Environmental Risk Assessment In The Sao Domingos Mine Biology Essay

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Soils are an essential resource in both managed and natural systems, and maintaining soil quality is critical to the development of sustainable human activities, in particular agriculture. The assessment of impacts of human activities on soil quality needs a universal practical approach that can be incorporated in global environmental assessments (Garrigues et al., 2012). Soil contamination is of increasing worldwide concern because of its effects on environmental quality, loss of soil productivity, and socioeconomic impacts (Conesa and Faz, 2011).

The environmental protection agencies stipulate the need for assessing contaminant bioavailability in soils to assist with the estimation of risk posed by such pollutants (Ming et al., 2012). Ecological risk assessment (ERA) is a process of collecting, organising and analysing environmental data to estimate the risk of contamination for ecosystems (Jensen and Mesman, 2006). ERA is a description or estimation of changes in populations or ecosystems at specific sites or areas already polluted and should hence be conveyed as impact assessment rather than risk assessment (Jensen and Mesman, 2006). Acording to Jensen and Mesman (2006), the ERA methods formulated are implemented in different stages: site characterisation and description of land-use; determination of ecological aspects; site-specific tiered assessment (the Triad).

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The triad approach (combining three lines of evidence - LoE; chemical, ecotoxicological and ecological) is based on a series of tiers that progressively allow collecting information in increasing detailed evaluations, improving the perception of the problem under assessment. It is a cost-effective process that integrates decision points (usually after each tier) that allows to stop the assessment (when negligible risk is found or immediate remediation actions are needed) or continuing to a higher tier (when an existing risk is considered) (Oliveira Filho et al., in press).

The Tier 1 comprises screening-level analyses (characterize the risk of contaminated soils) and when a risk is detected, Tier 2 (including more detailed site-specific investigations like acute and chronic tests) and, if necessary, Tier 3 (including more detailed in situ studies and complex models based on other ecological aspects such as comtaminant pathways and specific receptors) should follow (OLIVEIRA FILHO et al., in press). In Tier 2, when there is still uncertainty about the risk, the chemical LoE is based on previous caracterization of the total concentration of metals (i.e., held in Tier 1 or or acquired knowledge of the literature) more concentrations metals bioavailable to extract a more ecotoxicologically relevant fraction of the contamination than the total concentration, and the ecotoxicological LoE is based on previous data of the acute tests more chronic tests.

Wastes containing elevated metals and metalloids concentratrion have had particularly deleterious effects on soil organisms (Lefcort et al., 2010). To perform only a single species test could not provide a full picture of soil toxicity (Santorufo et al., 2012) and various biological tests have been developed to assess soil toxicity to complement the results from environmental chemistry, some of these tests have been applied to explore toxicity of post mining soils (Natal-Da-Luz et al., 2004; Boularbah et al., 2006; Alvarenga et al., 2008; Niemeyer et al., 2010; Frouz et al., 2011; Alvarenga et al., 2012; Oliveira Filho et al., in press).

Often bioavailability is estimated by using a variety of chemical and biological tests most of which still need to be tested and validated (Ming et al., 2012). Two of such common tests for assessing the bioavailability of contaminants in soil are acute toxicity and cronic test (Ming et al., 2012). Plants, enchytraeids and collembolans are very often used in ecological studies and in ecotoxicological risk assessment for terrestrial ecosystems (Alvarenga et al., 2008; Niemeyer et al., 2010; Frouz et al., 2011; Alvarenga et al., 2012; Oliveira Filho et al., in press). Plants have a relevant ecological role being primary producers and enchytraeids and collembolans play a key role in organic matter decomposition, nutrient cycling and energy flow (Santorufo et al., 2012).

Metals are amongst the most abundant soil pollutants and are not biodegradable (Wong et al., 2006), representing a problem of major interest in environmental (Van Gestel, 2008), and can accumulate strongly affecting biodiversity, functionality and activity of terrestrial ecosystems (Wong et al., 2006).

Mining activities have contributed to extensive soil contamination harming agricultural soils (Lefcort et al., 2010) and are known to be responsible for many soil contamination sites (Conesa et al., 2006; Wei et al., 2009; Oliveira Filho et al., in press). The acid mine drainage (AMD) caused by that activity is one of the most important responsible for the degradation of the soil (Zalack et al., 2010). This AMD pollution is caused when pyretic minerals, are exposed to oxygen and water during and after mining operations. Oxidation of these minerals generates sulfuric acid and produces conditions, in which many metals readily dissolve and flow through surface waters (Carroll et al., 2003; Zalack et al., 2010).

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Thus, AMD impacted soils are characterized by low pH, and high concentrations of metals and metalloids. Acidic pH, from AMD, may affect organisms directly (PROCURAR AUTORES), but it can also cause high availability of Al, Fe, and metals in post-mining soils (Van Gestel and Hoogerwerf, 2001; Loredo et al., 2006; Frouz et al., 2011; Oliveira Filho et al., in press).

