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CSG 15 (Rev. 12/99) 1

MINISTRY OF AGRICULTURE, FISHERIES AND FOOD CSG 15Research and Development

Final Project Report(Not to be used for LINK projects)

Two hard copies of this form should be returned to:Research Policy and International Division, Final Reports UnitMAFF, Area 6/011A Page Street, London SW1P 4PQ

An electronic version should be e-mailed to [email protected]

Project title Predicting exposure of benthic infauna to chemicals, particularly pesticides,bound to sediments

MAFF project code CSA 3907

Contractor organisationand location

WRc-NSFHenley RoadMedmenham, Marlow, Bucks, SL7 2HD

Total MAFF project costs £ 239 967

Project start date 01/01/97 Project end date 31/12/99

Executive summary (maximum 2 sides A4)

For those compounds that have the potential to accumulate in sediments, risk assessment should includesome measure of the likely exposure to, and effects on sediment-dwelling organisms. However, theassessment of exposure in sediment toxicity tests is problematic, as bioavailability of compounds to benthicorganisms is regulated by the physico-chemical properties of both the contaminants and the sediment tested.Consequently, for risk assessment purposes, the comparison of measured effects against actual or predictedenvironmental exposure concentrations is difficult.

Many of the current approaches for determining likely exposure of benthic organisms assume that the mainexposure route is via the sediment pore water. Whilst this is probably true for many neutral organiccompounds, for other contaminants (e.g. ionic and polar compounds, and metals) the approach is unsuitable,because they are accumulated via other exposure routes, for example from direct contact with the sediment orfrom ingestion of contaminated food and sediment. This project attempted to assess the route of exposure fora range of chemicals, primarily pesticides, and relate the exposure route to their structure.

Twenty seven chemicals, twenty of them pesticides, were selected covering a wide range of physico-chemicalcharacteristics and chemical classes (e.g. chlorinated phenols, pyrethroids, PAHs, surfactants, triazines,organophosphates, organochlorines etc). Radio-labelled analogues were either purchased or donated frommanufacturers and used for all experiments to provide high specificity and sensitivity of analysis.

All experiments utilised 14C-labelled compounds, all determinands which were extracted from sediment usingappropriate solvents (methanol, methanol:DCM, HCl:MeOH) in combination with utrasonification andcentrifugation. Tissue samples were dissolved in a tissue digester. All samples (water, sediment extracts,tissue extracts) were added to an appropriate scintillant and counted on a liquid scintillation counter (LSC) orby high performance liquid chromatography with radiodetector.

Projecttitle

Predicting exposure of benthic infauna to chemicals,particularly pesticides, bound to sediments

MAFFproject code

CSA 3907

CSG 15 (1/00) 2

An uncontaminated sediment of known characteristics was collected for use during the study. Partitioningexperiments were performed by spiking the sediment with between 1000 and 2000 Bq/g (wet weight) of thecompound of interest, 20g of the sediment was then placed in glass jars and 30ml of groundwater added.Three replicate samples of the overlying water, sediment and porewater were analysed at time intervalsranging from 1 to 14 days.

Uptake experiments were carried out using the oligochaete Lumbriculus variegatus. In order to differentiatebetween uptake from ingestion and uptake from absorption from porewater or sediment contact, adult wormswere decapitated, and duplicate experiments carried out with headed and headless worms. Decapitatedworms continue to survive and behave in an identical manner to their headed counterparts, but during the 8days that they take to re-grow a new head, they are unable to feed. By measuring the uptake of a contaminantinto headed and headless worms, it is possible to determine the significance of uptake by ingestion.

Data from the partitioning experiments showed that equilibrium concentrations were achieved between thesediment, overlying water and porewater within a few days and that five of the chosen compounds (phenol,fenthion, CDEA, pirimiphos-methyl and linuron) were not sufficiently persistent to be used for the worm uptakeexperiments. Calculated experimental organic carbon:water partition coefficients (log Koc) were in excellentagreement with previous data, where available.

The suitably persistent compounds (23) were used for worm uptake experiments. Calculated biota-sedimentaccumulation factors (BSAF) ranged from 0.008 ± 0.0002 (diquat) to 184 ± 20 (PCP). Substances with lowBSAFs were highly polar or ionic in nature which resulted in strong adsorption to the sediment and hencereduced bioavailability (e.g. DODMAC, diquat and paraquat). The substances with the highest BSAFs werechlorinated phenols, which may have displayed enhanced accumulation through hydrogen bonding withphospholipids present in cell membrane walls, resulting in increased membrane permeability of the phenols.The other test compounds exhibited BSAFs in the range 0.008 to 8.3, which was of the same order ofmagnitude as other literature values.

Significant uptake by ingestion (greater than 30%), was demonstrated by 7 compounds: quinclorac, lindane,DODMAC, pentachlorophenol, permethrin, cypermethrin and benzo(a)pyrene. Explanations for thesignificance of ingestion for these substances varied depending on their physico-chemical characteristics. Thepyrethroids (permethrin and cypermethrin) degraded to a small degree during the test (ca. 10%) to metaboliteswith carboxylic acid functional groups (e.g. phenoxy benzoic acid). These highly soluble products would havebeen protonated in the gut due to the low pH, and the neutral molecule is then accumulated. It is likelytherefore that the observed uptake via ingestion is not directly attributable to the pyrethroids alone. ForDODMAC a cationic surfactant, its strong affinity for particulates and low solubility resulted in such lowporewater concentrations that uptake from this route could be considered negligible, measurable uptake viaingestion was therefore calculated as significant. The chlorinated phenols and quinclorac dissociate in water tovarying degrees and their pKas showed an excellent correlation with uptake through ingestion, whichsuggested that the low pH encountered in the gut of the Lumbriculus resulted in the molecules becomingprotonated and accumulating. The PAH benzo(a)pyrene is a non-polar, high Kow hydrocarbon and so uptakevia ingestion is likely to occur through cell membrane interactions within the gut. Lindane(hexachlorocyclohexane), is another chlorine-rich substance which may as a consequence exhibit enhancedmembrane permeability within the gut, similar to the chlorinated phenols.

