The Challenges of Characterizing the Zooplankton Community Response Following Simulated Spills of Diluted Bitumen into Boreal Lake Limnocorrals.

We attempted to characterize zooplankton community response following spills of the unconventional crude oil, diluted bitumen (dilbit), into 10-m diameter, ~ 100 m3, ~ 1.5-m deep boreal lake limnocorrals, including two controls and seven dilbit treatments ranging from 1.5 to 180 L (1:100,000 to 1:1,000 v/v, dilbit:water). Community composition and abundances were monitored weekly to bi-weekly over three months. Total zooplankton biomass and abundance seemingly collapsed in all limnocorrals, regardless of treatment, though some rotifer species persisted. As a result, it was not possible to determine the impacts of dilbit. We theorize several potential non-oil-related reasons for the sudden community collapse - including elevated zinc levels, fish grazing pressures, and sampling biases - and provide guidance for future work using in-lake enclosures.

Fate of polycyclic aromatic compounds from diluted bitumen spilled into freshwater limnocorrals.

Diluted bitumens (dilbits) are produced by mixing highly viscous bitumen with lighter petroleum products to facilitate transport. The unique physical and chemical properties of dilbit may affect the environmental fate and effects of dilbit-derived chemical compounds when spilled. To further explore this, we monitored experimental spills of Cold Lake Winter Blend (CLWB) dilbit for 70 days in limnocorrals installed in a freshwater boreal lake. A regression design with 2 controls and 7 treatments was used to assess the fate and behaviour of polycyclic aromatic compounds (PACs) as they partitioned from the dilbit into the air, water column and sediments. Treatments ranged from 1.5 to 180 L of CLWB, resulting in oil:water ratios ranging between 1:71000 to 1:500 (v:v). We began to detect elevated concentrations of PACs as early as 6 h post-addition in the air, 12 h post-addition in the water column, and 15-28 d post-addition in the sediments. By the end of the experiment, concentrations of PACs had largely declined in the water column but remained elevated in the sediments. Our results demonstrate that under conditions typical of temperate boreal lakes, only a small proportion of PACs from dilbit enters the aquatic system, but even so, may produce concentrations of ecotoxicological concern, especially in the sediments, which is the ultimate sink for dilbit-derived PACs.

Surface oil is the primary driver of macroinvertebrate impacts following spills of diluted bitumen in freshwater.

The response of freshwater invertebrates following accidental releases of oil is not well understood. This knowledge gap is more substantial for unconventional oils such as diluted bitumen (dilbit). We evaluated the effects of dilbit on insect emergence and benthic invertebrates by conducting experimental spills in limnocorrals (10-m diameter; ~100-m3) deployed in a boreal lake at the IISD-Experimental Lakes Area, Canada. The study included seven dilbit treatments (spill volumes ranged from 1.5 L [1:66,000, oil:water, v/v] to 180 L [1:590, oil:water, v/v]), two controls, and additional lake reference sites, monitored for 11 weeks. Invertebrate emergence declined at the community level following oil addition in a significantly volume-dependent manner, and by 93-100 % over the 11 weeks following the spill in the highest treatment. Dilbit altered community structure of benthic invertebrates, but not abundance. One-year post-spill and following oil removal using traditional skimming and absorption techniques, benthic richness and abundance were greater among all treatments than the previous year. These results indicate that recovery in community composition is possible following oil removal from a lake ecosystem. Research is needed concerning the mechanisms by which surface oil directly affect adult invertebrates, whether through limiting oviposition, limiting emergence, or both. The response of benthic communities to sediment tar mats is also warranted.

Simulating diluted bitumen spills in boreal lake limnocorrals - part 2: Factors affecting the physical characteristics and submergence of diluted bitumen.