Toxicity to plants and soil organisms represents one of the most important problems in reconstructing functional ecosystems at post-mining sites (Bradshaw, 1997). Toxicity is complex because toxicity reflects not only the total content of pollutants but also pollutant bioavailability and pollutant interactions with different soil properties (Van Gestel, 1992; Spurgeon and Weeks, 1998; Crouau and Cazes, 2005; Frouz et al., 2011), sources of metals such as salts, sludge, aerial deposition (Van Gestel et al., 2012) and mineralogical characterization.

Open-cast mining has been accompanied by large-scale landscape intervention, resulting in strong alterations in terrain structures, waterways, microclimates, land uses, and communities of organisms (Sklenička et al. 2004). São Domingos Mine (SDM) was an open-cast mine that was operational during almost one century (Freitas et al., 2004), that it still suffers with problems of AMD. The risk assessment of soils from the SDM, for the effects of large mining operations, have been well documented (Lopes et al., 1999; Pereira et al., 1999; Pereira et al., 2000; Batista, 2000; Oliveira et al., 2002; Quental et al., 2002; Freitas et al., 2004; Natal-Da-Luz et al., 2004; Pereira et al., 2004; Matos and Martins, 2006; Pereira et al., 2006; Álvarez-Valero, et al., 2008; Pérez-López et al., 2008; Rosado et al., 2008; Abreu et al., 2009; Tavares et al., 2009; Alvarenga et al., 2012; Oliveira Filho et al., in press COLOCAR OS TRABALHOS FEITOS NA MINA DE SAO DOMINGOS).

Based on previously acquired knowledge from screening phase (Tier 1; Oliveira Filho et al., in press) developed in the whole impacted area, the main goal of the present study is to contribute for the more complex and precise tests, integrating information from chemical (metal analysis, concentrations metals bioavailable and calculation of toxic pressures) and chronic tests of ecotoxicological lines of evidence (Folsomia candida and Enchytraeus cripticus reproduction test and Avena sativa and Brassica napus seed germination and growth test) to evaluate some uncertainties that some samples showed in the SDM. Data obtained in Tier 1 (Oliveira Filho et al., in press) and in the study were used to calculate risk values of sampled sites.

2 Materials and Methods

2.1 Study area

São Domingos Mine (37°40'19.40"N/7°30'16.54"W) is located in southeast Portugal (in the Lower Alentejo) on the east margin of Guandiana River, near the Spanish border frontier (14 km), and the study area extended from Corte do Pinto (CP), a small village 5 km north of the mine, to Santana de Cambas (SC), in the south, is one of those historical mining centres that date from pre- and roman times.

The ore body of the SDM is predominantly composed by massive cupriferous pyrite from the Iberian Pyrite Belt. The massive ore body is 45 to 48% of sulphur, and is primarily in the form of pyrite (FeS2). In association with pyrite other minerals were found, such as chalcopyrite (CuFeS2), sphalerite (ZnS), galene (PbS), and blend [(Zn, Fe)S] (Pereira et al., 2004). The main activity of the mine was copper concentrate production, and as secondary activity, cupriferous pyrites were processed as an elementary source of sulphur.

SDM was an open-cast mine in operation between 1858 and 1957. During that period, more than 20 million tons of cupriferous pyrite were extracted and treated locally. Abandoned in 1965, after the mining activity became unproductive, mining wastes (25 Mm3) got dispersed in a large area (3,156,225 m2) and the entire area suffers from severe problems of acid mine drainage (Lopes et al. 1999; Pereira et al., 2004; Pérez-López et al., 2008), make it one of the most interesting abandoned mines in Portugal. This area actually is still characterized by high levels of metals in soils, marked by significant concentrations of As, Cu, Fe Mn, Pb, and Zn and others.

2.2 Soil sampling

In Tier 1 of a site-specific ERA in the SDM, a total of 62 soil samples were collected along six transepts (L0, L1, L2, L3, L4, L5, and L6) parallel between each other (distant by 1 to 2 km) and perpendicular to a central axis (CA). Each transept samples were taken at 0, 20, 50, 150, 400, and 1000 m distant from the CA, both on left (CA, A1, A2, A3, A4, and A5, respectively) and right sides (CA, B1, B2, B3, B4, and B5, respectively). The exception was in the transept further south (L6), where soil samples were taken only at 0, 20, 50, and 150 m distant from the CA on both sides. Aditional soil samples were collected upstream of the mine (at 15 to 20 km from the CA) and analysed for physico-chemical properties. Three of these soils were selected to be used as reference material (Refs. 1, 2, and 3) together with a soil from one of the transept points in a multireference system based on the physico-chemical properties of the soils collected. Soils were always collected from the top 10-cm layer, sieved (5 mm) and stored at room temperature in the laboratory before its use for physico-chemical characterization and in ecotoxicological tests.

