

Where Ri is the rugosity index and Rg is the

ratio between the chain length and the actual

length of the sector of the transect (Mendes et

al., 2006).

A group of 90 animals was randomly sam-

pled, on each campaign, for in loco biometry.

The following parameters were registered: total

length and width with contracted body, and

total weight after light pressure to expel the

liquid from the internal cavity. Linear measure-

ments were carried out with a precise ruler

(± 0.5 mm) and a field DC digital scale was

used to determine weights (± 0.1 g). Samplings

were non-destructive and the animals were

immediately returned to their habitat after

biometry. The moon phase and the following

environmental parameters were recorded in

each sampling occasion: air temperature, sky

coverage (clouds), water temperature and salin-

ity. Tidal information was obtained at CHN

tables (CHN, n.d.). This study was carried out

under license for collection of native fauna of

the Brazilian Ministry of Environment MMA/

SISBIO No. 68215.

Statistic approach: Normality and

homoscedasticity of the datasets were veri-

fied respectively with Shapiro-Wilk and Lev-

ene’s tests using StatisticaR, version 12 (Statsoft

Inc. USA), and density data was square root

transformed in order to meet those premises

(Zar, 1999). Two-Way ANOVA was used to

compare densities on spatial-temporal scale

(among transects, sectors and season) (Sokal

& Rohlf, 2000). Significant differences were

considered when P < 0.05 and Tuckey’s test

was used as post-hoc analysis. The comparison

Fig. 2. Plots of the transects at Armação do Itapocoroy, Penha (SC) (A - Google Earth); (B) aerial photo of the sampling area.

(Arrows indicate North).

Table 1

Surveyed months and respective seasons, transects lengths and the comparable sectors used for statistical analysis.

Month Season Transect distance (m) Comparable sectors

T1 T2 T3 T1 T2 T3

August/2019 Winter-19 33 50 50 A-BA-B-C A-B-C

October/2019 Spring-19 33 50 33 A-BA-B-C A-B

March/2020 Summer-20 50 50 50 A-B-C A-B-C A-B-C

October/2020 Spring-20 45 50 50 A-BA-B-C A-B-C

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

of mean length and weight of Holothuria (H.)

grisea among the sampling campaigns was

explored using One-Way ANOVA, however the

assumption for this analysis were not met, even

after data transformation, therefore the non-

parametric Kruskal-Wallis test was used in lieu

of ANOVA (Sokal & Rohlf, 2000).

Considering that it was not possible to

complete Sector C on T1 in 3 opportunities

and on T3 in one opportunity, only the sectors

fully sampled were included in the statisti-

cal analyses (Table 1). The comparison of the

mean densities on temporal scale was carried

out considering T2 (sectors A, B and C) for all

sampling occasions, as well as for all transects

using sector B only. The comparison among all

sectors and transects was carried out within the

summer-20 campaign and the comparison of

the densities among sectors B was carried out

considering all transects and seasons.

Aiming to explain the density of sea-

cucumbers based on both the mean depth of

the sectors and their rugosity indexes, linear

regression analyses were carried out. The den-

sity of animals was the response variable, while

the depth and rugosity were tested as explana-

tory variables, both separately and combined as

a multiple regression model, using the software

“R”, version 4.3.0 (R Core Team, 2023). The

relation between size and weight of the stud-

ied population was explored using the power

model regression:

Y = aXb

Where Y = weight, X = length, b = allometric

coefficient, a = intercept (Gould, 1966).

Considering that body measurements in

holothuroids is often imprecise due to body

wall elasticity, the compound index which com-

bines body length and width (SLW = square

root of the length-width product) was used

to further analyze its relationship with body

weight, as indicated for several species of sea-

cucumbers (see Poot-Salazar et al., 2014 for

details). To increase growth parameter accu-

racy, lengths were recalculated using a power

regression between length and SLW (a = 1.0731,

b = 0.6855).

Growth analyses of different SLW cor-

rected length classes (Le) were conducted by

Electronic Length Frequency Analysis (ELE-

FAN) in R 4.3.0 (R Core Team, 2023) using

the TropFishR package (Mildenberger et al.,

2017). The von Bertalanffy growth function

was applied as follows:

Where Let is the predicted corrected size at

age t; Le∞ is the asymptotic size; k is curvature

parameter per year, expressing the rate at which

Le∞ is approached; and to is the theoretical ‘‘age’’

if the organism were to have a size equal to zero.

Relative ages at certain lengths and weights

were generated by replacing the growth para-

meters with their respective values as follows:

RESULTS

Environmental variables: The environ-

mental variables recorded during the surveys

carried out from August 2019 to October 2020

are shown in Table 2. The sectors’ surveyed

areas, and the respective depths and rugosity

indexes are presented in Table 3.

