Biotreatability/Bioremediation Final Report

Home , Azovibrio, Coccobacillus, Nitrosomonadales, Proteiniphilum, Rhodoblastus, Rhodoblastus acidophilus

Prepared for Hercules Inc. Ashland Research Center, Bldg. 8139 500 Hercules Road Wilmington, Delaware 19808-1599

BIOTREATABILITY/BIOREMEDIATION FINAL REPORT

Former Hercules Higgins Plant Gibbstown, New Jersey

October, 2017

Prepared by

CSI Environmental, LLC 401 Headquarters Dr., Suite 203 Millersville, MD 21108 Biotreatabi/ity/ Bioremediatio11 Fi11al Report Hercules Incorporated, Gibbstown, New Jersey

. Stevens, PG Principal

r. Lowell Sieck, PhD Sr. Project Chemist

Dean Pinson, PE Sr. Project Engineer

Kevin Costello Sr. Project Scientist

TABLE OF CONTENTS

Contents

1.0. INTRODUCTION ...... 1

2.0. METHODS-DESCRIPTION OF PROJECT EXECUTION ...... 2

3.0 RESULTS ...... 3 3.1. Cumene in Soil ...... 3 3.2. Cumene Concentration in Groundwater...... 4 3.3. Chloride Concentration in Groundwater ...... 4 3.4. Dissolved Oxygen (DO) and Sulfate (SO4) as TEA Compounds for Microbial Growth and Metabolism ...... 5 3.4.1. DO Concentrations – Aerobic and Anaerobic Areas ...... 5 3.4.2. SO4 as a TEA at the Anaerobic Area ...... 5 3.5. Microbial Growth ...... 6 3.5.1. Aerobic Area ...... 6 3.5.2. Anaerobic Area ...... 6 3.5.3. Total Bacteria Counts ...... 6 3.5.4. Comparison of Aerobic and Anaerobic Microbial Activity ...... 7 3.5.5. Comparison of Aerobic and Anaerobic Microbial Community Composition ...... 7 3.6. Biological Oxygen Demand (BOD) Concentrations ...... 8 3.7. R-NAPL Considerations ...... 8

4.0. DISCUSSION AND SUMMARY ...... 9 4.1. Cumene Remediation ...... 9 4.2. Verifying the Distribution of TEA Reagents ...... 10 4.3. Verifying Sufficient Nutrient Availability for Microbial Growth ...... 11 4.4. Cumene Degradation Rate ...... 11

5.0. REFERENCES ...... 23

LIST OF FIGURES

Figure 1. Bioremediation Pilot Study Areas Figure 2. Cumene Concentrations in Soil - Aerobic Test Area Figure 3. Cumene Concentrations in Soil - Anaerobic Test Area Figure 4. Cumene Concentrations in Groundwater from Wells at the Aerobic Test Area Figure 5. Cumene Concentrations in Groundwater from Wells at the Anaerobic Test Area Figure 6. Chloride Concentrations in Groundwater from Wells at the Aerobic Test Area Rev. Date: 10/12/2017 i CSI Environmental, LLC

Figure 7. Chloride Concentrations in Groundwater from Wells at the Anaerobic Test Area Figure 8. Comparison of Mean DO Concentrations in Groundwater from Wells at the Aerobic Vs. Anaerobic Test Area Figure 9. Average DO over Time in Aerobic and Anaerobic Pilot Study Areas Figure 10. Sulfate Concentrations in Groundwater Resulting From the Injection of MgSO4 During the Pilot Test Figure 11. Comparison of SO4 and SO3 Concentrations in Soil During the Pilot Study Based on Statistical Means Among All Wells Figure 12. Microbial Growth in Well Water Sampled at the Aerobic Test Area in July 2016 Figure 13. Microbial Growth in Well Water Sampled at the Aerobic Test Area in July 2016 Figure 14. Microbial Growth in Well Water Sampled at the Anaerobic Test Area in July 2016 Figure 15. Microbial Growth in Well Water Sampled at the Anaerobic Test Area in July 2016 Figure 16. BOD Concentrations among Core Samples From the Aerobic Test Area Figure 17. BOD Concentrations among Core Samples From the Anaerobic Test Figure 18. Comparison of Kjeldahl Nitrogen Concentrations in Soils from the Aerobic and Anaerobic Test Areas During the Pilot Test

LIST OF TABLES

Table 1. Statistical Analyses Comparing Cumene Concentrations in Soil at the Aerobic Test Area During the Ten Month Pilot Test Table 2. Statistical Analyses Comparing Cumene Concentrations in Soil at The Anaerobic Test Area during The Ten Month Pilot Test Table 3. Comparison of Statistical Means for SO4 and SO3 Concentrations in Soil during the Pilot Test Table 4. BRT Agar Plate Count Results - Soil Table 5. BRT Agar Plate Count Results – Groundwater Table 6. Comparison of Statistical Means for BOD Concentrations in Soil at the Aerobic Test Area during the Pilot Test Table 7. Comparison of Statistical Means for BOD Concentrations in Soil at the Anaerobic Test Area during the Pilot Test Table 8. Pore Space Analysis Results

LIST OF ATTACHMENTS

Attachment I. Pace Analytical Laboratories Biotreatability Report

Rev. Date: 10/12/2017 i CSI Environmental, LLC 1.0. INTRODUCTION

The objective of the biotreatability/bioremediation in-field pilot project was to evaluate potential remedial alternatives for remediation of isopropylbenzene (cumene) encountered in soil and groundwater at the former Higgins Plant operated by Hercules, Inc. in Gibbstown, New Jersey (the Site). Specifically, the biotreatability/bioremediation pilot project was intended to evaluate alternatives to pumping and treatment (P&T) as discussed with the United States Environmental Protection Agency (USEPA) following the failure of pumping well PW-6 in June 2009. Historically, groundwater P&T at the Site has been ongoing since the mid 1980’s as an interim remedial measure (IRM) to attain hydraulic containment of dissolved Site-specific constituents including cumene.

Field installation of the biotreatability pilot infrastructure was initiated on May 3, 2016 following regulatory approval of the associated Work Plan1. Injection and monitoring wells were installed in the anaerobic and aerobic test areas, developed and ultimately sampled in preparation for the field testing. Reagent injections consistent with the procedures outlined in the approved Work Plan1 were initiated on June 21, 2016. The field work associated with the pilot project was completed in March 2017.

Rev. Date: 10/12/2017 1 CSI Environmental, LLC 2.0. METHODS-DESCRIPTION OF PROJECT EXECUTION

An injection well and five sampling wells concentrically located around each injection well were drilled at both an aerobic area (formerly designated as CPT-44) and a second location designated as an anaerobic area (formerly designated as CPT-28). Both test locations are located in the northern portion of the former Active Process Area (APA) as shown on Figure 1. The anaerobic and aerobic test areas were located along the western and eastern sections of the former APA, respectively, to eliminate potential for interference during testing.

For the aerobic area, oxygen was used as the terminal electron acceptor (TEA). Oxygen releasing compounds (ORC) and Regenox Part A (RegenOx) were injected as the TEA. RegenOx was injected as a solution and intended to provide oxygen to the subsurface quickly while ORC was injected as slurry and intended to provide a slow release oxygen source. For the anaerobic area, magnesium sulfate (Epsom Salt) was used as a substrate TEA to support microbial growth and cumene remediation in an anaerobic environment.

Prior to any injections, all wells were, developed and surveyed while select wells in each treatability area were slug tested to characterize hydraulic properties and ensure proper dosing. Injections of ORC, RegenOx, and magnesium sulfate began on June 21, 2016.

While the RegenOx mixture generally was injected without restriction, CSI encountered some fouling of the injection well during ORC slurry injections. Prior to injecting all ORC and RegenOx, the aerobic injection well became completely fouled and would not accept any more injection fluids. Completion of aerobic fluid injection was achieved via a one (1) day Geoprobe injection event on September 28, 2016. During this single day injection event, the remaining ORC and RegenOx was injected at five (5) injection points between the fouled injection well and the monitoring wells.

Similarly, the fine-grained formation in the anaerobic test area restricted injection rates of magnesium sulfate. Subsequent anaerobic injections occurred through November 2016. The total dose of ORC (960 lbs), RegenOx (480 lbs) and magnesium sulfate (4,900 lbs) was injected over a 6-month period in accordance with the approved work plan (CSI, 2016).

Rev. Date: 10/12/2017 2 CSI Environmental, LLC 3.0 RESULTS 3.1. Cumene in Soil

Initial concentrations of cumene in soil taken from six soil borings in the aerobic area in May, 2016 ranged from a few hundred milligrams per kilogram (mg/kg or ppm) to over 8,000 mg/kg, had a mean value of 6,060 mg/kg, and a standard deviation of 2,595 mg/kg (Figure 2 and Table 1). In contrast, the mean cumene concentrations taken in initial core samples from the anaerobic area in May 2016 were higher than those from the aerobic area on average and ranged from less than 5,000 mg/kg to in excess of 38,000 mg/kg (Figure 3). These variabilities in initial cumene concentrations between the aerobic and anaerobic areas are attributed to a combination of historical conditions and soil variability as documented in other letter reports (CSI, 2012 & CSI, 2016).

Soil sample cores obtained in October 2016 revealed that the mean cumene concentrations in soil samples from both the aerobic and anaerobic areas had decreased significantly (see Figures 2 and 3 plus Tables 1 and 2). The mean cumene concentration among soil cores sampled from the aerobic area decreased from an initial average of 6,060 mg/kg to 598 mg/kg (Figure 2). Similarly, the mean concentration of cumene samples collected from the anaerobic area in October decreased significantly from the mean of 11,907 mg/kg in May 2016 to a mean value of 1,210 mg/kg in October 2016 (Figure 3).

However, cumene concentrations in soil did not decrease significantly from October 2016 to March 2017 at either the aerobic or aerobic treatability area (Figures 2 and 3, and Tables 1 and 2). Specifically, the mean cumene concentration at the aerobic area in October 2016 was not significantly different from the concentration measured in March 2017 (i.e. 598 vs. 1,590 mg/kg, respectively). Similarly, the cumene concentration measured at the anaerobic area in October 2016 was not significantly different from the concentration measured in March 2017 (i.e. 1,210 vs. 1,971 mg/kg, respectively). Possible explanations for lack of significant soil concentration reductions from October 2016 to March 2017 include:

1) Initial threshold reached based on finite mass of reagents injected during short- term pilot test; 2) The dynamic equilibrium in subsurface is not fully understood and further soil reductions won’t be attained until R-NAPL mass declines. It’s even possible (as cited later in this report), that some reductions in R-NAPL were achieved during pilot testing that transferred cumene mass from R-NAPL to adsorbed phase while still effectively reducing overall cumene mass; 3) Other limiting factors such as nutrient limitations inhibited further cumene reductions in soil below initial reduced concentrations. However, no direct evidence of nutrient deficiencies were measured during the pilot study, though further analysis may be warranted; and, 4) Sample variability and overall in-situ heterogeneity may have contributed to observed results, though not likely to be solely responsible for lack of significant cumene reductions after October 2016.

Rev. Date: 10/12/2017 3 CSI Environmental, LLC However, despite the apparent threshold in cumene reductions noted for soil as of October 2016, the statistically similar post-injection data sets indicate a sustained drop in cumene mass compared to the pre-injection data.

3.2. Cumene Concentration in Groundwater

Cumene concentrations in groundwater sampled from all wells at the aerobic area decreased from the initial sampling in May 2016 through July 2016 (Figure 4). With the exception of a spike in concentrations in the last week of July, concentrations continued to decrease through October 2016. From October 2016 through May 2017, no further significant reductions in dissolved-phase cumene were observed in the aerobic area. Thus, much like with cumene concentrations in soils, it appears that some threshold was reached with cumene concentrations in groundwater. The same potential explanations provided previously for soils likely applies to observed groundwater trends as well. Cumene concentrations in groundwater at the anaerobic area decreased in wells ANMW- 3, ANMW-4, and ANMW-5 from the initiation of the project in May 2016 through October 2016 (Figure 5). Concentrations of cumene in ANMW-1 and ANMW-2, however, were observed to spike in concentration in August and October 2016. Similar to the aerobic area, cumene concentrations in groundwater did not decrease significantly from October 2016 to May 2017, likely for the reasons previously cited. Further testing is warranted to assess if this is a temporary plateau and/or to understand more fully how to achieve further reductions in cumene concentrations and mass more efficiently via microbial degradation. 3.3. Chloride Concentration in Groundwater

Sodium chloride (NaCl) solution was injected at both the aerobic and anaerobic areas as a means for tracing the nutrient injections. At the aerobic area, chloride concentrations were observed to increase in all monitoring wells within a month following initial injections (Figure 6). The intention of the NaCl tracer test was to evaluate the distribution of injected material for the injection wells and the five observation wells concentrically surrounding the injection well.

The concentration of NaCl tracer was observed to be significantly higher at aerobic sampling well AEMW-3 relative to the other four sampling wells (Figure 6). It is likely that some channelization and subsequent preferential pathway may have existed between the injection well and AEMW-3.

A clear response to NaCl tracer solution injection at the anaerobic area was also detected in all monitoring wells within a month following the initial injection at the centrally located injection well (Figure 7). Initial detections of NaCl were present at significantly higher concentrations in the anaerobic monitoring wells when compared to the aerobic area. The NaCl tracer solution results were more uniform in the anaerobic area suggesting that more uniform hydraulic communication may exist in the saturated zone immediately surrounding the anaerobic injection well, particularly in comparison to the aerobic area. The concentrations of NaCl tracer decreased to approximately pre-injection concentrations, following the observed peak, by the conclusion of the pilot test period in May, 2017.

Rev. Date: 10/12/2017 4 CSI Environmental, LLC 3.4. Dissolved Oxygen (DO) and Sulfate (SO4) as TEA Compounds for Microbial Growth and Metabolism 3.4.1. DO Concentrations – Aerobic and Anaerobic Areas

The statistical mean for DO concentrations in groundwater among wells at the aerobic area was significantly higher than for groundwater from wells at the anaerobic area (Figure 8). The average DO concentration among groundwater samples from wells at both the aerobic and anaerobic areas was nearly the same at the initiation of the pilot study (3.9 and 4.0 mg/L, respectively). As the pilot study proceeded and ORC oxygenating reagent was continually added to the injection well at the aerobic area, DO concentrations in groundwater at the aerobic area slightly increased or maintained levels similar to the initial DO concentrations. In contrast, post injection mean DO concentrations in groundwater at the anaerobic area remained significantly lower than compared to DO levels measured at the initiation of the pilot test, as anticipated (Figure 9).

3.4.2. SO4 as a TEA at the Anaerobic Area

Sulfate (SO4), introduced as magnesium sulfate or Epsom Salt, was used as the principal TEA for the anaerobic area. Sulfate was injected to bio-stimulate anaerobic microbial activity in the anaerobic test area. All wells showed a strong response after the injection of SO4 (Figure 10). Sulfide (SO3) is a byproduct of anaerobic degradation of SO4 and was also monitored to evaluate for evidence of increased anaerobic activity and is discussed further below.

SO4 concentrations in soil at the anaerobic area increased from an ambient mean concentration of 9.8 mg/kg by over an order of magnitude as SO4 solution was added during the pilot project (Figure 11). The statistical mean for SO4 concentrations among all soil core samples taken at the anaerobic area peaked at 515 mg/kg in October 2016, and decreased following SO4 injections to a statistical mean of 322 mg/kg in March 2017 (Table 3).

The increase in both SO4 and SO3 concentrations as measured in October 2016 and finally in March 2017 was significantly different from the ambient concentrations measured in May 2016 (Figure 11 and Table 3). Following initial and subsequent SO4 solution injections, SO4 concentrations remained at concentrations typically associated with acting as a TEA for microbial hydrocarbon remediation in soil (i.e. 100 – 250 mg/kg) (Kolhatkar & Taggart, 2004). Thus, the results observed indicate that sulfate was successfully introduced into the saturated zone surrounding the anaerobic injection well. Furthermore, review of the results suggests that the SO4 injections ultimately were successful in providing a TEA to the subsurface to enhance microbial activity as discussed in later sections of this report.

Sulfide concentrations from all soil core samples obtained at the anaerobic area also increased significantly following initial and subsequent injections of SO4 solution (Figure 11 and Table 3). The increase in the mean concentration of SO3 among all soil cores sampled may be indicative of stimulated microbial activity. Assuming that adequate concentrations of SO4 existed during the project, the reduction of SO4 via microbial

Rev. Date: 10/12/2017 5 CSI Environmental, LLC activity may directly correlate to the observed increase in SO3 concentrations, as anticipated.

3.5. Microbial Growth 3.5.1. Aerobic Area

Microbial growth was present in groundwater samples from most of the monitoring wells surrounding the aerobic area as indicated by using Hach® “Paddle Tester” kits. Wells monitored on July 21, 2016 had colonies averaging 1x103 to 1x104 colonies (Figure 12). Samples from aerobic monitoring wells collected on July 26, 2016 had a range of microbial colonies of 1x103 to 1x107 (Figure 13). 3.5.2. Anaerobic Area

A test kit designed for detecting sulfate reducing bacteria (SRB) designated as “BART” was used to test for the presence of SRB. The test kit, manufactured by Droycon Bioconcepts, Inc., consists of a solution contained in a vial that has an SRB incubation time of approximately 10 days. The “BART” test kit provided an indication of SRB microbial presence in composite samples collected from groundwater at the anaerobic test area through July 2016. In mid-July, review of the “BART” tests results indicated the presence of both aerobic and anaerobic SRB microbes (Figure 14). However, follow up samples collected on July 26, 2016 revealed that only anaerobic SRB microbial growth was present (Figures 15). This was a positive development indicating that injections in the anaerobic test area were favoring anaerobic microbial conditions as planned.

