Supplementary Data s24

Supplementary data

Title: Layer of organic pine forest soil on top of chlorophenol contaminated mineral soil enhances degradation

Authors: Aki Sinkkonen1,2*, Sari Kauppi1, Suvi Simpanen1, Anna-Lea Rantalainen1, Rauni

Strömmer1, Martin Romantschuk1

1 University of Helsinki, Department of Ecological and Environmental Sciences, Section of

Environmental Ecology, Niemenkatu 73, 15140 Lahti, Finland

2 University of Turku, Satakunta Environmental Research Institute, Konttorinkatu 1, 28900 Pori,

Finland

* Corresponding author, e-mail [email protected]

Contents: Page

Description of Vegetation S2

Table S1 S3

Description of Chemical Analysis S4

Table S2 S5

Table S3 S6

Table S4 S7

Pages in total: 7

Tables in total: 4

S1 Description of vegetation

The ground layer at the first site consists mainly of grasses Deschampsia flexuosa and

Calamagrostis arundinacea, dwarf shrubs Vaccinium vitis-idea and V. myrtillus, and a thick moss layer dominated by Pleurozium schreberi and Dicranum sp. (Rantalainen et al. 2006).

Vegetation at the second site consisted of grasses and herbaceous perennials with about 10 % patchy coverage of bare soil. The dominant species were Elymus repens (coverage > 50 %),

Alopecurus pratensis, Phleum pratense, Achillea millefolium, Trifolium repens, Festuca sp.,

Agrostis sp., Taraxacum sp. and Tripleurospermum inodorum.

There was no vegetation at the third site as industrial peat production takes place every summer.

At the fourth and fifth sites, the scattered tree layer consists of young birch (Betula pendula and

B. pubescens), mountain ash (Sorbus aucuparia) and aspen (Populus tremula) trees; the field layer is characterized by E. repens, Festuca sp., Poa sp. and Agrostis sp. Some herbaceous species like

Epilobium angustifolium and Fragaria vesca and small moss patches were also present.

S2 Table S1. A schematic representation of the treatments performed. Columns (left to right) show experiment name, soil origin, duration of the experiment, temperature (mean ± SE in °C) in the experiment, pot volume (L) and contaminant added in the beginning of the experiment.

Experiment Soils tested Duration Temperature Volume Spiking

Aeration Sawmill soils from 36 weeks 9.3 ± 0.0 10 - Löytö and Reposaari Max 19.1

Nutrient Sawmill soils from 20 weeks 9 10 - amendment Löytö and Reposaari

Soil type Pine forest soil, agri- 12 weeks 8.7 ± 0.0 10 2,4,6-TCP cultural soil and peat Max 11.0

Organic soil Sawmill soil from 24 d 20 .25 - addition 1 Reposaari (two contamination levels)

Organic soil Mineral soil Three weeks 16 .30 2,4,6-TCP, addition 2 PeCP

Field Anonymous 60 weeks -30 - +30 - - experiment sawmill soil Mean ca. 2

Field conditions: If winter months (December-March) are excluded, mean temperature in the experimental site is close to 10 °C. The mean during summer months (June-August) is ca. 16 °C, and the mean during the hottest month in 2010 was ca. 20 °C. Annual precipitation was ca. 600 mm. Surface soil in naturally reforested sites is typically relatively moist in Finland, except for the hottest days or weeks.

S3 Description of chemical analysis

The thawed soil samples (2 g fresh weight) were amended with internal standards (6 g 3,4,5-

TCP, 6 g 2,4,6-TBrP and 300 ng 2,4,6-TBrA) and extracted with 20 ml of hexane by sonicating

(Everest ultrasonic) for 15 min, and shaking for 1 hour (350 rpm) at room temperature. The samples were centrifuged for 2 min at 2000 rpm in 20 °C and the hexane was transferred to an Erlenmeyer flask. Extraction was repeated with 20 ml of fresh hexane for 15 min. in the sonicator and 30 min. in the shaker. Extracts were combined and then evaporated to a 3 ml volume with rotary evaporator

(Laborota 4000- efficient). The extract was quantitatively transferred to a 100 ml separation funnel and 50 ml of 0.1 M K2CO3 was added to the funnel. The funnel was manually shaken for 5 min. and the upper hexane layer was transferred to a Kimax tube. This fraction was concentrated and 50 ng deuterated anthracene was added in order to determine recovery of an internal standard. 2,4,6-TCA was analyzed from the above mentioned hexane fraction, whereas PCPs remained in the K2CO3- solution ready for derivatization.