Densities and spatial-temporal distribu-

tion: The total number of Holothuria (H.)

grisea counted during the study was 566 indi-

viduals out of which 360 were used for bio-

metrics. The highest density of H. (H.) grisea

recorded per unit area of the transects was 22.5

individuals*m-2 (T3-winter-09). The densities

varied along the transects for all sampling cam-

paigns (Fig. 3).

The mean densities of Holothuria (H.) gri-

sea within the different sectors of each transect

for all sampling campaigns are presented in

Fig. 4. It clearly shows that transect A (upper

intertidal) had lower densities than sectors

B (lower intertidal) and C (subtidal) in all

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

Table 2

Sampling months, tidal level at the beginning of the surveys, environmental observations (sea and air temperatures, salinity,

cloud cover) and moon phase.

Month Season Tide (m) Temperature (oC) Salinity (Kg.g-1)Cloud cover Moon phase

Sea Air

August/2019 Winter-19 0.0 18.5 18.0 35 Clear Full

October/2019 Spring-19 0.0 22.6 22.0 34 Clear New

March/2020 Summer-20 0.3 26.2 24.5 35 Clear Full

October/2020 Spring-20 0.0 22.8 23 33 Clear Full

Table 3

Surveyed area of the sectors A, B and C of each transect and respective depths and rugosity index (Ri).

Transect Sector Area (m2)Depth (m) Ri

1 A 32 0.4 0.006

B 34 0.6 0.029

C 34 0.7 0.029

2 A 32 0.45 0.023

B 34 0.55 0.023

C 34 0.65 0.029

3 A 32 0.45 0.018

B 34 0.6 0.006

C 34 0.65 0.029

Fig. 3. Densities of Holothuria (H.) grisea (ind*m-2) along the transects for the sampling campaigns carried out in winter-2019,

spring-2019, summer-2020 and spring-2020. Uppercase letters indicated the transect sectors: A. upper intertidal, B. lower

intertidal and C. subtidal.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

transects and samplings. The mean densities

for the samplings in Winter/2019, Spring/2019,

Summer/2020, and Spring/2020 considering

all the surveyed sectors and transects were

2.11, 1.21, 1.67, 1.94 individuals*m-2, respec-

tively. However, when only the lower inter-

tidal and subtidal sectors are taken into account

(excluded sector A where specimens were rare-

ly found), the mean densities for winter/2019,

spring/2019, summer/2020, and spring/2020

were 3.35, 2.02, 2.38 and 2.80 individuals*m-2,

respectively.

The comparison of the densities among

transects, sectors, and seasons are presented

in Table 4 (Two-Way ANOVA). The analyses

indicate no difference among the surveyed

seasons, in any of the compared sectors and

transects. On the other hand, significant dif-

ference among sectors was recorded in transect

2 (considering all seasons), with significantly

higher densities recorded in sector C, interme-

diate in sector B, and lowest densities in sec-

tor A (Tuckey test, p > 0.001). In Summer-19

densities recorded on sector C were also signifi-

cantly higher than sector B and A, respectively

(Tuckey test, p < 0.001), but there were no dif-

ferences among transects. Considering sector

B only, when all transects and seasons were

considered, there was significant difference

among transects, with lower densities recorded

in transect 3 (Tuckey test, p < 0.01).

The effect of bathymetry and rugosity

on the density of animals: Regression analyses

evidenced that the density of sea-cucumbers

is significantly correlated with both the depth

(p <0.001) and the rugosity (p <0.001) of the

studied sectors (Fig. 5). The levels of explained

variance, however, differed significantly, being

75 % and 35 %, respectively for models con-

sidering depth (Residual standard error: 0.7

ind.m-2) and rugosity (Residual standard error:

1.18 ind.m2) as explanatory variables. The for-

mer and best model evidence that the den-

sity of sea cucumbers tend to increase by

1.3 individuals.m-2 for the increment of every

Fig. 4. Densities of Holothuria (H.) grisea for each sector for all the transects and sampling campaigns. The boxes indicate

the interquartile range divided by the median and the whiskers indicate the lowest and highest results. A. upper intertidal,

B. lower intertidal and C. subtidal.

Table 4

Two-Way ANOVA outputs of comparisons of Holothuria (H.) grisea densities among sectors, transects and seasons.

Dependent variable Season Transect Sector Effect DF F p

Density All T2 A-B-C Season 3 1.726 0.16

Sector 2 90.459 > 0.01

Density Summer-19 All A-B-C Transect 2 1.10 0.33

Sector 2 38.17 > 0.01

Density All All BSeason 3 2.129 0.09

Transect 2 9.352 > 0.01

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

10 cm in depth (density = -5.66+13.5*depth).