3.5.3. Total Bacteria Counts

In addition to using bacteria test kits, soil and groundwater samples were also collected several times during the project and sent to Bioremedial Technologies Inc laboratory in Hermitage PA. Once at the lab, the samples were cultured on an agar plate. Additional plates were also made and dosed with either cumene or benzene to determine if the bacteria present could use those compounds as a carbon source.

After 7 days, the total colony forming units (CFU) of bacteria were counted. The CFU data for soil and groundwater collected as part of this pilot study is provided on Tables 4 and 5, respectively. Groundwater samples were not collected during the initial sampling period, but were obtained post injection. It is important to note the several uncertainties associated with culturing bacteria:

1. Not all bacteria present in the subsurface are colony forming or cultivable on an agar plate. 2. While steps were taken to ensure air was not mixed with samples, it cannot be stated with certainty that anaerobic samples were not slightly aerated during the sampling process. 3. The soil flushing technique works best on free-floating bacteria within the soil column, however those bacteria adsorbed onto soil grains are often missed in the sample.

Rev. Date: 10/12/2017 6 CSI Environmental, LLC With those uncertainties taken into account, the data can still be assessed qualitatively. There were no detections of CFUs in the pretest soils samples. Subsequently, in the post injection sampling round, only one sample, collected at the water table, from the aerobic area had a detection of total heterotrophs. In the final round of soil samples, total heterotrophs were detected in two samples in the aerobic area and one sample from the anaerobic area.

In groundwater (Table 5) CFUs were significantly higher in the final sample than in the post injection sample in both the aerobic and anaerobic pilot study areas. These data suggest that the injection of TEA, both aerobic and anaerobic, have led to an increase in the microbial biomass in the pilot study areas.

3.5.4. Comparison of Aerobic and Anaerobic Microbial Activity

Review of groundwater sampling results from April 26, 2017 at the termination of the pilot project confirmed that microbial activity was occurring at the aerobic and anaerobic areas. Samples from Wells AEMW-3 and ANMW-1 were analyzed by Pace Analytical, Inc., (Pace) using a polymerase chain reaction (qPCR) assay technique.

The report from Pace Analytical (Attachment I) states that “microbial biomass results were on the high end for groundwater and are indicative of a large amount of primary substrate (bacterial food) to support the observed bacterial population” in both the aerobic and anaerobic area. Furthermore, the report states that samples from the anaerobic area “ANMW-1 had the highest measured biomass and could be indicative of the response of the SO4 treatment over both the background and the ORC alternative treatment”.

3.5.5. Comparison of Aerobic and Anaerobic Microbial Community Composition

As described in the Pace report (Attachment I), species delineation among bacterial populations occurring in the groundwater from aerobic well AEMW-3 and anaerobic well ANMW-1 indicated significant populations of hydrocarbon degrading bacteria. Betaproteobacteria and Deltaproteobacteria are classes of bacteria noted for being well documented anaerobic hydrocarbon degraders (Attachment I).

Pace described the microbial community at well AEMW-3 as being composed of 45% Betaproteobacteria and Deltaproteobacteria while those two-species made up ~60% of the microbial population measured in groundwater at well ANMW-1. Looking at the results on a genus level provided further insight into the diverse bacteria communities in each of the pilot study areas.

Bacteria capable of aerobic and anaerobic degradation of hydrocarbons were found in both areas. Specifically, Acidovorax and Pseudomonos, bacteria noted for aerobic degradation of hydrocarbons and Geobacter and Clostridium, noted for anaerobic degradation of hydrocarbons, were identified in groundwater from well AEMW-3. Similarly, Acidovorax noted for aerobic hydrocarbon degradation and Geobacter and Desulfitobacterium, noted for anaerobic hydrocarbon degradation were found in groundwater collected from well ANMW-1. Collectively, the microbial community results reported by Pace are promising indicators regarding enhanced bacterial

Rev. Date: 10/12/2017 7 CSI Environmental, LLC colonization in both the anaerobic and aerobic test areas with the greatest biomass measured in the anaerobic test area.

3.6. Biological Oxygen Demand (BOD) Concentrations

BOD concentrations among all soil cores collected at the aerobic area remained constant throughout the pilot project at ~7 mg/kg (Figure 16). There were no statistical differences in mean BOD concentrations from soil samples collected in May and October 2016 and in March 2017 (Table 6). These results are indicative of a healthy aerobic microbial community.

In contrast, BOD concentrations measured in soil cores from the anaerobic area decreased significantly after the initiation of the project (Figure 17). The mean BOD concentration of 16.5 mg/kg at the initiation of the project was significantly higher than means of 6.28 and 4.00 measured in October 2016 and March 2017, respectively (Table 7). It is assumed that a decrease in BOD concentration was a response to a decrease in available oxygen (e.g. Figure 9), and an increase in the mass of anaerobic bacteria, and thus, likely indicative of a strong anaerobic microbial community.

3.7. R-NAPL Considerations

Cumene at the Site exists in multiple phases and media (e.g. vapor phase, dissolved phase and adsorbed phase). However, the most significant cumene mass has been observed in emulsified droplets of residual NAPL (R-NAPL). The R-NAPL principally occurs within saturated soil pore space or as dispersed R-NAPL droplets entrained within the subsurface environment. Discrete zones or lenses of NAPL at or above the water table have not been detected at the Site though isolated pockets of NAPL or NAPL ganglia may exist in areas associated with historic operations or prior releases from tanks in the former Tank Farm.

Within the subsurface/vadose environment, air filled soil pore space is displaced by any dispersed NAPL droplets. In addition, at or near the interface of the vadose zone and the water table, NAPL droplets is expected to displace water in soil pore space if capillary displacement pore pressure is exceeded. The appreciable mass of cumene in subsurface soil and groundwater (and as dispersed R-NAPL droplets entrained in saturated soil) is significant and likely approaches or exceeds 1 million pounds in the former Site process areas. Efforts to quantify R-NAPL and cumene mass at the Site are beyond the scope of this report but will be addressed within the Remedial Investigation (RI) Report.

As part of a previous study in the area, pore space tests were performed to determine the amount of R-NAPL present initially at each treatability area. In samples collected from the two injection point locations, it was determined that R-NAPL was present at 9.9% in the aerobic area and 12.4% in the anaerobic area. Due to infrastructure associated with the treatability test, these two injection locations could not be accessed for future sampling. However pre-injection samples were collected from AESO-2 (11.1% R-NAPL) and ANSO-2 (7.5 % R-NAPL). Post-injection samples were also collected and analyzed from AESO-2 (6.8% R-NAPL) and ANSO-2 (8.4 % R-NAPL). An additional post- injection sample was also collected from AESO-3 (5.6% R-NAPL) and ANSO-5 (6.7 % R-NAPL). In summary, it appears that some reduction in pore space R-NAPL was measured in post injection samples compared to pre-injection samples, which is favorable Rev. Date: 10/12/2017 8 CSI Environmental, LLC and promising (Table 8). However, further testing would be needed to confirm statistically that pore space R-NAPL was reduced as a result of bio pilot testing.

4.0. DISCUSSION AND SUMMARY

4.1. Cumene Remediation

Subsurface in situ bio-stimulation as a remedial alternative consisting of naturally degrading hydrocarbon contaminants in both groundwater and soils has been successfully applied at contaminated sites for many years and is well documented (Kolhatkar & Taggart, 2004). Bio-stimulation of hydrocarbon degradation has been demonstrated using enhanced levels of oxygen as the TEA in aerobic conditions or using a series of other TEAs in anaerobic soil conditions. As oxygen is used preferentially by soil microbes as an electron acceptor, most hydrocarbon contaminated soils become anaerobic before remediation is completed, provided sufficient hydrocarbon mass is present. Therefore, extensive research has been conducted on TEAs as agents for anaerobic remediation of hydrocarbon contaminated soils (Kolhatkar & Taggart, 2004).

The most effective electron acceptors that have been demonstrated in soil remediation studies are nitrate, ferric iron, manganese, and sulfate compounds. Of these electron acceptors, sulfate reduction has been shown to account for most of the hydrocarbon degradation in contaminated anaerobic groundwater and soils and its use has become more widespread. Anaerobic sulfate reduction to stimulate microbial degradation of hydrocarbon constituents is designated as sulfate enhanced natural attenuation (SENA) (Kolhatkar & Wilson, 2000 & Wiedemeier, Rafai,, Newell, and Wilson, 1999).

During the ten-month pilot project, the greatest reduction in cumene concentrations were observed during the period of sulfate enhancement at the anaerobic area (Figures 2 and 3). Significant reductions in cumene concentrations in subsurface soils were demonstrated at both the aerobic area using enhanced oxygen level augmentation and the anaerobic area using enhanced sulfate levels as a TEA (Figures 2 and 3). However, review of results from the ten-month bio-pilot test suggests that providing enhanced sulfate levels to the saturated soil stimulated greater cumene reduction relative to aerobic conditions. This finding holds true despite significant variations in the initial cumene concentrations in the anaerobic and aerobic test locations. Specifically, the mean cumene concentration among all wells at the aerobic area decreased by 5,462 mg/kg relative to a mean reduction of 10,697 mg/kg among wells at the anaerobic area. As noted previously, this result is similar to previous results indicating enhanced hydrocarbon degradation using sulfate augmentation in contaminated soils at other similar sites (Kolhatkar & Wilson, 2000).

The results of this pilot project indicate that biostimulation of microbial activity using either oxygen supplementing reagents in an aerobic environment or other TEA reagents in an anaerobic environment will enhance cumene degradation. This observation is supported by not only the measured decrease observed in cumene concentrations in ambient soil, but also by the increased microbial activity among species of bacteria noted to be hydrocarbon degraders (Attachment I) at both aerobic and anaerobic areas. Additional supporting observations of TEA reagents aiding in microbial degradation of cumene in the anaerobic area include: Rev. Date: 10/12/2017 9 CSI Environmental, LLC

1) cumene degradation increased at the anaerobic area even after DO concentrations decreased significantly, suggesting that oxygen was not the primary TEA, 2) as SO4 concentrations increased due to SO4 supplementation at the anaerobic area, significant increases in SO3 concentrations also occurred suggesting increased activity in anaerobic microbes; and, 3) BOD concentrations remained constant in soil at the aerobic area while BOD concentrations at the anaerobic area decreased, a positive development indicating the enhanced growth of anaerobic microbes due to TEA supplementation.

Although microbial activity and speciation associated with hydrocarbon degradation were detected in groundwater samples from both the aerobic and anaerobic areas (Pace Appendix I), decreases in cumene concentrations in groundwater were orders of magnitude less than those detected in soils (Figures 2, 3, 4, and 5). In addition, cumene concentrations noted for monitoring wells ANMW-1 and ANMW-2 at the anaerobic area increased during the study period. These observations concerning cumene concentrations in groundwater may have resulted from injection activities having transported groundwater through the test areas and mobilization of cumene from ambient soils and possibly from R-NAPL into the surrounding groundwater during the project period.

4.2. Verifying the Distribution of TEA Reagents

Based on the distribution of the NaCl tracer injected in both the aerobic and anaerobic injection wells, the distribution of reagents injected at the aerobic area may have been impeded due to channelization and flow restrictions (see Figures 6 and 7). For example, even with the addition of RegenOx and ORC, DO concentrations at the aerobic area did not significantly increase above initial concentrations until after the one day Geoprobe® injection event (Figure 9). However, it is likely that there were adequate DO concentrations for microbial growth and metabolism to initiate cumene remediation at the aerobic area during the pilot test. This contention is substantiated by the following findings:

1) DO concentrations at the aerobic area were considered sufficient to stimulate and support aerobic microbial activity; 2) elevated microbial activity and speciation among microbes considered sufficient to degrade hydrocarbons were observed at the aerobic area (Attachment I); and, 3) significant decreases in cumene concentrations in both groundwater and soil occurred at the aerobic area.

For the anaerobic area, significant spikes in chloride concentrations measured at all wells indicated that sulfate augmentation was well distributed across the test area (Figure 7). This conclusion is substantiated by spikes in the concentration of sulfate for all core soil samples following sulfate injections via the injection well. Additionally, anaerobic microbial populations were significantly higher at the anaerobic study area when compared to background and the aerobic study areas (Attachment I). Furthermore, DO concentrations decreased throughout the duration of the pilot program, as anticipated, consistent with the effects of a stimulated anaerobic microbial community. These results suggest the adequate availability of sulfate as a growth stimulant. Commensurate increases in sulfide levels concurrent to increased anaerobic microbial activity provides

Rev. Date: 10/12/2017 10 CSI Environmental, LLC further indication of enhanced anaerobic microbial activity. Available evidence from the pilot test suggests that microbial activity was using cumene as a carbon source as described within other sections of this report.

4.3. Verifying Sufficient Nutrient Availability for Microbial Growth

The availability of sufficient nitrogen for microbial growth was verified during this study, in addition to confirming the relatively uniform distribution of reagents as previously discussed. During the pilot project, the average total Kjeldahl nitrogen (TKN) concentration (i.e. biologically available nitrogen) among soil cores collected at the anaerobic site was 101.7 mg/kg (Figure 18). Mean TKN concentrations among soil cores taken at the aerobic site averaged 29.7 mg/kg. Although the average TKN concentration among cores collected at the aerobic site was approximately three times lower than the average for cores at the anaerobic site, TKN concentrations for both aerobic and anaerobic test locations exceeded the minimum concentrations considered to be limiting to microbial growth (Alden, Demoling, &Baath, 2001).

4.4. Cumene Degradation Rate

Natural microbial degradation of hydrocarbon contamination in general occurs through a variety of physical, chemical, and biological processes. Such processes include volatilization, dissolution, biodegradation, and sorption/desorption. Similar processes appear to be degrading hydrocarbons that are present as dispersed R-NAPL droplets entrained in subsurface saturated soil at the Site. However, there is uncertainty regarding the rate at which dispersed R-NAPL in the subsurface will degrade through stimulated microbial activity.

More extensive and longer duration in-situ bio-degradation testing is warranted to assess more fully the efficacy of aerobic and anaerobic microbial bio-stimulation and to account for the increased cumene mass present as dispersed R-NAPL. Thus, the significant cumene mass documented for the Site will likely require more extensive testing and application of anaerobic TEAs to verify that substantial mass reductions are attainable. However, the testing documented in this report provides a sound basis for refining procedures to document how best to achieve meaningful cumene mass reductions efficiently.

A decline in the rate of cumene degradation observed in the present biotreatability project (Figures 2 and 3) may be a result of chemical partitioning between R-NAPL droplets and both dissolved-phase and adsorbed phase cumene. Microbial degradation of hydrocarbon contaminants in both aerobic and anaerobic environments is well documented (Kolhatkar & Taggart, 2004). These studies have monitored subsurface hydrocarbon contamination that exist primarily in the aqueous and vapor phases.

Much less is known about microbial degradation of extensive, dispersed R-NAPL entrained in saturated interstitial soil pores. Although microbial degradation of R-NAPL has not been thoroughly documented, it is generally assumed that degradation of R- NAPL is limited by partitioning coefficients from pore space to the aqueous phase (Stout, & Lundegard. 1998) and, possibly, the adsorbed phase as well. This uncertainty is

Rev. Date: 10/12/2017 11 CSI Environmental, LLC significant given that appreciable cumene mass in the subsurface at the Site is associated with the R-NAPL.

The apparent threshold in further cumene reductions (R-NAPL, adsorbed, and dissolved) after the fifth month of testing (Figures 2 and 3) may be influenced by multiple factors including changes in cumene partitioning from R-NAPL droplets to adsorbed and dissolved phase. As previously noted in this report, the R-NAPL pore space mass may have declined slightly as a result of the pilot testing (Table 8) and could reflect accelerated partitioning of R-NAPL droplets. This potentially makes cumene available to partition to both the adsorbed and dissolved phase as the R-NAPL dissipates and breaks down.

Further extended testing is required to evaluate attainment of greater mass reductions and, specifically, optimal biodegradation conditions to maintain or increase the cumene reduction rates in R-NAPL and the adsorbed and dissolved-phase states. Extending biotreatability pilot testing also will help to determine or more closely estimate specific cumene half-life values necessary to make more accurate projections regarding cumene biodegradation and mass reduction rates at the Site.

Rev. Date: 10/12/2017 12 CSI Environmental, LLC

5.0. REFERENCES

CSI Environmental, LLC. (2012). Preliminary Focused Treatability Test Results Report. Former Hercules Higgins Plant, Gibbstown, New Jersey. Millersville, Maryland: Author.

CSI Environmental, LLC. (2016). Biotreatability/ Bioremediation Work Plan. Former Hercules Higgins Plant, Gibbstown, New Jersey. Millersville, Maryland: Author.

