Paper 2B-23, In: A.R

Paper 2B-23, in: A.R. Gavaskar and A.S.C. Chen (Eds.), Remediation of Chlorinated and Recalcitrant Compounds—2002. Proceedings of the Third International Conference on Remediation of Chlorinated and Recalcitrant Compounds (Monterey, CA; May 2002). ISBN 1-57477-132-9, published by Battelle Press, Columbus, OH, www.battelle.org/bookstore.

DEVELOPING PHYTOREMEDIATION FOR CHLOROFORM IN A TROPICAL AREA

Cyro Bernardes Junior ([email protected]), Marcia Sabbag, Cristina Simonetti, Paula Nogueira, Patrícia Oshiro, and Duva S. Brunelli (AMBITERRA Ltda, São Paulo, Brazil) Dave Vance (ARCADIS, Houston, TX, USA)

ABSTRACT: The objective of the phytoremediation system was to create a last barrier for a chloroform contamination plume (up to 16mg/l of chloroform) before the groundwater discharge reaches an artificial lake. The activities started in September of 2000 with 179 plants of 10 native species introduced in a 2175 m2 area. Four species are from seasonal (mesophytic) and/or tropical rain forests (wet areas) and the other six, from Cerrado (Brazilian savanna). Most of the species are deciduous or semi-deciduous pioneer and secondary species that grow fast and with relatively deep root systems. It were estimated that this system will evapotranspirate in a range from 4 to 170 m3/day. The specie with the highest growth rate, Peltophorum dubium (Leguminosae), has grown 1,24 m in 10 months. In August/2001 it was observed an average reduction of 0,15 m of the level of groundwater at the treatment area. The groundwater depth varies from 5,6 m in the upper parts to 1,0m nearby the lake. The groundwater in the area presented a growth in the concentration of dissolved carbon after 6 months, as well as the Fe2+ concentration. The results for chloroform are preliminary and not yet conclusive, even though a 43% reduction was observed at the wells within the treatment area, and also a metabolite, methylene chloride, appeared in the wells indicating biodegradation.

INTRODUCTION The study area is an industrial plant situated in the State of São Paulo, Brazil. The groundwater in the area is contaminated with chlorinated solvents, basically chloroform. The remediation approach used different techniques depending the concentration of chloroform. At the source area we used chemical oxidation with Fenton’s reagent (Bernardes Jr, 2001) and at the nearby plume, in situ reductive action with molasses and as a polishing system. A phytoremediation system was implemented as a barrier for the eventual contamination of an artificial lake. The goal of this study is to evaluate the effectiveness of the phytoremediation system as a polishing remediation technique and the use of tropical native species to do this. Phytoremediation acts through two fundamental remediation processes, enhancement of saturated zone in situ biodegradation and phytoextraction (ITRC,2001). The rhizosphere is the zone in the subsurface occupied by plants root system. Within the rhizosphere plants contribute to enhanced in situ biodegradation through the supply of carbonaceous substrate and oxygen transfer. Rhizodeposition is partially the result of the decay of dead roots and root hairs. Also important are carbonaceous root exudations such as leakage from epidermal cells, secretions resulting from metabolic activity, mucilages from root tips (which act as lubricants for root penetration), and lysates from sloughed cells. Exudates are composed of a wide range of chemicals that include sugars, amino acids, organic acids, fatty acids, and numerous other compounds. It is estimated that 7% to 27% of the total plant mass is annually deposited as carbonaceous material in the rhizosphere. This carbonaceous material stimulates overall bacterial activity as well as providing substrate to support cometabolic degradation of xenobiotic hydrocarbons. The primary in situ remediation potential of plants for hydrocarbons lies in their capacity to enhance oxidation rates in the subsurface and provide cometabolic substrate. However, plants also have the ability to remove compounds, a process termed phytoextraction, which can be applied to organic or metal contaminants. In the case of hydrocarbons, the compound must be water solubility and have a moderate degree of lipid solubility. Lipid solubility is a function of the octanol-water partition coefficient (Kow) for the compound. The compounds most readily mobilized by plants have log Kow values in the range of 1 to 3. Compounds with values of Kow in this range include BTEX hydrocarbons, chlorinated solvents. (the logKow for chloroform is 1,97). , and other short chain aliphatic hydrocarbons. Once in the interior of the plant the adsorbed hydrocarbons may be: stored via lignification; volatilized; partially degraded through metabolization; or completely mineralized. Compounds with values of log Kow higher than 3 such as PNA's are incapable of entering the root, those with log Kow values lower than 1 are rejected by the root membranes. In order to evaluate the effectiveness of the phytoremediation system, we performed soil and groundwater analysis, besides systematic measures of plant growth and mortality rates.