A gain of 4 % in terms of explained variance is

obtained for the multiple linear model consid-

ering both depth and rugosity as explanatory

variables (R2 = 0.79, p <0.001, model: Density =

-5.4+ 11.88*Depth + 34.16*Rugosity).

Length and weight distributions: The

length of the measured Holothuria grisea speci-

mens ranged from 5.2 to 22.5 cm and their

weight ranged from 6.0 to 230 g (Fig. 6). The

mean length of the population when data from

all sampling occasions were combined was

12.54 cm (SD = 2.53) (mode = 13 cm; median

= 12.4 cm). Approximately 75 % of the sampled

specimens were between 10 – 14 cm. The over-

all mean weight was 60 g and nearly 60 % of

the population was between 55 and 93 g. The

relationship between body length and weight of

H. (H.) grisea (Fig. 6) is well represented by the

power model (p < 0.001) with the variation in

length explaining 62 % of the weight variability.

When the same database is analyzed separately

by different sampling occasion, no difference

in length (Kruskal-Wallis, H = 5.27, p = 0.152)

and weight (Kruskal-Wallis, H = 3.31, p =

0.345) was observed (Fig. 7). Therefore, growth

analyses were represented for the entire dataset.

Growth analysis: The relationship between

the compound index SLW and body weight of

Holothuria (H.) grisea is presented in Fig. 8A.

The SLW calculated for Holothuria grisea speci-

mens ranged from 2.79 cm to 9.84 cm. The

SLW calculated explained 75 % of variation in

body weight, and was a more effective predictor

of weight than length (62 %).

The ELEFAN method (Electronic Length

Frequency Analysis) yielded the best-estimated

growth parameter results: Le∞ = 13.63, k = 0.72

y-1, to = 0.79. Graphic representations of the

growth curve of Holothuria (H.) grisea showed

that the youngest sea cucumber was one year-

old, and the oldest had approximately two

years and a half of age. Growth rates tended to

decrease between the first and the second year

of life (Fig. 8B).

DISCUSSION

The results of the present study evidence

that the densities of Holothuria (Halodeima)

grisea observed on the different sectors of the

transects are positively correlated with depth,

and therefore with the period and frequency at

which these areas are subject to air exposure due

to the tidal level variation. The coast of Santa

Catarina is characterized by a mixed microtidal

regime with predominance of astronomic semi-

diurnal tides, which means that there is a com-

plete tidal cycle every 12 hours, and a complete

lunar cycle every 29.5 days (Salles-de Araujo,

Fig. 5. Regression lines considering density of Holothuria (H.) grisea as response variable and A. rugosity and B. depth as

explanatory variables.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

2020). Tides are also frequently influenced by

meteorological factors which tend amplify the

predicted astronomical tidal range (de Souza &

Schettini, 2014; Salles-de Araujo, 2020). Dur-

ing new and full moons, the tidal amplitudes

are nearly 1.2 m (spring tides), whereas during

crescent and waning moons the neap tides dis-

play low amplitudes (~ 0.5 m) (Salles-de Araujo,

2020). In this manner, the sea cucumbers in the

upper intertidal level are subject to frequent air

exposure during the tidal cycles, whereas in the

subtidal sector, the frequency of air exposure

is negligible, and in the lower intertidal sector

the periods of air exposure tend to occur only

for a few hours during spring tide periods (full

and new moons). Air exposure and desiccation

represent a stressful situation for sea cucumbers

(Hou et al., 2019) and therefore they would

tend to avoid the areas prone to long periods

of emersion.

The similarity in densities observed among

the transects when all sectors were considered

is explained by the overall similarity in the

depth range and rugosity indexes when con-

sidering the total area of the transects. On the

contrary, comparing sector B alone, the lowest

density recorded in the transect 3, is clearly

related to the lower rugosity of this segment,

since the depth of sector B among transects is

similar. Therefore, within similar depths, the

rugosity tends to be an important factor affect-

ing the aggregation of H. (H.) grisea. In Canary

Islands, Navarro et al. (2013) also found sig-

nificant correlations relation between density

of sea cucumbers and rugosity of the substrate.

Mendes et al. (2006) found that H. (H.) gri-

sea occurred more densely in the subtidal stra-

tum and displayed an aggregated distribution

pattern, which was correlated to areas with high

substrate rugosity, which on its turn, was highly

correlated with rock coverage. Such observa-

tions were corroborated by the present study,

and furthermore, we demonstrate that depth

is a primary factor explaining the observed

Fig. 6. Length and weight histograms of Holothuria (H.) grisea as well as the length-weight regression pooled for all the

sampling campaigns.