CSI Environmental, LLC. (2016). Site-Specific Cumene Solubility and Chemical Saturation Values (Csat). Former Hercules Higgins Plant, Gibbstown, New Jersey. Millersville, Maryland: Author.

Kolhatkar, R. and Taggart, D. (2004). Enhanced Bioremediation Using Sulfate and/or Nitrate. GEM Group Environmental Management, Remediation Management Technology Meeting, Warrenville, Il January 22, 2004.

Kolhatkar, R. and Wilson, J. (2000). Evaluating Natural Biodegradation of MTBE at Multiple UST Sites. NGWA/API Petroleum Hydrocarbons and Organic Chemicals in Groundwater, Houston, 15-17 November, pp. 32-49.

Wiedemeier, T., Rafai, H., Newell C., and Wilson J. (1999). Natural Attenuation of Fuels and Chlorinated Solvents in the Subsurface. John Wiley & Sons, Inc. New York.

Stout, S.A. and Lundegard, P.D. (1998). Intrinsic Biodegradation of Diesel Fuel in An Interval Of Separate Phase Hydrocarbons. Applied Geochemistry 13(7): 851- 859.

Alden, L., Demoling, P.D., and Baath E. (2001). Rapid Method of Determining Factors Limiting Bacterial Growth in Soil. Applied Environmental Microbiology 67(4): 1830- 1838.

FIGURES

\ 1,, II = MW-AN-4 AN-S0-4 . 0 ~ ..., t IW-AN-1 MW-AN-3 .!I[)'~ =- - -4-AN-S0-6 \ AN-S0-3

Legend Biotreatability Study Areas • Aerobic Biotreatability Study Area • Anaerobic Biotreatability Study Area - Geoprobe Soil Location -$- Injection Well -$- Monitoring Well /

Bioremediation Pilot Study Areas CSI Environmental, LLC Figure 401 Headquarter Drive. Suite 203 Former Higgins Plant, Gibbstown, NJ Millersville, MD 21108 Hercules Incorporated 443-688-6453 Gibbstown, New Jersey 1 Figure 2 ‐ Cumene Concentrations in Soil ‐ Aerobic Test Area

9000 8300 8100 8000 Mean: 6060 Standard Deviation (SD): 2595 7000 6700 6800 I I Mean:1590 SD:1919 5900 6000 5400

5000 (mg/kg)

4000 Cumene Mean:598 3000 SD: 427

2000

1100 1100 370 970 1000 830 560 470 380 99 220 0 I n -• Inn 5-3-16 10-26-16 3-28-17 Sampling Dates

• Core 1 • Core 2 • Core 3 • Core 4 • Core 5 • Core 6

Figure 3 ‐ Cumene Concentrations in Soil ‐ Anaerobic Test Area 45000

40000 39000

35000 Mean: 11907 Standard Deviation (SD): 15931

30000 29000

25000

20000 Cumene. (mg/kg) Cumene.

15000 Mean:1971 SD:2085

10000 Mean:1210 SD:995 5700 5000 3900 3400 2900 1600 2000 7401300 790 880 339.8 28037 530 24 0 3-28-17 5-3-16 10-26-16 Sample Dates • Core 1 • Core 2 • Core 3 • Core 4 • Core 5 • Core 6

Figure 4 ‐ Cumene Concentrations in Groundwater from Wells at the Aerobic Test Area 100

60 MW‐AE 1 50 MW‐AE 2 mg/L Cumene MW‐AE 3 40 MW‐AE 4 MW‐AE 5 30

Figure 5 ‐ Cumene Concentrations in Groundwater from Wells at the Anaerobic Test Area

1000

100 MW‐AN 1 MW‐AN 2

mg/L MW‐AN 3 Cumene MW‐AN 4 10 MW‐AN 5

Figure 6 ‐ Chloride Concentrations in Groundwater from Wells at the Aerobic Test Area 1000

100

10 MW‐AE‐1 Mg/L

Chloride MW‐AE‐2 MW‐AE‐3 MW‐AE‐4

1 MW‐AE‐5

0.1

Sample Date

Figure 7 ‐ Chloride Concentrations in Groundwater From Wells At The Anaerobic Site 10000

1000

100 MW‐AN‐1 mg/L

Chloride MW‐AN‐2 MW‐AN‐3 MW‐AN‐4 MW‐AN‐5 10

Sample Date

Figure 8 ‐ Comparison of Mean DO Concentrations in Groundwater from Wells at the Aerobic vs. Anaerobic Site 4

3.5

2.5

2 (mg/L)

1.5

0.5

0 Well 1Well 2 Well 3Well 4Well 5 WELL IDENTIFICATION

AEROBIC ANAEROBIC

Figure 9 - Average DO over Time in the Aerobic and Anaerobic Pilot Study Areas 25

15 DO (mg/L)

0 3/21/2016 5/10/2016 6/29/2016 8/18/2016 10/7/2016 11/26/2016 1/15/2017 3/6/2017 4/25/2017 6/14/2017

Aerobic DO Anaerobic DO

Figure 10. Sulfate (SO4) Concentrations in Groundwater Resulting From the Injection of MgSO4 During the Pilot Test 2500

2000

1500

MW‐AN 1 mg/L) (

4 MW‐AN 2 SO 1000 MW‐AN 3 MW‐AN 4 MW‐AN 5

500

SAMPLE DATES

Figure 11. Comparison Of SO4 And SO3 Concentrations In Soil During The Pilot Study Based On Statistical Means Among All Wells Sampled. 600

515 500

400

322 (mg/kg) 300 • SO3 242 • SO4 SO3/SO4

200

100 68 65

9.8 0 I. I 5/1/2016 6/1/2016 7/1/2016 8/1/2016 9/1/2016 10/1/2016 11/1/2016 12/1/2016 1/1/2017 2/1/2017 3/1/2017 Sample Dates

Figure 12 ‐ Microbial Growth in Well Water Sampled at the Aerobic Test Area on July 2016

7-~l-J<,

Figure 13 ‐ Microbial Growth in Well Water Sampled at the Aerobic Test Area in July 2016

,o.,fJ~-

Figure 14 ‐ Microbial Growth in Well Water Sampled at the Anaerobic Test Area in July 2016

Figure 15 ‐ Microbial Growth in Well Water Sampled at the Anaerobic Test Area in July 2016

flN - _; P/'l~Roo1c o "'Y 9 rt 4,....,,i c. CJtnl 'f ffrl- ~ 7-~f.- )(o R"'",to lfll, onr 7...-1-C.- I(.. ,7-,2."- '"

Figure 16 ‐ BOD Concentrations among Core Samples From the Aerobic Test Area

12 Mean: 7.23

10 Mean: 7.17 Mean: 7.16 ~ • Core 1 • Core 2 8 • Core 3

(Mg/Kg) Core 4 • Core 5

BOD 6 • lf • Core 6

0 5‐3‐16 10‐26‐16 3‐28‐17 Sampling Dates

Figure 17 ‐ BOD Concentrations among Core Samples From the Anaerobic Test Area

25 Mean: 16.5 Standard Deviation (SD): 9.0 20

15 (Mg/Kg)

BOD Mean: 6.28 Mean: 4.00 10 I SD: 1.26

0 1111 I ml 5/1/2016 6/1/2016 7/1/2016 8/1/2016 9/1/2016 10/1/2016 11/1/2016 12/1/2016 1/1/2017 2/1/2017 3/1/2017

5‐3‐16 10‐26‐16 3‐28‐17 Sampling Dates • Core 1 • Core 2 • Core 3 • Core 4 • Core 5 • Core 6

Figure 18 ‐ Comparison of Kjeldahl Nitrogen Concentrations in Soils from the Aerobic and Anaerobic Test Areas During the Pilot Test 140

120

100

80 (mg/kg) 2 N

60 Mean

0 I I I ~

Sampling Dates

• Aerobic • Anaerobic

Tables

Table 1 - Statistical Analyses Comparing Cumene Concentrations in Soil at the Aerobic Test Area During the Ten Month Pilot Study Former Hercules Higgins Plant; Gibbstown, NJ

CORE Sample Date # 5/3/2016 10/26/2017 3/28/2017 1 560 1100 830 2 6700 470 380 3 8300 1100 370 4 8100 99 5400 5 5900 220 570 6 6800 NA NA

Mean 6060 598 1590 SD 2595 427 1919 Statistically Statistically Significant as significant as compared to 5‐3‐16 data compared to 5‐3‐16 @p<0.05 data @p<0.05 Not statistically significant as compared to 10‐26‐17 data @p<0.05 Notes: All cumene concentrations are presented in milligrams per kilogram (mg/kg) NA = Not Applicable

Rev Date: 09/20/2017 CSI Environmental, LLC Print Date: 10/12/2017 Page 1 of 1 www.contactcsi.com Table 2 - Statistical Analyses Comparing Cumene Concentrations in Soil at the Anaerobic Test Area During the Ten Month Pilot Test Former Hercules Higgins Plant; Gibbstown, NJ

CORE Sample Date # 5‐3‐16 10‐26‐17 3‐28‐17 1 3400 2900 530 2 39000 2000 5700 3 33 280 790 4 9.8 37 24 5 1600 740 880 6 29000 1300 3900

Mean 11907 1210 1971 SD 15931 995 2085 Not statistically Not statistically significant compared significant compared to 5‐ to 5‐3‐16 data 3‐16 data @p<0.05 @p<0.05 Not statistically significant compared to 10‐26‐17 data @p<0.05

Notes: All units are in milligrams per kilogram (mg/kg)

Rev Date: 09/20/2017 CSI Environmental, LLC Print Date: 10/12/2017 Page 1 of 1 www.contactcsi.com Table 3 - Comparison of Statistical Means for SO4 and SO3 Concentrations in Soil During the Pilot Test Former Hercules Higgins Plant; Gibbstown, NJ

CORE Sample Date # 5/3/2016 10/26/2017 3/28/2017

SO4 SO3 SO4 SO3 SO4 SO3 1 9.5 54 34 660 610 59 2 9.6 66 180 71 280 68 3 9.4 58 1300 68 90 63 4 9.5 67 940 340 570 70 5 11 96 120 70 60 66 6 Mean 9.8 68 515 242 322 65 SD 0.6 15 509 234 232 4

SO4 & SO3 Statistically SO4 Statistically significant compared significant compared to 5‐3‐16 data to 5‐3‐16 data @p<0.05 @p<0.05 SO4 Not statistically significant compared to 10‐26‐17 data @p<0.05 SO3 Not statistically significant compared to 5‐3‐16 data @p<0.05 SO3 statistically significant Notes: d t 10 26 17 All units are in milligrams per liter (mg/L) SD = Standard Deviation

Rev Date: 09/20/2017 CSI Environmental, LLC Print Date: 10/12/2017 Page 1 of 1 www.contactcsi.com Table 4 ‐ BRT Agar Plate Count Results ‐ Soil Former Hercules Higgins Plant; Gibbstown, NJ Total Cumene Benzene Heterotrophs Degraders Degraders Sample ID Date CFU/ml CFU/ml CFU/ml Aerobic Study Area AE‐SO‐1 (17) 5/3/2016 <2.0E+02 <2.0E+02 <2.0E+02 AE‐SO‐2 (17) 5/3/2016 <2.0E+02 <2.0E+02 <2.0E+02 AE‐SO‐3 (17) 5/3/2016 <2.0E+02 <2.0E+02 <2.0E+02 AE‐SO‐4 (17) 5/3/2016 <2.0E+02 <2.0E+02 <2.0E+02 AE‐SO‐5(17) 5/3/2016 <2.0E+02 <2.0E+02 <2.0E+02 AE‐SO‐6 (17) 5/3/2016 <2.0E+02 <2.0E+02 <2.0E+02 AE‐SO‐1 (17.5) 10/26/2016 <2.0E+02 <2.0E+02 <2.0E+02 AE‐SO‐2 (17.5) 10/26/2016 <2.0E+02 <2.0E+02 <2.0E+02 AE‐SO‐3 (17.5) 10/26/2016 <2.0E+02 <2.0E+02 <2.0E+02 AE‐SO‐4 (17.5) 10/26/2016 <2.0E+02 <2.0E+02 <2.0E+02 AE‐SO‐5(17.5) 10/26/2016 <2.0E+02 <2.0E+02 <2.0E+02 AE‐SO‐6 (9) 10/26/2016 4.5E+04 <2.0E+02 <2.0E+02 AE‐SO‐1 (17) 3/28/2017 3.2E+02 <2.0E+02 <2.0E+02 AE‐SO‐2 (17) 3/28/2017 <2.0E+02 <2.0E+02 <2.0E+02 AE‐SO‐3 (17) 3/28/2017 <2.0E+03 <2.0E+02 <2.0E+02 AE‐SO‐4 (17) 3/28/2017 <2.0E+04 <2.0E+02 <2.0E+02 AE‐SO‐5(17) 3/28/2017 2.0E+04 2.0E+03 1.0E+02 Anaerobic Study Area AN‐SO‐1 (17.5) 5/3/2016 <2.0E+02 <2.0E+02 NS AN‐SO‐2 (17.5) 5/3/2016 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐3 (17.5) 5/3/2016 <2.0E+02 <2.0E+02 NS AN‐SO‐4 (17.5) 5/3/2016 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐5 (18) 5/3/2016 <2.0E+02 <2.0E+02 NS AN‐SO‐6 (17.5) 5/3/2016 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐1 (17) 10/26/2016 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐2 (9) 10/26/2016 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐2 (14) 10/26/2016 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐2 (17) 10/26/2016 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐3 (17) 10/26/2016 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐4 (17) 10/26/2016 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐5 (17) 10/26/2016 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐6 (17) 10/26/2016 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐1 (17.5) 3/28/2017 2.9E+03 <2.0E+02 <2.0E+02 AN‐SO‐2 (17.5) 3/28/2017 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐3 (17.5) 3/28/2017 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐4 (17.5) 3/28/2017 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐5 (17.5) 3/28/2017 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐6 (17.5) 3/28/2017 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐6 (17.5) 3/28/2017 <2.0E+02 <2.0E+02 <2.0E+02 AN‐SO‐6 (17.5) 3/28/2017 <2.0E+02 <2.0E+02 <2.0E+02

Rev Date: 10/05/2017 CSI Environmental, LLC Print Date: 10/12/2017 Page 1 of 1 www.contactcsi.com Table 5 ‐ BRT Agar Plate Count Results ‐ Groundwater Former Hercules Higgins Plant; Gibbstown, NJ

Total Cumene Benzene Heterotrophs Degraders Degraders Sample ID Date CFU/ml CFU/ml CFU/ml Aerobic Study Area AE‐MW‐1 10/27/2016 8.1E+02 <2.0E+02 <2.0E+02 AE‐MW‐2 10/27/2016 <2.0E+02 <2.0E+02 <2.0E+02 AE‐MW‐3 10/27/2016 1.0E+04 1.0E+03 2.0E+03 AE‐MW‐4 10/27/2016 <2.0E+02 <2.0E+02 <2.0E+02 AE‐MW‐5 10/27/2016 <2.0E+02 <2.0E+02 <2.0E+02 AE‐MW‐2 3/12/2017 5.0E+04 7.6E+02 4.1E+02 AE‐MW‐3 3/12/2017 3.7E+02 <2.0E+02 <2.0E+02 AE‐MW‐4 3/12/2017 4.3E+04 8.8E+03 6.3E+03 AE‐MW‐5 3/12/2017 5.6E+05 2.0E+04 4.2E+04 Anaerobic Study Area AN‐MW‐1 10/27/2016 9.5E+02 <2.0E+02 <2.0E+02 AN‐MW‐2 10/27/2016 <2.0E+02 <2.0E+02 <2.0E+02 AN‐MW‐3 10/27/2016 <2.0E+02 <2.0E+02 <2.0E+02 AN‐MW‐4 10/27/2016 <2.0E+02 <2.0E+02 <2.0E+02 AN‐MW‐5 10/27/2016 8.4E+02 <2.0E+02 <2.0E+02 AN‐MW‐1 3/12/2017 3.8E+04 <2.0E+02 <2.0E+02 AN‐MW‐2 3/12/2017 2.5E+04 2.7E+03 3.8E+03 AN‐MW‐5 3/12/2017 6.7E+04 <2.0E+02 3.2E+03

Rev Date: 10/05/2017 CSI Environmental, LLC Print Date: 10/12/2017 Page 1 of 1 www.contactcsi.com Table 6 - Comparison of Statistical Means for BOD Concentrations in Soil at the Aerobic Test Area During the Pilot Test Former Hercules Higgins Plant; Gibbstown, NJ

CORE Sample Date # 5‐3‐16 10‐26‐17 3‐28‐17 1 9.4 7.2 4.3 2 4.2 5.2 4.5 3 8.6 6.3 4.7 4 7.2 9.1 9.5 52 8 8 612 12

Mean 7.23 7.16 7.17 SD 3.31 1.35 2.91 Not statistically Not statistically significant compared significant compared to 5‐ to 5‐3‐16 data 3‐16 data @p<0.05

Not statistically significant compared to 10‐26‐17 data @p<0.05 Notes: All units are in milligrams per kilogram (mg/kg)

Rev Date: 09/20/2017 CSI Environmental, LLC Print Date: 10/12/2017 Page 1 of 1 www.contactcsi.com Table 7 - Comparison of Statistical Means for BOD Concentrations in Soil at the Anaerobic Test Area During the Pilot Test Former Hercules Higgins Plant; Gibbstown, NJ

CORE Sample Date # 5‐3‐16 10‐26‐17 3‐28‐17 1 23 6.1 3.8 2 2 7.4 8.2 3 6 5.7 2 4 20 4.6 2 5 23 5.5 2 6 25 8.4 6

Mean 16.5 6.28 4 SD 9 1.26 2.37 Statistically Statistically significant significant compared compared to 5‐3‐17 data to 5‐3‐17 data @p<0.05 @p<0.05

Not statistically significant compared to 10‐26‐17 data @p<0.05

Notes: All units are in milligrams per kilogram (mg/kg)

Rev Date: 09/20/2017 CSI Environmental, LLC Print Date: 10/12/2017 Page 1 of 1 www.contactcsi.com

ATTACHMENT I (Pace Analytical Laboratory Biotreatability Report)

Pace Analytical Energy Services LLC 220 William Pitt Way Pittsburgh, PA 15238

Phone: (412) 826-5245 Fax: (412) 826-3433

June 27, 2017

Kevin Costello CSI Environemental 401 Headquarters Pt Suite 203 Millersville, MD 21108

RE: BIOTREATABILITY / A15-005 Pace Workorder: 22444 Dear Kevin Costello: Enclosed are the analytical results for sample(s) received by the laboratory on Thursday, April 27, 2017. Results reported herein conform to the most current NELAC standards, where applicable, unless otherwise narrated in the body of the report. If you have any questions concerning this report, please feel free to contact me.