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The need for a more detailed evaluation at some points on which still remains an uncertainty of risk, meant that the points were selected: Group 1 (Ref.2, L1A5, L2A5), Group 2 (L6A3, L2A2, L2B4, L5B3), Group 3 (CP, L1A4, L3A1, L3A2, L3C, L5A1), Group 4 (Ref.1, L1A2, L1A3, L1B3, L2A1, L2C, L3B3, L3B4, L5B1, L6C).

All sampling sites of Tier 1 were georeferenced in a total of 65 points (more details can be seen in Oliveira Filho et al., in press), and the 23 points of this study are showed in the Figure 1.

2.3 Soil physico-chemical characterization

Total As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn concentrations were measured and measurements were performed in the field using a portable X-ray fluorescence spectrometry (XRF). Validation of XRF measurements was achieved by measuring metals in laboratory (by aqua regia digestions) from randomly selected soil samples (about 10% of scan sites), using Inductively Coupled Plasma (OES Axial 730 ES, Varian Scientific Instruments) following the United States Environmental Protection Agency (USEPA; 2001) for As, Cr, Cu, Ni and Zn and USEPA (1994) for Cd and Pb. The accuracy of extractions was checked using XXXXX as reference material and the analysis were considered valid with quality control recoveries between 95 to 115%.

The other soil physico-chemical parameters measured were soil moisture (loss on drying at 105 °C for 12h), soil pH (1M KCl, 1:5, w:v), conductivity in soil water suspension (1:5, w:v), water holding capacity (WHC; ISO 1998), organic matter (OM) content (loss on ignition at 500 °C for 6h) and soil texture (silt/clay, fine sand, and coarse sand contents; LNEC 1970).

As part of the chemical analyzes of Tier 2, for 23 selected points, pseudo-total (As, Cd, Cr, Cu, Ni, Pb and Zn) concentrations were determined by either flame or electrothermal atomic absorption spectrometry (by aqua regia digestions) according to ISO 11466 (1995).

2.4 Reproduction of Folsomia candida

For springtails, the reproduction test followed the ISO 11267 (1999a) guideline, with duration of the 28 days. Each replicate consisted of a glass container (4 cm diameter, 7 cm height) filled with 30 g (dry weight) of each soil tested, in which 10 synchronized juvenile sprigtails (10-12 days old) were introduced. Animals were fed at the beginning of the tests and at 7, 14 and 21 days with 2 mg of granulated dry yeast and the glass containers were opened twice a week for aeration. The initial moisture content of the soil was adjusted and controlled each week by replenishing weight loss with the appropriate amount of distilled water. At the end of the assay, the container was emptied to another vessel, water was added and coloured with some drops of blue ink, if necessary. After the floating organisms, the adult's were counted, digital photographs were taken and the numbers of juveniles appearing on the surface were counted, using the ImageTool software. The test was conducted at 20 ± 2 °C with a photoperiod of 16 h light and 8 h darkness. Five replicates were prepared for each soil tested, plus one replicate without animals for pH and moisture determination. At the beginning and at the end of the assay, soil pH and moisture content were determined.

2.5 Reproduction of Enchytraeus cripticus

The assays to determine the reproduction of E. cripticus were performed in accordance to the standardized ISO Guideline 16387 for enchytraeids reproduction testing (ISO, 2004). Because of the smaller size and shorter reproductive cycle of this species, the test duration was reduced to 28 d, and the adults were maintained in the vessels for this period of time (Chelinho et al., 2011). Ten adult worms with well developed clitellum were introduced in vessels, each containing 20 g dry soil (pre-moistened at 40-60% of the maximum water holding capacity) plus food supply (50 mg of finely ground and autoclaved rolled oats, being half of the amount supplied every week). The initial moisture content of the soil was adjusted and controlled each week by replenishing weight loss with the appropriate amount of distilled water. Five replicates were prepared for each soil tested, plus one replicate without animals for pH and moisture determination. At the end of the test, the number of enchytraeids was assessed after fixation with alcohol (80%), staining with Bengal rose (1% solution in ethanol), facilitates observation and counting, and wet sieving (103-mm mesh). All tests were performed at 20 °C ± 2 °C with a photoperiod of 16 h light and 8 h darkness. Also, soil pH and soil moisture were measured at the beginning and at the end of the exposure period.

2.6 Plant germination and growth test

The germination and growth test were carried out according to ISO guideline 11269-2 (ISO, 1999b), using a monocotyledonous and a dicotyledonous plant, Avena sativa (Poaceae) e Brassica napus (Brassicaceae), respectively. Each replicate, four for each species, consisted of a plastic container (9 cm length, 12 cm width, 6 cm height) filled with 400 g (dry weight) of test soil. To ensure suitable test substrate moisture along the experimental time, each container was perforated and connected to a vessel filled with deionised water by a glass fibre wick. Before the introduction of plants seeds, each test container was left during 24 h to allow the moistening of the test substrate. Thereafter, 10 seeds were buried at a maximum depth of 1 cm from the surface. After 50% seed germination of the controls (Ref.2, L6A3, CP and Ref.1) expected to seven days and counted the number of seedlings. Then, they were left five plants per container (where possible) and growth was allowed for 18 days in A. sativa and B. rapa. At the end of the assay, the aerial part of each plant were cut, was measured the length and dried during 48 h at 60 ± C, and weighted. After separation of the aerial part, all the roots of each container were washed, dried during 48 h at 60 ± C, and weighted The assays were carried out in a greenhouse, at an average temperature of 29 °C (maximum 38.5 °C and minimum 19.5 °C), at an average air relative humidity of 49.6% (maximum 69.5% and minimum 30%) and with a photoperiod of 16 h light and 8 h darkness. Soil pH and moisture content were determined at the beginning and at the end of the assays.