Ageing tests carried out on sediment spiked with pyrene and left for varying periods up to 7 months showedthat the bioavailability of pyrene reduced significantly (by 40-50%) over a period of 24 hours and by 70% at theend of 7 months, compared with a decrease of only 20% in the chemical extractability.

It was concluded that the dissociation constant (pKa) could be used to predict uptake via ingestion forionisable compounds, but none of the other molecular descriptors (e.g. HOMO, LUMO, dipole, lipole,molecular volume, log Kow) examined were able to predict uptake by ingestion for the other test compounds.

Projecttitle


MAFFproject code

CSA 3907

CSG 15 (1/00) 3

Scientific report (maximum 20 sides A4)

Table of Contents

INTRODUCTION 6

TEST COMPOUNDS 8

SEDIMENT PORE WATER PARTITIONING STUDIES 9INTRODUCTION 9TEST SEDIMENT 9EXPERIMENTAL PROCEDURE 9RESULTS AND DISCUSSION 10

BIOACCUMULATION EXPERIMENTS 12INTRODUCTION AND APPROACH 12METHODS 13EXPERIMENTAL PROCEDURE 14CONTROLS 14STATISTICAL ANALYSIS 15RESULTS AND DISCUSSION 15CONTROLS 18

SEDIMENT AGEING 21MATERIALS AND METHODS 21RESULTS AND DISCUSSION 21

PREDICTING UPTAKE ROUTES OF CHEMICALS 23MATERIALS AND METHODS 24RESULTS AND DISCUSSION 24

CONCLUSIONS 25

RECOMMENDATIONS 25

REFERENCES 26

APPENDIX A Summary Datasheets For Test Compounds

APPENDIX B Table of physico-chemical properties of test compounds

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MAFFproject code

CSA 3907

5

List of Tables

Table 1 Compound Properties 8

List of Figures

Figure 1 Overview of information generated for each chemical 7

Figure 2 Experimental log Koc values plotted against values 10predicted using the SRC software.

Figure 3 Experimental design for assessing the relative contributions 13of the dietary uptake route to bioaccumulation

Figure 4 Bioaccumulation of contaminants from natural sediment into 15the worm L.variegatus.

Figure 5 Comparison of uptake from pore water only with that from all 16routes of uptake.

Figure 6 Ratio of accumulation in feeding worms over accumulation in non-feeding worms 17

Figure 7 Percent uptake through the dietary route plotted against pKa 18for the phenols and quinclorac.

Figure 8 Three different worm groups (headed, headless and tail-less) 19exposed to pyrene for two days in water and water/sand.

Figure 9 Bioaccumulation of pyrene from sediment by headed, headless 19and tail-less worms.

Figure 10 Log BCF vs log Kow for the study compounds. 21

Figure 11 The effect of sediment ageing on the accumulation of pyrene by 21feeding and non-feeding Lumbriculus.

Figure 12 Schematic representation of the accumualtion of organic contamiants in a sediment 22dwelling-organism

Figure 13 The effect of sediment ageing on the chemical extractability of 23pyrene from the sediment.

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MAFFproject code

CSA 3907

6

IntroductionOrganic compounds with lipophilic or ionic properties have the potential to sorb to the organic and/or inorganicmaterial in aquatic sediments and accumulate there. Such compounds may pose a risk to benthic organisms ifthey are toxic or bioaccumulative. Risk assessment of these compounds should include some measure of thelikely exposure to, and effects of chemicals on sediment-dwelling organisms. Test methods for the directassessment of effects on selected benthic species are currently being drafted by the Organisation forEconomic Co-operation and Development (OECD). However, the assessment of exposure in these tests isdifficult, as bioavailability of compounds to benthic organisms is regulated by the physico-chemical propertiesof both the contaminants and the sediments tested. Consequently, for risk assessment purposes, thecomparison of measured effect concentrations against actual or predicted environmental exposureconcentrations is problematic.

Many of the current approaches for determining likely exposure of benthic organisms to the sediment assumethat the main exposure route is via the sediment pore water. The foundation for this assumption is theequilibrium partitioning theory (EqP) which states that the chemical activity in the sediment, pore water andbiota is the same and can be predicted from the concentration in any one matrix given the appropriatecoefficients (Di Toro et al. 1991). Whilst this is probably true for many neutral hydrophobic organic compounds,for other contaminants (e.g. ionic and polar organic compounds) the approach is unsuitable, because theymay also be accumulated to a significant extent via other exposure routes. For example accumulation mayoccur from direct contact with the sediment or from ingestion of contaminated food and sediment.

The Ministry of Agriculture Fisheries and Food (MAFF) commissioned WRc-NSF to carry out a researchprogramme to develop improved approaches for assessing exposure of sediment dwelling organisms tosediment-associated contaminants. A parallel study, funded by NERC, looking at the effects of sedimentproperties on compound bioavailability was undertaken at the University of Reading. The scientific objectivesof this research programme (a) and the extent to which these objectives have been met (b) is outlined below:

1. a) Assess current approaches for determining exposure to sediment-associated contaminants.b) A review of the current understanding of the importance of different exposure routes and sediment,contaminant and biological characteristics that influence exposure to sediment-associated contaminantshas been given by Boxall et al., (1997).

2. a) Identify classes of pesticides that may sorb to sediments and select representative compounds fromeach class for further study.b) Study compounds were chosen to be persistent, of low toxicity, non-volatile and exhibit a range ofphysical and chemical properties. A preliminary list of compounds was produced and radiolabelledanalogues were sought for experimental purposes.