SITE DESCRIPTION The site is near the 22oS latitude. The phytoremediation system is located downhill between the contamination source area and an artificial lake, where the groundwater discharges (Figure 1). The slope has a 11% gradient, covered only with grass. All the phytoremediation area is within the plant limits, so there is no direct recipients of contamination nearby. According to IAC, 2002, the average monthly temperature in area ranges from 17oC to 24oC, and the monthly precipitation from 37 mm rain in July to 238 mm in January. The evaporation ranges from 46,1 mm in June up to 125 mm in January. Geological substratum is characterized by the palaeozoic sediments of the Itararé Group and the mesozoic basalts of the Serra Geral Group. Those rocks originate interbeded soils of silt and sand layers. Figure 1 shows the location of the phytoremediation area, and the soil profile. The average hydraulic conductivity in the area is 9.5E-05 cm/s .The groundwater is 5,6 m deep at the higher portion of the area to 1,7m nearby the lake. Soil characteristics are shown in Table 1, and contamination before the implementation of the plants are shown in Table 2.

TABLE 1. Soil Characteristics S RESULTS FOR THE SITE (*) N% 0,009 P AVAILABLE (mg/l soil) 3,17 ORGANIC MATTER (mg/L soil) 13,6 pH 4,44 BULK DENSITY( g/cm3) 1,35 % CLAY 41 (*) sample collected at 0,5m depth, average of 5 samples TABLE 2. Initial Site Contamination PARAMETER \ WELLS PZF1 PZF2 PZF3 PZF4 PZF5 PZF6 PZF7 DATE ago/03/00 ago/03/00 ago/03/00 ago/03/00 ago/03/00 ago/03/00 ago/03/00 HOUR 12:30 12:20 12:40 12:50 14:20 14:30 14:40 GROUNDWATER DEPTH(m) 5,16 5,62 3,59 4,50 1,56 1,72 7,1 ORP (mV) -5 155 105 155 -60 90 170 PH 6,5 6,1 6,8 6,3 6,3 7 4,7 TOC (mg/L) 10 11,9 10,2 12,7 12,4 11,6 12,6 CONDUCTIVITY (mS) 0,5 0,6 0,1 0,3 0,4 0,2 0,3 TEMPERATURE (ºC) 24,5 23,9 25,0 25,1 24,2 23,0 22,3 FE +2(mg/L) 7,46 0,25 nd nd 17,2 0,95 5,4 FE +3(mg/L) 1,72 0,17 nd nd 7,9 0,29 0,2 TOTAL FE(mg/L) 9,18 0,42 nd nd 25,1 1,24 5,6 CL(mg/L) 1835 210 35 115 150 30 130 SO4(mg/L) 6 nd 11 6 nd nd nd NITRATE(mg/L) nd nd nd nd nd nd nd ALKALINITY 70 40 70 78 47 96 0 BICARBONATES(mgCACO3/L) CHLOROFORM( g/L) 75 10000 4250 4600 2170 2300 16 1,2-DICHLOROETHANE( g/L) <10 212 212 530 <10 <10 87 BENZENE( g/L) 12 310 44 258 59 59 59

FIGURE 1. Site and Soil Profile According to Chapell (1997) concentration of chlorinated solvents ranging from 50 to 200 mg/l are toxic to plants, so the levels found at the site are not phytotoxic.

