Polycyclic aromatic hydrocarbon concentrations,mutagenicity, andMicrotox® acute toxicity testing of Peruviancrude oil and oil-contaminated water and sediment

Evelyn G. Reátegui-Zirena & Paul M. Stewart &Alicia Whatley & Fred Chu-Koo & Victor E. Sotero-Solis &Claudia Merino-Zegarra & Elías Vela-Paima

Received: 13 June 2013 /Accepted: 6 November 2013 /Published online: 30 November 2013# Springer Science+Business Media Dordrecht 2013

Abstract The oil industry is a major source of contam-ination in Peru, and wastewater and sediments contain-ing oil include harmful substances that may have acuteand chronic effects. This study determined polycyclicaromatic hydrocarbon (PAH) concentrations by GC/MS, mutagenicity using TA98 and TA100 bacterialstrains with and without metabolic activation in theMuta-ChromoPlate™ test, and Microtox® 5-min EC50

values of Peruvian crude oil, and water and sedimentpore water from the vicinity of San José de Saramuro onthe Marañón River and Villa Trompeteros on theCorrientes River in Loreto, Peru. The highest totalPAH concentration in both areas was found in water(Saramuro=210.15 μg/ml, Trompeteros=204.66 μg/ml). Total PAH concentrations in water from San Joséde Saramuro ranged from 9.90 to 210.15 μg/ml (mean=66.48 μg/ml), while sediment pore water concentrations

ranged from 2.19 to 70.41 μg/ml (mean=24.33 μg/ml).All water samples tested from Saramuro andTrompeteros sites, and one out of four sediment porewater samples from Trompeteros, were found to bemutagenic (P<0.001). One sediment pore water samplein Saramuro was determined to have a measurable tox-icity (Microtox EC50=335.1 mg/l), and in Trompeteros,the EC50 in water and sediment pore water ranged from25.67 to 133.86 mg/l. Peruvian crude oil was mutagenicusing the TA98 strain with metabolic activation, and theEC50 was 17.18 mg/l. The two areas sampled had veryhigh PAH concentrations that were most likely associ-ated with oil activities, but did not lead to acute toxiceffects. However, since most of the samples were mu-tagenic, it is thought that there is a greater potential forchronic effects.

Keywords Peruvian crude oil . PAH concentrations .

Mutagenicity . Microtox acute toxicity

Introduction

Oil industry activities, such as exploration, transporta-tion, storage, use, and disposal, are sources of majorcontamination problems in the Amazon Region ofSouth America. For instance, in 2003, one of the world'slargest integrated energy companies (Texaco), laterbought by Chevron, was sued by Ecuadorian residentsfor dumping and spilling toxic waste and oil,abandoning waste pits, and burning gases in theEcuadorian Amazonian rainforest in the 1970s. This

Environ Monit Assess (2014) 186:2171–2184DOI 10.1007/s10661-013-3527-2

E. G. Reátegui-Zirena : P. M. Stewart (*) :A. WhatleyDepartment of Biological and Environmental SciencesTroy University,Troy, AL 36082, USAe-mail: mstewart@troy.edu

E. G. Reátegui-ZirenaDepartment of Environmental Toxicology, The Institute ofEnvironmental and Human Health, Texas Tech University,Lubbock, TX 79409, USA

F. Chu-Koo :V. E. Sotero-Solis : C. Merino-Zegarra :E. Vela-PaimaInstituto de Investigaciones de la Amazonía Peruana (IIAP),Programa para el Uso y Conservación del Agua y susRecursos–AQUAREC,Iquitos, Perú

case was settled, pending appeal (2010), when Chevronwas ordered to pay an $8.6 billion fine (WSJ 2011).

Oil-contaminated wastewater contains polycyclic ar-omatic hydrocarbons (PAHs) and other harmful sub-stances that may have chronic effects includinggenotoxic impacts on DNA structure (Bohne andCathomen 2008). Genotoxicity studies in Ecuador, onthe Amazonian human population close to crude oilextraction zones have shown DNA damage such as typeB nuclei fragmentation and chromosomal aberrations(Paz-y-Miño et al. 2012). Numerous spills and leakagesinvolving petroleum have occurred in Brazilian rivers,and genotoxicity assays have also been performed. Forinstance, chromosomal aberration assays on onionAllium cepa exposed to petroleum polluted watershowed breaks in chromosomes and changes in chro-mosome number (Leme et al. 2008). Nuclear degenera-tion and bi-nucleated hepatocytes were found in marinepejerrey Odontesthes argentinensis exposed to water-soluble fractions (WSFs) of diesel and gasoline(Rodrigues et al. 2010).