3. a) Develop approaches for analysis of study compounds (selected in objective 2) in whole sediment,interstitial water and biota.b) Methods for the analysis of all study compounds in water, sediment and a freshwater oligochaete(L.variegatus) are summarised in this report.

4. a) Identify suitable techniques for spiking sediment with study compounds (selected in objective 2).b) Boxall et al., (1997), reviewed the different techniques available for sediment spiking. The adoptedmethod which is outlined in this report was chosen because it was fast and effective in producing ahomogeneously spiked study sediment.

5. a) Determine the major uptake routes for compounds (selected in Objective 2) using bioaccumulation testsin water/sediment and water only systems.b) Boxall et al., (1998b) describe the experimental limitations of the initial approach adopted to discerndifferent uptake routes. The novel approach used to determine uptake routes for all study compounds isoutlined in Conrad et al., 2000.

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MAFFproject code

CSA 3907

7

6. a) Determine how the magnitude and route of exposure is influenced by a compounds ‘physico-chemical’properties.b) A number of approaches were used to predict a variety of physico-chemical properties for the studycompounds and statistical analysis methods were used to search for correlations with experimentalparameters to assess which properties are important in determining exposure (this report).

7. a) Develop a model to predict the likely exposure of organisms to sediment-associated contaminants.b) Bioaccumulation and route of uptake data was generated for 23 chemicals (mostly pesticides) tested aspart of the project. Most of the pesticides were not taken up through the dietary route and thereforeconsidering ingestion as an additional uptake route for these compounds was unnecessary. A significantcontribution to uptake from ingestion was found for the ionic compounds and a relationship wasestablished between pKa and dietary uptake.

8. a) Integrate the approach developed in Objective 7 with the results of a study performed at ReadingUniversity into the effects of sediment surface properties on exposure.b) A summary of the research carried out at Reading University is annexed to this report. A joint paper is inpreparation.

9. a) Transfer the technology developed in the study to MAFF for use in risk assessment of pesticides, anddisseminate the results through publication in the scientific literature.b) A novel technique has been developed to determine the significance of digestive uptake ofcontaminants into a benthic invertebrate, which has lead to one publication, and it is anticipated that theproject will generate at least two more publications in the scientific literature. Sediment exposure data hasbeen generated for over 20 compounds, belonging to 7 chemical classes, for some of which there was littleprevious sediment accumulation data available.

This report summarises the work that has been performed over the past three years to address the aboveobjectives. Over 20 compounds were identified as being suitable candidates for testing. For each substance aseries of experiments were performed to assess its sorption and chemical stability characteristics within awater sediment system. For compounds that did not degrade over the test period bioaccumulation studieswere carried out on feeding and non-feeding worms to establish total accumulation, and the contribution fromingestion of sediment particles. This report focuses on the findings of that work, more detailed descriptions ofoptimisation of experimental design, method development and analytical methods can be found in Boxall et al.(1998 a,b).

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MAFFproject code

CSA 3907

8

Test CompoundsA number of criteria were identified to be important for the selection of test compounds in order to meet thestudy objectives. They should be:

• non-degradable over 15 days;• able to be accurately and precisely determined analytically;• non-toxic to oligochaete worms;• non-volatile• available as radiolabelled analogues;• covering a range of physical and chemical properties.

Physical and chemical properties that have been identified as important in governing bioavailability includehydrophobicity, polarity, solubility, hydrogen bonding characteristics, electrostatic interactions and the topologyof a chemical. Table 1 shows the compounds chosen for this study and some of their associated properties. Abroad range of different compounds was covered (log octanol-water partition coefficient (Kow) from –2.7 to7.4, molecular weight from 94 to 586, dipole moment 0.01 to 23.6, solubility (mg/l) 0.001 to 1000000 andHenry’s Law Constant (HLC) (atm.m3.mole-1) 3.2 x 10-13 to 1.3 x 10-4. Figure 1 gives an overview of theexperimental work carried out for each chemical. Information on structure, source and nature of radiolabelledanalogues for each of the study compounds can be found in Appendix B.

Figure 1 Overview of information generated for each chemical

new compound develop analyticaltechnique

partitioning study

whole sediment test

ingesting worms non-ingesting worms

Compare Uptake

yes biodegradation

no biodegradation

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CSA 3907

9

Sediment pore water partitioning studies

Introduction

The bioaccumulation potential of a chemical in sediment is controlled by a number of factors, including degreeof sorption of the chemical to sediment (influenced by several physico-chemical factors including organiccarbon and sulphide content, particle size distribution, pH etc), and the assimilation efficiency of the chemicalby the benthos.

As a consequence, the partitioning behaviour of a chemical will strongly influence its bioavailability andpotentially, its route of uptake. The main purposes of the partitioning experiments was to generate sediment-water partition coefficients (Kp):

It has been well established that organic compounds are mainly associated with the organic carbon fraction ofthe sediment, therefore the partition coefficient is often described in terms of the organic carbon-water partitioncoefficient (Koc) where:

where foc is the fraction of organic carbon.

As well as providing information on how rapidly equilibration was achieved for each compound (i.e. thekinetics), the tests were used to assess the stability of the compound in the sediment/water system over thetime period of the subsequent Lumbriculus uptake experiments.

Test sediment

A natural sediment, collected in bulk, sieved to 1 mm, homogenised and stored at 4 °C was chosen for thisstudy. The sediment was collected from the ARC Study Centre, Wolverton Road, Great Lindford, MiltonKeynes which is on a nature reserve, well away from industrial activity, road or agricultural runoff or any othersources of contamination. The mean total organic carbon concentration of the sediment was 1.73 % (SE =0.06, n=6), the mean cation exchange capacity was 22.8 meq/100 g (SE = 1.59, n=6) and the pore waterdissolved organic carbon concentration was 27.96 mg/l (SE = 1.26, n=6). The sediment particle size rangedfrom 0 - 500 µm with the bulk of the particles being less than 63 µm (68%). The water content of the sedimentwas 50% (measured by drying sub-aliquots of the bulk sample). Exposure of L. variegatus to the sediment for28 days resulted in no mortality.