We examined the fate and behaviour of diluted bitumen (dilbit) as it weathered for 70 days in freshwater limnocorrals (10 m diameter × 1.5 m depth) installed in a boreal lake to simulate dilbit spills in a natural aquatic environment. We added seven different dilbit spill volumes, ranging from 1.5 to 180 L, resulting in oil-to-water ratios between 1:71,000 (v/v, %) and 1:500 (v/v, %). Volatile hydrocarbons in the dilbit slick decreased rapidly after the dilbit was spilled on the water's surface, and dilbit density and viscosity significantly increased (>1 g mL-1 and >5,000,000 mPa s, respectively). Dilbit sank to the bottom sediments in all treatments, and the time to sinking was positively correlated with spill volume. The lowest dilbit treatment began to sink on day 12, whereas the highest dilbit treatment sank on day 31. Dilbit submerged when its density surpassed the density of freshwater (>0.999 g mL-1), with wind, rain, and other factors contributing to dilbit sinking by promoting the break-up of the surface slick. This experiment improves our ability to predict dilbit's aquatic fate and behaviour, and its tendency to sink in a boreal lake. Our findings should be considered in future pipeline risk assessments to ensure the protection of these important aquatic systems.

Forensic source differentiation of petrogenic, pyrogenic, and biogenic hydrocarbons in Canadian oil sands environmental samples.

To facilitate monitoring efforts, a forensic chemical fingerprinting methodology has been applied to characterize and differentiate pyrogenic (combustion derived) and biogenic (organism derived) hydrocarbons from petrogenic (petroleum derived) hydrocarbons in environmental samples from the Canadian oil sands region. Between 2009 and 2012, hundreds of oil sands environmental samples including water (snowmelt water, river water, and tailings pond water) and sediments (from river beds and tailings ponds) have been analyzed. These samples were taken from sites where assessments of wild fish health, invertebrate communities, toxicology and detailed chemistry are being conducted as part of the Canada-Alberta Joint Oil Sands Monitoring Plan (JOSMP). This study describes the distribution patterns and potential sources of PAHs from these integrated JOSMP study sites, and findings will be linked to responses in laboratory bioassays and in wild organisms collected from these same sites. It was determined that hydrocarbons in Athabasca River sediments and waters were most likely from four sources: (1) petrogenic heavy oil sands bitumen; (2) biogenic compounds; (3) petrogenic hydrocarbons of other lighter fuel oils; and (4) pyrogenic PAHs. PAHs and biomarkers detected in snowmelt water samples collected near mining operations imply that these materials are derived from oil sands particulates (from open pit mines, stacks and coke piles).

Fingerprinting of petroleum hydrocarbons (PHC) and other biogenic organic compounds (BOC) in oil-contaminated and background soil samples.

Total petroleum hydrocarbons (TPH) or petroleum hydrocarbons (PHC) are one of the most widespread soil contaminants in Canada, the United States and many other countries worldwide. Clean-up of PHC-contaminated soils costs the Canadian economy hundreds of millions of dollars annually. In Canada, most PHC-contaminated site evaluations are based on the methods developed by the Canadian Council of the Ministers of the Environment (CCME). However, the CCME method does not differentiate PHC from BOC (the naturally occurring biogenic organic compounds), which are co-extracted with petroleum hydrocarbons in soil samples. Consequently, this could lead to overestimation of PHC levels in soil samples. In some cases, biogenic interferences can even exceed regulatory levels (300 µg g(-1) for coarse soils and 1300 µg g(-1) for fine soils for Fraction 3, C(16)-C(34) range, in the CCME Soil Quality Level). Resulting false exceedances can trigger unnecessary and costly cleanup or remediation measures. Therefore, it is critically important to develop new protocols to characterize and quantitatively differentiate PHC and BOC in contaminated soils. The ultimate objective of this PERD (Program of Energy Research and Development) project is to correct the misconception that all detectable hydrocarbons should be regulated as toxic petroleum hydrocarbons. During 2009-2010, soil and plant samples were collected from over forty oil-contaminated and paired background sites in various provinces. The silica gel column cleanup procedure was applied to effectively remove all target BOC from the oil-contaminated sample extracts. Furthermore, a reliable GC-MS method in combination with the derivatization technique, developed in this laboratory, was used for identification and characterization of various biogenic sterols and other major biogenic compounds in these oil-contaminated samples. Both PHC and BOC in these samples were quantitatively determined. This paper reports the characterization results of this set of 21 samples. In general, the presence of petroleum-characteristic alkylated PAH homologues and biomarkers can be used as unambiguous indicators of the contamination of oil and petroleum product hydrocarbons; while the absence of petroleum-characteristic alkylated PAH homologues and biomarkers and the presence of abundant BOC can be used as unambiguous indicators of the predominance of natural organic compounds in soil samples.