Sincerely,

Ruth Welsh 06/27/2017 [email protected]

Customer Service Representative

Enclosures

As a valued client we would appreciate your comments on our service. Please email [email protected]. Total Number of Pages ____

Report ID: 22444 - 939522 Page 1 of 13

CERTIFICATE OF ANALYSIS This report shall not be reproduced, except in full, without the written consent of Pace Analytical Energy Services LLC. Pace Analytical Energy Services LLC 220 William Pitt Way Pittsburgh, PA 15238

Phone: (412) 826-5245 Fax: (412) 826-3433

LABORATORY ACCREDITATIONS & CERTIFICATIONS

Accreditor: Pennsylvania Department of Environmental Protection, Bureau of Laboratories Accreditation ID: 02-00538 Scope: NELAP Non-Potable Water and Solid & Hazardous Waste

Accreditor: West Virginia Department of Environmental Protection, Division of Water and Waste Management Accreditation ID: 395 Scope: Non-Potable Water

Accreditor: South Carolina Department of Health and Environmental Control, Office of Environmental Laboratory Certification Accreditation ID: 89009003 Scope: Clean Water Act (CWA); Resource Conservation and Recovery Act (RCRA)

Accreditor: NELAP: New Jersey, Department of Environmental Protection Accreditation ID: PA026 Scope: Non-Potable Water; Solid and Chemical Materials

Accreditor: NELAP: New York, Department of Health Wadsworth Center Accreditation ID: 11815 Scope: Non-Potable Water; Solid and Hazardous Waste

Accreditor: State of Connecticut, Department of Public Health, Division of Environmental Health Accreditation ID: PH-0263 Scope: Clean Water Act (CWA) Resource Conservation and Recovery Act (RCRA)

Accreditor: NELAP: Texas, Commission on Environmental Quality Accreditation ID: T104704453-09-TX Scope: Non-Potable Water

Accreditor: State of New Hampshire Accreditation ID: 299409 Scope: Non-potable water

Accreditor: State of Georgia Accreditation ID: Chapter 391-3-26 Scope: As per the Georgia EPD Rules and Regulations for Commercial Laboratories, PAES is accredited by the Pennsylvania Department of Environmental Protection Bureau of Laboratories under the National Environmental Laboratory Approval Program (NELAC).

Report ID: 22444 - 939522 Page 2 of 13

Phone: (412) 826-5245 Fax: (412) 826-3433

SAMPLE SUMMARY

Workorder: 22444 BIOTREATABILITY / A15-005

Lab ID Sample ID Matrix Date Collected Date Received

224440001 AE-MW-3 Water 4/26/2017 11:30 4/27/2017 11:00 224440002 AE-MW-1 Water 4/26/2017 08:40 4/27/2017 11:00

Report ID: 22444 - 939522 Page 3 of 13

Phone: (412) 826-5245 Fax: (412) 826-3433

PROJECT SUMMARY

Workorder: 22444 BIOTREATABILITY / A15-005

Sample Comments

Lab ID: 224440002 Sample ID: AE-MW-1 Sample Type: N The analysis for volatile fatty acids, method AM23G, was reported at dilution for sample due to the measured chloride concentration within the sample; matrix interfering compound.

Batch Comments

Batch: DISG/6085 - AM20GAX Water QC The matrix spike and/or spike duplicate, recovery or relative percent difference; accuracy influenced by the concentration of the reference sample 224330008. Analyte Ethane and Ethene. Batch acceptance based on laboratory control sample recovery.

Batch: DISG/6088 - AM20GAX Water QC The matrix spike and/or spike duplicate, recovery or relative percent difference; accuracy influenced by the concentration of the reference sample 224800015. Analyte Methane. Batch acceptance based on laboratory control sample recovery.

Report ID: 22444 - 939522 Page 4 of 13

Phone: (412) 826-5245 Fax: (412) 826-3433

ANALYTICAL RESULTS

Workorder: 22444 BIOTREATABILITY / A15-005

Lab ID: 224440001 Date Received: 4/27/2017 11:00 Matrix: Water Sample ID: AE-MW-3 Date Collected: 4/26/2017 11:30

Parameters Results Units PQL MDL DF Analyzed By Qualifiers RegLmt

Molecular Diagnostics - PAES Analysis Desc: 16S Analytical Method: 16S DNA Sequencing Complete 1 6/2/2017 00:00 YD n

RISK - PAES Analysis Desc: AM20GAX Analytical Method: AM20GAX Methane 21 ug/l 0.50 0.027 1 5/3/2017 17:50 BW n

Report ID: 22444 - 939522 Page 5 of 13

Phone: (412) 826-5245 Fax: (412) 826-3433

ANALYTICAL RESULTS

Workorder: 22444 BIOTREATABILITY / A15-005

Lab ID: 224440002 Date Received: 4/27/2017 11:00 Matrix: Water Sample ID: AE-MW-1 Date Collected: 4/26/2017 08:40

Parameters Results Units PQL MDL DF Analyzed By Qualifiers RegLmt

EDonors - PAES Analysis Desc: AM23G Analytical Method: AM23G Lactic Acid <2.0 mg/l 2.0 0.060 10 5/2/2017 06:06 KB d,B Acetic Acid <1.0 mg/l 1.0 0.070 10 5/2/2017 06:06 KB d,B Propionic Acid <1.0 mg/l 1.0 0.090 10 5/2/2017 06:06 KB d Formic Acid <1.0 mg/l 1.0 0.050 10 5/2/2017 06:06 KB d,B Butyric Acid <1.0 mg/l 1.0 0.070 10 5/2/2017 06:06 KB d Pyruvic Acid <1.0 mg/l 1.0 0.070 10 5/2/2017 06:06 KB d i-Pentanoic Acid <1.0 mg/l 1.0 0.070 10 5/2/2017 06:06 KB d Pentanoic Acid <1.0 mg/l 1.0 0.060 10 5/2/2017 06:06 KB d i-Hexanoic Acid <2.0 mg/l 2.0 0.040 10 5/2/2017 06:06 KB d Hexanoic Acid <2.0 mg/l 2.0 0.070 10 5/2/2017 06:06 KB d

Molecular Diagnostics - PAES Analysis Desc: 16S Analytical Method: 16S DNA Sequencing Complete 1 6/2/2017 00:00 YD n

RISK - PAES Analysis Desc: AM20GAX Analytical Method: AM20GAX Methane 1200 ug/l 0.50 0.027 1 5/4/2017 07:55 BW n,M5

Report ID: 22444 - 939522 Page 6 of 13

Phone: (412) 826-5245 Fax: (412) 826-3433

ANALYTICAL RESULTS QUALIFIERS

Workorder: 22444 BIOTREATABILITY / A15-005

DEFINITIONS/QUALIFIERS MDL Method Detection Limit. Can be used synonymously with LOD; Limit Of Detection.

PQL Practical Quanitation Limit. Can be used synonymously with LOQ; Limit Of Quantitation.

ND Not detected at or above reporting limit.

DF Dilution Factor.

S Surrogate.

RPD Relative Percent Difference.

% Rec Percent Recovery.

U Indicates the compound was analyzed for, but not detected at or above the noted concentration.

J Estimated concentration greater than the set method detection limit (MDL) and less than the set reporting limit (PQL).

n The laboratory does not hold NELAP/TNI accreditation for this method or analyte.

B The analyte was detected in the associated blank.

d The analyte concentration was determined from a dilution.

M5 The matrix spike duplicate sample recovery was outside laboratory control limits.

Report ID: 22444 - 939522 Page 7 of 13

Phone: (412) 826-5245 Fax: (412) 826-3433

QUALITY CONTROL DATA

Workorder: 22444 BIOTREATABILITY / A15-005

QC Batch: EDON/3320 Analysis Method: AM23G QC Batch Method: AM23G Associated Lab Samples: 224440002

METHOD BLANK: 48482

Blank Reporting Parameter Units Result Limit Qualifiers

EDonors Lactic Acid mg/l <0.20 0.20 B Acetic Acid mg/l <0.10 0.10 B Propionic Acid mg/l <0.10 0.10 Formic Acid mg/l <0.10 0.10 B Butyric Acid mg/l <0.10 0.10 Pyruvic Acid mg/l <0.10 0.10 i-Pentanoic Acid mg/l <0.10 0.10 Pentanoic Acid mg/l <0.10 0.10 i-Hexanoic Acid mg/l <0.20 0.20 Hexanoic Acid mg/l <0.20 0.20

LABORATORY CONTROL SAMPLE: 48483

Spike LCS LCS % Rec Parameter Units Conc. Result % Rec Limits Qualifiers

EDonors Lactic Acid mg/l 2 2.0 99 70-130 B Acetic Acid mg/l 2 2.0 100 70-130 B Propionic Acid mg/l 2 2.0 103 70-130 Formic Acid mg/l 2 1.8 89 70-130 B Butyric Acid mg/l 2 2.0 101 70-130 Pyruvic Acid mg/l 2 2.1 107 70-130 i-Pentanoic Acid mg/l 2 2.0 100 70-130 Pentanoic Acid mg/l 2 2.0 100 70-130 i-Hexanoic Acid mg/l 2 2.1 104 70-130 Hexanoic Acid mg/l 2 1.8 92 70-130

MATRIX SPIKE & MATRIX SPIKE DUPLICATE: 48484 48485 Original: 224410001

Original Spike MS MSD MS MSD % Rec Max Parameter Units Result Conc. Result Result % Rec % Rec Limit RPD RPD Qualifiers

EDonors Lactic Acid mg/l 0.034 2 1.8 1.9 90 91 70-130 1.1 30 B

Report ID: 22444 - 939522 Page 8 of 13

Phone: (412) 826-5245 Fax: (412) 826-3433

QUALITY CONTROL DATA

Workorder: 22444 BIOTREATABILITY / A15-005

MATRIX SPIKE & MATRIX SPIKE DUPLICATE: 48484 48485 Original: 224410001

Original Spike MS MSD MS MSD % Rec Max Parameter Units Result Conc. Result Result % Rec % Rec Limit RPD RPD Qualifiers

Acetic Acid mg/l 0.12 2 2.1 2.1 101 100 70-130 1 30 B Propionic Acid mg/l 0.0036 2 2.1 2.0 105 103 70-130 1.9 30 Formic Acid mg/l 0.037 2 1.8 1.8 86 86 70-130 0 30 B Butyric Acid mg/l 0.002 2 2.1 2.0 104 102 70-130 1.9 30 Pyruvic Acid mg/l 0 2 2.0 2.0 102 101 70-130 0.99 30 i-Pentanoic Acid mg/l 0 2 2.1 2.0 106 101 70-130 4.8 30 Pentanoic Acid mg/l 0 2 2.2 2.1 112 104 70-130 7.4 30 i-Hexanoic Acid mg/l 0 2 2.4 2.0 119 102 70-130 15 30 Hexanoic Acid mg/l 0 2 2.3 1.8 114 91 70-130 22 30

Report ID: 22444 - 939522 Page 9 of 13

Phone: (412) 826-5245 Fax: (412) 826-3433

QUALITY CONTROL DATA

Workorder: 22444 BIOTREATABILITY / A15-005

QC Batch: DISG/6085 Analysis Method: AM20GAX QC Batch Method: AM20GAX Associated Lab Samples: 224440001

METHOD BLANK: 48540

Blank Reporting Parameter Units Result Limit Qualifiers

RISK Methane ug/l <0.50 0.50 n

LABORATORY CONTROL SAMPLE & LCSD: 48541 48542

Spike LCS LCSD LCS LCSD % Rec Max Parameter Units Conc. Result Result % Rec % Rec Limit RPD RPD Qualifiers

RISK Methane ug/l 750 740 740 98 100 80-120 2 20 n

MATRIX SPIKE & MATRIX SPIKE DUPLICATE: 48577 48578 Original: 224330008

Original Spike MS MSD MS MSD % Rec Max Parameter Units Result Conc. Result Result % Rec % Rec Limit RPD RPD Qualifiers

RISK Methane ug/l 4900 1500 6000 6000 75 72 70-130 4.1 20 d,n

Report ID: 22444 - 939522 Page 10 of 13

Phone: (412) 826-5245 Fax: (412) 826-3433

QUALITY CONTROL DATA

Workorder: 22444 BIOTREATABILITY / A15-005

QC Batch: DISG/6088 Analysis Method: AM20GAX QC Batch Method: AM20GAX Associated Lab Samples: 224440002

METHOD BLANK: 48555

Blank Reporting Parameter Units Result Limit Qualifiers

RISK Methane ug/l <0.50 0.50 n,M5

LABORATORY CONTROL SAMPLE & LCSD: 48556 48557

Spike LCS LCSD LCS LCSD % Rec Max Parameter Units Conc. Result Result % Rec % Rec Limit RPD RPD Qualifiers

RISK Methane ug/l 750 730 730 97 98 80-120 1 20 M5,n

MATRIX SPIKE & MATRIX SPIKE DUPLICATE: 48589 48590 Original: 224800015

Original Spike MS MSD MS MSD % Rec Max Parameter Units Result Conc. Result Result % Rec % Rec Limit RPD RPD Qualifiers

RISK Methane ug/l 740 750 1300 1200 74 66 70-130 11 20 n,M5

Report ID: 22444 - 939522 Page 11 of 13

Phone: (412) 826-5245 Fax: (412) 826-3433

QUALITY CONTROL DATA QUALIFIERS

Workorder: 22444 BIOTREATABILITY / A15-005

QUALITY CONTROL PARAMETER QUALIFIERS

B The analyte was detected in the associated blank.

M5 The matrix spike duplicate sample recovery was outside laboratory control limits.

d The analyte concentration was determined from a dilution.

n The laboratory does not hold NELAP/TNI accreditation for this method or analyte.

Report ID: 22444 - 939522 Page 12 of 13

Phone: (412) 826-5245 Fax: (412) 826-3433

QUALITY CONTROL DATA CROSS REFERENCE TABLE

Workorder: 22444 BIOTREATABILITY / A15-005

Analysis Lab ID Sample ID Prep Method Prep Batch Analysis Method Batch

224440002 AE-MW-1 AM23G EDON/3320

224440001 AE-MW-3 AM20GAX DISG/6085

224440002 AE-MW-1 AM20GAX DISG/6088

224440001 AE-MW-3 16S MOLE/1148 224440002 AE-MW-1 16S MOLE/1148

Report ID: 22444 - 939522 Page 13 of 13

CERTIFICATE OF ANALYSIS This report shall not be reproduced, except in full, without the written consent of Pace Analytical Energy Services LLC. -ace AnalyJicar £Energy Servires'"

June 23, 2017

22253 and 22444 Combined Report Summary

From the data included in this report it appears that the AN-MW-1 had the most impact on the microbial community in regards to enhancing hydrocarbon degradation.

Background for Biologically-Induced Hydrocarbon Degradation Processes

Biological hydrocarbon degradation is well documented in the subsurface. There are four encompassing factors that control in-situ biodegradation of hydrocarbons and include carbon, energy, biotic community, and hydrogeologic conditions.

Carbon

Subsurface microbes are readily capable of using hydrocarbon contaminants as substrates for energy and growth. During this process the oxidation of the hydrocarbons are coupled to the reduction of electron acceptors such as oxygen, nitrate, or sulfate. The process converts the contaminants into harmless products (principally CO2 and water), cell mass, and inorganic salts. In dissolved-phase subsurface systems impacted by LNAPL, carbon in the form of hydrocarbons is delivered via the dissolution of soluble components.

Energy

Microbes derive energy via aerobic, anaerobic and fermentation/methanogenic pathways. In the dissolved phase oxygen and nitrate, which are high energy terminal electron acceptors, are consumed preferentially over alternate lower energy terminal electron acceptors. After oxygen is consumed, anaerobic microorganisms use a series of terminal electron acceptors including nitrate, iron (III), manganese (IV), and sulfate. Under fermentative conditions (when other terminal electron accepters are limiting) no external electron acceptor is needed because microbes use the hydrocarbon itself as both an electron donor and acceptor. The products of fermentation include CO2, organic acids, alcohols, and hydrogen gas. The fermentation products are degraded by other microbes, ultimately resulting in

CO2, methane, and water.