Mutagenicity is a critical step in genotoxic carcino-genesis development, and several PAHs have beenfound to be mutagenic, leading to possible human car-cinogenesis (IARC 1983). A very common test to iden-tify environmental mutagens and potential carcinogensis the Muta-ChromoPlate™ test, which uses a mutantstrain of Salmonella typhimurium that carries mutationin the operon coding for histidine biosynthesis (Zeigerand Mortelmans 1999). Specific strains may be used todetect either frameshift mutations (strain TA98, TA97)or base pair substitutions (strain TA100, TA102; Maronand Ames 1983). While there are data on the mutagenic/carcinogenic potential of specific PAHs, there is notsimilar information on Peruvian crude oil and oil-contaminated water and sediment from the PeruvianAmazon.

Not only is there a lack of information on PAHconcentrations and mutagenic activities in Peruviancrude oil, but also there is no acute toxicity informationthat can be used to prescreen polluted samples in thisregion. The Microtox® system, an assay based on theinhibition of light emitted by the bioluminescent marinebacteria Vibrio fischeri, has been successfully used as ascreening system to detect the relative toxicity of manycontaminants from disparate areas including indus-trial waste, WSFs of crude oil (Ziolli and Jardim2002), and oil contaminated soil and sediment(Loureiro et al. 2005).

There is a definite lack of information regardingacute and chronic toxicity data that can be used to formthe scientific basis for regulatory development, riskassessment, and management for potential oil contami-nation problems in the Peruvian Amazon. This studydetermined PAH concentrations, mutagenicity, andEC50 values of Peruvian crude oil and water and sedi-ment from two contaminated areas in proximity to oilextraction and transportation in Loreto Region, Peru.

Methods

Study area

Water and sediment samples were collected from twoareas near oil-related activities, both about 200 km fromIquitos, the main Amazonian city in the Loreto Region,Peru (Fig. 1). Five sites were selected on the MarañónRiver near the town of San José de Saramuro, southwestof Iquitos (Fig. 2): site 1 (S4°42′37.0″, W 074°56′33.2″), site 2 (S 4°43′06.4″, W 074°55′33.6″), site 3 (S4°43′37.7″, W 074°55′08.5″), site 4 (S 4°44′28.3″, W074°54′34.1″), and site 5 (S 4°53′57.2″, W 074°54′41.7″). The Marañón River originates in the PeruvianAndes and its width varies from 800 to 2,600 m (about500 m at the sampling site). The bottom is composed ofsand, lime and clay, and depth varies seasonally from3 m in August to 8 m in April (IIAP 2002). San José deSaramuro (~2,000 inhabitants) is the first station of theNorth Peruvian oil pipeline (854 km long) that belongsto PetroPeru. The pipeline goes to the west across theAndes to the north coast of Peru, finally arriving atSechura Bay, on the Pacific coast (PetroPeru 2000).

Six sites were selected on the Corrientes River nearthe town of Villa Trompeteros, west of Iquitos (Fig. 3):site 1 (S 3°48′44.4″, W 075°04′29.9″); sites 2 and 3 (S3°48′51.6″, W 075°04′05.7″); site 4 (S 3°48′24.6″, W075°03′27.6″); site 5 (S 3°48′26.9″, W 075°01′47.9″);and site 6 (S 3°48′26.3″, W 075°01′31.6″) TheCorrientes River has its origins in the Ecuadorian high-lands, and it was about 100 m wide at the sampling site.Both the Marañón and Corrientes River have whitewater (i.e., high concentrations of sediments on thesurface, total suspended solids about 109 mg/l and highconductivity>150 μS; Barthem et al. 2003). TheCorrientes River drains to the Tigre River, which drainsto the Marañón River, a main tributary of the AmazonRiver. Villa Trompeteros is the nearest town to the oil

2172 Environ Monit Assess (2014) 186:2171–2184

activities complex called “Block 8” that belongs to theArgentinian oil and gas company, Pluspetrol Peru.Block 8 contains 29 native communities and 3,900inhabitants (Ministerio de Energía y Minas 2009).

Water sampling and analysis

Certified 1 L low-density polyethylene (LDPE) collaps-ible cubitainers (VWR International) were used for wa-ter sampling and rinsed with native water before use.

Grab samples were collected at ~15 cm depth. Sampleswere taken to the laboratory on ice and stored in the darkat 4 °C until PAH analysis, within 3 weeks. Sampleextracts were prepared using EPA method 550(USEPA 1990). Water samples were extracted usingmethylene chloride and a Kuderna–Danish (K–D) con-centrator in a hot bath and stored in a Teflon-sealedscrew-cap borosilicate vial wrapped with aluminum foilto protect it from light, and stored at 4 °C, until used foranalysis.

Fig. 1 Map of Peru showing the location of the collecting sites on the Marañón and the Corrientes River in Loreto, Peru, sampled duringsummer 2011

Environ Monit Assess (2014) 186:2171–2184 2173

Fig. 2 Map of San José de Saramuro and five collection sites on the Marañón River in Loreto, Peru, sampled during summer 2011. Waterflows from west to east

Fig. 3 Map of Villa Trompeteros and six collection sites on the Corrientes River in Loreto, Peru, sampled during summer 2011.Water flowsfrom west to east

2174 Environ Monit Assess (2014) 186:2171–2184

Sediment sampling and analysis

A stainless steel bottom sampling Ekman dredge (Code 1097,LaMotte®) was used for collection of sediments. Each bottomsample was mixed and placed in a 1 L glass jar sealed with alid andParafilm®. Sampleswere taken to the laboratory on iceand stored in the dark at 4 °C until use, within 3 weeks(Shelton and Capel 1994).