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

density patterns while rugosity of the substrate

is secondary. Leite-Castro et al. (2016) observed

a clear correlation between aggregative behav-

ior and the reproductive cycle of H. (H.) gri-

sea in northeast Brazil, with strong negative

correlation between aggregation and gonad

maturity. The highest degree of aggregation

(groups of > 3 individuals under the same rock)

occurred when the gonads were poorly devel-

oped or post-spawned. Conversely, the high-

est frequency of solitary individuals occurred

when most of them were mature. In that region,

the reproductive peak of gametogenic activity

is from December to February when aggrega-

tive behavior was minimal. In southern Brazil,

information about the reproductive cycle of this

species is still limited. The only study available

(Bueno et al., 2015) suggests that mature indi-

viduals are found year round with increased

sexual activity in February, however this study

was carried out with limited samplings and

organisms. The reproductive pattern of H. (H.)

grisea, may affect its aggregation behavior in

southern Brazil, however further investigations

on reproduction of this species are required to

examine such hypothesis.

Information about the status of the wild

populations of Holothuria (H.) grisea is scarce.

Souza Jr. et al. (2017) refers to densities up to

40 ind.m-2 in the locality of Bitupitá (Ceará)

Fig. 7. Length and weight histograms of Holothuria (H.) grisea as well as the length-weight scatterplots recorded on the

surveys carried out in winter 2019 (WI-19), spring 2019 (SP-19), summer 2020 (SU-20) and spring 2020 (SP-20).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

in northeast Brazil before the onset of sea

cucumber fisheries, whereas subsequently to

the establishment of fishing pressure, Farias-

Dias (2012) records mean densities of 0.54

ind.m-2 in the same area, which indicates a

dramatic drop (nearly 98 %) in sea cucumber

densities. The characterization of that fishery is

presented by Ponte and Feitosa (2019), which

indicates that in some areas the catch could

reach up to 6 600 Kg*yr-1. However, no infor-

mation is presented about the status of the wild

populations on those fishing sites. Rupp et al.

(2023) found densities ranging from 0.11 to

3.66 ind.m-2 at the intertidal region on differ-

ent sites along Santa Catarina coast, and Rupp

and Marenzi (2021) inform the occurrence of

eventual unregulated and unreported fishery

of sea cucumbers also in the state of Santa

Catarina. Mendes et al. (2006) reports mean

densities ranging from 0.74 to 8.04 ind*m-2 in

the same region of the present study. Although

that study does not present the absolute densi-

ties recorded per sampled metric unit as in the

present one (Fig. 3), a comparison of the mean

densities (ind*m-2) presented in that study,

carried out 16 years before the present one, is

interesting. The mean densities obtained in the

former study at the lower intertidal and subtidal

strata were respectively 3.05 and 6.65 ind*m-2;

and for winter-03, spring-03 and summer-04

the mean densities were respectively 5.68; 5.34

and 4.14 ind.m-2, respectively. In the present

study the mean densities recorded in the lower

intertidal and subtidal regions were 2.17 and

3.31 ind*m-2, respectively; and for winter-19,

Spring-19 and summer-20 were 3.35, 2.02, 2.38

ind*m-2, respectively. The comparison of both

studies suggests that the densities recorded in

the present study are remarkably lower than

those recorded in the previous one for equiva-

lent strata and seasons (28 % to 62 % drop

depending on strata end season). However, it

is not possible to infer whether the differences

from both studies are due to biological factors

or due to an eventual effect of undercovered

fishing activity. In any case, differently from the

northeastern Brazil, where a dramatic drop in

the population densities were found after the

onset of the sea cucumber fisheries (Souza Jr.

et al., 2017), the reduction in densities of H.

(H.) grisea observed in Santa Catarina does not

seem to be so striking, but it certainly rises a

Fig. 8 A. Relationship between the compound index SLW (square root of the length-width product) and body weight (g) of

Holothuria (H.) grisea. B. Individual growth of Holothuria (H.) grisea expressed by the von Bertalanffy growth function in

cm*y-1 fitted to the corrected lengths (Le), and the relative ages generated by replacing the growth parameters calculated by

ELEFAN in the inverted von Bertalanffy growth function.

12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

concern about eventual anthropic influence on

wild sea cucumber stocks.

Juveniles of most sea cucumber species

are seldom observed in great numbers in the

wild (Shiell, 2004; Shiell, 2005) as also noted

for H. (H.) grisea by Leite-Castro et al. (2016)

in northeast Brazil. In the present study there

was a low frequency of juveniles smaller than

8 cm and no individual was smaller than 5.2

cm. The reasons for such pattern in holo-

thuroids comprise two main hypotheses: that

young juveniles occur alongside with adults but

displaying a cryptic behavior, therefore being

difficult to find (Cameron & Fankboner, 1989;

Rogers et al., 2021); or else, juveniles settle

in specific habitats outside adult areas (Mer-

cier et al., 2000a; Mercier et al., 2000b). In fact,

Aquino-Souza and Gomes-Filho (2023) found

high densities of H. (H.) grisea juveniles (< 7.5

cm) associated with seagrass beds (Halodule

wrightii) in northeast Brazil, favoring the latter

hypothesis, however only one juvenile below

5 cm was found. Consequently, the habitat for

juveniles smaller than 5 cm is still to be found

for H. (H.) grisea along its distribution range.