KALSCH, W.; JUNKER, T. & RÃ-MBKE, J. A Chronic Plant Test for the Assessment of Contaminated Soils. Part 1: Method development. Soils & Sediments 6 (1) 37 - 45 (2006)

Com 5 dias já havia 50% de germinação dos solos de referência

PROCURAR OS VALORES DE RISCO PARA BIOMASSA DEVE TER NO PROTOCOLO OU PERGUNTAR AO TIAGO

2.7 Statistical analysis

2.7.1 Ecotoxicological tests

2.7.2 Risk calculations

Risk calculations followed the approach proposed by Jensen and Mesman (2006) where risk values are expressed in a scale ranging from zero ("no risk") to one ("high risk"). This method assumes that the risk value of the reference soils is zero and that the risk of the contaminated soils (soils being tested) is calculated according to the value of the respective reference soil. Therefore, data on different parameters across the two LoE (chemistry - ChLoE - and ecotoxicology - EcLoE) should be made comparable (expressed under the same scale). For each soil sample, the calculation of the risk value was done in three steps: (1) scaling the results of each test and chemical analysis within each LoE; (2) integrating scaled information and calculating the contribution of each LoE to the overall risk; (3) integrate the information from the two LoEs and calculate the integrated risk.

3 Results and discussion

Soil invertebrates can adapt themselves to toxic chemicals physiologically and/or by developing genetic defence mechanisms when being exposed for longer periods of time to certain chemicals. Well-known examples are the adaptation of earthworms and collembolans to heavy metals when living at sites with a long history of mining (e.g. Posthuma, 1990).

Posthuma, L., 1990. Genetic differentiation between populations of Orchesella cincta (Collembola) from heavy-metal contaminated sites. J. Appl. Ecol. 27, 609-622.

Roembke, J., 2008. Bioavailability in soil: the role of invertebrate behaviour. Developments in Soil Science, v.32, Elsevier, p.245-260.

It is often assumed that the toxicity of metals and metalloids to soil organisms is determined by the concentration of metal ions in the soil solution, although it appears that other biotic and abiotic factors contribute (Smolders et al., 2009; Van Gestel et al., 2012).

3.1

The soils used in the tests had different levels of metal contamination and low pH.

Results from the multivariate factor analysis indicated the variables that mostly contributed to separate samples along first axis (33.0% of variation explained) were silt/clay, WHC and coarse sand. Along the second axis (22.4% of variation explained) samples were separated mainly due to pH and conductivity. In the third axis (17.2% of variation explained) samples were separated essencially by fine sand content.

Therefore, four soil groups were defined with their respective references, in agreement with their physico-chemical characterization: Group 1 - Ref.2, with 31 soils (soil samples were characterized by high silt/clay, low coarse sand, high WHC, high pH); Group 2 - L6A3, with 8 soils (high silt/clay, low coarse sand, high WHC, low pH); Group 3 - CP, with 8 soils (low silt/clay (main proportion), high coarse sand (main proportion), low WHC, medium pH); Group 4 - Ref.1, with 18 soils (medium silt/clay, medium/high coarse sand, low/medium WHC, low pH).

Table 1. Values of the physical and chemical characterization of each soil.

Soil

Silt/Clay

(%)

Fine sand

(%)

Coarse sand

(%)

OM

(%)

WHC

(%)

Cond.

(µS cm-1)