Experimental Procedure

Wet sediment (ca. 180 g) was spiked with an aliquot of a standard solution of test compound in methanolusing a calibrated syringe (typically between 10 and 1000 µl of radio-labelled standard was used), to give aspecific activity of between 900 and 2000 Bq/g wet weight of sediment. The spiked sediment was stirred for 1hr using a using a Kenwood Crypto Peerless mixer.

Subsamples (20 +/- 0.5 g) of the spiked sediment were then placed in 12, 30 ml screw top glass vessels and30 ml of fresh groundwater was added. Two holes were drilled into the lids of the vessels, the first allowedaeration of the test system, the second was connected to a 2 M NaOH solution and acted as a ‘trap’ for any

Concentration in water (Bq/ml)

Concentration of substance in the sediment (Bq/g)Kp =

foc

KpKoc =

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MAFFproject code

CSA 3907

10

14CO2 that was produced during the test. The vessels were kept in a constant temperature room (20oC +/- 2oC)and pH and dissolved oxygen measurements showed that the overlying water remained oxygenated and at astable pH throughout the course of the experiments.

Three vessels were removed at intervals throughout the study (typically 2, 5, 8 & 12 days). Samples ofsediment (100 +/- 10 mg) were extracted using either methanol, dichloromethane:methanol or methanolichydrochloric acid depending on the compound (details available in Appendix A) and added to 10ml ofscintillant supplied by Canberra Packard (Instagel Plus for methanol and methanol:DCM extracts, and HionicFluor for the acidified extracts). Pore water was extracted via centrifugation (15 minutes at 6000 rpm).Overlying water (1+/- 0.01ml) and sediment-pore water (1+/- 0.01ml) were added to 10ml of scintillant (InstagelPlus) and analysed using liquid scintillation counting (LSC; Beckmann LS 6000SE). Sediment was analysedusing both LSC and HPLC (Varian Star System 9010) with radiodetection (Canberra Packard FlowRadiometer).

The pyrethroids (permethrin, cypermethrin and cyfluthrin) were found to degrade slightly (approximately 10%degradation over 14 days) to a highly water soluble product which resulted in an under-estimation of the Kocvalue. The overlying water and pore water samples were therefore extracted into hexane (the breakdownproduct remained in the water phase), and counted by LSC.

Results and discussion

Pore water-sediment partitioning experiment results are presented in Appendix A, and show that equilibriumconcentrations in the sediment, porewater and overlying water were achieved between 4-6 days.

Figure 2 compares the experimental organic carbon-water partition coefficients (Koc) with values that werepredicted using modelling software (SRC, 1994 see also Appendix B). In general there is good agreementbetween the experimental and predicted data, at the low Koc range. For compounds in the higher Koc range,experimental values tended to be lower than predicted. The accuracy of the predicted log Koc could accountfor deviations between predicted and observed results. The SRC software uses a chemical’s molecularconnectivity index (MCI) to predict Koc. The compound’s molecular skeleton is dissected into its constituentbonds, a value is calculated for each bond and the sum of these bond values over the entire molecule is usedto provide the index (Meylan et al., 1992). Calculating a chemical’s MCI is based on a sound mathematicalpremise that works well for nonpolar organic compounds with few if any functional groups (e.g. pyrene). Afragment correction factor is used in addition to the MCI for polar and ionic compounds, with functional groupspresent (e.g. carboxyl, hydroxyl, chloride groups). These statistically derived correction factors are subject toerror especially for more complex molecules as is the case for most compounds that fall within the high Kocrange in this study. However, when comparing the experimental data from this study against otherexperimentally derived Koc values for soil-water partitioning (Gerstl, 1990), there is a much better agreement.Our data further supports the fact that the current understanding of Koc prediction is poor for ionic, and highlypolar organic compounds.

Because of the heterogeneity of sediments, sorption partition coefficients cannot be fully normalised withrespect to a single sediment characteristic, like organic carbon (Podoll and Mabey 1987). The partitioningbehaviour of a compound can also be affected by other sediment properties like particle size, pH, clay contentand redox potential. This was the case for paraquat and diquat which sorbed to the sediment to a muchgreater extent than predicted. Their ionic nature meant that their affinity for the whole sediment was enhancedthrough electrostatic interactions. For these positively charged compounds partitioning was not dominated bysorption to the organic carbon fraction of the sediment via non-specific van der Waals interactions. Instead,they exhibited a strong affinity for the negatively charged clay fraction of the sediment, and so their behaviourcould be better described by normalisation to another measurable sediment property, such as cation exchangecapacity.

Linear relationships between log Kow and Log Koc have been extensively reported in the literature (Karickhoff1979, 1981, Spacie 1994, Gobas and Zhang 1994, DiToro et al 1991) and Koc is often estimated using Kow.DiToro et al. (1991) reported that log Koc is equal to log Kow whereas Karickhoff (1981) found that log Koc isequal to 0.99 times log Kow minus 0.35. The slope of the line (slope = 0.58) calculated in this study was less

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11

steep demonstrating that Koc values were lower than expected from the above relationships. Variability in theobserved relationship could be caused by the uncertainty surrounding log Kow measurements as well asdiscrepancies in experimental log Koc values as described above.