MATERIAL AND METHODS – PROJECT DESCRIPTION The criteria used to choose the native species were: high growth rates, deep root penetration, high transpiration rates and a morphology of elongate cylinder. We selected species from the seasonal (mesophytic) and tropical rain forests for the higher groundwater table area and from the Cerrado (Brazilian savanna) for the deeper water table area. Pioneer and secondary species, preferentially non-deciduous, that grow relatively fast and have wind-dispersed seeds (i. e. fruits not consumed by birds or mammals) were prioritized. Other criteria was to avoid uniform plantation, mixing all species, and to simulate the natural process of secondary succession. Doing so we expect to reconstitute the natural vegetation, originally forested, and to minimize the action of plagues. The area was divided into three zones according to the water table depth; for each zone the most adequate species were chosen. The plants were planted in 3 m distant rows, with a final distance of 2,5m between each individual. All plants were cultivated in a hole with 0,6*0,6*0,6m filled with a prepared substratum (1/3 sand, 2/3 fertilized soil for wet area plants or 1/3 fertilized soil for the cerrado plants). In the first month all saplings were irrigated daily. The initial heights varied from 0,1 m (Qualea dichotoma, Vochysiaceae) to 1 m (Lafoensia pacari, Lythraceae). Table 3 shows the characteristics of the species, their location and the number of individuals planted.

TABLE 3. Species Used at Beginning, Number, Habitat, Size and Location

CODE SPECIE NAME HABITAT ADULT QUANTITY SIZE(M)* ZONE 1- NEAR THE LAGOON A1 Croton urucurana Baill. Sangra-d’água (**) 7-14m 27 B1 Inga marginata Willd. Ingá-feijão (**) 5-15m 24 C3 Anaderanthera columbrina (Vell.) Brenan Angico-branco-da-mata CERRADO 12-15m 01 Total Zone 1 52 ZONE 2 INTERMEDIARY ZONE A2 Kielmeyera coriacea (Spreng.) Mart. Pau-santo CERRADO 03-08m 14 B2 Qualea dichotoma (Warm.) Stafl. Pau-terra-mirim CERRADO 10-18m 13 C2 Senna macranthera (Collad.) Irwin et Manduirana (**) 06-08m 14 Barnaby D2 Peltophorum dubium (Spreng.) Taub. Canafístula (**) 15-25m 12 Total Zone 2 53 ZONE 3 DEEPER WATER TABLE A3 Anaderanthera macrocarpa (Benth.) Brenan Angico-vermelho (**) 13-20m 22 B3 Anaderanthera falcata (Benth.) Spreng. Angico-do-cerrado CERRADO 08-16m 19 C3 Anaderanthera columbrina (Vell.) Brenan Angico-branco-da-mata (**) 12-15m 15 D3 Lafoensia pacari St. Hil. Dedaleiro CERRADO 10-18m 14 C2 Senna macranthera (Collad.) Irwin et Manduirana (**) 06-08m 01 Barnaby D2 Peltophorum dubium (Spreng.) Taub. Canafístula (**) 15-25m 01 B1 Inga marginata Willd. Ingá-feijão TROPICAL RAIN 5-15m 02 FOREST Total Zone 3 74 TOTAL 179 (*)- Barros et al., 1991; Siqueira, 1994; Lorenzi, 1992 (*) –TROPICAL SEASONAL FOREST

To estimate the transpiration rates we used as a parameter the transpiration rate obtained by Rawitscher (1994) of 13 mg water/min/100cm2 of leaf area for a cerrado specie. The canopy areas were estimated using the average size of the adult plant (Lorenzi,1992). The results indicated that the system could remove from the water table a water volume ranging from 4 m3/day (at the beginning) up to 170 m3/day when the trees were mature. This represents an average per tree from 0,02 m3/day/tree up to 0,95 m3/day/tree.In ITRC, 2001 for adult poplar trees the rate ranges from 0,076 m3/day/tree to 0,15 m3/day/tree.