pH

Group 1

Ref.2

49.03

18.16

32.81

37.07

3.30

295

3.80

L1A5

38.24

28.07

33.68

50.89

3.08

278

5.31

L2A5

34.47

29.70

35.82

34.11

3.03

161

5.62

Group 2

L6A3

51.10

31.43

17.46

48.30

4.08

361

4.28

L2A2

46.76

33.44

19.80

49.58

5.56

599

3.50

L2B4

63.53

11.69

24.78

45.23

4.69

596

2.95

L5B3

62.50

26.79

10.71

77.02

1.79

298

2.83

Group 3

CP

29.74

0.67

28

5.81

L1A4

6.24

17.74

76.02

34.90

4.23

482

6.71

L3A1

0.00

0.16

99.74

12.53

0.00

355

4.63

L3A2

1.39

22.16

76.45

12.67

0.00

76

5.65

L3C

3.08

22.19

74.74

15.92

1.27

348

3.79

L5A1

9.79

26.19

64.02

54.40

21.31

427

4.21

Group 4

Ref.1

41.73

2.34

55.93

52.87

4.77

610

4.10

L1A2

26.05

26.43

47.52

75.15

14.55

559

4.50

L1A3

33.85

27.83

38.32

67.30

8.16

588

3.51

L1B3

40.80

11.91

47.29

35.73

2.74

315

3.73

L2A1

36.59

12.40

51.01

33.55

6.15

778

2.79

L2C

38.95

20.48

40.56

30.42

5.08

689

3.17

L3B3

47.29

20.35

32.35

36.67

7.69

3950

2.92

L3B4

40.00

21.72

38.28

48.20

4.62

431

3.23

L5B1

30.85

20.60

48.55

40.90

27.59

381

3.35

L6C

34.81

19.84

45.35

30.25

9.72

2380

4.01

WHC: water holding capacity; OM: Organic matter; Cond.: Conductivity; Max: maximal value; Min: minimal value; S.D.: standard deviation; n: number of values.

3.2 Soil metal and arsenic concentrations

Table 2. Total concentrations of metals and arsenic for each soil (mg kg-1 dry weight, except Fe - g kg-1 dry weight).

Soil

As

Cd

Cr

Cu

Mn

Ni

Pb

Zn

Fe

Group 1

Ref.2

15.5

UDL

160.3

42.3

21.3

56.7

107.3

29.0

L1A5

532.8

134.3

1432.9

168.6

671.8

43.1

L2A5

36.1

25.6

28.4

133.7

64.6

956.2

29.6

Group 2

L6A3

15.7

73.2

47.2

22.8

49.27

489.6

25.6

L2A2

1465.2

25.5

319.8

3532.9

149.2

83.6

L2B4

343.7

166.4

191.7

296.7

153.6

L5B3

7987.7

0.5

54.2

11595.9

924.7

11.5

Group 3

CP

276.2

43.4

19.6

34.6

151.3

10.2

L1A4

517.5

39.3

1406.2

65.6

485.4

42.4

L3A1

267.9

1.0

1523.0

6163.4

256.1

18557.2

1032.1

393.5

L3A2

546.7

1.4

2429.9

8154.5

18436.7

860.3

393.9

L3C

619.9

1721.3

6114.9

7644.4

794.1

336.4

L5A1

201.0

344.2

701.4

29.2

162.2

553.7

65.5

Group 4

Ref.1

18.0

99.3

35.7

17.1

57.8

37.5

408.4

28.6

L1A2

441.7

871.8

2903.6

42.6

395.8

460.1

75.9

L1A3

8057.1

166.5

26000.1

148.1

766.5

98.5

L1B3

1810.2

367.8

5692.7

135.7

159.2

121.8

L2A1

1096.4

142.2

1165.7

129.7

140.5

L2C

1157.8

211.1

1302.2

157.7

102.1

L3B3

1893.4

289.2

5257.5

266.0

193.0

102.0

L3B4

935.0

0.6

27.7

215.6

2576.5

62.5

102.3

L5B1

806.5

487.8

2979.4

148.6

140.1

L6C

674.6

388.1

1056.9

57.5

99.9

247.9

127.2

Max: maximal value; Min: minimal value; S.D.: standard deviation; n: number of values; UDL - under detection limit (see section 2.3).

3.3 Reproduction of Folsomia candida

Reproduction tests with soil invertebrates are more sensitive that the acute tests (Achazi, 2002). The sensitivity of the test species decreases in the following way: Eisenia fetida > Folsomia candida > Enchytraeus crypticus (Achazi, 2002). This difference in sensitivity reflects the different habitat requirements, and feeding habits (Achazi, 2002). In E. fetida, exposure to pollutants takes place via pore water and food, in E. crypticus almost entirely through pore water, and in E candida through the air in the soil pores (Achazi, 2002). The plant germination and growth test was performed with two species, Brassica rapa and Avena sativa (Achazi, 2002). The former is more sensitive than the latter (Achazi, 2002).

ACHAZI, R.K. Invertebrates in Risk Assessment: Development of a Test Battery and of Short Term Biotests for Ecological Risk Assessment of Soil. J Soils and Sediments, 2(4):174-178, 2002.

Ecological and anatomical differences among soil invertebrates influence exposure to and effects of metals present in the soils (USEPA, 2004).

U.S. Environmental Protection Agency 2004. Framework for Inorganic Metals Risk Assessment. Peer Review Draft, EPA/630/P-04/068B, Washington. 344p.

In general, for metals, the lower the pH and OM content of the soil, the more negative the effect on reproduction of F. candida at a given concentration (Fountain and Hopkin, 2005).

FOUNTAIN, M.T. and HOPKIN, S.P. Folsomia candida (Collembola): A "Standard" Soil Arthropod. Annu. Rev. Entomol. 50:201-22, 2005.