Several compounds (phenol, fenthion, CDEA, pirimiphos-methyl and linuron) were found to degradesignificantly (>30%) over the course of the partitioning experiment (12 days). HPLC analysis revealed theformation of breakdown products and decreases in concentrations of the parent compound. Pirimiphos-methyl,an organo-phosphorus pesticide, degraded through oxidation of the P-O-C linkage to produce O,O-demethylphosphorothioate and O-2-ethylamino-6-methylpyrimidin-4-yl (RSC 1991). Fenthion, anotherorganophosphorus compound was initially oxidised to sulphoxide and sulphone, and further oxidised to thesulphone phosphate which was then hydrolysed (RSC 1991). Linuron biodegrades in freshwater sediments byseveral mechanisms including demethylation to produce desmethoxy linuron (Henze 1993). The pyrethroidswere hydrolysed to a small degree (ca. 10%), and their degradation products (primarily 3-phenoxybenzaldehyde and 3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropane carboxylic acid (DCVA)) werevery soluble in water (Tyler et al. 2000). As a consequence, liquid scintillation counting resulted in overestimates of the ‘dissolved’ pyrethroid in the sample. This problem was overcome by selectively extracting theparent compound into hexane prior to liquid scintillation counting. Phenol was rapidly mineralised insediment:water mixtures to carbon dioxide and water (Heim 1995).

Figure 2 Experimental log Koc values plotted against values predicted using the SRCsoftware

CDEA

pirmiphosmethylsimazine

phenolethofumesate

atrazinequinclorac

linuron

DCPTCP fenthion

trifluralin

diquatparaquat

b(a)p

fenpropadin

cyproconazole

cyfluthrinpermethrin

pyrenefenazaquin

cypermethrin

difenconazoleMGK264

lindane

PCP

0

1

2

3

4

5

6

7

0 1 2 3 4 5 6 7

Log Koc predicted

Log

Koc

exp

erim

enta

l

1:1 line

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12

Bioaccumulation experiments

Introduction and approach

A sediment-associated contaminant can potentially enter an organism via:

• partitioning from the pore water;• ingestion of contaminated sediment;• direct contact with sediment.

The relative importance of these uptake routes and the dependence of route of uptake on chemical structureare poorly understood with results of studies to date being potentially contradictory. A number of wholeorganism studies using polychaetes, fish and earthworms have demonstrated that contaminants can beaccumulated from food (Forbes et al, 1998, Driscoll and McElroy, 1996, Belfroid et al., 1994, Kolok et al. 1996)and that uptake from ingestion is a significant source of exposure (Kaag et al., 1997). In contrast, other studieshave shown that the contribution from dietary uptake is insignificant due to low absorption efficiency (Jiminezet al., 1987, Niimi and Dookhran, 1989).

The total body burden of an organism is the sum of uptake from all three routes (Equation 1), less loss fromdepuration and metabolism.

Total Uptake = Uptake Pore water + Uptake Ingestion + Uptake Direct contact (Eq 1)

Standard sediment bioaccumulation tests (ASTM, 1995) give information on the total exposure the oligochaeteworm Lumbriculus variegatus receives from a given contaminant. Risk assessment for compounds to thesediment compartment assumes that the major contribution to the body burden of benthic organisms proceedsthrough contaminant partitioning from the pore water (DiToro et al., 1991), however using standard bioassaysthis cannot be validated. Therefore to determine the relative contributions of uptake routes by sediment-dwelling organisms new approaches were needed.

Leppaenen and Kukkonen (1998) have investigated the significance of ingestion as an uptake route using L.variegatus. This species reproduces by architomy, in which new individuals bud off the anterior end of theparent and after fragmentation, individuals regenerate fresh segments for tail and head. The formation of anew head takes about 6-7 days during which time the worm is unable to feed.

Isolating and identifying recently budded non-feeding worms is, however, a skilled and very labour-intensiveexercise requiring the separation of a large number of worms from sediment. It is therefore impractical to carryout intensive uptake tests requiring the use of large numbers of recently budded worms (up to 300 worms aretypically needed for each uptake test). It is also difficult to determine the precise age of headless individualswhich are used in the sediment test. Ideally non-feeding worms should be freshly fragmented to prolong thenon-feeding phase.

An alternative technique was developed and tested during this project whereby the heads of adult worms wereremoved. Decapitation provides a viable organism for uptake tests which is unable to feed for the 7 day periodduring which a new head generates.

Uptake by feeding and headless worms exposed to whole sediment was examined to determine thecontribution of ingestion to total uptake. Based on these measurements the relative contribution of the dietaryuptake route could be determined (Figure 3). This approach can be applied to test chemicals with differentproperties in order to improve the understanding of bioaccumulation processes in addition to partitioning ofchemicals between the biota and aqueous phase.

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CSA 3907

13

Figure 3 Experimental design for assessing the relative contributions of the dietary uptake routeto bioaccumulation

WHOLE SEDIMENT

Ingesting (headed) worms Non-ingesting (headless) worms

⇓ ⇓

TISSUE ANALYSIS

⇓ ⇓Uptake from pore water (Upw) Uptake from pore water (Upw)

+ +

Direct uptake from sed. (Ud) Direct uptake from sed. (Ud)

+

Uptake from ingestion (Ui)

Uptaketotal= Upw + Ud + Ui Uptakeingestion=Utotal-(Upw+Ud)

In addition, for each compound, the biota-sediment accumulation factor (BSAF) was calculated. The meanconcentration (total 14C) in the headed worms at the end of the test was divided by the average sedimentconcentration throughout the experiment. Concentrations of the test chemical in the worm were expressed inBq/g wet weight and concentrations in the sediment in Bq/g dry weight. BSAFs were calculated by normalisingthe worm weight to lipid content and the sediment to organic carbon content (Equation 2).