Forensic fingerprinting and source identification of the 2009 Sarnia (Ontario) oil spill.

This paper presents a case study in which integrated forensic oil fingerprinting and data interpretation techniques were used to characterize the chemical compositions and determine the source of the 2009 Sarnia (Ontario) oil spill incident. The diagnostic fingerprinting techniques include determination of hydrocarbon groups and semi-quantitative product-type screening via gas chromatography (GC), analysis of oil-characteristic biomarkers and the extended suite of parent and alkylated PAH (polycyclic aromatic hydrocarbon) homologous series via gas chromatography-mass spectrometry (GC-MS), determination and comparison of a variety of diagnostic ratios of "source-specific marker" compounds, and determination of the weathering degree of the spilled oil, and whether the spilled oil hydrocarbons have been mixed with any other "background" chemicals (biogenic and/or pyrogenic hydrocarbons). The detailed chemical fingerprinting data and results reveal the following: (1) all four samples are mixtures of diesel and lubricating oil with varying percentages of diesel to lube oil. Both samples 1460 and 1462 are majority diesel-range oil mixed with a smaller portion of lube oil. Sample 1461 contains slightly less diesel-range oil. Sample 1463 is majority lubricating-range oil. (2) The diesel in the four diesel/lube oil mixture samples was most likely the same diesel and from the same source. (3) The spill sample 1460 and the suspected-source sample 1462 have nearly identical concentrations and distribution patterns of target analytes including TPHs, n-alkane, PAHs and biomarker compounds; and have nearly identical diagnostic ratios of target compounds as well. Furthermore, a perfect "positive match" correlation line (with all normalized ratio data points falling into the straight correlation line) is clearly demonstrated. It is concluded that the spill oil water sample 1460 (#1, from the water around the vessel enclosed by a boom) matches with the suspected source sample 1462 (#3, from the vessel engine room bilge pump). (4) From the n-alkane and PAH analysis, it appears that the oil in the spill sample 1460 is slightly more weathered in comparison with sample 1462. The minor differences in fingerprints of two samples were most likely caused by weathering effects. (5) Sample 1461 (#2, from the vessel engine room bilge) and sample 1463 (#4, from the vessel bilge waste collection tank) demonstrated significantly different fingerprints and diagnostic ratios of target compounds from that of spill sample 1460. This was caused most likely by percentages of diesel to lube oil in these two samples different from that in spill sample 1460.

Forensic differentiation of biogenic organic compounds from petroleum hydrocarbons in biogenic and petrogenic compounds cross-contaminated soils and sediments.