Sample extracts were prepared using the NorthwestTotal Petroleum Hydrocarbon Identification analyticalmethod (NWTPH-HCID; Oregon Department ofEnvironmental Quality 1996). Sediment samples wereextracted using methylene chloride and a sonic bath andstored in a Teflon-sealed screw-cap vial wrapped withaluminum foil to protect it from light, and stored at 4 °Cuntil used for analysis.

Analysis of PAHs

PAHs fromwater and sediment pore water samples wereanalyzed in triplicate using a gas chromatograph/massspectrometer (GC/MS) VARIAN 450 (detection limit:1 μg/l) following EPA method 827 °C (USEPA 1996).Quantification was done using the EPA 610-N PAHmixfrom the company Sigma-Aldrich to determine 16PAHs. The mix included naphthalene, acenaphthylene,acenaphthene, fluorene, phenanthrene, anthracene, fluo-ranthene, pyrene, benz[a]anthracene, chrysene,benzo[b]fluoranthene, benzo[k]f luoranthene,benzo[a]pyrene (BaP), dibenzo[a,h]anthracene,indeno[1,2,3-cd]pyrene, and benzo[ghi]perylene (orga-nized by molecular weight).

Preparation of water accommodated fraction

Peruvian crude oil (obtained from PetroPeru) is a heavy,sour variety with 1.2 % sulfur content and 20° AmericanPetroleum Institute gravity (API). API is an inversemeasure of petroleum and water (Kuramoto 2008).The Peruvian crude oil water accommodated fraction(WAF)—a solution free of particles of bulk material(i.e., droplets≥1 μm diameter) derived from mixing(no vortex) test material and water–was prepared inaccordance with the procedure described by Aurandand Coelho (1996). A 2-L borosilicate glass aspiratorbottle (Thomas Scientific) was used, with the sidearmclosed off with silicone tubing and a clamp. The bottlewas filled with 1 L of dilution water adding 200 g ofPeruvian crude oil, leaving a 20 % headspace. A stir bar

was used to stir the mix on a magnetic stir plate for 22 hin darkness. The mixed WAF was used for the mutage-nicity and Microtox® tests immediately after prepara-tion (Singer et al. 2001).

Muta-ChromoPlate™

Mutagenicity was tested using the Muta-ChromoPlate™kit, which is a liquid culture assay based on the Ames testthat uses mutant S. typhimurium strains that revert toamino acid histidine independence upon exposure tomutagens (EBPI 2005). Materials and chemicals werepurchased from Environmental Biodetection Products(EBPI in Canada). All samples (water, sediment porewater and crude oil) were prepared in duplicate, usingS. typhimurium test strain TA98, which detects frameshiftmutations, and TA100, which detects base pair substitu-tions following the Muta-ChromoPlate™ Basic kit pro-tocol (EBPI 2005). However, not all samples were testeddue to lack of reagents and plates. Samples were chosenaccording to proximity to main pipelines.

The reaction mixture was prepared mixing 21.62 mlDavis Mingioli medium, 4.75 ml D-glucose, 2.38 mlbromocresol purple, 1.19 ml D-biotin, and 0.06 mlL-histidine. About 30 ml of the aqueous sample was filtersterilized using a 0.22 μm sterile filter. For sedimentsamples, 0.1 g of the sample was mixed with 0.5 mldimethyl sulfoxide (DMSO) and 17.5 ml of distilledwater, and then sterile-filtered. Samples were mixedwith water, reaction mixture, and bacterial suspension(TA98 and TA100) from the culture grown overnight.For each sample mixture, 200 μl aliquots were dis-pensed into each well of a 96-well microtitration plate.Prepared plates were covered with lids and sealed inairtight plastic bags to prevent evaporation. Two nega-tive controls (background samples), one for TA98 andanother for TA100, containing the reaction mixture,water, and the bacteria, were used in order to makecomparisons with the treatment plate. A blank, andpositive controls containing sodium azide (NaN3) and2-nitrofluorine (2-NF), two known direct-acting muta-gens, were also used. For the Peruvian crude oil, the S9fraction (a crude rat liver extract to activate metabolism)was added to the treatment plates, and a positive controlusing 2-amino anthracene (2-AA, requires enzymaticactivation) was used. Plates were incubated at 37 °Cfor 5 days. After the incubation period, plates werescored visually by counting yellow or turbid wells aspositives and purple wells were scored as negatives.