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Figure 1. Reproduction and survival of Folsomia candida (average and standard deviation) after exposure for 28 days. Reference soil (black bars), test soil (white bars). Asterisk indicates a statistically significant difference from test soil and corresponding reference soil, for *juveniles (p < 0.05; ANOVA one-way followed by Dunnett's test).

The low reproduction rate can be explained by the low OM content of the soil, which inhib its microbial activity and food production for F. candida (BUR et al., 2010)

BUR, T.; PROBST, A.; BIANCO, A.; GANDOIS, L.; CROUAU, Y. Determining cadmium critical concentrations in natural soils by assessing Collembola mortality, reproduction and growth. Ecotoxicology and Environmental Safety, 73:415-422, 2010.

De acordo com os resultados de Hund-Rinke and Wiechering (2001), os ensaios de comportamento com amostras de áreas contaminadas mostraram significativamente maior sensibilidade do que os ensaios com amostras contaminadas artificialmente.

HUND-RINKE, K.; WIECHERING, H., 2001, Earthworm avoidance test for soil assessments: an alternative for acute and reproduction tests. Journal Soils & Sediments, 1:15-20.

Results obtained with the collembola F. candida differed some what from those obtained with the other organisms, probably because collembola are less exposed to soil solution than the other organisms. Unlike the other organisms (vascular plants, enchytraeids), which are in close contact with the soil solution, collembola live mostly on the soil surface or in large pores that are usually drained, and collembola are covered by a cuticle with only limited permeability to liquids. Thus, the pH and chemical substances in soil solution are likely to have a smaller direct effect on collembola than on other organisms (Frouz, et al., 2011).

For example, the most important factor for most organisms in the laboratory tests was pH but the most important chemical properties in the field varied between groups (As for fauna, and Cd and conductivity for vascular plants) (FROUZ, et al., 2011).

FROUZ, J.; HRÄŒKOVÁ, K.; LANA, J.; KRIÅ TÅ®FEK, S., MUDRÁK, O; LUKEÅ OVÁ, A. and MIHALJEVIÄŒ, M. Can laboratory toxicity tests explain the pattern of field communities of algae, plants, and invertebrates along a toxicity gradient of post-mining sites? Applied Soil Ecology 51:114-121, 2011.

3.4 Reproduction of Enchytraeus cripticus

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*

*

*

*

*

*

*

*

*

*

Figure 2. Reproduction and survival of Enchytraeus crypticus (average and standard deviation) after exposure for 28 days. Reference soil (black bars), test soil (white bars). Asterisk indicates a statistically significant difference from test soil and corresponding reference soil, for *juveniles (p < 0.05; ANOVA one-way followed by Dunnett's test).

Reproduction and biochemical responses in Enchytraeus albidus (Oligochaeta) to zinc or cadmium exposures REFERÊNCIA

Effects on endpoints have been observed from soil concentrations whereas sublethal effects on springtails, earthworms, and plants generally start above 100-200 mg Cu kg-1 (Spurgeon et al. 1994; Kjær and Elmegaard 1996; Scott-Fordsmand et al. 2000a). As one exception to this, Scott-Fordsmand et al. (1997) reported effects on reproduction of springtails from approximately 40mgkg-1 in spiked soil.

JENSEN, J.; PEDERSEN, M.B. Ecological Risk Assessment of Contaminated Soil. Rev Environ Contam Toxicol 186:73-105, 2006. TIRADO DAQUI

3.5 Plant germination and growth test

Populations of a variety of higher plant species are able to colonise degraded mine soils in which other cultivated plants cannot survive (Freitas et al., 2004).

B. rapa was more sensitive than A. sativa to this type of soil contamination, but they both allowed the classification of soil C as the most toxic.

GYURICZA, V., FODOR, F.; SZIGETI, Z. Phytotoxic Effects of Heavy Metal Contaminated Soil Reveal Limitations of Extract-Based Ecotoxicological Tests. Water Air Soil Pollut 210:113-122, 2010. REFERÊNCIA

On soils containing heavy metals, colonisation is assisted, or even permitted, by the development of local metal-tolerant populations due to natural selection, such as in Agrostis and Festuca spp. (Shaw, 1989). These can grow normally, and the organic matter they produce can lead to reduced toxicity by complexing the available metals (BRADSHAW, 1997).

All this suggests that soil development and the restoration of mine workings can be left to natural processes. But in most situations the process of natural succession is slow, and it is common for 50 or 100 years to elapse before a satisfactory vegetation cover develops, particularly on mine wastes (BRADSHAW, 1997).

BRADSHAW, A. Restoration of mined lands - using natural processes. Ecol. Eng., 8:255-269, 1997.

3.6 Lines of evidence and integrated risk

The results arising from the investigation support the idea defended by other authors: when evaluating the potential risk posed by metals in soil, results from analytical environmental chemistry should be combined with results from bioassays in order to provide a more complete and relevant information on the bioavailability of contaminants (van Gestel et al., 2001; Loureiro et al., 2005a, 2005b; Alvarenga et al., 2008; Antunes et al., 2008). TIRADO DE Alvarenga et al., 2012.