Microbial Community

Microbial hydrocarbon degradation activity in subsurface is catalyzed by different functional guilds of aerobic and anaerobic microbes. In aerobic systems hydrocarbon mineralization is coupled via the microbial community to the reduction of oxygen. In the dissolved phase once oxygen is consumed nitrate reducing microbes capable of hydrocarbon degradation will predominate. As nitrate is depleted, iron and sulfate reducing microbes capable of using hydrocarbons as a carbon and energy source flourish. Finally, fermentative bacteria synthesize hydrolytic enzymes (e.g. amylases, lipases, cellulases, and proteases) that degrade polymers to soluble monomers. The soluble products are then fermented to H2, CO2, simple alcohols, and fatty acids (including a significant amount of acetate). The acetogenic bacteria then catalyze the oxidation of alcohols and fatty acids to H2, CO2, and acetate. Lastly, methanogenic Archaea and sulfidogenic bacteria use the H2, CO2, and acetate to generate CH4 and H2S.

Once CH4 is produced, much of it may then be oxidized by CH4 oxidizing bacteria in the vadose zone where molecular oxygen is present.

Hydrogeologic Environment

. The hydrogeologic environment plays a major role in the activity of subsurface microbial communities, as: . Most bacteria living in the subsurface ecosystem do so as attached communities and so the surface area of the subsurface media can influence how much bacterial biomass can exist in a given system; . pH and temperature are critical parameters in microbial activity; . The hydrogeologic environment can regulate the amount and bioavailability of hydrocarbons through sorption to subsurface matrices; and . The hydrogeologic environment can influence toxicity via sorption of toxic constituents.

Analytical Approach for Assessment of the Microbial Community

The report contains three parts. Part one includes a summary of the results. Part two contains the biomass (qPCR) results. Part three presents the microbial community analysis which contains descriptions of all abundant (> ~1%) genus level identifications.

The purpose of this project was to characterize the bacteria in the samples and identify any groups that may be involved in the degradation of the aromatic hydrocarbon isopropyl benzene (cumene) under the two treatments. For this effort one background sample was previously analyzed, and in this project two additional samples were analyzed representing two treatments. Each of the samples were analyzed for biomass using quantitative a polymerase chain reaction (qPCR) assay which essentially counts the bacterial genes and can be used as a surrogate for total bacterial biomass. The second analysis identified the bacteria in the samples via genetic sequencing of the bacterial DNA.

The samples analyzed were designated by the lab as 224440001 (AE MW-3 by client) and 224440002 (AN-MW-1 by client). The report summary includes the sample 222530001 (MW-10R) which was being used as a background sample.

Results

The total 16s gene copies (biomass estimate Exhibit 1) in the samples ranged from 2.23e10 in AE-MW-3 to 1.53e12 in AN-MW-1. The biomass results were on the high end for groundwater and are indicative of a large amount of primary substrate (bacterial food) to support the observed bacterial population. AN-MW-1 had the highest measured biomass and could be indicative of the response of the treatment over both the background and the alternative treatment.

Exhibit 1.

Microbial Biomass (gene copies/Liter)

1.00E+12

1.00E+ 10I

1.00E+08

1.00E+06

1.00E+04

1.00E+02

1.00E+OO MW-10R AE-MW-3 AN-MW-1

Comparison of Microbial Community Composition

Exhibit 2 shows a comparison at the Class level for the three samples. There are noticeable differences in several Class groups. The largest differences are in Bacteroidia, Betaproteobacteria, Deltaproteobacteria, Gammaproteobacteria and Spirochaetia. Based on the paper from Rabus et al (2016) there are several classes of bacteria that contain members that can anaerobically degrade hydrocarbons and the two most identified Classes of anaerobic hydrocarbon degraders included Betaproteobacteria and Deltaproteobacteria. Exhibit 3 shows the portion of the profile (percent of reads) for Betaproteobacteria and Deltaproteobacteria. About 32 percent of the profile of the background sample (MW-10R) was Betaproteobacteria and Deltaproteobacteria while AE-MW-3 was 45% of the profile and AN-MW-1 was ~60 percent of the profile. Exhibit 2. Community Composition (percent of reads Class Level)

60.00

so.oo

40.00

30.00 • MW,!OR • A(~MW-3 • AN-MW-1 20.00

10.00

Exhibit 3. Selected Class Level IDs

Class MW-lOR AE-MW-3 AN-MW-1 be tap roteobacte ri a 15.0 26.8 49.7 deltaproteobacteria 17.3 18.3 8.9 Total 32.3 45.1 58.6

Microbial Community Composition MW-10R

The sample contained bacteria known to degrade hydrocarbons anaerobically. The abundant phylotypes were dominated by anaerobic Gram-negative bacteria. Key microbial metabolic groups associated with aerobic and anaerobic hydrocarbon degradation have been identified and described. Genetic sequencing of the sample identified many phylotypes associated with anaerobic degradation of hydrocarbons. In a recent Synopsis Rabus et al. (2016) described several anaerobic hydrocarbon degraders both at Class and lower levels. The two dominant Classes that contain anaerobic hydrocarbon degraders are Betaproteobacteria and Deltaproteobacteria. The results from the genetic profiling show that these are the two most abundant Classes in the sample representing over 32% of the profile (Exhibit 4).

Exhibit 4. Class level phylotypes MW-10R Class Percent of Profile deltaproteobacteria 17.26 betaproteobacteria 15.02 spirochaetia 14.48 clostridia 10.84 bacteroidia 8.94 alphaproteobacteria 8.53 gammaproteobacteria 8.44 anaerolineae 5.30 dehalococcoidia 4.45 thermotogae 2.34 caldisericia 1.26 epsilonproteobacteria 0.50 actinobacteria 0.47 nitrospira 0.41 synergistia 0.29 cloacimonetes 0.25 bacilli 0.20 verrucomicrobiae 0.15 flavobacteriia 0.13 solibacteres 0.09 cyanobacteria 0.08 holophagae 0.08 sphingobacteriia 0.07 elusimicrobia 0.06 planctomycetia 0.06 gloeobacteria 0.03 cytophagia 0.03 chlamydiia 0.03 opitutae 0.03 negativicutes 0.02 chloroflexia 0.02 gemmatimonadetes 0.01 deinococci 0.01 chlorobia 0.01 thermoleophilia 0.01

The Rabus paper also identifies several bacterial groups at the Genus level that contain members that are capable of anaerobic or microaerophilic hydrocarbon degradation. These included Magnetospirillum spp. (~8% of the profile), Geobacter spp. (~7% of the profile), and Clostridia spp. (~2%) of the profile (see Genus level report). Other bacteria in the sample that contain members known to degrade hydrocarbons aerobically included Pseudomonas spp. The most abundant phylotype in the profile was Treponema spp. There may be a relationship with the presence of this bacteria and cumene hydroperoxide since these bacteria are known to have a robust mechanism to handle oxidative stress (Parsonage et al. 2010).

Microbial Community Composition AE-MW-3

The sample contained bacteria known to degrade hydrocarbons both aerobically and anaerobically. The abundant phylotypes were dominated by Gram-negative bacteria. Genetic sequencing of the sample (Class level Exhibit 5.) identified Betaproteobacteria and Deltaproteobacteria as 45 percent of the profile. At the Genus level phylotypes associated with aerobic degradation (Acidovorax spp. and Pseudomonas spp.) and anaerobic degradation (Geobacter spp. and Clostridium spp.) of hydrocarbons were identified (see Genus level report). Exhibit 5. Class level phylotypes AE-MW-3 Class Percent of Profile bacteroidia 28.57 betaproteobacteria 26.80 deltaproteobacteria 18.29 spirochaetia 8.12 clostridia 4.03 alphaproteobacteria 3.93 anaerolineae 3.27 gammaproteobacteria 2.32 synergistia 1.12 bacilli 0.82 cyanobacteria 0.54 actinobacteria 0.44 holophagae 0.29 erysipelotrichia 0.26 acidobacteriia 0.25 verrucomicrobiae 0.19 epsilonproteobacteria 0.14 opitutae 0.14 chlamydiia 0.12 nitrospira 0.09 sphingobacteriia 0.06 caldisericia 0.05 dehalococcoidia 0.04 lentisphaeria 0.02 ignavibacteria 0.02 cytophagia 0.01 negativicutes 0.01 elusimicrobia 0.01 gemmatimonadetes 0.01

Microbial Community Composition AN-MW-1

The sample contained bacteria known to degrade hydrocarbons both aerobically and anaerobically. At the Class level over 58 percent of the profile was Betaproteobacteria and Deltaproteobacteria (Exhibit 6). The abundant phylotypes were dominated by Gram-negative bacteria. Genetic sequencing of the sample at the Genus level identified phylotypes associated with aerobic degradation (Acidovorax spp.) and anaerobic degradation (Geobacter spp. and Desulfitobacterium spp.) of hydrocarbons (see Genus level report). Exhibit 6. Class level phylotypes AN-MW-1 Class Percent of Profile betaproteobacteria 49.70 alphaproteobacteria 8.88 deltaproteobacteria 8.88 clostridia 7.12 sphingobacteriia 3.97 gammaproteobacteria 2.60 cyanobacteria 2.59 actinobacteria 2.34 holophagae 2.13 bacteroidia 1.74 verrucomicrobiae 1.66 spirochaetia 0.92 chlamydiia 0.85 opitutae 0.83 bacilli 0.76 mollicutes 0.65 gloeobacteria 0.60 oligosphaeria 0.56 acidobacteriia 0.55 dehalococcoidia 0.53 epsilonproteobacteria 0.36 planctomycetia 0.31 elusimicrobia 0.28 gemmatimonadetes 0.25 ignavibacteria 0.24 anaerolineae 0.18 cytophagia 0.14 nitrospira 0.13 negativicutes 0.06 lentisphaeria 0.04 flavobacteriia 0.04 solibacteres 0.03 caldisericia 0.03 erysipelotrichia 0.02 acidobacteriia 0.01 synergistia 0.01 nitrospinia 0.01 chloroflexia 0.01

From the data included in this report it appears that the AN-MW-1 had the most impact on the microbial community in regards to enhancing hydrocarbon degradation. References

Parsonage et al. 2010. Broad specificity AhpC-like peroxiredoxin and its thioredoxin reductant in the sparse antioxidant defense system of Treponema pallidum. PNAS vol. 107 no. 14 6240-6245.

Rabus et al. 2016. Anaerobic Microbial Degradation of Hydrocarbons: From Enzymatic Reactions to the Environment. J Mol Microbiol Biotechnol 2016; 26:5–28. Molecular Detection Quantitative Polymerase a'Ce.AnalyJical ® Energy Services .. Chain Reaction Assay

Pace Analytical ID: 22444 Analyst: Y. Diao / A. Peacock 220 William Pitt Way Pittsburgh, PA 15238 412-826-5245

Company: CSI Environmental Client: CSI Environmental Date Collected: 4/26/2017 Job Number: BIOTREATABILITY / A15-005 Date Received: 4/27/2017 Project Name: Date Reported: 5/4/2017

Pace Energy ID: 224440001 Matrix: Water Sample ID: AE-MW-3 Description: gene copies per liter of water

Total Eubacteria: 2.23E+10

Pace Energy ID: 224440002 Matrix: Water Sample ID: AE-MW-1 Description: gene copies per liter of water

Total Eubacteria: 1.53E+12

QA/QC NIC UNC IPC UPC Pass Pass Pass Pass

Page 1 of 1 Pace Analytical Energy Services ace Ana/yJicar' 220 William Pitt Way Energy Services·· Pittsburgh, PA 15238 (412) 826-5245 Genomic Sequencing Report Project: A15-005 6/2/2017 Sample ID: AE-MW-3 Complete profile data was transmitted to Kevin Costello [email protected] Summary:

Results: Phylotype Information In microbiology, a phylotype is an environmental DNA sequence or group of sequences sharing more than an arbitrarily chosen level of similarity of a particular gene maker. Abundant phylotypes are defined as any identified phylotype that comprised ~1% or more of the total profile. The results that are presented below are derived from the phylotypes and are reported at the genus level (closest match). Table 1 shows what percentage of the total profile consisted of abundant phylotypes.

Table 1. Percentage of Profile Represented by Abundant Phylotypes Sample ID: AE-MW-3 % of Profile: 87.52 # of Abundant ID's: 17 # of Total ID's: 261

Gram-Stain Distribution Gram staining is a common method used to distinguish two large groups of bacteria based on the properties of their cell wall. The two categories in Gram-stain are positive and negative. Gram-negative bacteria tend to be fast growing and early colonizers of disturbed environments. Gram-negative bacteria can also be more sensitive to perturbation and environmental stress. Gram-positive bacteria on the other hand tend to grow more slowly but can be more resilient to environmental stress. The following table and graph reports the distribution of Gram-positive and negative bacteria from the samples.

Figure 1. Gram Stain Distribution of Phylotypes % of Profile Table and Graph

Sample ID: AE-MW-3 Negative: 83.73 • Negative Positive: 2.51 • Positive Varied: 0 • Varied Not Characterized: 13.76 • NotCharacterized Pace Analytical Energy Services 220 William Pitt Way Results Continued: Pittsburgh, PA 15238 Sample ID: AE-MW-3 (412) 826-5245

Aerobic/Anaerobic Metabolism Bacteria can use a number of terminal electron acceptors other than oxygen. Aerobic bacteria use oxygen as a terminal electron accepter while anaerobic bacteria use terminal electron acceptors other than oxygen (e.g. nitrate, iron, sulfate) or through fermentation. The following table and graph show the distribution of aerobic and anaerobic bacteria from the identified phylotypes.

Figure 2. Metabolism Distribution of Phylotypes % of Profile Table and Graph

Sample ID: AE-MW-3 Aerobic: 23.82 • Aerobic Anaerobic: 58.28 • Anaerobic Varied: 3.03 • Varied Not Characterized: 14.87 • NotCharacterized

Dominant phylotypes that represented more than 1% of the community profile in any sample (Genus level closest match)

Respiration Key: Aerobic - Uses oxygen as a terminal electron acceptor SOX - Sulfur oxidizing bacteria Anaerobic - Does not use oxygen as a terminal electron accepter MOB - Methane oxidizing bacteria Variable - Members of the genus have multiple capabilites IOB - Iron oxidizing bacteria MRB - Metal reducing bacteria NOB - Nitrate oxidizing bacteria Fermentive - Capable of fermentation SRB - Sulfate reducing bacteria Pace Analytical Energy Services 220 William Pitt Way Abundant Phylotypes Pittsburgh, PA 15238 Sample ID: AE-MW-3 (412) 826-5245

Genus (Closest Match): % of Profile: Metabolism: Gram: proteiniphilum 24.57 Anaerobic Negative geobacter 15.19 Anaerobic/MRB Negative acidovorax 12.75 Aerobic/IOB Negative azovibrio 7.44 Aerobic/Nitrate Negative treponema 4.67 Anaerobic/microaerophilic Negative bacteroides 3.98 Anaerobic Negative spirochaeta 3.45 Anaerobic/Fermenter Negative rhodoferax 3.03 Varied Negative longilinea 2.42 Anaerobic Negative pseudomonas 1.75 Aerobic Negative desulforhabdus 1.49 Anaerobic/SRB Negative sedimentibacter 1.49 Anaerobic/fermentive Positive nordella 1.28 cloacibacillus 1.11 Anearobic/Fermenter Negative desulfovibrio 1.02 Anaerobic/SRB Positive magnetospirillum 0.96 Aerobic Negative comamonas 0.92 Aerobic Negative Total Abundant Phylotypes: 87.52 Pace Analytical Energy Services ace Ana/yJicar' 220 William Pitt Way Energy Services·· Pittsburgh, PA 15238 (412) 826-5245 Genomic Sequencing Report Project: A15-005 6/2/2017 Sample ID: AN-MW-1 Complete profile data was transmitted to Kevin Costello [email protected] Summary:

Table 1. Percentage of Profile Represented by Abundant Phylotypes Sample ID: AN-MW-1 % of Profile: 77.24 # of Abundant ID's: 23 # of Total ID's: 303

Figure 1. Gram Stain Distribution of Phylotypes % of Profile Table and Graph

Sample ID: AN-MW-1 Negative: 69.42 • Negative Positive: 6.15 • Positive Varied: 0 • Varied Not Characterized: 24.43 • NotCharacterized Pace Analytical Energy Services 220 William Pitt Way Results Continued: Pittsburgh, PA 15238 Sample ID: AN-MW-1 (412) 826-5245

Figure 2. Metabolism Distribution of Phylotypes % of Profile Table and Graph

Sample ID: AN-MW-1 Aerobic: 58.76 • Aerobic Anaerobic: 17.48 • Anaerobic Varied: 1 • Varied Not Characterized: 22.76 • NotCharacterized

Dominant phylotypes that represented more than 1% of the community profile in any sample (Genus level closest match)

Genus (Closest Match): % of Profile: Metabolism: Gram: curvibacter 14.26 Aerobic Negative acidovorax 12.79 Aerobic/IOB Negative geobacter 7.25 Anaerobic/MRB Negative azovibrio 4.65 Aerobic/Nitrate Negative nitrosovibrio 4.32 Aerobic/Ammonia oxidizer Negative comamonas 4.09 Aerobic Negative rhodoblastus 3.87 Aerobic Negative sediminibacterium 3.14 Aerobic Negative desulfitobacterium 2.73 Anaerobic Positive pelotomaculum 2.51 Anaerobic Positive prochlorococcus 2.47 Aerobic Negative sulfuricella 2.28 Aerobic/SOB Negative geothrix 1.67 Anaerobic/MRB Negative methylocystis 1.67 Aerobic MOB verrucomicrobium 1.32 Anaerobic Negative rhodanobacter 1.23 Aerobic Negative bacteroides 1.20 Anaerobic Negative bordetella 1.20 Aerobic Negative rhodoferax 1.00 Varied Negative gallionella 0.99 Aerobic/IRB Negative actinomadura 0.91 Aerobic Positive bradyrhizobium 0.89 Aerobic Negative opitutus 0.80 Anaerobic Negative Total Abundant Phylotypes: 77.24 Phylotype Descriptions

Acidovorax Straight to slightly curved rods, 0.2–1.2 × 0.8–5.0 µm, occurring singly, in pairs, or in short chains. Gram negative. Motile by means of one or rarely two or three polar flagella. Aerobic, having a strictly oxidative type of metabolism with O2 as the terminal electron acceptor; some strains of two species (Acidovorax delafieldii and Acidovorax temperans) are capable of heterotrophic denitrification of nitrate. Most strains do not produce pigments on nutrient agar, but some phytopathogenic strains may produce a yellow to slightly brown diffusible pigment. Oxidase positive; urease activity varies among strains. Chemoorganotrophic, although strains of two species (A. facilis and A. delafieldii) can grow lithoautotrophically, using the oxidation of H2 as an energy source. Good growth occurs on organic acids, amino acids, and peptone, but organisms show less versatile growth on carbohydrates. Fatty acids present always include 3-hydroxyoctanoic acid (C8:0 3OH) and 3-hydroxydecanoic acid (C10:0 3OH); 2-hydroxy-substituted fatty acids are absent. Acidovorax strains can be isolated from soil, water, clinical samples, activated sludge, and infected plants. Proteobacteria / Betaproteobacteria / Burkholderiales / Comamonadaceae / Acidovorax Willems, A. and Gillis, M. 2015. Acidovorax. Bergey's Manual of Systematics of Archaea and

Bacteria. 1–16.