Environ Monit Assess (2014) 186:2171–2184 2175

Microtox®

The toxicity is expressed in terms of EC50 (half maximaleffective concentration; Doherty 2001). The Microtox®bacterial assay was used to determine 5-min EC50 valuesusing the Azur Environmental Basic Test protocol and aMicrobics M500 toxicity analyzer. Water samples wereanalyzed without extraction and sediment samples werecentrifuged for 1 h and the pore water produced wasanalyzed without extraction. Freeze-dried bacteria(available from Azur Environmental, previouslyMicrobics) were rehydrated immediately prior to usein testing (Doherty 2001). Phenol was used as a stan-dard, and the samples were run in triplicate.

Data analysis

Average and standard deviations for PAH concentra-tions were calculated from three replicates of each waterand sediment pore water sample. The mutagenicity ofthe sample was determined by comparing the number ofwells scored as positive in the background plate to thenumber of positive wells in the treatment plate (Zeigerand Mortelmans 1999). Statistical differences were de-termined using the table for analysis of results of fluc-tuation tests developed by Gilbert 1980; (EBPI 2005).The mutagenic ratio (MR) was determined as the num-ber of histidine revertants in a test plate divided by thenumber of spontaneous revertants of the negative con-trol (Lupi et al. 2009). The EC50 (half maximal effectiveconcentration) was determined by calculating the expo-sure concentration at which the ratio of the light lost bybioluminesent bacteria to the light remaining equals one(Azur Environmental Basic Test protocol).

Results

PAH concentrations

This study analyzed the concentration of 16 priorityPAHs and the total PAH concentration in water andsediment pore water samples from San José deSaramuro (S1–S5) on the Marañón River (Table 1)and Villa Trompeteros (T1–T6) on the CorrientesRiver (Table 2). Each of the 16 priority PAHs weredetected in at least one of the sites. The PAH concentra-tions in water samples from both sites ranged from 7.54

to 210.15 μg/ml and in sediment pore water samplesvaried from 2.19 to 70.41 μg/ml.

San José de Saramuro

No PAHs were detected in water at S1 and the total PAHconcentration in the rest of the sites ranged from7.54 μg/ml at S5 to 210.15 μg/ml at S3 (Table 1). ThePAH that contributed the most to the total concentrationin water from S2, S3, and S4 was dibenzo[a,h]anthra-cene; and from S5 was BaP. Total sediment pore waterPAH concentrations ranged from 2.19 μg/ml at S2 to70.41 μg/ml at S5. BaP was detected in all sedimentpore water samples with the highest concentration at S5,and at S1 and S2, it was the only PAH detected. The lowmolecular weight PAHs found were naphthalene, ace-naphthylene, acenaphthene, fluorene, phenanthrene, an-thracene, and fluoranthene, and these contributed about12% (water) and 23% (sediment pore water) of the totalPAH concentration in the samples from the area.

Villa Trompeteros

No PAHs were detected in water at T1, T2, T3, and T5,while fluoranthene was the only PAH detected at T4 at20.71 μg/ml. At T6, all 16 priority PAHs were detectedwith a total PAH concentration of 204.66 μg/ml, fromwhich anthracene contributed the most with 70.08 μg/ml (Table 2). On average, the PAHs with low molecularweight contributed the most in this sample, about 64 %.The PAHs detected in sediment pore water sampleswere fluoranthene, pyrene, benzo[k]fluoranthene, BaP,and dibenzo[a,h]anthracene. The total PAH concentra-tion in sediment pore water samples ranged from3.59 μg/ml at T1 to 67.33 μg/ml at T4. BaP was detect-ed in all sediment pore water samples, the highest con-centration was found at T4, and it was the most abun-dant PAH at T4, T5, and T6.

Muta-ChromoPlate™

Themutagenic profiles of samples in the two study areasare shown (Table 3). The revertant colonies in negativecontrol plates were six for TA98 and ten for TA100. Themutagenicity ratio (MR: number of histidine revertantsin a test plate divided by the number of spontaneousrevertants of the negative control) was higher for TA98in all the samples compared to TA100, except sedimentpore water samples from T1, T5, and T6. The three