BSAF = Concentration in biota / lipid content in biota (g/glipid ) (eqn 2) Concentration in sediment / organic carbon content of sediment (g/gOC)

Normalisation to the lipid content and organic carbon fraction essentially provides a measure of thecompound’s solubility within an organic phase. As a consequence, the BSAF should be similar for allchemicals and across all species. If it assumed that the organic phase in both the lipid and sediment hassimilar physico-chemical properties then, in theory, the BSAF should equal 1 for all substances. In reality,however, this is rarely the case as the lipid phase within an organism can be considered non-polar, whereasthe organic carbon fraction for sediment is a much more heterogeneous mixture of polar and non-polarcompounds of varying molecular weight and physico-chemical parameters. As a consequence, based onpartitioning, a value of roughly 1.7 would be expected in the absence of food chain biomagnification (Di Toro etal. 1991). Most BSAFs have been generated for hydrophobic compounds such as PCBs and PAHs (e.g. Ma etal. 1998; Ferguson and Chandler 1998), rather than more soluble organic compounds and pesticides.

Methods

The test organism Lumbriculus variegatus was maintained in culture under light and temperature controlledconditions with strict protocols (Conrad et al. 2000).

Experimental procedure

Test worms were retrieved with a glass pipette from a vessel containing around 500 worms which had beenrandomly selected from the culture. Intact adult worms (i.e., fully formed posterior and anterior ends) weighing

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between 5-10 mg were used in the experiment. Non-feeding worms were prepared by removing the head endof the worm (2-4 mm) with a scalpel. The head end of the worm can be identified as it is slightly thicker andgreener than the tail end.

A sediment exposure test was carried out to assess the significance of ingestion as an uptake route for allpersistent test compounds. Standard sediment (see Section 3.2) was spiked with a radiolablelled analogue ofthe compound of interest to give a sediment concentration of between 1000-2000 Bq/g wet weight. To spikethe sediment, the test compound was added dropwise in a methanol solution to the sediment. The sedimentwas then thoroughly stirred with a mixer for an hour and left overnight to equilibrate.

Thirty glass jars (60 ml) were prepared by adding either 6 headed or six headless worms to each jar. Spikedsediment (20 g) was added to each vessel covering the worms. Each vessel was topped up with 30 ml of WRcgroundwater and the jar sealed and fitted to an aeration system venting into a solution of 2 M sodiumhydroxide. Three replicates each of headed and headless worms were removed after 2, 4, 6, 8, and 12 days.

At each time interval the worms were removed from the sediment and depurated overnight in a beaker ofgroundwater and sand (10 g) to remove any sediment particles contained in the gut of the feeding worms.After depuration the worms were removed from the water, blotted dry with tissue paper, weighed and placed ina liquid scintillation counter (LSC) vial with 2 ml of tissue solublizer (Soluene, Canberra Packard) and allowedto dissolve overnight. Next day, 10 ml of LSC cocktail (Hionic Fluor, Canberra Packard Ltd) was added to thevial and mixed. At each time interval the overlying water concentration and sediment concentration wasanalysed.

Controls

A control, unspiked sediment with worms added as described above, was run alongside each exposure test tocheck for contamination.

A period of non-feeding in worms could result in a lower total lipid content of the headless worms which mayaffect their ability to bioaccumulate the compounds of interest. Total lipid content of L.variegatus was thereforeanalysed by a gravimetric method at the Scottish Crop Research Institute. Four different worm groups wereanalysed: feeding and headless worms taken directly from culture as well as headed and headless worms thathad been exposed to clean sediment for a duration of seven days, the time it takes for non-feeding individualsto regrow their heads. Three replicates of each group were prepared.

A tail-less control group was prepared to establish if the action of removing the worms’ head could lead to anyphysiological changes in the worm that facilitated or reduced the ability of the worms to take up thecontaminant, pyrene. Tail-less worms were prepared in much the same way as headless individuals exceptthat their posterior rather than their anterior end was removed.

The three groups of worms headed, headless and tail-less were exposed to pyrene in a water only and awater-sand environment for two days. The objective was to establish if the magnitude of uptake differed inheaded, headless and tail-less individuals. Thirty-six glass jars were filled with 30 mL of spiked ground water(10 Bq/mL of labelled 14C- pyrene, specific activity 10.7 MBq/mg). Clean sand (10 g, <100 µm) was added to18 of the test systems and ten worms were placed in each jar. Six replicates of headed, headless and tail-lessworms were prepared for the water and water-sand system.

In a separate experiment with pyrene-spiked sediment the same worm groups as above were exposed. Theexperimental set-up mirrored that for the sediment exposure studies described in the previous section, the onlydifference being the addition of the tail-less group.

Statistical Analysis

For each time interval, the mean and standard deviation of the body burden data were calculated for both theHeaded (H) and Headless (HL) worms. The H/HL ratio RH/HL was then determined, together with anapproximate 95% confidence interval as shown in Equations 3 and 4.

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SE(RH/HL) = (RH/HL)√[(SH2/MH + SHL

2/MHL)/n] (eqn 3)and hence

95% confidence interval is RH/HL ± t × SE(RH/HL) (eqn 4)

where:SE is the standard error of the RH/HL,MH and SH are the mean and standard deviation of the headed worms,MHL and SHL are the mean and standard deviation of the headless worms,n is the number of replicates per worm type (usually 3), andt is Student’s t at the two-sided 0.05 significance level with 2(n-1) degrees of freedom.If the lower confidence limit on the ratio RH/HL was greater than 1, this indicated that the contribution from thedietary route was statistically significant.

Results and Discussion

Plotting BSAFs for all compounds showed a wide range of values from 0.008 for diquat to 184 for PCP (Figure4).

Based on equilibrium partitioning theory, a BSAF around 1.7 should be observed for all non-ionic compounds(DiToro et al. 1991). In reality though, a range of BSAFs have been reported, both below and above thetheoretical value based on partitioning. For highly hydrophobic compounds such as dioxins most BSAFs havebeen reported as less than 1.7 owing to retarded uptake of dioxins (Batterman et al. 1989) and othersuperhydrophobic chemicals (Gobas et al. 1989). Higher BSAF values have been reported for caged musselsand crayfish in lake water experiments, which had been exposed to dioxins for a 10 to 21 day periodimmediately after the addition of the chemicals. The higher values may have reflected initial non-steady stateconditions due to sedimentation and resuspension of the added particles to which the dioxins were adsorbed(Muir et al. 1992). The few studies available that have measured BSAF for pesticides and polar compounds(Ferraro et al., 1990, Tracey and Hansen, 1996) report values in excess of 1 (ca. 1 – 8).