Actinomadura Ac.ti.no.ma.du'ra. Gr. n. actis actinos a ray; N.L. n. Madura Madura, name of a province in India; N.L. fem. n. Actinomadura referring to a micro-organism first described as the causative agent of “Madura foot” disease. Actinobacteria / Actinobacteria / Streptosporangiales / Thermomonosporaceae / Actinomadura. Gram-stain- positive, non-acid–alcohol-fast, nonmotile actinomycetes that form an extensively branched non-fragmenting, substrate mycelium. Aerial mycelium moderately developed or absent. When present, aerial hyphae carry up to 50 arthrospores. Aerial mycelium at maturity forms short or occasionally long chains of arthrospores. Spore chains straight, hooked (open loops), or irregular spirals (1–4 turns). Spore surface folded, irregular, rugose, smooth, spiny, or warty. Color of mature sporulated aerial mycelium: blue, brown, cream, gray, green, pink, red, white, or yellow. Colonies have a leathery or cartilaginous appearance when aerial mycelium is lacking. Aerobic, chemoorganotrophic with an oxidative type metabolism. Temperature growth range 10–60°C. Cell wall contains meso- 2,6-diaminopimelic acid as the major diamino acid and N-acetylated muramic acid. Whole-cell hydrolysates contain galactose, glucose, madurose, mannose, and ribose. The major phospholipids are diphosphatidylglycerol and phosphatidylinositol. Menaquinones are predominantly hexahydrogenated with nine isoprene units saturated at sites II, III, and VIII. Complex fatty acid profile is rich in branched saturated and unsaturated fatty acids, including tuberculostearic acid. Mycolic acids are absent. Widely distributed in soil. Some strains are pathogenic for animals, including man. DNA G+C content (mol%): 66–73 (Tm, HPLC). Type species: Actinomadura madurae (Vincent 1894) Lechevalier and Lechevalier 1970a, 400AL. Trujillo, M. E. and Goodfellow, M. 2015. Actinomadura. Bergey's Manual of Systematics of

Archaea and Bacteria. 1–32.

Azovibrio A.zo' vi.bri.o. Fr. n. azote nitrogen; L. v. vibrare move rapidly to and fro, vibrate; M.L. masc. n. Azovibrio nitrogen- fixing organism which vibrates. Proteobacteria / Betaproteobacteria / Rhodocyclales / Rhodocyclaceae / Azovibrio Gram-negative somewhat curved motile rods (0.6–0.8 × 1.5–3.6 μm). One polar flagellum. Chemoorganoheterotrophic and microaerophilic. Strictly respiratory; O2 and nitrate are electron acceptors. Oxidase positive. Fix N2. Grow on acetate, ethanol, fumarate, l-glutamate, dl-lactate, l-malate, propionate, and succinate. The mol% G + C of the DNA is: 64–65. Type species: Azovibrio restrictus Reinhold-Hurek and Hurek 2000, 657. The Editorial Board 2015. Azovibrio. Bergey's Manual of Systematics of Archaea and

Bacteria. 1–2.

Bacteroides Genus of gram-negative, obligate anaerobic bacteria. Possible syntrophic relationship with Dehalococcoides via fermentation. Bacteroidetes / Bacteroidia / Bacteroidales / Bacteroidaceae / Bacteroides. Rod-shaped cells with rounded ends. Gram-stain-negative. Cells are fairly uniform if smears are prepared from young cultures on blood agar. Nonmotile. Anaerobic. Colonies are 1–3 mm in diameter, smooth, white to gray, and nonhemolytic on blood agar. Chemo-organotrophic. Saccharolytic. Weakly proteolytic. Most species grow in the presence of 20% bile, but are not always stimulated. Esculin is usually hydrolyzed. Nitrate is not reduced to nitrite. Indole variable. Major fermentation products are succinate and acetate. Trace to moderate amounts of isobutyrate and isovalerate may be produced. Predominant cellular fatty acid is C15:0 anteiso. DNA G+C content (mol%): 39–49. Song, Y., Liu, C. and Finegold, S. M. 2015. Bacteroides. Bergey's Manual of Systematics of Archaea and Bacteria. 1–24.

Bordetella Bor.de.tel' la. M.L. dim ending -ella; M.L. fem. n. Bordetella named after Jules Bordet, who with O. Gengou first isolated the organism causing pertussis. Proteobacteria / Betaproteobacteria / Burkholderiales / Alcaligenaceae / Bordetella. Minute coccobacillus, 0.2–0.5 μm in diameter and 0.5–2.0 μm in length, often bipolar stained, and arranged singly or in pairs, more rarely in chains. Gram negative. Nonmotile or motile by peritrichous flagella. Strictly aerobic. Optimal temperature, 35–37°C. Colonies on Bordet–Gengou medium are smooth, convex, pearly, glistening, nearly transparent, and surrounded by a zone of hemolysis without definite periphery. Respiratory metabolism. Chemoorganotrophic. Require nicotinamide, organic sulfur (e.g., cysteine), and organic nitrogen (amino acids). Utilize oxidatively glutamic acid, proline, alanine, aspartic acid, and serine, with production of ammonia and CO2. Litmus milk is made alkaline. Mammalian and avian parasite and pathogen. Most species localize and multiply among the epithelial cilia of the respiratory tract. The mol% G + C of the DNA is: 66–70. Type species: Bordetella pertussis (Bergey, Harrison, Breed, Hammer and Huntoon 1923a) Moreno-López 1952, 178 (Microbe de coqueluche Bordet and Gengou 1906, 731; Haemophilus pertussis Bergey, Harrison, Breed, Hammer and Huntoon 1923a, 269.) Sanden, G. N. and Weyant, R. S. 2015. Bordetella. Bergey's Manual of Systematics of

Archaea and Bacteria. 1–20.

Bradyrhizobium Bra.dy.rhi.zo' bi.um. Gr. adj. bradus slow; M.L. neut. n. Rhizobium a bacterial generic name; M.L. neut. n. Bradyrhizobium the slow (growing) rhizobium. Proteobacteria / Alphaproteobacteria / Rhizobiales / Bradyrhizobiaceae / Bradyrhizobium Rods 0.5–0.9 × 1.2–3.0 μm. Commonly pleomorphic under adverse growth conditions. Usually contain granules of poly-β-hydroxybutyrate that are refractile by phase-contrast microscopy. Nonsporeforming. Gram negative. Motile by one polar or subpolar flagellum. Fimbriae have not been described. Aerobic, possessing a respiratory type of metabolism with oxygen as the terminal electron acceptor. Optimal temperature 25–30°C. Optimal pH, 6–7, although lower optima may be exhibited by strains from acid soils. Colonies are circular, opaque, rarely translucent, white, and convex, and tend to be granular in texture; they do not exceed 1.0 mm in diameter in less than 5–6 days incubation on A1EG medium. Turbidity develops only after 3–4 days in agitated broth. Generation times are 9– 18 h. Chemoorganotrophic, utilizing a range of carbohydrates and salts of organic acids as carbon sources, without gas formation; arabinose and other pentoses are preferred carbon sources. Cellulose and starch are not utilized. Produce an alkaline reaction in mineral salts medium containing mannitol and/or many other carbohydrates. Growth on carbohydrate media is usually accompanied by extracellular polysaccharide slime production particularly with glycerol, gluconate, or mannitol. Some strains can grow chemolithotrophically in the presence of H2, CO2, and low levels of O2. Ammonium salts, usually nitrates, and some amino acids, can serve as nitrogen sources. Peptone is poorly utilized (except for strains isolated from Lotononis). Casein and agar are not hydrolyzed. There is usually no requirement for vitamins with the rare exception of biotin, which also may be inhibitory to some strains. 3-Ketoglycosides are not produced (Bernaerts and De Ley, 1963). The organisms are characteristically able to enter the root hairs of tropical-zone and some temperate-zone leguminous plants (family Leguminosae) and incite the production of root nodules, in which the bacteria occur as intracellular nitrogen-fixing symbionts. Some strains, especially B. elkanii, fix nitrogen in the free-living state when examined under special conditions. The mol% G + C of the DNA is: 61–65. Type species: Bradyrhizobium japonicum (Kirchner 1896) Jordan 1982, 137 (“Rhizobacterium japonicum” Kirchner 1896, 221; Rhizobium japonicum (Kirchner 1896) Buchanan 1926, 90.)

Comamonas Co.ma.mo' nas. L. n. coma lock of hair; Gr. n. monas a unit, monad; M.L. fem. n. Comamonas cell with a polar tuft of flagella. Proteobacteria / Betaproteobacteria / Burkholderiales / Comamonadaceae / Comamonas Straight or slightly curved rods or spirilla, 0.3–0.8 × 1.1–4.4 µm; occasionally longer (5–7 µm), irregularly curved cells or spirilla may occur. Cells occur separately or in pairs and are motile by means of polar or bipolar tufts of 1–5 flagella except for C. koreensis, which is nonmotile. Gram negative. No diffusible pigments are produced on nutrient agar. Oxidase and catalase positive. Aerobic. Chemoorganotrophic, oxidative carbohydrate metabolism with oxygen as the terminal electron acceptor. C. nitrativorans is also capable of denitrification. Good growth on media containing organic acids, amino acids, or peptone; few carbohydrates are used. Major fatty acids are hexadecanoic acid (C16:0), hexadecenoic acid (C16:1) and octadecenoic acid (C18:1); 3-hydroxydecanoic acid (C10:0 3OH) is always present. The major quinone is ubiquinone Q-8. The mol% G + C of the DNA is: 60–69 Willems, A. and Gillis, M. 2015. Comamonas. Bergey's Manual of Systematics of Archaea and Bacteria. 1–17.

Cloacibacillus Gram negative, anaerobic amino-acid-utilizing bacteria. Fermenter of arginine, histidine, lysine and serine and showed growth on yeast extract, brain-heart infusion (BHI) medium and tryptone, but not on carbohydrates, organic acids or alcohols. The end products of degradation were: acetate, butyrate, H2 and CO2 from arginine; acetate, propionate, butyrate, H2 and CO2 from lysine; and acetate, propionate, butyrate, valerate, H2 and CO2 from histidine, serine, BHI medium, Casamino acids and tryptone.

Curvibacter A Gram-negative aerobic or microaerophillic spirillum. Ding L., and Yokota, A. 2006. Int J Syst Evol Microbiol, November 2004 54: 2223-2230.

Desulfitobacterium De.sul.fi.to.bac.te'ri.um. L. pref. de from, off, away; N.L. n. sulfis sulfite; N.L. masc. n. bacter rod; N.L. neut. n. Desulfitobacterium rod-shaped bacterium that reduces sulfite. Firmicutes / “Clostridia” / Clostridiales / Peptococcaceae / Desulfi tobacterium. Cell wall is of Gram-positive structure but may stain Gram-negative or -positive depending on the strain. Exponential-growth phase cells are straight or curved rods, 2–5 µm in length, depending on the species and strain. Although the physiology is obligately anaerobic; certain species tolerate microaerophilic culture conditions (<5% air in N2 head gas phase); mesophilic and heterotrophic. Desulfitobacterium species use a variety of chlorinated phenols and/or alkenes as electron acceptors during dehalorespiration (also called halorespiration or chloridogenesis). Furthermore, they reduce sulfite, thiosulfate, sulfur, fumarate (forming succinate), and nitrate, but not sulfate, in the presence of various electron donors. Yeast extract as growth supplement is required; some strains grow only using pyruvate (forming lactate + acetate + CO2), others can utilize various sugars. Four species are validly published, Desulfitobacterium dehalogenans, Desulfitobacterium chlororespirans, Desulfitobacterium hafniense, and Desulfitobacterium metallireducens. For differentiation of the species, see Table 181. DNA G+C content (mol%): 45–48.8. Type species: Desulfitobacterium dehalogenans Utkin, Woese and Wiegel 1994, 615VP. Lupa, B. and Wiegel, J. 2015. Desulfitobacterium. Bergey's Manual of Systematics of

Archaea and Bacteria. 1–13.

Desulforhabdus De.sul.fo.rhab' dus. L. pref. de from; L. neut. n. sulfur sulfur; Gr. fem. n. rhabdus rod; M.L. fem. n. Desulforhabdus a rod-shaped sulfate reducer. Proteobacteria / Deltaproteobacteria / Syntrophobacterales / Syntrophobacteraceae / Desulforhabdus. Cells are rod-like to ellipsoid, 1.4–1.9 × 2.5–3.4 μm. Occur singly, in pairs, or in long chains. Spore formation is not observed. Gram negative and nonmotile. Strictly anaerobic, having a respiratory type of metabolism. Chemoautotrophic or chemoorganotrophic, using H2 (+ CO2), formate, acetate, propionate, butyrate, isobutyrate, lactate, pyruvate, ethanol, 1-propanol, and 1-butanol as electron donors and carbon sources. Organic substrates are completely oxidized to CO2 via the anaerobic C1-pathway (carbon monoxide dehydrogenase pathway, Wood-pathway) as indicated by carbon monoxide dehydrogenase activity, but transient excretion of acetate, isobutyrate, and propionate can occur. For chemoautotrophic growth, H2 serves as the electron donor and CO2 as the carbon source. Sulfate, sulfite, and thiosulfate serve as terminal electron acceptors and are reduced to H2S. Sulfur is not reduced. No growth by fermentation in the absence of an external electron acceptor. The mol% G + C of the DNA is: 52.5. Type species: Desulforhabdus amnigena Oude Elferink, Maas, Harmsen and Stams 1997, 1274 (Effective publication: Desulforhabdus amnigenus (sic) Oude Elferink, Maas, Harmsen and Stams 1995, 123.) Kuever, J., Rainey, F. A. and Widdel, F. 2015. Desulforhabdus. Bergey's Manual of

Systematics of Archaea and Bacteria. 1–3.