2176 Environ Monit Assess (2014) 186:2171–2184

Table1

Sixteenpolycyclicarom

atichydrocarbons

(PAHs)and∑PA

Hconcentrations

inwater

andsedimentsamples

(chemically

extractedpore

water)from

five

collectionsiteson

the

Marañón

River

near

SanJosé

deSaramuroin

Loreto,Peru,sampled

during

summer

2011

PAHs

Water

Sedim

ent

S1

S2

S3

S4S5

S1

S2

S3

S4S5

Naphthalene

ndnd

ndnd

ndnd

Nd

ndnd

nd

Acenaphthylene

ndnd

ndnd

ndnd

Nd

ndnd

nd

Acenaphthene

ndnd

0.46

±0.79

ndnd

ndNd

ndnd

nd

Fluorene

ndnd

33.11±31.27

ndnd

ndNd

ndnd

nd

Phenanthrene

ndnd

3.82

±1.70

ndnd

ndNd

ndnd

22.14±38.35

Anthracene

ndnd

0.37

±0.64

3.13

±5.42

ndnd

Nd

ndnd

0.84

±1.45

Fluoranthene

ndnd

ndnd

ndnd

nd4.32

±3.96

1.12

±1.93

nd

Pyrene

ndnd

3.47

±0.74

4.03

±2.08

ndnd

nd3.86

±3.96

ndnd

Benz[a]anthracene

ndnd

1.19

±2.06

ndnd

ndnd

ndnd

11.80±20.44

Chrysene

ndnd

2.00

±0.99

12.48±6.44

ndnd

nd0.55

±0.96

nd0.39

±0.68

Benzo[b]fluoranthene

ndnd

18.61±10.29

13.87±12.21

1.41

±2.44

ndnd

0.44

±0.76

ndnd

Benzo[k]fluoranthene

ndnd

21.62±13.95

2.56

±2.98

ndnd

nd7.77

±0.75

nd23.33±21.31

Benzo[a]pyrene

nd1.23

±1.16

41.59±18.10

21.07±9.99

6.14

±2.09

2.56

±2.44

2.19

±1.91

16.42±1.40

12.03±5.05

20.95±10.89

Dibenzo[a,h]anthracene

nd8.67

±15.02

95.69±31.16

38.86±8.36

ndnd

ndnd

nd13.09±8.22

Indeno[1,2,3-cd]pyrene

ndnd

22.37±13.95

4.43

±1.57

ndnd

ndnd

ndnd

Benzo[ghi]perylene

ndnd

3.25

±1.57

6.38

±2.68

ndnd

ndnd

ndnd

∑PA

Hconcentration

nd9.90

210.15

104.81

7.54

2.56

2.19

33.36

13.14

70.41

Result(μg/ml)isthemeanof

threereplicates±standard

deviation

ndnotd

etected(detectio

nlim

it:1μg/l),∑

sum

Environ Monit Assess (2014) 186:2171–2184 2177

water samples tested from San José de Saramuro and thetwo water samples from Villa Trompeteros were muta-genic (P<0.001) with strain TA98 and TA100. One offour sediment pore water samples from VillaTrompeteros was mutagenic (P<0.001) for both strains.None of the water and sediment pore water sampleswere tested with S9 fraction (metabolic activation).Moreover, not all samples were tested due to lack ofreagents and plates.

The mutagenic profile of the WAF using Peruviancrude oil is shown in Table 4. Water-accommodatedsamples were tested in TA98 and TA100 strains, withand without S9 fraction (metabolic activation), and theMR varied from 0.13 to 1.46. Peruvian crude oil wasfound to be mutagenic (P<0.001) in bacterial strainTA98 containing S9 fraction.

Microtox®

The 5-min EC50 values for 11 water samples and ninesediment pore water samples are shown (Table 3). Ingeneral, samples ranged from 25.67 to 335.1 mg/l. One

water sample of the total 11 had an EC50 of 133.86 mg/l(T4), and the EC50s of three of the total nine sedimentpore water samples were S4=335.10 mg/l, T4=25.67 mg/l, T5=69.38 mg/l, respectively. No toxicitywas detected at the concentrations tested for most sam-ples. The EC50 for WAF using Peruvian crude oil was17.18 mg/l, the mean of three replicates (Table 4).

Discussion

Polycyclic aromatic hydrocarbons

The persistence of PAHs is related directly to theirmolecular weight. Most of the low molecular weightPAHs were present in water from S3 (also highest∑PAH concentration), which was the nearest collectionsite from the main pipeline in Saramuro. PAHs withgreater molecular weight were found in either water orsediment pore water samples from both rivers, Marañón(San José de Saramuro) and Corrientes (VillaTrompeteros), posing a possible threat to human

Table 2 Sixteen polycyclic aromatic hydrocarbons (PAHs) and ∑PAH concentrations in water and sediment samples (chemically extractedpore water) from six collection sites on the Corrientes River near Villa Trompeteros in Loreto, Peru, sampled during summer 2011

Water Sediment

PAHs T1 T2 T3 T4 T5 T6 T1 T4 T5 T6

Naphthalene nd nd nd nd nd 2.7±2.21 nd nd nd nd

Acenaphthylene nd nd nd nd nd 5.69±1.65 nd nd nd nd

Acenaphthene nd nd nd nd nd 5.76±4.22 nd nd nd nd

Fluorene nd nd nd nd nd 10.60±4.42 nd nd nd nd

Phenanthrene nd nd nd nd nd 8.30±13.58 nd nd nd nd

Anthracene nd nd nd nd nd 70.08±56.46 nd nd nd nd

Fluoranthene nd nd nd 20.71±1.80 nd 27.62±22.57 nd 10.62±3.62 2.36±2.18 3.82±3.35