Whereas seagrasses forms extensive meadows

in northeast Brazil, they are rare in Santa Cata-

rina (Marques & Creed, 2008) and Halodule

wrightii is non-existing, therefore other habitats

should be searched as settlement area for H.

(H.) grisea. However, it must be pointed out

that, in the preset study, we avoided substrate

disturbance, and rocks were not removed and

sediments not revolved, therefore the hypoth-

esis that the juveniles are more cryptic, hiding

deeper under the rocks, crevices and cracks,

could not be ruled out at this moment.

The temperatures recorded in the present

study (both air and water) are in accordance

with the expected seasonal variation in this

region. Salinity displayed little variation among

the campaigns, as all of the samplings were

carried out with fair weather, even though

severe rainstorms may often occur, decreasing

the salinity in coastal areas of Santa Catarina

(de Souza et al., 2016). All the surveys of the

present study were carried out in periods of full

and new moons, when the lower tidal levels are

recorded. Although studies demonstrate that

moon phase is an important factor affecting

holothurian reproduction (Mercier et al., 2007),

it is not clear whether it directly influences sea

cucumber abundance and densities.

Sea cucumber fishery is an important

activity in northeast Brazil generating income

for fisherman of coastal communities, however

it is an unregulated, not declared, and unman-

aged activity, and a severe decrease of wild

stocks have been reported (Leite-Castro, 2016;

Ponte & Feitosa, 2019; Souza Jr. et al., 2017).

Ponte and Feitosa (2019) provide information

about the size distribution of the catch in cer-

tain localities in the state of Ceará. The average

length of captured H. (H.) grisea varied among

fishing sites, ranging from 10.93 cm (SD = 2.01,

median = 10.80 cm) in the Conflict Zone to

14.43 cm (SD = 2.36, median = 15.67 cm) in

Caraúbas, with and average size among sites

of 12.25 cm. Additionally, Farias-Dias (2012)

reports a mean body length of 12.59 cm (SD =

2.42) in Bitupitá. The mean length of H. (H.)

grisea found at Armação do Itapocoroy in the

present study (12.54 cm, SD = 2.53, median =

12.4 cm) suits within the range of those report-

ed for northeast Brazil. It was not possible,

however, to compare the weights of the sea

cucumbers from both regions, since the infor-

mation presented by Ponte and Feitosa (2019)

refers to the eviscerated organisms, whereas our

study evaluated the whole body weight.

The relationship between body length

and weight of H. (H.) grisea is well repre-

sented by the power model (P < 0.000001)

with the variation in length explaining 62 %

of the weight variability, and the obtained “b”

value was 1.8765. Farias-Dias (2012), using

the same model found a determination coef-

ficient of 54 % and a “b” value of 1.225 for the

same species in northeast Brazil. The obtained

“b” value, different from 3, indicate negative

allometry (Gould, 1966), which means that the

body length of the species grows faster than

its weight. Negative allometry is commonly

observed in other sea cucumbers (Khodja &

Mezali, 2023; Poot-Salazar et al., 2014). The

regression of the compound index SLW (square

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

root of the length-width product) and weight

indicates that the use of this index enhance the

weight estimation accuracy by 75 % rather than

the Le. On the other hand, the determination

of Le allowed the calculation of growth param-

eters and age determinations. The curvature

parameter per year (K) also approximate other

determinations conducted for sea cucumbers

in Mexico and Portugal (0.20-0.88) using ELE-

FAN (Olaya-Restrepo et al., 2017; Poot-Salazar

et al., 2014). Finally, sea cucumbers studied

in Mexico presented a much longer life than

Holothuria (H.) grisea, as demonstrated by the

reconstructed relative ages (Poot-Salazar et al.,

2014). This difference is probably associated to

specific characteristics of the genus Isosticho-

pus, since Holothuria arguinensis in Portugal

partially overlapped the relative ages (Olaya-

Restrepo et al., 2017) of the hereby study.

The sea cucumber Holothuria (H.) grisea

is a slow moving and unprotected invertebrate

easily found in intertidal and subtidal zones

along the Brazilian coast, therefore being sus-

ceptible to effortless manual collection dur-

ing low tides or by snorkeling. It seems that

in Santa Catarina these wild populations are

still abundant but the threats of unregulated

and unreported fishery are present. Therefore,

urgent actions are required to protect these

greatly important elements of marine ecosys-

tems before the stocks become over-harvested

or depleted, as reported for several species

in various countries (Conand, 2017; Conand,

2018; Purcell et al., 2013). Once the wild stocks

have collapsed there is little capacity for natural

recovery (Purcell et al., 2023). We advocate that

the development of sea cucumber aquaculture

in Brazil is the only sustainable way to supply

these highly prized organisms to the avid Asian

markets while preventing wild stocks from

being depleted.