"Total petroleum hydrocarbons" (TPHs) or "petroleum hydrocarbons" (PHCs) are one of the most widespread soil pollutants in Canada, North America, and worldwide. Clean-up of PHC-contaminated soils and sediments costs the Canadian economy hundreds of million of dollars annually. Much of this activity is driven by the need to meet regulated levels of PHC in soil. These PHC values are legally required to be assessed using standard methods. The method most commonly used in Canada, specified by the Canadian Council of Ministers of the Environment (CCME), measures the total hydrocarbon concentrations in a soil by carbon range (Fraction 1: C(6)-C(10); Fraction 2: C(10)-C(16), Fraction 3: C(16)-C(34): and Fraction 4: C(34)+). Using the CCME method, all of the materials extractible by a mixture of 1:1 hexane:acetone are considered to be petroleum hydrocarbon contaminants. Many hydrocarbon compounds and other extractible materials in soil, however, may originate from non-petroleum sources. Biogenic organic compounds (BOCs) is a general term used to describe a mixture of organic compounds, including alkanes, sterols and sterones, fatty acids and fatty alcohols, and waxes and wax esters, biosynthesized by living organisms. BOCs are also produced during the early stages of diagenesis in recent aquatic sediments. BOC sources could include vascular plants, algae, bacteria and animals. Plants and algae produce BOCs as protective wax coating that are released back into the sediment at the end of their life cycle. BOCs are natural components of thriving plant communities. Many solvent-extraction methods for assessing soil hydrocarbons, however, such as the CCME method, do not differentiate PHCs from BOCs. The naturally occurring organics present in soils and wet sediments can be easily misidentified and quantified as regulated PHCs during analysis using such methods. In some cases, biogenic interferences can exceed regulatory levels, resulting in remediation of petroleum impacts that are not actually present. Consequently, reliance on these methods can trigger unnecessary and costly remediation, while also wasting valuable landfill space. Therefore, it is critically important to develop new protocols to characterize and differentiate PHCs and BOCs in contaminated sediments. In this study, a new reliable gas chromatography-mass spectrometry (GC-MS) method, in combination with a derivatization technique, for characterization of various biogenic compounds (including biogenic alkanes, sterols, fatty acids and fatty alcohols) and PHCs in the same sample has been developed. A multi-criteria approach has been developed to positively identify the presence of biogenic compounds in soil and sediment samples. More than thirty sediment samples were collected from city stormwater management (SWM) ponds and wetlands across Canada. In these wet sediment samples, abundant biogenic n-alkanes, thirteen biogenic sterols, nineteen fatty carboxylic acids, and fourteen fatty alcohols in a wide carbon range have been positively identified. Both PHCs and BOCs in these samples were quantitatively determined. The quantitation data will be used for assessment of the contamination sites and toxicity risks associated with the CCME Fraction 3 hydrocarbons.

Effects of three biodiesels and a low sulfur diesel in male rats--a pilot 4-week oral study.

Because of the accessible and renewable nature of feedstock and the potential for the reduction of harmful combustion emissions and greenhouse gases, biodiesels have received increasing interest as an alternate fuel. Oral exposure to biodiesels is a concern because of contact during refuelling, accidental ingestion and exposure through ground water contamination. Although biodiesels from various feedstock are in use commercially and experimentally, very little is known about their potential adverse effects and no data is available on their potential for ground water contamination. A study was performed on male rats following oral treatment with experimental biodiesels (dissolved in corn oil) derived from canola oil (Bio-C), soy oil (Bio-S) and fish oil (Bio-F), at 500 mg/kg body weight/day, 5 days per week, for 4 weeks. Separate groups of animals were treated with low sulfur diesel (LSD) for comparison purpose, and with corn oil alone to serve as control. The potential for ground water contamination by biodiesels was investigated by the preparation of water-accommodated fractions (WAF) followed by gas chromatographic analysis. WAF from Bio-F and Bio-S was found to have the highest level of dichloromethane extractable materials. Gas chromatographic analysis indicated that the extractable materials from biodiesels contained much higher proportion of C15-C30 materials than LSD. Increased liver weight was observed in animal treated with Bio-C, Bio-S and LSD and decreased thymus weight was found in those treated with Bio-S. Histopathological changes typical of male-rat specific hyaline-droplet nephropathy were detected in kidney tubules of animals treated with LSD, Bio-S and Bio-C. Mild adaptive changes were observed in thyroids of animals treated with LSD, Bio-S and Bio-F. Clinical chemical and biochemical changes were confined to Bio-S and LSD treated rats and included elevation in some hepatic phase-I and phase-II drug metabolizing enzymes and hepatic palmitoyl Co-A oxidase, and elevated urinary concentrations of ascorbic acid and albumin. At the given dose level of 500 mg/kg bw/day, the overall treatment-related effects of biodiesels and LSD are mild, and the severity of the treatment effects may be ranked as: LSD>Bio-S>Bio-C>Bio-F. Considered together with the presence of a higher level of water extractable materials, Bio-S may be more of a concern for potential human health than Bio-C and Bio-F in an oral exposure scenario. Further studies are needed to identify and characterize the constituents contributing to the treatment-related effects specific to these experimental biodiesels.