Pyrene nd nd nd nd nd 2.90±1.14 nd 0.68±1.18 nd 0.48±0.83

Benz[a]anthracene nd nd nd nd nd 0.21±0.23 nd nd nd nd

Chrysene nd nd Nd nd nd 5.84±6.65 nd nd nd nd

Benzo[b]fluoranthene nd nd Nd nd nd 2.58±1.33 nd nd nd nd

Benzo[k]fluoranthene nd nd Nd nd nd 2.56±2.55 nd nd 0.90±1.55 0.87±1.50

Benzo[a]pyrene nd nd Nd nd nd 0.93±0.43 3.59±6.22 44.90±9.38 8.36±1.50 9.73±4.24

Dibenzo[a,h]anthracene nd nd Nd nd nd 47.67±33.88 nd 11.13±14.63 nd nd

Indeno[1,2,3-cd]pyrene nd nd Nd nd nd 7.48±4.85 nd nd nd nd

Benzo[ghi]perylene nd nd Nd nd nd 3.76±4.53 nd nd nd nd

∑PAH concentration nd nd Nd 20.71 nd 204.66 3.59 67.33 11.62 14.90

Result (μg/ml) is the mean of three replicates±standard deviation

nd not detected (detection limit: 1 μg/l), ∑ sum

2178 Environ Monit Assess (2014) 186:2171–2184

inhabitants of these oil-impacted areas. In this study,when comparing samples taken upstream to down-stream, higher concentrations of PAHs were founddownstream in sediment pore water samples taken inSan José de Saramuro and water samples in VillaTrompeteros, suggesting that the oil companies in theseareas might be responsible for these higherconcentrations.

Towns along these rivers lack infrastructure fordrinking water, and rural people have to collect theirdomestic water directly from the river. Neither Peru nor

USEPA has standard limits for PAHs as a class indrinking water, but Europe does, which is 0.0001 μg/ml (European Communities 2007). This study foundthat all the PAH concentrations found in water (7.54 to210.15 μg/ml) exceeded this limit.

Several other studies in South America have deter-mined PAH concentrations in water and sediment porewater samples related to oil contamination (Table 5).The PAH concentrations in water samples in theUruguay and Plata River, the Patagonia coastline(Barra et al. 2007) and Chaco, Bolivia (González

Table 3 Mutagenic profiles andMicrotox® median effective con-centrations (EC50) of water andsediment samples (centrifugedsediment pore water) collectedfrom San José de Saramuro andVilla Trompeteros using the Sal-monella fluctuation test

NTACT no toxicity at concentra-tion tested, SD standard deviation(if 0.00: all 96-well plate wasconverted), MR mutation ratio,NS not significant, − not done

EC50

(mg/l)Bacteriastrain(Salmonella)

Test platepositives(SD)

Negativecontrolplatepositives

MR Significance

Water samples

Saramuro 1 NTACT – – – – –

Saramuro 2 NTACT TA98 96 (0.00) 6 16 <0.001

TA100 96 (0.00) 10 9.6 <0.001

Saramuro 3 NTACT TA98 94.5 (0.71) 6 15.75 <0.001

TA100 94 (0.00) 10 9.4 <0.001

Saramuro 4 NTACT – – – – –

Saramuro 5 NTACT TA98 95.5 (0.71) 6 15.92 <0.001

TA100 96 (0.00) 10 9.6 <0.001

Trompeteros 1 NTACT – – – – –

Trompeteros 2 NTACT TA98 96 (0.00) 6 16 <0.001

TA100 96 (0.00) 10 9.6 <0.001

Trompeteros 3 NTACT TA98 96 (0.00) 6 16 <0.001

TA100 96 (0.00) 10 9.6 <0.001

Trompeteros 4 133.86 – – – – –

Trompeteros 5 NTACT – – – – –

Trompeteros 6 NTACT – – – – –

Sediment samples

Saramuro 1 NTACT – – – – –

Saramuro 2 335.1 – – – – –

Saramuro 3 NTACT – – – – –

Saramuro 4 NTACT – – – – –

Saramuro 5 NTACT – – – – –

Trompeteros 1 NTACT TA98 3 (1.41) 6 0.5 NS

TA100 11.5 (3.54) 10 1.15 NS

Trompeteros 4 25.67 TA98 76 (1.41) 6 12.67 <0.001

TA100 67 (1.41) 10 6.7 <0.001

Trompeteros 5 69.38 TA98 3.5 (0.71) 6 0.58 NS

TA100 9.5 (3.54) 10 0.95 NS

Trompeteros 6 NTACT TA98 3 (1.41) 6 0.5 NS

TA100 10.5 (7.78) 10 1.05 NS

Environ Monit Assess (2014) 186:2171–2184 2179

Alonso et al. 2010) were all found to be lower than theones found in this study. However, the PAH concentra-tions in sediment pore water samples in this study arewithin the ranges of similar studies in Santos, Brazil(Nishigima et al. 2001) and Cartagena Bay, Colombia(Parga-Lozano et al. 2002).