Acetylation of PCPs was performed by adding 1 ml of acetic anhydride (C4H6O3) to the K2CO3- solution followed by 5 min. shaking. Thereafter, the carbonate solution was extracted twice with 5 ml of hexane. The hexane extracts were evaporated to 0.3 ml volume and 600 ng of 2,4,6-TBrA was added for determination of internal standard recovery. All samples were stored in –20 °C prior to analysis with a gas chromatograph-mass spectrometer (GC-MS).

PCPs and 2,4,6-TCAs were analyzed with a GC (Shimadzu GC-17A) equipped with a MS- detector (Shimadzu GCMS-QP5000) and Phenomenex capillary column ZB-5MS (30 m length,

0.25 mm i.d. and 0.25 μm phase thickness). For both PCP- and 2,4,6-TCA-analysis, the GC oven temperature was first held in 100 °C for 1 min and then raised to 220°C at rate 4 °C min -1 and to

270 °C at rate 8 °C min-1. The standards for the quantitative analyses are presented in Table S2.

S4 Table S2. Standards for the quantitative analyses of chlorophenols and chloroanisoles. First mentioned mass of ion is used for quantitative analysis and the second one to confirm qualitative analysis. compound abbreviation relative m/z retention time 4-monochlorophenol 4-MCP 0.3851 128, 130 analyte 2,6-dichlorophenol 2,6-DCP 0.5093 162, 164 analyte 2,4-dichlorophenol 2,4-DCP 0.5466 162, 164 analyte 2,4,6-trichlorophenol 2,4,6-TCP 0.6957 196, 198 analyte 2,4,5-trichlorophenol 2,4,5-TCP 0.7950 196, 198 analyte 2,3,4,6-tetrachlorophenol 2,3,4,6-TeCP 1.0124 232, 230 analyte Pentachlorophenol PeCP 1.3106 266, 264 analyte 2,4,6-trichloroanisole 2,4,6-TCA 0.5404 195, 197 analyte 3,4,5-trichlorophenol 3,4,5-TCP 0.9006 196, 198 internal standard 2,4,6-tribromoanisole 2,4,6-TbrA 1.0000 344, 342 internal standard, recovery standard 2,4,6-tribromophenol 2,4,6-TBrP 1.1863 330, 328 internal standard Deuterated anthracene D-Anthracene 1.3602 188, 189 recovery standard

S5 Table S3. Within-subjects and between-subjects effects in Repeated measures ANOVA on 2,4,6- trichlorophenol concentrations (ng g-1) in mesocosms containing either organic forest soil, peat or agricultural mineral soil.

F df p

Time 246.1 2 < .0005

Time x Soil Type 54.6 4 < .0005

Intercept 7182.6 1 < .0005

Soil Type 39.8 2 < .0005

S6 Table S4. Effect of soil type on 2,4,6-trichlorophenol concentration (µg g-1) on weeks 0, 4 and 12 in mesocosms containing either organic forest soil, peat or agricultural mineral soil.

Week 0 F df p

Corrected model 1.63 2 .272

Intercept 24370 1 < .0005

Soil type 1.63 2 .272

Week 4 F df p

Corrected model 17.89 2 .003

Intercept 2198 1 < .0005

Soil type 17.89 2 .003

Week 12 F df p

Corrected model 143.1 2 < .0005

Intercept 4245 1 < .0005

Soil type 143.1 2 < .0005