Ethical statement: the authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

ACKNOWLEDGMENTS

The authors thank Robson Cardoso da

Costa, Mariana Lopes, Giovani Oliveira da

Costa and Gilberto C. Manzoni for their sup-

port during field surveys.

REFERENCES

Aquino-Souza, R., & Gomes-Filho, J. G. F. (2023). Where

are the juveniles of the gray sea cucumber Holothuria

(Halodeima) grisea? Latin American Journal of Aqua-

tic Research, 51(2), 316–323. https://doi.org/10.3856/

vol51-issue2-fulltext-2938

Bueno, M. L., Tavares, Y. A. T., Domenico, M. D., & Borges

M. (2015). Gametogenesis and weight change of body

organs of the sea cucumber Holothuria (Halodeima)

grisea (Aspidochirotida: Holothuriidae) in southern

Brazil. Revista de Biología Tropical, 63(S2), 285–296.

http://dx.doi.org/10.15517/rbt.v63i2.23163

Cameron, J. L., & Fankboner, P. V. (1989). Reproductive bio-

logy of the commercial sea-cucumber Parastichopus

californicus (Stimpson) (Echinodermata: Holothuroi-

dea). II. Observations on the ecology of development,

recruitment, and the juvenile life stage. Journal of

Experimental Marine Biology and Ecology, 127, 43–67.

https://doi.org10.1016/0022-0981(89)90208-6

Centro de Hidrografia e Navegação. (n.d.). Marinha do Bra-

sil. https://www.marinha.mil.br/chm/tabuas-de-mare

Conand, C. (2017). Expansion of global sea cucumber

fisheries buoys exports. Revista de Biología Tropi-

cal, 65(S1), S1–S10. https://doi.org/10.15517/rbt.

v65i1-1.31661

Conand, C. (2018). Tropical sea cucumber fisheries:

Changes during the last decade. Marine Pollution

Bulletin, 133, 590–594. https://doi.org/10.1016/j.

marpolbul.2018.05.014

de Souza, M. F., & Schettini, C. A. F. (2014). Assessment

of tide and wind effects on the hydrodynamics and

interactions between tijucas bay and the adjacent

continental shelf, Santa Catarina, Brazil. Revista

Brasileira de Geofisica, 32(3), 515–529. https://doi.

org/10.22564/rbgf.v32i3.506

de Souza, R. V., Novaes, A. L., Garbossa, L. H., & Rupp G.

S. (2016). Variações de salinidade nas Baías Norte

e Sul da Ilha de Santa Catarina: implicações para o

cultivo de moluscos bivalves. Revista Agropecuária

Catarinense, 29, 45–48.

14 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

Empresa de Pesquisa Agropecuária e Extensão Rural de

Santa Catarina. (n.d.). Centro de Informações de

Recursos Ambientais e de Hidrometeorologia de

Santa Catarina. https://ciram.epagri.sc.gov.br/index.

php/maregrafos/

Farias-Dias, I. (2012). Distribuição espaço temporal e densi-

dade populacional de Holothuria (Halodeima) grisea

(Holothuroidea: Aspidochirotida) na praia de Bitupita,

Ceará, nordeste do Brasil [Master’s Thesis, Federal

University of Ceará]. DSpace. https://repositorio.ufc.

br/bitstream/riufc/18355/1/2012_dis_ifdias.pdf

Gould, S. J. (1966). Allometry and size in ontogeny and

phylogeny. Biological Reviews, 41(4), 587–640. https://

doi.org/10.1111/j.1469-185X.1966.tb01624.x

Hou, S., Jin, Z., Jiang, W., Chi, L., Xia, B., & Chen, J. (2019).

Physiological and immunological responses of sea

cucumber Apostichopus japonicus during desiccation

and subsequent resubmersion. PeerJ, 7, e7427. http://

doi.org/10.7717/peerj.7427

Khodja, I., & Mezali, M. (2023). Biometric study of the

royal sea cucumber, Parastichopus regalis (Cuvier,

1817), from Algeria’s west coast. Beche-de-mer Infor-

mation Bulletin, 43, 46–51.