Several tests have been performed in order to deter-mine the carcinogenicity of PAHs, and the followinghydrocarbons are carcinogenic or are possible humancarcinogens: benzo[a]anthracene, benzo[b]fluoranthene,benzo[k]fluoranthene, BaP, dibenzo[a,h]anthracene, andindeno[1,2,3-cd]pyrene (IARC 1983). In Saramuro, thesechemicals contributed up to 88 % of the ∑PAH concen-trations for all the water samples together, 91 % forsediment pore water samples, and in Trompeteros, 82 %in sediment pore water samples. BaP was found in allsediment pore water samples from both San José deSaramuro and Trompeteros; while in water, it was foundin four out of five samples in San José de Saramuro, and

one out of six samples in Trompeteros. All BaP concen-trations found exceeded the standard limits for drinkingwater set by Europe of 0.00001 μg/ml (EuropeanCommunities 2007), the USA of 0.0002 μg/ml (USEPA2011), and Peru of 0.0007 μg/ml (El Peruano 2008). Thisresult is worth noting since BaP has been identified as apromutagen in fish (i.e., requires metabolic activation tobecome a DNA-damaging agent; Hawkins et al. 1990).

Pluspetrol Peru is not the only oil company pollutingthe Corrientes River. An important and constant sourceof contamination is the upstream Ecuadorian oil indus-try, since the river originates in this neighboring country(Laraque et al. 2007). Crude oil extraction began inEcuador more than 40 years ago and has become amajor source of income for the country, as well as amajor source of environmental contamination and hu-man health problems (San Sebastián and Hurtig 2004).Studies on the Ecuadorian Amazon have reported skinmycosis, ear pain, gastritis (San Sebastián et al. 2001),

Table 4 Mutagenic profile and Microtox® median effective concentration (EC50) of water accommodated fraction with 200 g/l Peruviancrude oil using the Salmonella fluctuation test, strains TA98 and TA100 with and without metabolic activation (S9 fraction)

EC50 (mg/l) S9 Bacteria strainSalmonella

Test platepositives (SD)

Negative controlplate positives

MR Significance

Crude oil 17.18 None TA98 6 (2.12) 20 0.30 NS

Yes TA98 95 (0.71) 65 1.46 <0.001

None TA100 3 (1.41) 12 0.25 NS

Yes TA100 11 (1.41) 86 0.13 NS

EC50 value is the average of three replicates

SD standard deviation, MR mutation ratio, NS not significant

Table 5 Total polycyclic aro-matic hydrocarbon (PAH) con-centrations (μg/ml) in water andsediment pore water from differ-ent locations in South America

Sample Location Total PAHconcentration ranges(μg/ml)

Reference

Water Uruguay and Plata River 0.0018–0.012 Barra et al. 2007

Patagonia coastline 0.008–0.041 Barra et al. 2007

Chaco, Bolivia 0.002–2.99 González Alonso et al. 2010

Corrientes River upstream 0.222 Goldman et al. 2007

San José de Saramuro nd–210.15 Present study

Villa Trompeteros nd–204.66 Present study

Sediment Santos, Brazil 0.08–42.39 Nishigima et al. 2001

Cartagena Bay, Colombia 100.00–1415.00 Parga-Lozano et al. 2002

San José de Saramuro 2.56–70.41 Present study

Villa Trompeteros 3.59–67.33 Present study

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increase of spontaneous abortions (San Sebastián et al.2002), child leukemia (Hurtig and San Sebastián 2004),and elevated incidence of stomach, rectal, kidney andcervical cancer (Hurtig and San Sebastián 2002), allrelated to living in the vicinity of oil activities.

Muta-ChromoPlate™

Water and sediment samples

The positive mutagenic responses of this study suggeststhat the water in San José de Saramuro, and water andsediment pore water in Villa Trompeteros contain mu-tagens that may pose risks of unknown magnitude toorganisms and people along the river. A higher mutationratio in TA98 suggests that the water samples containmostly frameshift mutagens, even compared to the mu-tation ratio in sediment pore water samples. Frameshiftmutation or framing error is the insertion or deletion of abase or bases into the genome causing a change in thereading frame (Streisinger et al. 1966). Other studieshave shown that samples related to oil and aromatichydrocarbons have higher mutation ratios using TA98.In Alaska, Prudhoe Bay crude oil was found to bemutagenic using strain TA98 (Sheppard et al. 1983).Despite that no hydrocarbons were detected in the watersamples from Trompeteros, two samples (T2 and T3),tested positive for mutagenicity, indicating that theremay be other mutagenic pollutants at these sites. Insediment pore water, T4 (Trompeteros) was the onlysample that tested positive, most likely due to the high∑PAH concentration.