Elevated BSAFs above values based on partitioning alone (Figure 5), may be accounted for by contributionsfrom ingestion. However, for many of the higher BSAF values reported, ingestion is not a significant uptakeroute. In these cases other explanations need to be sought to explain contaminant accumulation.

A reason for the relatively high uptake of the chlorophenols into the worm may be that phospholipids such aslecithin and cephalin, major components of the cell wall, play an important role in membrane permeability ofchemicals. Kishino and Kobayashi (1995) have found that intermolecular hydrogen bonding between 1-octanoland lecithin and the hydroxyl group of the chlorophenols plays an important role in their transfer from water tomembranes.

Figure 4. Bioaccumulation of contaminants from natural sediment into the worm L.variegatus after14 days exposure. Error bars are 95% confidence intervals.

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BSAFs for the pesticides tested ranged between 0.008 (diquat) and 8.3 (simazine). Paraquat and diquat areionic substances with positive charges on the nitrogen atoms within the ring structures. They therefore have avery strong affinity for the sediment, illustrated by their high experimental Koc, and subsequently exhibitgreatly reduced bioavailability. Simazine and atrazine on the other hand are taken up to a much larger extentthen would be expected from their respective log Kow of 2.4 and 2.8.

DODMAC, a cationic surfactant, is a very large molecule (molecular weight > 500) and it has been proposedthat these chemicals are not able to cross biological membranes by passive diffusion (Opperhuizen et al.,1985). It has been hypothesised that hydrophobic chemicals with long chain length are poorly, if at all, takenup from water (Opperhuizen 1990). For this chemical which is practically insoluble in water very little will beavailable for uptake from the pore water therefore ingestion becomes the only route whereby the chemical canenter the organism.

Plotting bioaccumulation from all routes against bioaccumulation from pore water shows that most compoundsfall on or near the line of equal uptake illustrating that the major route of uptake for the study compounds is byadsorption through the pore water (Figure 5).

A better illustration of the dietary contribution to the body burden for each of the study compound is given inFigure 5. For three compounds uptake from ingestion was the main route (i.e. contribution from the dietaryroute was over 50%), namely DODMAC, permethrin and quinclorac. Dietary contribution was significant (i.ethe lower confidence limit on the RF/NF was greater than 1) for lindane, PCP, cyfluthrin, fenazaquin,benzo(a)pyrene, cypermethrin. Percent accumulation from feeding for these compounds ranged from 27%(fenazaquin) to 44% (benzo(a)pyrene). For the remaining sixteen compounds dietary uptake contributed lessthan 25% to the total body burden and was not statistically significant. No relationship between percent uptakethrough ingestion and a chemical’s log Kow was found (Figure 4).

1849952

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Uptake with time for headed (feeding) and headless (non-feeding) worms for each compound can be found inAppendix A.

Figure 5 Comparison of uptake from pore water only with that from all routes of uptake.

The low uptake of DODMAC has been explained above. The high contribution from ingestion is thereforeattributable to the fact that the compound is not present in the dissolved phase within the pore water and thisuptake route then becomes negligible.

Results suggested that the pyrethroid permethrin was taken up to a significant extent by ingestion. Pyrethroidshave log Kow values in excess of 6.4, and will therefore exhibit strong tendencies to bioaccumulate. This initself however, does not explain why permethrin had a significant uptake via ingestion. One possibility is thatduring the course of the experiments a proportion of the pyrethroid is biodegraded to compounds withcarboxylic acid functional groups, including 3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropane carboxylic acid(DCVA) which is highly soluble, and did not partition into a non polar phase (hexane) during the partitioningexperiments. It is therefore unlikely that any DCVA would be taken up into the Lumbriculus via contact with thesediment or porewater. However, the high concentration present in the porewater would lead to ingestion bythe feeding worms, and at the low pH in the gut the DCVA would become protonated forming a neutralcompound which can be accumulated, hence resulting in a significant uptake through feeding.

Figure 6. Ratio of accumulation in feeding (H) worms over accumulation in non-feeding worms(HL). If the lower confidence interval of the RatioH/HL was greater than 1 contribution fromthe dietary route was significant. Error bars are 95% confidence intervals. Values arepercentage uptake through the dietary route.

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The pesticides quinclorac and PCP were accumulated to a significant extent through dietary uptake.Quinclorac and PCP were also the only pesticides tested that showed any degree of dissociation, with arespective pKa of 4.34 (Weber, 1994) and 5.12 (Davies et al., 1999). At a pKa of 4.34 half the molecules are inthe associated form (non-charged) and the other half are dissociated. The pore water in the test system had apH of around 7.0 (6.5-7.3), and so quinclorac and PCP were mostly dissociated and hence not available foruptake through absorption. Once the molecule was ingested, however, and travelled through the gut of theworm (which had a much lower pH) protonation led to the formation of the neutral molecule which was readilyabsorbed by the worm, thus explaining the high proportion of uptake through the dietary route.

DCP, TCP were the other compounds tested that exhibited a degree of dissociation. Plotting the pKa againstpercent uptake through ingestion (Figure 7) showed a negative trend. The lower the pKa of the substance therelatively more important ingestion became as an uptake pathway. For substances with a pKa below 6 it istherefore advisable to consider uptake through feeding. It is well established that the toxicity of compoundsthat dissociate depends significantly on the pH of the test system (Saarikoski and Viluksela 1981, Kishino andKobayashi, 1995). Holcombe et al. (1980) reported that a decrease in toxicity of 2,4-dichlorophenol to fatheadminnow with an increase of pH, is mainly attributed to the decrease of the concentration of the undissociatedform.