Leite-Castro, L. V., de Souza Junior, J., Salmito-Vanderley,

C. S. B., Nunes, J. F., Hamel, J. F., & Mercier, A.

(2016). Reproductive biology of the sea cucumber

Holothuria grisea in Brazil: importance of social

and environmental factors in breeding coordina-

tion. Marine Biology, 163, 67. https://doi.org/10.1007/

s00227-016-2842-x

Luckhurst, B. E., & Luckhurst, K. (1978). Analysis of the

influences of substrate variables on coral reef fish

communities. Marine Biology, 49, 317–323. https://

doi.org/10.1007/BF00455026

Marques, L. V., & Creed, J. C. (2008). Marques e Creed -

2008 - Biologia e ecologia das fanerogamas marinhas

do Brasil. Oecologia Brasiliensis, 12(2), 315–331.

Martins, L. (2012). Estudo taxonômico dos Holothuroidea

(Echinodermata) de águas rasas da costa Brasileira

[Master’s Thesis, Federal University of Bahia]. DSpa-

ce. https://repositorio.ufba.br/handle/ri/12840

Martins, L., Marques, A., Fukuda M., & Tavares M. (2022).

An annotated catalogue of Echinodermata types in

the Museu de Zoologia, Universidade de São Paulo,

Brazil. Papéis Avulsos De Zoologia, 62, e202262015.

https://doi.org/10.11606/1807-0205/2022.62.015

Martins, L., & Souto, C. (2020). Taxonomy of the Brazilian

Apodida (Holothuroidea), with the description of two

new genera. Marine Biology Research, 16(4), 219–255.

https://doi.org/10.1080/17451000.2020.1761027

Martins, L., & Tavares, M. (2021). A new species of Parath-

yone (Holothuroidea: Dendrochirotida: Cucumarii-

dae) from northeastern Brazil, with a key to species.

Zootaxa, 4985(2), 245–252. https://doi.org/10.11646/

zootaxa.4985.2.7.

Mendes, F., Marenzi, A. W. C., & Domenico, M. D.

(2006). Population patterns and seasonal observa-

tions on density and distribution of Holothuria grisea

(Holothuroidea: Aspidochirotida) on the Santa Cata-

rina Coast. SPC Beche-de-mer Information Bulletin,

23, 5–10.

Mercier, A., Battaglene, S. C., & Hamel, J. F. (2000a).

Periodic movement, recruitment and size-related dis-

tribution of the sea cucumber Holothuria scabra in

Solomon Islands. Hydrobiologia, 440, 81–100. https://

doi.org/10.1023/a:1004121818691

Mercier, A., Battaglene, S. C., & Hamel, J. F. (2000b). Settle-

ment preferences and early migration of the tropical

sea cucumber Holothuria scabra. Journal of Experi-

mental Marine Biology and Ecology, 249(1), 89–110.

https://doi.org/10.1016/s0022-0981(00)00187-8

Mercier, A., Ycaza, R. H., & Hamel, J. F. (2007). Long-term

study of gamete release in a broadcast-spawning

holothurian: predictablelunar and diel periodicities.

Marine Ecology Progress Series, 329, 179–189. https://

doi.org/10.3354/meps329179

Mildenberger, T. K., Taylor, M. H., & Wolff, M. (2017).

“TropFishR: an R package for fisheries analy-

sis with length-frequency data”. Methods in Ecolo-

gy and Evolution, 8(11), 1520–1527. http://dx.doi.

org/10.1111/2041-210X.12791

Navarro, P. G., García-Sanz, S., & Tuya, F. (2013). Patro-

nes de abundancia y talla de Holothuria sanctori,

Holothuria mammata y Holothuria arguinensis (Echi-

nodermata: Holoturoidea) en la isla de Gran Canaria,

Atlántico oriental. Revista de Biologia Marina y Ocea-

nografia, 48(2), 273–284. https://doi.org/10.4067/

S0718-19572013000200007

Olaya-Restrepo, J., Erzini, K., & González-Wangüemert,

M. (2017). Estimation of growth parameters for the

exploited sea cucumber Holothuria arguinensis from

South Portugal. Fishery Bulletin, 116, 1–8. https://doi.

org/10.7755/FB.116.1.1

Pawson, D., Pawson, D., & King, R. (2010). A taxonomic

guide to the Echinodermata of the South Atlan-

tic Bight, USA: 1. Sea cucumbers (Echinodermata:

Holothuroidea). Zootaxa, 2449, 1–48. https://doi.

org/10.5281/zenodo.195134

Ponte, I. de A. R., & Feitosa, C. V. (2019). Evaluation of an

unreported and unregulated sea cucumber fishery in

eastern Brazil. Ocean and Coastal Management, 167,

1–8. https://doi.org/10.1016/j.ocecoaman.2018.09.016

Poot-Salazar, A., Hérnandez-Flores, A., & Ardisson, P. L.

(2014). Use of the SLW index to calculate growth

function in the sea cucumber Isostichopus badionotus.

Scientific Reports, 4, 5151. https://doi.org/10.1038/

srep05151.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58623, marzo 2024 (Publicado Mar. 01, 2024)

Purcell, S., Conand, C., Uthicke, S., & Byrne, M. (2016).