Forensic fingerprinting of diamondoids for correlation and differentiation of spilled oil and petroleum products.

Diamondoids (adamantanes and diamantanes) are rigid, three-dimensionallyfused cyclohexyl-ring alkane compounds that can be found in almost all crude oils and in most petroleum products. Forforensic environmental investigations, the most commonly used biomarkers are high molecular weight (MW) tri- to pentacyclic terpanes and steranes. Most of these high MW biomarkers, however, are removed from the original crude oil feedstocks during the refining processes, while smaller biomarkers including diamondoids are concentrated in petroleum products. Fingerprinting diamondoids could thus provide another diagnostic means for correlation and differentiation of spilled oils and be particularly valuable for light to midrange distillates, such as jet and diesel fuels, the source of which may be difficult to identify using routine biomarker techniques. In this work, a reliable GC-MS analytical method has been developed for characterization and quantitation of diamondoids. The method detection limits for five target diamondoids were determined to be in the range of 0.06-0.14 microg/g oil. Distributions of diamondoids in over 100 different oils and refined products were quantitatively compared. The concentrations of four groups of target biomarkers were found, in general, to decrease in the order of sesquiterpanes > terpanes and steranes > adamantanes > diamantanes in both crude oils and refined products. A number of indices of admantanes and diamantanes have been developed and assessed as source indicators using their diagnostic powers (DP). The effects of evaporative weathering and biodegradation on alteration of diamondoid distributions have been quantitatively investigated. Finally, a spill case study by statistical evaluation of diagnostic ratios using the "two-tailed" Student's tapproach is presented to illustrate the unique utility of diamondoids for correlation and differentiation of unknown spilled diesels.

Characterization and identification of the Detroit River mystery oil spill (2002).

In this paper, a case study of the Detroit River mystery oil spill (2002) is presented that demonstrates the utility of detailed and integrated oil fingerprinting in investigating unknown or suspected oil spills. The detailed diagnostic oil fingerprinting techniques include determination of hydrocarbon groups and semi-quantitative product screening, analysis of oil-characteristic biomarkers and the extended suite of parent and alkylated polycyclic aromatic hydrocarbons (PAHs), and quantitative determination of a variety of diagnostic ratios of "source-specific marker" compounds. The detailed chemical fingerprinting data and results highlight the followings: (1) The spill samples were largely composed of used lube oil mixed with smaller portion of diesel fuel. (2) The diesel in the samples had been weathered and degraded. (3) Sample 3 collected from N. Boblo Island on 14 April was more weathered (most probably caused by more evaporation and water-washing) than samples 1 and 2. (4) All fingerprinting results clearly demonstrated oils in three samples were the same, and they came from the same source. (5) Most PAH compounds were from the diesel portion in the spill samples, while the biomarker compounds were largely from the lube oil. (6) Input of pyrogenic PAHs to the spill samples was clearly demonstrated. The pyrogenic PAHs were most probably produced from combustion and motor lubrication processes, and the lube oil in these spill samples was waste lube oil.

Review of behaviour of oil in freezing environments.