RESULTS AND DISCUSSION

Plants. The heights of the plants were measured monthly. One month after the initial planting, 20 plants died and were substituted; eleven of them were ingá-feijão (Inga marginata, Leguminosae), for most of the saplings were not healthy. Up to January/2002, 27 plants died (15% of the total). Manduirana (Senna macranthera, Leguminosae; C2) and pau-terra-mirim (Qualea dichotoma, Vochysiaceae; B2) presented the highest mortality rates, (67% and 58% of the plants died). Those with no registered deaths were pau-santo (Kielmeyera coriacea, Guttiferae; A2), canafístula (Peltophorum dubium, Leguminosae; D2), angico-do-cerrado (Anaderanthera falcata, Leguminosae; B3), and dedaleiro (Lafoensia pacari, Lythraceae; D3). As to the depth of the groundwater, there were higher mortality rates at the Zone 1, near the lake (26 % of the total), than in Zone 3, with only 7 %. This is rather a consequence of the poor adaptation of the species to the wetter substratum than the toxicity, once the wells with higher levels are situated at the Zone 3. The species with highest growth rate was canafístula (Peltophorum dubium, Leguminosae; D2), with a growth of 1,24 m in 8 months, followed by angico-do-cerrado (Anaderanthera falcata, Leguminosae; B3), with 1,0 m after 8 months (Figure 1). The specie with lower growth rate was ipê-amarelo-do-brejo (Tabebuia umbellata, Bignoniaceae), with only 0,05 m in 8 months. These preliminary results reveal that considering both growth rate and mortality, canafístula (Peltophorum dubium, Leguminosae) and angico-do-cerrado (Anaderanthera falcata, Leguminosae) are the most adequate species for phytoremediation among the tem selected species. Angico- branco-da-mata (Anadenanthera colubrina, Leguminosae; C3) could also be an option because of its good development and low mortality rate, only 6,7 %. It is interesting to note that Peltophorum dubium is a characteristic species of the mesophytic forest, while Anaderanthera falcata occurs mainly in the Cerrado or in its transitional zones to the seasonal forest (ecotones). The last options would be manduirana (Senna macranthera, Leguminosae), pau-terra-mirim (Qualea dichotoma, Vochysiaceae), and ipê-amarelo-do- brejo (Tabebuia umbellata, Bignoniaceae), due to slow development or high death rates.

Groundwater behavior. Before the plantation of the saplings 7 monitoring wells were built at the site (see figure 1), two of them up gradient (PF01 and PF02) but near the plants, two at the middle of the system (PF03 and PF04) and three down the gradient (PF05, PF06, PF07). Soil samples were taken to analyze for moisture. These samples were collected before the plantation and one year after. The sampling used hand auger. The objective was to evaluate how the plants change the soil moisture profile; groundwater depth was also measured. After one year using a hand auger soil samples were taken from the same depths nearby the monitoring wells and the moisture analyzed. The results indicate the action of the plants, at least in the wells PF01, PF02, PF03 and PF04 were we registered a increase in the soil moisture from the water table up to the ground level. At the wells PF05, PF06 and PF07 this effect is not very clear as the water level is higher. Figures 3 and 4 show the results for PF04 and PF02. 1,40

1,20

1,00

0,80

0,60 Growth /Specie (m) /Specie Growth

0,40

0,20

0,00

17/1 17/1 17/0 17/0 17/0 17/0 17/0 17/0 1/00 2/00 1/01 2/01 3/01 4/01 5/01 6/01

A1 - Sangra d'água B1 - Ingá feijão C1- Ipê Amarelo do Brejo A2 - Pau santo B2 - Pau terra mirim C2 - Manduirana D2 - Canafístula A3 - Angico vermelho B3 - Angico do cerrado C3 - Angico branco da mata D3 - Deadaleiro

FIGURE 2. Growth Since Planting (Actual Height - Initial One)

Moisture (%) 0 5 10 15 20 25 30 0

2 jul/00

ago/01 4 Depth of the sample (m)

6 FIGURE 3. Moisture Variation With Depth Pf04

Mositure (%) 0 5 10 15 20 25 30 35 0

2 jul/00

3 ago/01

5 Depth of the sample (m) the of Depth 6

7 FIGURE 4. Moisture Variation With Depth Pf02

Even for the brief period of observation, we registered a change in the water table, probably due to the effects of evatranspiration of the plants. Considering the levels observed at the same month (August) to avoid the hydrologic cycle effects, an average drop in the water level for the seven monitoring wells were 0,15 m. This represents one year after planting. Figure 5 with data up to December 2001 shows this effect more clearly. From August to December 2000 the line indicated that the the aquifer is filling up (the water level is rising) while in December 2001 the water level is dropping. It is important to observe that in August 2001 we had a problem with the measurement of PF06.