This study is a contribution towards a risk assessment of the São Domingos Mine area (Portugal), integrating information from: soil physicochemical characteristics, pseudo-total and bioavailable trace elements (As, Cd, Cr, Cu, Ni, Pb and Zn), ecotoxicological evaluation, and microbial indicators. The bioassays using soil eluates (seed germination, luminescent inhibition of Vibrio fischeri and Daphnia magna immobilization) confirmed the soil toxicity categorization obtained with the bioassays using soil (plant growth tests, Eisenia fetida mortality and avoidance behaviour). However, the soil identified as the most toxic using bioassays, was different from the expected when considering the results from pseudototal and effective bioavailable trace elements.. TIRADO DE Alvarenga et al., 2012.

Risk values EcLoE

Risk values ChLoE

Integrated Risk

Table 3. Combined risk for ecotoxicological and chemical lines of evidence and integrated risk values.

Transepts

Sampling points

A5

A4

A3

A2

A1

CA

B1

B2

B3

B4

Ecotoxicological line of evidence

L0

L1

0.255

± 0.524

0.318

± 0.511

0.095

± 0.257

0.136

± 0.354

0.328

± 0.461

L2

0.007

± 0.020

0.537

± 0.557

0.451

± 0.286

0.353

± 0.377

0.435

± 0.604

L3

0.319

± 0.683

0.325

± 0.691

0.301

± 0.529

0.948

± 0.938

0.489

± 0.429

L5

0.035

± 0.100

0.072

± 0.200

0.703

± 0.458

L6

0.000

0.419

± 0.803

Risk values for chemical line of evidence

L0

L1

0.646

0.725

0.990

0.860

0.915

L2

0.012

0.856

0.751

0.781

0.352

L3

1.000

0.999

0.998

0.888

0.788

L5

0.631

0.795

0.976

L6

0.000

0.743

Integrated risk values

L0

L1

0.486

± 0.478

0.567

± 0.497

0.904

± 1.095

0.652

± 0.886

0.761

± 0.718

L2

0.010

± 0.007

0.742

±0.391

0.630

± 0.367

0.624

± 0.524

0.395

±0.103

L3

0.984

± 0.833

0.981

± 0.825

0.963

± 0.853

0.924

± 0.073

0.671

± 0.366

L5

0.403

±0.730

0.564

± 0.885

0.915

± 0.334

L6

0.000

0.613

± 0.395

CA = central axis; L0, L1, L2, L3, L5, L6 = transepts perpendicular to the CA; A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 = soil samples from each transept distant from the AC by 20, 50, 150, 400 and 1000 m both on left and right sides, respectively.

Green background: low integrated risk (RI < 0.5); yellow background: moderate integrated risk (0.5 < RI < 0.75); red background: high integrated risk (0.75 < RI < 1.00).

Values in boldface correspond to the soils selected for Tier 2.

4 Conclusions

To the best of our knowledge, this paper reports the first approach in estimating regional coal-mining damages, specifically using environmental risk assessment indicators in an environmental valuation framework. Coal-mining impacts have been identified using soil chemistry and chronic tests.

Acknowledgments We would like to thank Miriam Bianchi Oliveira for helping in the ecotoxicological assays and the Brasilian institution "Coordenação de Aperfeiçoamento de Pessoal de Nível Superior" (CAPES/GRICES) for financial support through the Doctoral-sandwich scholarship of Luís Carlos I. Oliveira Filho.

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Appendix 1. Physical and chemical characterization of the soils (arsenic and metals in soil - mg kg-1 dry weight, except Fe - g kg-1 dry wt).

Soil group

Silt/Clay (%)

ps <63 µm

Fine Sand (%)

63 µm <ps <250 µm

Coarse Sand (%)

250 µm <ps <2 mm

WHC

(%)

OM

(%)

Cond.

(µS cm-1)

pH (KCl 1:5 v:v)