Desulfovibrio Gram-negative sulfate-reducing bacteria. Desulfovibrio species are commonly found in aquatic environments with high levels of organic material, as well as in water-logged soils, and form major community members of extreme oligotrophic habitats such as deep granitic fractured rock aquifers. Sulfate reducing bacteria can aid in the production of sulfide minerals that can degrade chlorinated solvents abiotically. Commonly found in conjunction with Dehalococcoides bacteria. Members have been shown to degrade TCE and also have shown sustainable syntrophic growth with Dehalococcoides. Proteobacteria / Deltaproteobacteria / Desulfovibrionales / Desulfovibrionaceae / Desulfovibrio. Curved or occasionally straight rods, sometimes sigmoid or spirilloid, 0.5–1.5 × 2.5–10.0 μm. The morphology is influenced by age and environment; descriptions refer to freshly grown cultures in anoxic sulfate media. Spore formation is absent. Gram negative. Motile by means of a single or lophotrichous polar flagella. Obligately anaerobic growth in pure cultures. Possess mainly a respiratory type of metabolism with sulfate or other sulfur compounds as the terminal electron acceptors, being reduced to H2S; however, the metabolism is sometimes fermentative. Media containing a reducing agent are required for growth. In a few cases, a vitamin requirement has been reported. Some species and subspecies are moderately halophilic. Optimal growth temperature, usually 25–35°C; upper limit normally 44°C. No thermophilic species have been reported. Thermophilic Desulfovibrio species formerly described have been reclassified and currently belong to the genera Thermodesulfovibrio and Thermodesulfobacterium. Chemoorganotrophic. Most species oxidize organic compounds such as lactate incompletely to acetate, which cannot be oxidized further. Carbohydrates are utilized by few species. One species, D. inopinatus, can use hydroquinone as electron donor and carbon source for growth. Cells contain c-type cytochromes (such as c3) and usually b-type cytochromes. All members of the genus Desulfovibrio contain desulfoviridin. Hydrogenase is usually present. Strains of some species may show chemolithoheterotrophic growth, using H2 as electron donor and assimilating acetate and CO2, or yeast extract, as carbon sources. Gelatin is not liquefied. Nitrate is sometimes reduced to ammonia. Some species can reduce oxygen or metal ions, but growth has never been observed with these electron acceptors in pure cultures. Molecular nitrogen is sometimes fixed. Species generally show some degree of antigenic cross reaction. Habitats: anoxic mud of fresh and brackish water and marine environments; intestines of animals; manure and feces. The mol% G + C of the DNA is: 46.1–61.2. Kuever, J., Rainey, F. A. and Widdel, F. 2015. Desulfovibrio. Bergey's Manual of Systematics of Archaea and Bacteria. 1–17.

Gallionella Gal.li.o.nel' la. M.L. dim. ending -ella ; M.L. fem. n. Gallionella named for B. Gaillon, a customs agent and zoologist (1782–1839) in Dieppe, France. Proteobacteria / Betaproteobacteria / Nitrosomonadales / Gallionellaceae / Gallionella. Gram-negative, bean-shaped cells, usually 0.5–0.8 × 1.6–2.5 µm, that secrete an extracellular twisted stalk from the concave side, 0.3–0.5 µm in width and up to 400 µm or more in length. The stalk is composed of numerous 2 nm-wide fibers and is produced under microaerophilic conditions when cells are in late exponential or stationary growth phase. Motile by means of a polar flagellum. Microaerophilic; chemolithotrophic growth can be obtained in vitro using oxygen and ferrous iron concentration gradients in a salt medium with CO2 as sole carbon source (Table BXII.β.108). Mixotrophic metabolism has been demonstrated with glucose, fructose, and sucrose. Can be found where anaerobic groundwater with ferrous iron reaches an oxygen-containing environment. Belongs to the Betaproteobacteria, family Gallionellaceae, with one known species, G. ferruginea. Most closely related species according to 16S rDNA sequence analysis is the chemolithotroph Nitrosospira multiformis, distantly related with a 16S rDNA sequence similarity of 90%. The mol% G + C of the DNA is: 51–54.6 (Hanert, 1989). Type species: Gallionella ferruginea Ehrenberg 1838, 166. Hallbeck, L. E.-L. and Pedersen, K. 2015. Gallionella. Bergey's Manual of Systematics of

Archaea and Bacteria. 1–10.

Geobacter A well known metal reducing group of microbes (anaerobic). Many members of the genus are capable of hydrocarbon degradation and iron reduction. At least one Geobacter species (lovleyi) is capable of reductive dechlorination of PCE and TCE to cis-DCE. Others have been implicated in supplying Dehalococcoides with nutrients for growth. Sung Youlboong, Kelly E. Fletcher, Kirsti M. Ritalahti, Robert P. Apkarian, Natalia

Ramos-Hernández, Robert A. Sanford, Noha M. Mesbah and Frank E. Löffler. Geobacter lovleyi sp. nov. Strain SZ, a Novel Metal-Reducing and Tetrachloroethene-Dechlorinating

Bacterium. Appl. Environ. Microbiol. April 2006 vol. 72 no. 4 2775-2782.

Yan et al. 2012. Unexpected Specificity of Interspecies Cobamide Transfer from Geobacter spp. to Organohalide-Respiring Dehalococcoides mccartyi Strains. Appl. Environ. Microbiol.

2012, 78(18):6630.

Geothrix Ge'o.thrix. Gr. n. gê earth; Gr. fem. n. thrix hair; N.L. fem. n. Geothrix hair of earth, refers to the cell morphology under fumarate-reducing conditions. Acidobacteria / Holophagae / Holophagales / Holophagaceae / Geothrix. Rod-shaped cells 0.1 µm × 1–2 µm. Nonphotosynthetic. Nonsporeforming. Cells occur singly and in chains. Anaerobic, having an obligately anaerobic metabolism that can be either fermentative or respiratory using nitrate, Mn(IV), anthraquinone-2,6-disulfonate (AQDS), poorly crystalline iron(III) oxide, and iron(III) chelated with nitrilotriacetic acid [Fe(III)-NTA] or citrate as alternative electron acceptors. Chemoorganotrophic. Growth on yeast extract and organic acids including acetate and lactate, but not on alcohols including ethanol. Capable of fermenting organic acids including citrate and fumarate. End products of citrate fermentation are acetate and succinate. Optimal growth is 35–40°C. No growth at or below 25°C. Nonmotile. Contains c-type cytochromes. DNA G+C content (mol%): unknown. Type species: Geothrix fermentans Coates, Ellis, Gaw and Lovley 1999, 1620VP. Thrash, J. C. and Coates, J. D. 2015. Geothrix . Bergey's Manual of Systematics of Archaea and Bacteria. 1–2.

Longilinea Strictly anaerobic, filamentous bacteria of the phylum Chloroflexi isolated from methanogenic propionate-degrading consortia. Yamada T, Imachi H, Ohashi A, Harada H, Hanada S, Kamagata Y, Sekiguchi Y. Bellilinea caldifistulae gen. nov., sp. nov. and Longilinea arvoryzae gen. nov., sp. nov., strictly anaerobic, filamentous bacteria of the phylum Chloroflexi isolated from methanogenic propionate-degrading consortia. Int J Syst Evol Microbiol. 2007 Oct;57(Pt 10):2299-306.

Magnetospirillum Gram-negative, microaerophilic genus of magnetotactic bacterium. This is a biologically, ecologically, and commercially relevant group of microbes that synthesizes high quality single-domain magnetite crystals. Abiotic reductive dechlorination of chlorinated ethylenes (tetrachloroethylene (PCE), trichloroethylene (TCE), cis-dichloroethylene (cis-DCE), and vinyl chloride (VC)) is facilitated by magnetite. Lee W, and Batchelor B. 2002. Abiotic reductive dechlorination of chlorinated ethylenes by iron-bearing soil minerals. Pyrite and magnetite. Environ Sci Technol. 2002 Dec 1;36(23):5147-54.

Methylocystis Me.thyl.o.cys' tis. Fr. methyle the methyl radical; cystis bag; M.L. n. Methylocystis methyl bag. Proteobacteria / Alphaproteobacteria / Rhizobiales / Methylocystaceae / Methylocystis. Cells are small, rod-like to reniform in shape, 0.3–0.5 × 0.5–1.5 µm, usually arranged singly. Reproduce by binary division. Nonmotile. Cells contain type II intracytoplasmic membranes, which are arranged as multiple layers along the periphery of the cell wall. May form cylindrical spinae. Cells may contain a desiccation-resistant lipoidal cyst resting stage. Form inclusions of poly-β-hydroxybutyrate. Aerobic, possessing a strictly respiratory type of metabolism with oxygen as the terminal electron acceptor. Obligately methanotrophic, utilizing only methane and methanol as sole carbon and energy sources. C2+ and other C1 compounds are not utilized. Fixes formaldehyde for cell carbon via the serine pathway. Enzymes for the Benson-Calvin cycle pathway are absent and the tricarboxylic acid pathway is complete. Fixes atmospheric nitrogen. Mesophilic, neutrophilic, and nonhalophilic ecophysiology with optimal growth at about 25–30°C and pH 7.0. Major habitats include rice paddy soils, sewage, and freshwater sediments. Primary fatty acids are C18:1 ω8c and C18:1 ω7c. Primary quinone is ubiquinone-8 (Q-8). Member of the family Methylocystaceae in the Alphaproteobacteria. The mol% G + C of the DNA is: 61– 67 (Tm). Type species: Methylocystis parvus (ex Romanovskaya, Malashenko and Bogachenko 1978) Bowman, Sly, Nichols and Hayward 1993c, 751. Bowman, J. P. 2015. Methylocystis. Bergey's Manual of Systematics of Archaea and Bacteria.

1–7.

Nitrosovibrio Ni.tro.so.vib' ri.o. M.L. adj. nitrosus nitrous; L. v. vibrio to move rapidly to and from, to vibrate; M.L. masc. n. Nitrosovibrio a vibrio producing nitrite. Proteobacteria / Betaproteobacteria / Nitrosomonadales / Nitrosomonadaceae / Nitrosovibrio. Slender, curved rods (Figure 1a), 0.3–0.4 × 1.1–3.0 µm. Spherical forms, 1.0–1.2 µm in diameter, may occur in cultures. Gram-negative cell wall. Intracytoplasmic membranes are rare (Figure 1b), but when present, appear as tubular invaginations. Motile cells possess 1–4 subpolar to lateral flagella (Figure 2), about 18 nm wide and 4.2–7.5 µm long. Carboxysomes observed in one strain. Most, but not all, strains are urease positive. Commonly distributed in oligotrophic soils, such as grasslands, heath, and forest soils, as well as in mountainous environments. Some isolates originate from acid tea soils and from building stones. The mol% G + C of the DNA is: 53.9. Type species: “Nitrosovibrio tenuis” Harms, Koops and Wehrmann 1976, 110. Koops, H.-P. and Pommerening-Röser, A. 2015. “Nitrosovibrio”. Bergey's Manual of

Systematics of Archaea and Bacteria. 1–2.

Nordella Genus contains three species and was isolated via amoebal co-cultures . La Scola, B., Barrassi, L., and Raoult, D. ."A novel alpha-Proteobacterium, Nordella oligomobilis gen. nov., sp. nov., isolated by using amoebal co-cultures.." Res. Micriobiol.

(2004) 155:47-51.

Opitutus O.pi.tu'tus. L. fem. n. Ops, Opis a Roman Earth and harvest goddess; L. part. adj. tutus protected; N.L. masc. n. Opitutus the one protected by Ops. Verrucomicrobia / Opitutae / Opitutales / Opitutaceae / Opitutus. Cocci or coccobacilli. Stain Gram-negative. Motile with flagellum. No endospores. Anaerobe; media containing a suitable reductant shorten the lag phase. Chemo-organotrophic metabolism. Monosaccharides, disaccharides, and polysaccharides are fermented, but alcohols, amino acids, and organic acids are not. Acetate, propionate, CO2, and H2 are the fermentation end products; the ratios of these are dependent on the partial pressure of H2. Nitrate is reduced to nitrite. Sulfate, sulfur, thiosulfate, and fumarate are not used as terminal electron acceptors. DNA G+C content (mol%): 65. Type species: Opitutus terrae Chin, Liesack and Janssen 2001, 1968VP. Janssen, P. H. 2015. Opitutus. Bergey's Manual of Systematics of Archaea and Bacteria. 1–3.

Pelotomaculum Group of anaerobic Gram-positive, spore-forming, syntrophic propionate-oxidizing bacterium. Is an obligate syntroph with methanogenic bacteria. de Bok et al. 2005. The first true obligately syntrophic propionate-oxidizing bacterium,

Pelotomaculum schinkii sp. nov., co-cultured with Methanospirillum hungatei, and emended description of the genus Pelotomaculum. Int J Syst Evol Microbiol. 55(Pt 4):1697-703.

Prochlorococcus a genus of very small (0.6 µm) gram negative marine cyanobacteria with an unusual pigmentation (chlorophyll b). These bacteria belong to the photosynthetic picoplankton and are probably the most abundant photosynthetic organism on Earth. Microbes of the genus Prochlorococcus are among the major primary producers in the ocean, responsible for a large percentage of the photosynthetic production of oxygen.

Proteiniphilum Strictly anaerobic, Gram-negative. Isolated from the granule sludge of an upflow anaerobic sludge blanket reactor treating brewery wastewater. Chen S, Dong X. Proteiniphilum acetatigenes gen. nov., sp. nov., from a UASB reactor treating brewery wastewater. Int J Syst Evol Microbiol. 2005 Nov;55(Pt6):2257-61.

Pseudomonas A genus of Gram-negative, aerobic gammaproteobacteria, belonging to the family Pseudomonadaceae containing 191 validly described species. The members of the genus demonstrate a great deal of metabolic diversity. They have been identified in hydrocarbon degrading and other xenobiotics. Some members can degrade TCE (Chen et al 2007) and cis-DCE (Lu et al 2015). Chen YM, Lin T-F, Huang C, Lin J-C, and Hsieh F-M. 2007. Degradation of phenol and TCE using suspended and chitosan-bead immobilized Pseudomonas putida. Journal of Hazardous

Materials V 148, Issue 3, pp 660-670.

Lu Q, de Toledo RA, Xie F, Li J, Shim H. 2015. Combined removal of a BTEX, TCE, and cis-DCE mixture using Pseudomonas sp. immobilized on scrap tyres. Environ Sci Pollut

Res Int. 22(18):14043-9. doi: 10.1007/s11356-015-4644-y.

Rhodanobacter Gram negative, aerobic, at least one member can degrade lindane; γ-HCH. Type strain isolated from a wood treatment site. Rhodanobacter have been identified as acid tolerant denitrifiers. Green SJ, Prakash O, Jasrotia P, et al. Denitrifying Bacteria from the Genus Rhodanobacter

Dominate Bacterial Communities in the Highly Contaminated Subsurface of a Nuclear Legacy

Waste Site. Applied and Environmental Microbiology. 2012;78(4):1039-1047. doi:10.1128/AEM.06435-11.

Rhodoblastus Rho.do.blas' tus. Gr. n. rhodon the rose; Gr. n. blastos bud shoot; M.L. masc. n. Rhodoblastus the budding rose. Proteobacteria / Alphaproteobacteria / Rhizobiales / Bradyrhizobiaceae / Rhodoblastus. Cells are rod shaped, motile by means of flagella; show polar growth, budding, and asymmetric cell division. Gram negative and belong to the Alphaproteobacteria. Internal photosynthetic membranes appear as lamellae underlying and parallel to the cytoplasmic membrane. Photosynthetic pigments are bacteriochlorophyll a and carotenoids. Straight-chain monounsaturated C18:1, C16:1, and saturated C16:0 are the major cellular fatty acids. Contain ubiquinones, rhodoquinones, and menaquinones with 10 isoprene units (Q-10, MK-10, and RQ-10). The mol% G + C of the DNA is: 62.2–66.8. Type species: Rhodoblastus acidophilus (Rhodopseudomonas acidophila Pfennig 1969a, 601) Imhoff 2001, 1865. Imhoff, J. F. 2015. Rhodoblastus. Bergey's Manual of Systematics of

Archaea and Bacteria. 1–11.

Rhodoferax: Gram-negative rods, ranging in diameter from 0.5 to 0.9 µm with a single polar flagellum. The first two species described for the genus, R. fermentans and R. antarcticus, are facultative photoheterotrophs that can grow anaerobically when exposed to light and aerobically under dark conditions at atmospheric levels of oxygen. R. ferrireducens is a non-phototrophic facultative anaerobe capable of reducing Fe(III) at temperatures as low as 4°C. All Rhodoferax species possess ubiquinone and rhodoquinone derivatives with eight unit isoprenoid side chains.

Sedimentibacter Se.di.men.ti.bac'ter. N.L. masc. n. sedimentum sediment; N.L. n. bacter masc. equivalent of Gr. Neut. Dim. N. bakterion rod or staff; N.L. masc. n. Sedimentibacter rod from sediment, referring to its origin. Firmicutes / “Clostridia” / Clostridiales / incertae Sedis - Family I / Sedimentibacter Slightly curved rod-shaped cells found singly or in chains, 0.35 × 7 μm, oval spores formed from terminal swollen sporangia. Gram-stain-positive or -negative. Motile by means of peritrichous flagella. Strict anaerobe. Growth between 12 and 41°C with optimal growth at 33–37°C. pH optimum 7–8.2. Growth requires yeast extract and is supported by the fermentation of pyruvate or of amino acids in a Stickland-type reaction. H2 not produced. Carbohydrates not fermented. Purines including uric acid, adenine, hypoxanthine, guanine, and xanthine are not utilized. Catalase and urease are absent. The cell-wall type is A1α (l-lysine, direct). Menaquinones are not present. The genus represents a separate line of descent within the Peptostreptococcaceae according to 16S rRNA gene sequence analysis. DNA G+C content (mol%): 34–35.5. Type species: Sedimentibacter hydroxybenzoicus (Zhang, Mandelco and Wiegel 1994) Breitenstein, Wiegel, Haertig, Weiss, Andreesen and Lechner 2002, 806VP (Clostridium hydroxybenzoicum Zhang, Mandelco and Wiegel 1994, 218).