The current knowledge of the physical fate and behaviour of crude oil and petroleum products spilled in Arctic situations is reviewed. The fate and final deposition of oil in marine conditions is presented as based on the extant literature. Spreading models were evaluated for oil on ice, under ice, in snow, in brash ice, and between blocks of ice. Models of oil transport under sheet and broken ice were considered, both for sea and river conditions. The ability of ice sheets to trap oil is discussed in relation to oil storage capacity. The effects of oil on a growing ice sheet were examined, both in terms of ice formation and the thermal effects of oil inclusions in ice. The migration of oil through ice was reviewed, focussing primarily on the movement through brine channels. The effects of oil on the surface of ice were considered, with emphasis on the effects of surface pools on ice melt. Similar consideration was given to the effects of oil on snow on the surface of ice. The few quantitative studies of oil in open and dynamic ice conditions are reviewed. Observations of intentional small-scale spills in leads and ice fields are reviewed and compared with observations from real spills. The conditions under which "oil pumping" from leads occurs were quantified. The most common ultimate fate of oil in an ice field is to be released onto the water surface.

A thermodynamic QSAR analysis of the polysubstrate monooxygenase responses to xenobiotic chemicals.

Empirical quantitative structure activity relationships (QSAR) predict risk from exposure to xenobiotic chemicals as a dependence of inverse concentration on oil-water partition and electronic structural factors. This is equivalent, term by term to standard kinetic molecular transition state theory. When this transformed version is applied to the behaviour of the polysubstrate monooxygenase (PSMO) enzyme system, the rate of unintended behaviour depends directly on only two electronic factors. One is negative dependence on the strength of the weakest carbon-hydrogen (C-H) bond, a factor common to both ionic and free radical mechanisms. The second is a strong negative dependence on the polarization of this bond towards positive charge on the carbon, a factor relevant to ionic reactions. The risk of unintended oxidation reactions, in contrast, depends directly on these two factors because as the rate of intended hydroxylation is inhibited by strong or polar C-H bonds, alternative essentially 'accidental' oxidations occur. These are shown to lead to several different disease end points depending on the ratio of C-H bond strength to polarity.

Evidence of differential hematopoietic compensation to lead intoxication in blood, liver and kidney.

The dependence of fluorescent porphyrin levels on the concentration of lead in blood, liver and kidney has been examined as functions of both the level and duration of dosing. Individually housed male Wistar COBS rats of 200 g were randomly selected for 3, 7, 21 and 35 day dosing periods in groups of: control, 50, 100 and 1000 ppm dosing in drinking water. The plot of all data points for porphyrin concentration against measured lead burden covers the same range of levels and closely resembles the scatter of data observed in humans. However, subsets of this plot defined by individual dose levels and durations yield well-defined linear relationships. At all dose levels at 7 days the correlation of porphyrin concentration to lead tissue burden is negative in all tissues, showing a direct inhibition of hemopoiesis with lead burden. Depending on the tissue the slope becomes less negative, or, as in blood, positive at 21 and 35 days at all doses. This compensation is most rapid in blood, then liver and is least evident in kidney. The time at which compensation is observed is the same for all doses in each tissue and seems therefore, to depend on the rate of protein turnover in different tissue types. These results suggest that a direct correlation of porphyrin concentration to lead burden is not valid without knowledge of the dosing history. As well, evidence of compensation in one tissue does not imply successful compensation in other affected tissues where regeneration rates are slower.(ABSTRACT TRUNCATED AT 250 WORDS)

A correlation of induced mixed-function oxidase specific activity to C-H bond strengths in partially chlorinated monocyclic hydrocarbons.

The nature of structure-function relationships between the mixed-function oxidase (MFO) system and certain chemically related xenobiotics has been studied. The total hepatic induction index has been modified and used to investigate whether the induction of hepatic MFO is primarily a response to molecular shape (entropic) or to the strength of specific carbon-hydrogen bond strength (enthalpic). Using both Sprague-Dawley male rats and ring doves (Streptopelia risoria) and testing with benzene, 1,2,4-trichlorobenzene, cyclohexane and gamma-1,2,3,4,5,6-hexachlorocyclohexane as both inducers and substrates, a response of the MFO system to substrate C-H bond strength has been found.

The relative induction of mixed-function oxidase specific activity to C-H and C-C1 bond strengths in polychlorinated derivatives of dibenzo-p-dioxin (PCDDs).