Ecological roles of exploited sea cucumbers. Ocea-

nography and Marine Biology: An Annual Review, 54,

367–386. https://doi.org/10.1201/9781315368597-8

Purcell, S.W., Lovatelli, A., González-Wangüemert, M.,

Solís-Marín, F.A., Samyn, Y., & Conand, C. (2023).

Commercially important sea cucumbers of the world

– Second edition. Food and Agriculture Organization

of the United Nations (FAO). https://doi.org/10.4060/

cc5230en

Purcell, S. W., Mercier, A., Conand, C., Hamel, J. F.,

Toral-Granda, M. V., Lovatelli, A., & Uthicke, S.

(2013). Sea cucumber fisheries: Global analysis of

stocks, management measures and drivers of over-

fishing. Fish and Fisheries, 14(1), 34–59. https://doi.

org/10.1111/j.1467-2979.2011.00443.x

Purcell, S. W., Ngaluafe, P., Lalavanua, W., & Ceccarelli,

D. M. (2018). Market price trends of Latin Ameri-

can and Caribbean sea cucumbers inform fisheries

management. Regional Studies in Marine Science, 17,

127–132. https://doi.org/10.1016/j.rsma.2017.12.003

R Core Team. (2023). R: A Language and Environment

for Statistical Computing [Computer software]. R

Foundation for statistical computing. http://www.R-

project.org

Rogers, A., Hamel, J. F., Quetzal, J., & Mercier, A. (2021).

Unique reproductive biology of the broadcast

sea cucumber Holothuria floridana: facultative

recruitment on adults inside nursery grounds. Inver-

tebrate Reproduction & Development, 65(2), 141–153.

https://doi.org/10.1080/07924259.2021.1900936

Rupp, G. S., & Marenzi, A. C. (2021). Holotúrias do litoral

de Santa Catarina (Brasil): captura ilegal e potencial

para a aquicultura. Proceedings Foro Iberoamericano

de los Recursos Marinos y la Acuicultura, 10, 607–618).

Rupp, G. S., Marenzi, A. W. C., De Souza, R. V., & Martins,

L. (2023). Sea Cucumbers (Echinodermata: Holothu-

roidea) from Santa Catarina Coast, Southern Brazil,

with Notes on Their Abundance and Spatial Distri-

bution. Journal of Shellfish Research, 42(1), 143–153.

https://doi.org/10.2983/035.042.0115

Salles-de Araujo, C. E. (2020). Análise das flutuações do

nível do mar no litoral De Santa Catarina. Agro-

pecuária Catarinense, 33(1), 61–67. https://doi.

org/10.52945/rac.v33i1.535

Shiell, G. (2004). Field observations of juvenile sea cucum-

bers. SPC Bêche-de-Mer Information Bulletin, 20,

6–11.

Shiell, G. (2005). Information on juvenile sea cucumbers:

a contribution by Dr. D.B. James. SPC Bêche-de-Mer

Information Bulletin, 21, 26–27.

Sokal, R. R., & Rohlf, F. J. (2000). Biometry: The Principles

and Practices of Statistics in Biological Research (3rd

ed.). W. H. Freeman and Co.

Sonnenholzner, J. I. (2021). ¿Hacia dónde va la acuicultura

de equinodermos en América Latina ? Potencial, retos

y oportunidades. Revista de Biología Tropical, 69(S1),

514–549.

Souza Jr., J., Ponte, I., Melo Coe, C., Lobo Farias, W. R., Viei-

ra Feitosa, C., Hamel, J. -F., & Mercier, A. (2017). Sea

cucumber fisheries in Northeast Brazil. SPC Beche-de-

Mer Information Bulletin, 37, 43–47.

Tiago C. G., & Ditadi, A. S. (2001). Holothurians from

the Brazilian coast: a historical survey. In M. Barker.

(Ed.), Echinoderms 2000 (pp. 379–384). Swets &

Zeitlinger.

Tommasi, L. R. (1969). Lista do Holothuroidea recentes do

Brasil. Contribuições Avulsas Do Instituto Oceanográ-

fico, 15, 1–29.

Ventura, C. R., Borges, M., Campos, L. S., Costa-Lotufo, L.

V., Freire, C. A., Hadel, V. F., Manso, C. L., Silva, J. R.,

Tavares Y., & Tiago G. C. (2013). Echinoderm from

Brazil: Historical Research and the Current State of

Biodiversity Knowledge. In J. J. Alvarado & F. A. Solís-

Marín (Eds.), Echinoderm Research and Diversity in

Latin America (pp. 301–344). Springer. https://doi.

org/10.1007/978-3-642-20051-9_9

Zar, J. H. (1999). Biostatistical analysis. Prentice Hall.