• Ana Alexandre
  • Maria Francisca
  • Ana Beatriz
Agrupamento de Escolas de Arouca
Irakaslea / tutorea
Carminda Gomes Santos
Arouca Portugal
Lanaren izena
Wildfires Ecotoxicology
Lanaren deskribapena
This work proposes to contribute to the establishment of cause-effect relationships in aquatic environments submitted to runoff from the burned areas. Ecotoxicological assays were developed from aqueous-extracts of ashes (AEA) of a forest fire, using as test-organisms two species of different trophic levels: the moss Sphagnum denticulatum Brid. and the freshwater snail Lymnaea stagnalis L. Experimental studies have shown that higher concentrations of AEA are highly toxic to both organisms. The lower concentrations also show high toxicity to the snail embryos, causing a delay in the embryonic development, hindering the development of the shell and leading to the appearance of malformations. The moss proved to be a less sensitive species for lower concentrations of AEA.
Lanaren eremua
ingurumena / Ekologia
Proiektu mota
Ikerketa proiektua
Lanaren plangintza


Ash sample collection and preparation of aqueous-extracts of ashes

Ash sampling occurred in October of 2017, immediately after a wildfire in Arouca, Aveiro, in the north of Portugal. The burned area was mostly composed by pines, oak trees and cork oak. The geological substrate is composed by the Arouca granodiorite. To be representative, the ash sampling was executed by defining a 15 meters long line and collected along that line at three equidistant points. About 2L of ash were collected at each point. Ash samples were sieved at 2 mm, weighed and preserved at 4º C in the dark until further use for aqueous extracts preparation.

The different test solutions were prepared by dilution of the aqueous-extract of ashes (AEA). This extract was prepared with the mixture of the three ash samples (same weight of ash at each sampling point) in a 1:4 (v/v) ratio with mineral sterilized water for the treatments with Lymnaea stagnalis (L.) and with sterilized water from the spring of the Caima River for the treatments of Sphagnum denticulatum Brid.

These mixtures were wrapped in aluminium foil for protection against light and placed on an orbital shaker for 2 hours at 200 rpm. After 12 hours of deposition, the aqueous fractions  were collected by decanting and filtration on paper with 5-13 µm pore.

The aqueous extracts of ashes and their dilutions (75%, 50%, 25% and 12.5%) were stored at 4 ° C until use in the bioassays (maximum preservation time: 24 hours).

1- The test organism Sphagnum denticulatum Brid.

Sphagnum denticulatum Brid is a relatively common species in Portugal and extremely variable, whose form depends on the type of habitat it develops in; from a medium size to a very large one until 20cm high (Mendes e Dias, 2002), with a round and well developed capitulum, in the terrestrial forms. The capitulum is green, yellow-brown to copper brown; they form pure mats or combine with other species.

It is a species of easy access, as it can be taken directly from its natural ecosystem. For this study, the moss was taken from peat bogs on the Freita moutain (at the spring of Caima river), in Arouca. After the permission given by ICNF (Nature and Forest Conservation Institute; Licence nº511/2018-ICNF). Due to the big amount of available material, it’s possible to select stems with identical characteristics, with the same development degree, of green colour and without bifurcations. The observation of chloroplasts in the surface cells is very easy because the leaves only have one layer of cells (Clymo, R.S. 2012).

1.1.- Sphagnum culture

Sphagnum has been collected in the peat bogs of Freita mountain. The stems were   kept in mineral water in 5L glass vats, in a climatized room at 20 ± 1°C, under a 14:10h light-dark cycles, for 2 weeks before the beginning of the tests. Weekly, the culture’s mineral water was renewed. To ensure the good development of the stems, the mineral water of the culture was renewed every week.


1.2- Stems selection

The Sphagnum stems were selected from the culture by their degree of development, shape and colour. Each stem was cut on top of a moisture absorbent paper sheet, with a scalpel and a scaled ruler, so as to show 5 cm of length. All the stems were examined to exclude the existence of morphological differences and replace them with others.

Five branch leaves were collected from the terminal branches from each treatment for observation and initial count of chloroplasts under the microscope.