The structure-activity relationships between the mixed-function oxidase (MFO) system and six polychlorinated derivatives of dibenzo-p-dioxin (PCDDs) were studied using Sprague Dawley rats. The study was set up in accordance with previous work in these laboratories involving monocyclic hydrocarbons. Three possible mechanisms at the MFO active site are proposed. C-H bond hydroxylation in low chlorine substituted PCDDs, and probable expoxidation in the intermediate chlorinated species including 2378 tetrachlorodibenzodioxin. Benzo[a]pyrene activity induced by some PCDDs appears irrelevant to the desired metabolic result.

A total hepatic induction index of metabolism for lipophilic xenobiotics.

A model for monitoring the hepatic ability to metabolize lipophilic xenobiotics is proposed. By identifying the behaviour of the mixed-function oxidase system as the rate determining feature of the response, an index can be devised from three independent factors: (i) the rate of the catalyzed reaction at each enzyme site, (ii) the concentration of such enzyme sites per hepatocyte and (iii) the total number of active hepatocytes available. Use of the model was tested by administering an acute oral dose of 1,2-benzanthracene to Sprague-Dawley rats. The above factors were measured in practical terms, these being (i) enzyme activity per time per mg protein, (ii) the concentration of enzyme per unit microsomal protein as well as the concentration of microsomal protein per unit liver weight and (iii) ratio of liver weight to body weight. After statistical analysis, it was found that each factor obeyed a different response curve characterized by a different ratio of growth to decay exponents. The product of these four measurable and physiologically discrete phenomena at any dose is the Total Hepatic Induction index. Together, the parameters that make up our model provide a means for inter- and intra-species comparisons as a response to various chemicals.

Comparative inhibition of hepatic hydroxymethylbilane synthase by both hard and soft metal cations.

The in vitro inhibition of hydroxymethylbilane synthase (EC 4.3.1.8, uroporphyrinogen I synthetase) obtained from livers of Sprague-Dawley rats has been studied with a wide range of di- and tri-valent metal ions. After purification by cell lysis, heat treatment, and centrifugation, the stable, soluble enzyme yielded sigmoidal inhibition curves with increasing concentrations of each of the 16 test ions. Using the negative logarithm of metal concentration for 50% inhibition (the pM50 value), the metal ions could be classified according to their Klopman hardness values. Very soft ions including Hg2+, intermediate ions including Cr3+, and very hard ions including Al3+ all yielded large pM50 values indicating strong inhibition. In comparison to known metal-ion chemical behaviour, these three ions could indicate three different types of inhibitory binding sites at or near the active site: Hg2+ corresponding to sulfur in cysteine, Cr3+ corresponding to nitrogen in histidine, and Al3+ corresponding to oxygen in carboxyl groups. The presence of the first two sites is also indicated by the pH dependence of activity.

The in vitro inhibition of hepatic ferrochelatase by divalent lead and other soft metal ions.

A solubilized protein with ferrochelatase activity has been extracted from hepatic mitochondria of Sprague-Dawley rats. Under anerobic conditions in the presence of sodium ascorbate the ferrochelatase velocity was typically 1.8 nM X min-1 X mg-1. The extract also displayed zinc chelatase activity of 1.2 nM X min-1 X mg-1 without either anerobic conditions or ascorbate. In both cases substrate inhibition occurred for metal ion and deuteroporphyrin, but in the linear range a noncompetitive two-site mechanism was observed. The ferrochelatase activity is inhibited by divalent copper, mercury, and lead ions and the sodium salt of p-chloromercuribenzoate and is replaced by zinc chelation activity. This evidence suggests that the metal-binding site includes a thiol group. The inhibition of the site is greatest with Cu2+ and decreases with increasing ionic radius to Pb2+. This observation implies that the binding site is stereochemically adapted to the small Fe2+ ion and to some extent protected from larger, sulfur-binding ions which can inhibit ferrochelatase activity.