As

Cd

Cr

Cu

Pb

Ni

Zn

Mn

Fe

Group 1

Ref.2

49.03

18.16

32.81

37.07

3.30

295

3.80

15.5

UDL

160.3

42.3

21.3

56.7

107.3

29.0

L1A5

38.24

28.07

33.68

50.89

3.08

278

5.31

532.8

134.3

1432.9

168.6

671.8

43.1

L2A5

34.47

29.70

35.82

34.11

3.03

161

5.62

36.1

25.6

28.4

133.7

64.6

956.2

29.6

Group 2

L6A3

51.10

31.43

17.46

48.30

4.08

361

4.28

15.7

73.2

47.2

22.8

49.27

489.6

25.6

L2A2

46.76

33.44

19.80

49.58

5.56

599

3.50

1465.2

25.5

319.8

3532.9

149.2

83.6

L2B4

63.53

11.69

24.78

45.23

4.69

596

2.95

343.7

166.4

191.7

296.7

153.6

L5B3

62.50

26.79

10.71

77.02

1.79

298

2.83

7987.7

0.5

54.2

11595.9

924.7

11.5

Group 3

CP

29.74

0.67

28

5.81

276.2

43.4

19.6

34.6

151.3

10.2

L1A4

6.24

17.74

76.02

34.90

4.23

482

6.71

517.5

39.3

1406.2

65.6

485.4

42.4

L3A1

0.00

0.16

99.74

12.53

0.00

355

4.63

267.9

1.0

1523.0

6163.4

256.1

18557.2

1032.1

393.5

L3A2

1.39

22.16

76.45

12.67

0.00

76

5.65

546.7

1.4

2429.9

8154.5

18436.7

860.3

393.9

L3C

3.08

22.19

74.74

15.92

1.27

348

3.79

619.9

1721.3

6114.9

7644.4

794.1

336.4

L5A1

9.79

26.19

64.02

54.40

21.31

427

4.21

201.0

344.2

701.4

29.2

162.2

553.7

65.5

Group 4

Ref.1

41.73

2.34

55.93

52.87

4.77

610

4.10

18.0

99.3

35.7

17.1

57.8

37.5

408.4

28.6

L1A2

26.05

26.43

47.52

75.15

14.55

559

4.50

441.7

871.8

2903.6

42.6

395.8

460.1

75.9

L1A3

33.85

27.83

38.32

67.30

8.16

588

3.51

8057.1

166.5

26000.1

148.1

766.5

98.5

L1B3

40.80

11.91

47.29

35.73

2.74

315

3.73

1810.2

367.8

5692.7

135.7

159.2

121.8

L2A1

36.59

12.40

51.01

33.55

6.15

778

2.79

1096.4

142.2

1165.7

129.7

140.5

L2C

38.95

20.48

40.56

30.42

5.08

689

3.17

1157.8

211.1

1302.2

157.7

102.1

L3B3

47.29

20.35

32.35

36.67

7.69

3950

2.92

1893.4

289.2

5257.5

266.0

193.0

102.0

Appendix 1. (Continued)

Soil group

Silt/Clay (%)

ps <63 µm

Fine Sand (%)

63 µm <ps <250 µm

Coarse Sand (%)

250 µm <ps <2 mm

WHC

(%)

OM

(%)

Cond.

(µS cm-1)

pH (KCl 1:5 v:v)

As

Cd

Cr

Cu

Pb

Ni

Zn

Mn

Fe

L3B4

40.00

21.72

38.28

48.20

4.62

431

3.23

935.0

0.6

27.7

215.6

2576.5

62.5

102.3

L5B1

30.85

20.60

48.55

40.90

27.59

381

3.35

806.5

487.8

2979.4

148.6

140.1

L6C

34.81

19.84

45.35

30.25

9.72

2380

4.01

674.6

388.1

1056.9

57.5

99.9

247.9

127.2

ps: Particle size; WHC: water holding capacity; OM: Organic matter; Cond.: Conductivity. UDL - under detection limit (see section 2.3).

Appendix 2. Individual risk values for each parameter and combined risk for each line of evidence in Tier 2.

Soil group

ChLoE

Combined ChLoE (msPAF)

EcLoE

Combined EcLoE

As

Cd

Cr

Cu

Pb

Ni

Zn

Group 1

Ref.2

L1A5

L2A5

Group 2

L6A3

L2A2

L2B4

L5B3

Group 3

CP

L1A4

L3A1

L3A2

L3C

L5A1

Group 4

Ref.1

L1A2

L1A3

L1B3

L2A1

L2C

L3B3

Appendix 2. (Continued)

Soil group

ChLoE (PAF individual)

Combined ChLoE (msPAF)

EcLoE

Combined EcLoE

As

Cd

Cr

Cu

Pb

Ni

Zn

L3B4

L5B1

L6C

For each sampling point in the EcLoE, values are scaled between 0 and 1 and are given in relation to the respective reference soil (risk for reference soil is set to 0).

ChLoE chemical line of evidence, EcLoE ecotoxicological line of evidence.

* indicates standard deviation > 0.4 (Jensen & Mesman, 2006).

Appendix 3. Risk values for combined risk for each line of evidence and integrated risk in Tier 1 and 2.

Soil group

Tier 1

Tier 2

Combined ChLoE (msPAF)

Combined EcLoE

Integrated risk

Combined ChLoE (msPAF)

Combined EcLoE

Group 1

Ref.2

0.00

L1A5

0.65

L2A5

0.01

Group 2

L6A3

0.00

L2A2

0.86

L2B4

0.35

L5B3

0.98

Group 3

CP

0.00

L1A4

0.72

L3A1

1.00

L3A2

1.00

L3C

1.00

L5A1

0.63

Group 4

Ref.1

0.00

L1A2

0.86

L1A3

0.99

L1B3

0.91

L2A1

0.75

L2C

0.78

L3B3

0.89