Spirochaeta Group of free-living, Gram negative, saccharolytic non-pathogenic, obligate or facultative anaerobic helical shaped bacteria. Sphaerochaeta bacteria extract energy from sugars by fermentation, generating a mixture of waste products that include acetate and H2. Dehalococcoides have a strict requirement for acetate as a carbon source, and they must use hydrogen as the electron donor for anaerobic respiration of organic chlorides. Members of Sphaerochaeta may provide these critical substrates to Dehalococcoides.

Sulfuricella Facultatively anaerobic, sulfur-oxidizing bacterium.

Treponema Spiral-shaped bacteria. Spirochaetes / Spirochaetia / Spirochaetales / Spirochaetaceae / Treponema Host-associated, helical cells 0.1–0.7 μm in diameter and 1–20 μm in length. Cells have tight regular or irregular spirals and one or more periplasmic flagella (axial fibrils or axial filaments) inserted at each end of the protoplasmic cylinder. Cytoplasmic filaments are seen in the protoplasmic cylinder just under the cytoplasmic membrane and running parallel with the periplasmic flagella. Under unfavorable cultural or environmental conditions, spherical cells are formed. These can also be seen in old cultures. Gram-stain-negative. Cells stain well with silver impregnation methods. Most species stain poorly, if at all, with Gram or Giemsa stain. Best observed with darkfield or phase-contrast microscopy. Motile. Cells have rotational movement in liquid media, and translational motion in media with high viscosity [e.g., those containing 1% (w/v) methyl cellulose]. In a semisolid or solid medium, cells exhibit a serpentine type movement, sometimes referred to as creeping motility. Strictly anaerobic or microaerophilic. Frank pathogens (Treponema pallidum subspecies, Treponema carateum, and the rabbit pathogen Treponema paraluiscuniculi) represent a closely related subset within this genus and are considered microaerophiles. Limited multiplication of Treponema pallidum subsp. pallidum strains has been obtained in a tissue culture system, but none of the pathogenic Treponema have been cultivated continuously in artificial media or in tissue culture. Chemo-organotrophs, using a variety of carbohydrates or amino acids for carbon and energy sources. Cultivated anaerobic species are catalase- and oxidase-negative. Some require long-chain fatty acids found in serum for growth, while other cultivated species require short-chain volatile fatty acids for growth. Host-associated. Pathogenic Treponema pallidum subspecies cause skin lesions, and Treponema pallidum (particularly subspecies pallidum) can cause systemic infections that, if untreated, can last for years to decades. Other species are found in the oral cavity, intestinal tract, and genital areas of humans or other mammals, and in the gut contents of wood-feeding insects. DNA G+C content (mol%): 37– 54. Norris, S. J., Paster, B. J. and Smibert, R. M. 2015. Treponema. Bergey's Manual of

Systematics of Archaea and Bacteria. 1–42.

Verrucomicrobium Heterotrophic and can ferment sugars, anaerobic, Gram-negative, nonmotile bacteria with appendages called prosthecae that can be wart-like or long and extended in shape (MicrobeWiki 2016).

Requested Requested

Address: Address: Phone: Phone:

Email Email

Company: Company:

Required Required

Section Section

f f

aeAnalytical aeAnalytical

12 12

10 10

11 11

!:: !::

w w

:E :E

'It 'It

9 9

7 7

2 2

4 4

6 6

8 8

3 3

5 5

1 1

To: To:

Required Required

Section Section

443-688-6453 443-688-6453

A A

Client Client

Due Due

Sample Sample

*Important *Important

Millersville, Millersville,

[email protected] [email protected]

401 401

CSI CSI

Information: Information:

Date/TAT: Date/TAT:

SAMPLE SAMPLE

D D

Client Client

ADDITIONAL ADDITIONAL

IDs IDs

Headquaters Headquaters

Environmental Environmental

(A-Z, (A-Z,

Note: Note:

Information Information

MUST MUST

p,'J(;":;/_.,/;;, p,'J(;":;/_.,/;;,

0-9/ 0-9/

By By

MD MD

signing signing

BE BE

lFax: lFax:

,-) ,-)

COMMENTS COMMENTS

Standard Standard

t."t,J~I t."t,J~I

ID ID

AN-MW-1 AN-MW-1

AE-MW-3 AE-MW-3

21108 21108

UNIQUE UNIQUE

Drive Drive

this this

443-688-6725 443-688-6725

form form

Suite Suite

you you

WATER WATER

WIPE WIPE

TISSUE TISSUE OTHER OTHER

WASTEWATER WASTEWATER

AIR AIR

OIL OIL

SOIUSOLIO SOIUSOLIO

DRINKING DRINKING

PRODUCT PRODUCT

MATRIX MATRIX

Valid Valid

are are

203 203

accepting accepting

Matrix Matrix

WATER WATER

Codes Codes

Pace's Pace's

AR AR

WP WP

OT OT

WT WT OL OL

SL SL

TS TS

WW WW

OW OW

p p

Project Project

Purchase Purchase

Copy Copy

Report Report

Required Required

Section Section

CODE CODE

NET NET

To: To:

/XN1{}'1 /XN1{}'1

To: To:

Name: Name:

Number: Number:

30 30

, ,

Order Order

B B

Project Project

RELl~QUISHED RELl~QUISHED

day day

CSI CSI

WT WT

u u

::,; ::,;

0 0

I- <( <(

a:: a::

X X

0 0

w w

e e

u u

i i

~ ~

u u

ID ID

~ ~ 0 0

0 0 u u

payment payment

No No

Environmental Environmental

A A

Biotreatability Biotreatability

Information: Information:

(J) (J)

::;; ::;;

<( <(

Q_ Q_

~ ~

0 0 w w ('.) ('.)

~ ~

Q_ Q_

w w

...J ...J 0 0

::;; ::;;

0 0

ii:" ii:"

0:: 0::

<( <(

(D (D

G G

II II

.. ..

II II

15-005 15-005

terms terms

DATE DATE

xxxxx xxxxx

xxxxx xxxxx xxxxx xxxxx

COMPOSITE COMPOSITE

and and

BY/ BY/

)kLtt )kLtt

START START

agreeing agreeing

~ ~

AFFILIATION AFFILIATION

SAMPLER SAMPLER

xxxxx xxxxx

TIME TIME

COLLECTED COLLECTED

to to

late late

PRINT PRINT

SIGNATURE SIGNATURE

The The

CHAIN-OF-CUSTODY/ CHAIN-OF-CUSTODY/

charges charges

04/26/17 04/26/17

NAME NAME

DATE DATE

COMPOSITE COMPOSITE

Chain-of-Custody Chain-of-Custody

ENO/GRAB ENO/GRAB

Name Name

of of

AND AND

1.5% 1.5%

,f/~£1 ,f/~£1

of of

'1/J.l/1'1 '1/J.l/1'1

11.jLJ 11.jLJ

',.;; ',.;;

,.,lff ,.,lff

TIME TIME

SIGNATURE SIGNATURE

SAMPLER: SAMPLER:

per per

DATE DATE

month month

\ \

is is

(J) (J)

r r

0 0

w w ::;; ::;; 0 0

::;; ::;;

<( <( w w

r r

0 0 Q_ Q_ <( <(

...J ...J

Q_ Q_

0 0

z z

...J ...J

i= i=

w w

for for

a a

any any

LEGAL LEGAL

Manager: Manager:

Pace Pace

Reference: Reference:

Attention: Attention:

&'?a &'?a Address: Address:

Pace Pace

Company Company

Invoice Invoice

Section Section

1<,i.iJL 1<,i.iJL

0 0

'It 'It

0 0

u u

1,111 1,111

er: er: (f) (f)

z z

~ ~

LJ.. LJ..

z z

w w

6 6

4 4

invoices invoices

1,-\Yl 1,-\Yl

TIME TIME

Project Project

Profile Profile

Quote Quote

::JI ::JI

"O "O

X X

(/) (/)

X X

Q) Q)

C C 0. 0.

~ ~

Q) Q)

DOCUMENT. DOCUMENT.

Information· Information·

( (

Name: Name:

C C

(/) (/)

0 0

not not

#: #:

" "

N N

j''\, j''\,

paid paid

Preservatives Preservatives

V}'\ V}'\

0 0

I I

z z

401 401

Ruth Ruth

Kevin Kevin

Analytical Analytical

within within

u"' u"'

CSI CSI

I I -

H H

Headquaters Headquaters

Welsh Welsh , ,

All All

Costello Costello

0 0

Z Z

I I

l, l,

30 30

r~ r~

Environmental Environmental

relevant relevant

L, L,

days. days.

ACCEPTED ACCEPTED

0 0

z z

c!J c!J

cE' cE'

C-ft'i>_ C-ft'i>_

t t

.c .c ~o ~o

0 0

C C

"' "'

Q) Q)

fields fields

.c .c

Q) Q)

~ ~

X X

Drive Drive

Request Request

......

...... BY/ BY/

iij iij

<( <(

·;;; ·;;;

I- ......

> >

must must

s:: s::

z z

rn rn

>, >,

rn rn

Q) Q)

Suite Suite

AFFILIATION AFFILIATION

.c .c

___J ___J

~ ~

ai ai

cu cu

C C

Q) Q) X X

X X

DATE DATE

(MM/DD/YY): (MM/DD/YY):

be be

Requested Requested

completed completed

:.a :.a

m m

Cl) Cl)

~ ~

·u ·u

Q) Q)

e e

0 0

Q) Q)

O" O"

C C

:J :J

C C

Ol Ol X X X X

X X

203 203

Signed Signed

; ;

___J ___J

Document Document

C C

accurately. accurately.

' '

Analysis Analysis

'I/.).{//"/ 'I/.).{//"/

REGULATORY REGULATORY

I I

Site Site

-·· -··

IY IY

Jct,;, Jct,;,

NPDES NPDES

UST UST

Location Location

DATE DATE

STATE: STATE:

, ,

Filtered Filtered

/F-/7 /F-/7

AGENCY AGENCY

(Y/N) (Y/N)

TIME TIME

GROUND GROUND

RCRA RCRA

(Page: (Page:

NJ NJ

F-ALL-Q-020rev.08, F-ALL-Q-020rev.08,

·-

r r

~ ~

C C

E E

(I) (I)

a. a.

WATER WATER

rn rn :c :c

·;;; ·;;; 0::: 0:::

u u

"O "O

z z ~ ~

-~ -~

:J :J

C C

Q) Q)

N N

"O "O

0:: 0::

·m ·m

~ ~

(I) (I)

u u

0 0

C C

Pace Pace

SAMPLE SAMPLE

z z

u u Q) Q)

' '

f;t f;t

of of

Project Project

DRINKING DRINKING

12-0ct-2007 12-0ct-2007

![ ![

0 0

"O "O

"O "O ~8 ~8

CONDITIONS CONDITIONS

OTHER OTHER

(I)- >, >,

0 0 0 0

~ ~

No./ No./

WATER WATER

Lab Lab

;z ;z

(J) (J)

~ ~

t5 t5

~ ~

"' "'

E E

a.-

:;:: :;:: I.D. I.D. \_:_,

,,~

-(.,-l

r-1 r::

C) ~-j

Cl(II•

1!:I

!! \'I ''I 1! ,, [:j 1!1

(, i\ \''

11 t \' ~ II l

11 )I

II I:

·u

..G .Y.

0 ./' :?1

{lJ

(0 ,_ L 0 ·o L.

•

>-J u ~~: ,_ UJ

S,J

C: [: rl\ CU

-1-'

,,_ '.i.:i U\ UJ ~ C 0 U\ CL ..c ,_ ,u l]J

(fl l

:~ CL ,,,c LI 0. -~I (\J

LL v, u ~ ,u

Ll to C I 0 0 ,_

r: /0 (\J ~

C CJ .. 0 C - CL

-0-

(~ _C:\

-1-'

1/\ 0 ,u

,[\_ L ,;, (\l

<\l 0 C

,,J

u, LI)

Q :::J u ,,J Lil

uJ Tl :=i

LL {U L

'C:

(lJ u C

)( ,u

0 :i {)J ,_

_ti

I- _.::.~

L E (\J

C .:i

[ll lLO

_"]

.1-1

Lil 0

...... U\ 7

l\J ,';,- ,\1 1;

C: CL .i-i {[J

ill 'll CD

0 u,

(l) l/\ LI

11, LlJ

)( LU -J-l C ,_ 0

UJ C [~!

QJ 0 m

;:i ::1 0

l/1

...

-1:-. 6

.I:l

J:,

,IJ

(t) L .D ...L:t <( 3

{jj E _[~ Ill

.,.J U1 C 2: UJ 0 L r~

I\) CL [l.. u::, LI

L. :i

fl! ,u llJJ

:i(

C /0

0 u

r'.l ,ll

-c.1

?. ';

.j.J ·8

w -, ,,,

L,_

-:;: U)

CLJ ,,_ > rn Ul

Q.1 1:- C

u ,JJ

C: 0 t!J :J

f--' ill

,u u 0 ill

Cl.

'I··

~L:_ CL

u ,,J

u -1-'

L) l.U

C U\ ql

>- ,u 0 LC C ~J 0 :r.J [/J '"

[,' :\l u,

u ,u LU

C UI C u :o 1-- ,u

C_) u u

1lJ 1-\·) \J ,_ .J ,_ E cu ' !.'L IU 0 (L\ 0

I..J .:~

E :IJ 0 E c::

. -. 'C L

.:..:

._J

(ij u;

C ,_ C1. C

-1-' U\

m {L,' ,u

.L1 <]. ,__ CL CL 0

[L) __ u CL fl_) u ,JJ l]J

IUJ C

L 0 . f:: UJ lUJ c· v, ll) C

/1J

IU 0 0 ,_ ...J

----1----l-~-f------1------l--l·

+----'

>- Lf\ I.LI z

C. E Ill

,['' L)

/.c

L' ,l,

[ i"l~ () C: u C C 0 ,_ C LU C u l\.J (U _,

----1--,1

______

--/~-

-o li=

+-·

,,_

_(_

,,J CJ ::J (\J 0 ,.,..

0 0. ll.l ~">- ,_ 0,

::J 0

0 l/\

~ c

.1.:

_L_ "O

(_l

U\ il.J ::i

o· ~~ C

D ::J

---

--1--

[Cl. I

---1,-

0 u u

L,')

z --

L. ,,: ,_, 0 ::i

(IJ (\J L11l

L\J ru E

::C) /U

-!----/-~/

----.-~

---·1

,,~ ·l-·

(_}

L' ,n

C !3 C "-1------

l(J \_

t_)

I l'>

,~ 10 Ltil

ill 0 L1

CL m ,1J C UI

U\ JO

U\ OJ CL

l]J t: ~

--1------

·1-··---~1--1--·---1-··------.---

1; Cl u LI

1..1}

I/\ u ---!- LO t: JU

J~ ~

0 m C

CU /0 CL U I::

---1·---!------1----

-~,,

'D __

·1-l

.J___l

v·1 8 '.1: Qj

;~~ E: OJ

Ill

1:: J}J LQ f::

(ll

CL C

,--

LJ -o

_.:.~

U) ·c

I\J 6

E Q, llJ

,- '? t.U

::J

-<--

l/1 ,,(

CL -1-J

I/': (IJ L ::i V1

{O LU 0 C u '

(c,

-o

-1-> '+:._ .1:= ·o -,~ ,,J

C::

(\/j

C U\

(lJ

L\J ,u ill u, :..J 1J CT

L ill

-1-1 U\ 2: CU (\) <( >· CL ,u

l\J L Ci_

VI L Ct. 0 L\J L sc:;;··c:i 0 ro

L, Um_ilJU\·L l]J

- .D

Gl u11m /[j

'.::i .0 J~

·,. ~/\ ~I :Jj D

{

_c ~ i::.1

,\J

OJ t;

./...J .~ ~-. '--1- .X c1J u I. r..: u ,1, C OJ (0 Ill 0

L (\j

1\1 (1J

[ 0 ~ll - !~ C

=t i.l\

= L C

~l 'i '-!-

·- _ 0 ---..

L_L/

~ f:: __;. u

:~ O )-( C U

-o

c,=

~o ·I-' l~

-~:-..'. TJ -o'

2 ~~ '-1-- ·!__l 0

-l....J C ...::...

l:'I (IJ

iLl o C (IJ

11J -::::-

L\J) C UJ

(lJ (lj L11

E>i'.l C)

(IJ 0

CU > -J

~, ·1-

·o

-~:: -o .

.L-.

ro (U C

_cu ('-. c Q l_J Q u ll ...::: 01 w LL. L u, ill

C ((}

u llJ ,flJ >

d.1

[ (IJ ::J

Cl U\

J-l llJ

l_j

1 C

.. I

0 SJ ·---.._ _[)

7"]

.r:".

-a

f(J

>---

,J- ~j JJ

LI Lll

' aJ

di u

:,( C

((\ E

0 C (1) ,u c

(i u

C.:., -1-1

·-, !]J 10

~ t 3:.

ru bo m

f(J ~

ili 0 L

(:, 1·-;.--

(·,\ -~1-

C> ('.\ ·~' !,~)

n-1 I cY ,_, Cf\ j (.;\

~cl

nl )·-,

' ' I

~1 p_t•£S -::;./ ork 016eJ ~ · ------

F:ecc ~ ·/-:-r: ______'. ...J:3..'LC: ------