Crude oil

In the present study, two S. typhimurium test strainswere used for assessing Peruvian crude oil, TA98 andTA100, with and without metabolic activation of the testcompound (S9), and the sample was found mutagenicusing TA98 with S9 activation. Mutagens requiringmetabolic activation by microsomal (S9) enzymes tobecome genetically active are called promutagens. Themutagenicity of water and sediment pore water samplesand the low mutagenicity of the WAF using Peruviancrude oil suggest that the sampled areas contain addi-tional contaminants affecting the rivers. Vandermeulenet al. (1985) tested different oils such as Saran Gachfrom Iran and Kuwait crude, diesel 25, and Bunker C, aresidual fuel. The WSFs of oil products showed low

mutagenicity, suggesting that the toxicity of some com-ponents might be masking the mutagenic activity ofothers, something that could be happening in theserivers. This contradiction in results could also be dueto the potential problem that crude oil is a complexchemical mixture and sensitivity may be lost, sincemutagenicity of the whole material could be less thanindividual components (Pelroy and Petersen 1979).

Microtox®

Water and sediment samples

In the present study, one water sample (T4) was foundacutely toxic, which was located on a stream where oilcontaminated water and untreated wastewater isdischarged. This result is also consistent with the fluo-ranthene concentration found in the sample; it was thehighest of all, and aqueous solutions of aromatic hydro-carbons such as fluoranthene (three-ring) have greataqueous solubilities that may be acutely toxic (Di Toroet al. 2007). In addition, three sediment pore watersamples were found acutely toxic, compared to onewater sample. The sediment pore water sample fromT4 had the lowest EC50 of all the samples, and thehighest ∑PAH concentration in the Trompeteros areasamples. The EC50 values for sediment pore water in thepresent study ranged from 25.67 to 335.1 mg/l, andaccording to Doe et al. (2005), sediments with EC50

values≤1000 mg/l are toxic. For future studies, it wouldbe appropriate to run the amphipod survival test.Amphipods are widely used to measure sediment toxic-ity; using this test Long et al. (1998) found that total andindividual PAH concentrations were highly toxic.

Crude oil

The Microtox toxicity of the WAF using Peruvian crudeoil (EC50=17.18 mg/l) was lower than the EC50 valuesfor water and sediment pore water samples tested, pos-sibly because the sample of crude oil was more concen-trated (200 g oil/l water) and sediment pore water sam-ples contained other potential contaminants. Whencrude oil is extracted, “produced water” is brought upwhich is the highest volume waste generated in associ-ation with oil production that contains sodium and cal-cium sulfates, cadmium, mercury, chromium, arsenicand lead (Goldman et al. 2007). Depending on the typeof crude oil, toxicity varies. Hokstad et al. (1999) tested

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WAF (25 g oil/l seawater) from Statfjord and Trollcrude oil and found the 5-min EC50 to be 2.08 and1.08 mg/l, respectively. Faksness et al. (2012)found a similar EC50 value for Troll crude oil,1.1 mg/l. All these studies found EC50 values lower thanthe value found for Peruvian crude oil, therefore, weremore toxic.

Conclusions

Although oil companies have operated for decades inthese areas, few studies have tested contamination in thearea for toxicity and mutagenicity like this study did.Studies neither had determined concentrations of spe-cific PAHs nor had toxicological tests been used toprescreen polluted samples.

The results in the present study suggest that eventhough the water and sediment pore water samplescollected contained PAHs, the toxic effects were morechronic (as in mutagenicity) than acute in nature. Thisconfirms that wastewater containing oil is comprised ofharmful substances including those with genotoxic andcarcinogenic effects and that the oil industry in Peru hasthe potential to adversely affect aquatic organisms andhuman health. These results should be considered sincethere are indigenous people that depend on these riversand its tributaries.

Intensive field monitoring and additional assays arenecessary in the future in order to determine all thecontaminants present in the water and sediment of theserivers such as heavy metals (specifically cadmium, mer-cury, chromium, arsenic and lead). In addition, concen-tration of the 16 priority PAHs in fish samples fromdifferent sites in both rivers should be determined.There is also a great variety of standardized toxicitytests such as acute and chronic tests on invertebratesand vertebrates that are highly recommended.Microcosms and mesocosms would also be appropriatesince we can evaluate the effects of chemicals on a largenumber of species with different sensitivities simulta-neously. Finally, in situ caged organisms could be usedto increase environmental realism and have a betterunderstanding of the life history of resident species.The present research is only one of several studies thatwould be needed to make a complete hazard evaluationto predict the effects of oil and implement bioremedia-tionmethods to alleviate the damage that the oil industryhas caused in this part of Peru.

Acknowledgments Special thanks to Dr. Carmen García, andDr. Dennis Del Castillo of the Peruvian Amazon Research Institute(Instituto de Investigaciones de la Amazonía Peruana–IIAP).Thanks to Bijay Niraula, Murray Hyde, and Luciano Chu forassistance with data collection and in many other areas. Financialsupport for this project was provided by the ALFA Fellowship andthe Peruvian Amazon Research Institute [Instituto deInvestigaciones de la Amazonía Peruana (IIAP)].

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