3 Depth of water table (m) 2

1 ago-00 out-00 dez-00 fev-01 abr-01 jun-01 ago-01 out-01 dez-01 Data

PZF01 PZF02 PZF03 PZF04 PZF05 PZF06 PZF07 FIGURE 5. Variation of the Depth of the Water Table at the Site Degradation of chemicals. Even if its early to get significant alterations in the groundwater due to the action of the plants, some changes in TOC, sulfate and Fe2+ were observed. The TOC results showed a increase soon after the plantation of the plantings in all wells. For wells PF03 and PF04 in the middle of the phytoremediation area, we observed an increase in the Fe2+ concentrations, which is consistent with the increase in the carbon that increases the reducing conditions of the environment. We have also registered metabolites of chloroform decomposition (methylene chloride) in the wells PF03 and PF04, consistent with the increase in the reducing environment. On the other side, the results of Oxidation Reduction Potential do not show an increase in the reducing environment at these wells, only at wells PF06 and PF07, downstream of the phytoremediation area. The results of conductivity tests and chloroform indicated that a higher concentrated area of chloroform is approaching the area (see results for PF02). Regarding the chloroform the results of wells PF03, 04, 05, 06 and 07 indicated an average reduction of 44% since August 2000. As a new plume is showing up at well PF02, the results of wells PF01 and 02 were not considered. The figures below show the results. 80

PZF1 PZF2 60

PZF3 PZF4 50

PZF5 PZF6

40 PZF7

Concentration(mg/L C) 30

0 ago/00 out/00 dez/00 fev/01 abr/01 jun/01 ago/01 out/01 dez/01

FIGURE 6. Results of Total Organic Carbon

50 PZF1 PZF2 PZF3 PZF4 40 PZF5 PZF6 PZF7 30 Concentration (mg/L Fe2+) (mg/L Concentration 20

0 ago/00 out/00 dez/00 fev/01 abr/01 jun/01 ago/01 out/01 dez/01 FIGURE 7. FE2+ Concentration in Groundwater

20 18 16 14 12 10 8

Concentration mg/L 6

4 2 0 ago/00 out/00 dez/00 fev/01 abr/01 jun/01 ago/01 out/01 dez/01 PZF1 PZF2 PZF3 PZF4 PZF5 PZF6 PZF7 FIGURE 8. Chloroform Results 2

1,5

concentration mg/L concentration 0,5

0 ago/00 out/00 dez/00 fev/01 abr/01 jun/01 ago/01 out/01 dez/01 PZF1 PZF2 PZF3 PZF4 PZF5 PZF6 PZF7

FIGURE 9. Methylene Chloride

CONCLUSIONS Based on the above results, we concluded that: S There is a mortality of 15% of total plantings, which indicated that at the concentrations (10 mg/L of chloroform) found there is no observed toxicity. S The most adequate species among those used in this study were canafístula (Peltophorum dubium, Leguminosae) and angico-do-cerrado (Anaderanthera falcata, Leguminosae) that showed higher growth and lower death rates. S The less adequate were manduirana (Senna macranthera, Leguminosae), pau- terra-mirim (Qualea dichotoma, Vochysiaceae), and ipê-amarelo-do-brejo (Tabebuia umbellata, Bignoniaceae). S canafístula (Peltophorum dubium, Leguminosae) growth 1,24 m in 8 months. S Even with a small period of study, the effect in the groundwater was already observed with an average drop in groundwater depth of 0,15 m S The pumping effect was observed also in the change of the soil moisture profile S Considering the wells in the middle and downstream of the area, it was observed an average reduction of 44% in the chloroform concentration, even though the results are not very conclusive, S The effect of the plants seems to increase in the dissolved organic carbon which increase the conditions for a reductive decomposition of the chloroform. The increase of TOC, Fe2+ and the show up of metabolites as methylene chloride are the evidence lines that this could be happening.

ACKNOWLEDGMENTS This study was possible due to the support of AMBITERRA Ltda.

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