1.3- Toxicity tests

The assays were made in 50mL beakers, previously rinsed with sterile distilled water. In each beaker there were 10 stems and 20mL of one of the test solutions. For each treatment, 3 replicates were made (total of 30 analyzed stems). The experiment was carried out for 28 days (4 weeks), at 20 ± 1°C, under a 14:10 h light-dark cycle. The test solutions were renewed whenever necessary.

1.4- Observations

The stems exposed to different treatments were observed every 2 weeks. Its growth was measured with a scaled ruler and then registered. Changes were exhibited at the branch leaves and observed through a stereoscopic microscope. Five branch leaves were collected from the most representative stems of each assay to observe and count the chloroplasts under the microscope.

2- The test organism Lymnaea stagnalis L.

Lymnaea stagnalis (L.) is a snail detritivore species that eats plants, algae and microorganisms (Van Duivenboden, 1983). It has a wide distribution in Europe and North America, where it colonizes small lakes of fresh and brackish water. These snails reach sexual maturity about 3 months after egg hatching when grown at 20 ± 1°C. By that time the shell measures about 2 cm, but in the older adults (2 to 3 years old) can reach 7 cm of length. They are hermaphrodites with preferential cross-fertilization, being, however, capable to self-fertilize (Van Duivenboden, 1983). During the copulation, only the snail that assumes the female role is fertilized and produces a jelly mass, which contains several dozens of transparent eggs, in the same embryonic development stage (Van Duivenboden, 1983).

The embryo development occurs inside the egg, making it easy to distinguish the main stages: morula – rounded embryo, with yellow appearance and slow movement; trochophore – rounded embryo, with granular appearance and uninterrupted rotative movement; veliger – the embryo loses its rounded shape, becoming evident the foot and the shell in formation; Hypo – completely developed embryo, with clear eyes, shell and foot and visible heartbeat.

2.1- Snail husbandry

The progenitors of the snail population used in this study were acquired from a well established laboratory culture at Kingston University of London, UK. Snails were maintained in a covered rectangular 36 L aquarium supplied with continuously aerated standard tap water and a room temperature of 20ºC ± 1 ºC, under a 14:10 h light-dark regime/ cycle. The snails were fed ad libitum with organic green lettuce leaves previously washed in clean water or fish food spayed with Tetraphyll. The aquarium has a substrate of ground oyster shell to ensure the development of the snails shell. Faeces and remaining food was removed by siphoning twice a week.

2.2- Synchronized culture of isolated eggs

Freshly laid egg masses, no older than 12 h, were used. Eggs were isolated from their gelatinous matrix using a scalpel and disposable pipette. Isolated egg capsules were completely freed from the jelly by rolling them over moist filter paper with the aid of a plastic micropipette tip and transferred to a 6 cm Petri dish previously rinsed in deionised water. The eggs were examined under a stereomicroscope to check for possible abnormalities. All eggs showing signs of multifertilisation or damaged egg capsule were discarded.

  2.3- Embryo toxicity tests

In the tests, eggs less than 24h old were used (from egg masses produced during the same night).

In order to exclude the possibility of the effects resulting from genetic differences, eggs from 5 to 6 different egg masses were always used in each treatment.

In order to exclude the possibility of the effects resulting from genetic differences, the eggs used in each treatment were always from 5 to 6 different egg masses.

All experiments were conducted in 6 multi-well plates, previously rinsed with sterile distilled water. A total of 6 eggs were randomly transferred with a disposable pipette into each well of the multi-well plate containing 6 mL of the test solutions. For each treatment, 6 replicates were made (total of 36 analysed embryos). The test had the duration of 21 days, at 20 ± 1°C under a 14:10 h light-dark regime/cycle. The test solutions were renewed once a week. Embryonic stages were established based on the morphological and behaviour characteristics of the embryos.

2.4- Observations

Embryo viability was accessed and recorded every 24 hours, with embryos determined non-viable (dead) after showing signs of coagulation, immobility, and loss of heartbeat (depending on their embryonic development stage).


Proiektuaren txostena

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