The larger tissue concentrations in sediment ingesting worms for some compounds could be due to digestiveenzymes which solubilise the compound and/or the gut architecture which allows more efficient penetration ofcontaminant through outer membranes than outer integument (Leppanen and Kukkonen, 1998). However, themechanisms underlying this are not easily discernible. For compounds that dissociate this seems to be relatedto pH change in the gut flora compared to the outer environment. Uptake of other chemicals might beexplained by enzymatic processes.

84%

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27%35%

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Simazine

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Lindane

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Trifluralin

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/HL

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Figure 7 Percent uptake through the dietary route plotted against pKa for the phenols andquinclorac. Test system was at pH 7.

Controls

None of the unspiked sediment vessels run alongside the sediment bioaccumulation studies showed anycontamination with 14C.

No differences in total lipid content (ANOVA: F=2.1, df=3,8, p=0.17) were found between the headed (H) andheadless (HL) worms taken directly from culture and those exposed for seven days in sediment (lipid contentin percent of wet weight: Hculture=1.39±0.4, HLculture=1.6±0.1, Hsed=1.63±0.3 and HLsed= 1.77±0.5). The averagelipid value of 1.6±0.3% compared well with the value of 1.36±0.32% reported by West et al., (1997) for thesame species.

Figure 8 shows the accumulation factors (AF) (concentration in the worm/ concentration in the water) for theheaded, headless and tail-less worms exposed in the water and sand/water systems. There was no differencebetween the groups for the water exposure (ANOVA: F=2.2, df=2,9, p=0.17) and the water/sand exposure(ANOVA: F=0.11, df=2,9, p=0.9). The slightly higher AF measured for the samples containing sand could beexplained by a small amount of adsorption of the 14C-pyrene to the particulate phase. This data confirmed thatheaded, headless and tail-less worms behaved similarly in water-only tests where the sole route of exposurewas by uptake from the aqueous phase.

Figure 8 Accumulation factors for three different worm groups (headed, headless and tail-less)exposed to pyrene. Error bars are 95% confidence intervals.

quinclorac

DCP (2,4)

TCP (2,4,5)

PCPy = -19.2x + 145

R2 = 0.92

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In the sediment bioaccumulation study with these three groups (Figure 9) there was again no statisticallysignificant difference in uptake between them.

Figure 9 Bioaccumulation of pyrene from sediment by headed, headless and tail-less worms.Error bars are 95% confidence intervals.

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Sediment Ageing

Bioavailability of sediment-sorbed compounds has been shown previously to decrease with increasing contacttime between the sediment and contaminant (Gunnarsson et al. 1996, Landrum et al., 1992, Kukkonen andLandrum 1989 and Loonen et al., 1997). This reduction in bioavailability has been observed for laboratory-dosed sediments tested for differing lengths of time. The most likely mechanism for these observations is thepresence of a two stage sorption process; for a short time after spiking a sediment the chemical is readilyavailable on the surface of particles, however, with time there is a slow migration into the pores of thesediment making the chemical less bioavailable. Even though reductions in accumulation have beenobserved, the potential impact of this process has not generally been recognised.

As a consequence, newly deposited contaminants may be more bioavailable than older buried material. In arisk assessment context, data from sediment tests are usually for the freshly spiked material assuming a worstcase scenario. Ageing or increased contact time between sediment and contaminants however, might be morerelevant for the field situation, where long incubation periods occur.

If the process of sediment ageing can affect its bioavailability then it could also alter the relative contributionfrom each uptake route. To investigate the relationship between sediment contact time and uptake route, theaccumulation study with headed and headless worms was conducted after incubating sediment with pyrene forup to 7 months.

Materials and Methods

Sediment (5 +/- 0.5 kg) was spiked to give a concentration of 500 Bq/g wet weight of radio-labelled pyrene.The sediment was then transferred to a polyethene bucket and kept at room temperature (20-22 °C).Accumulation experiments with headed and headless Lumbriculus were prepared as described in section4.2.1 with sediment incubated for 0, 1, 14, 28, 70 and 220 days.

Pyrene was extracted from the sediment (ca. 0.1 g) with 4 ml of methanol and counted on the LSC. Sediment(2 +/- 0.02 g) was extracted with methanol, and the pyrene determined by hexane extraction and HPLC with aradio-detector to confirm that no degradation had occurred with sediment ageing.

Results and Discussion

Decreased bioavailability of pyrene with increased contact time with the sediment was observed (Figure 10).The average body burden for days 5,6 and 7 of each worm test with aged sediment is shown in Figure 10. Thelargest decline in bioavailability of sediment-bound pyrene occurs at the beginning of the ageing processbetween days 0 and 1. The overall reduction in bioavailability of pyrene over the 7 months test period wasaround 70%. However, the chemical extractability of pyrene (which was shown by HPLC, to be stable in thesediment over the course of 7 months), using methanol, was only reduced by about 20% over the test period(Figure 11). The decrease in chemical extractability seems to be linear with time whereas the decrease inbioavailablity was rapid within the first 24 hours. Previous studies have indicated that bioavailability decreasesfaster than chemical extractability (Landrum 1989, Landrum et al., 1992).

These results highlight the potential overestimation of risk by equilibrium based models which use data oncontaminant concentrations from chemical extraction methods carried out immediately after sediment spiking.It also highlights the need for precise protocols when assessing sediment toxicity in the laboratory. Forexample, in standard sediment bioassays with Lumbriculus variegatus and Chironomus riparius (ASTM 1996)the sediment is left overnight to settle before adding the test organisms. From our experiments this 24 hour“settling” period is shown to be essential in order for the test chemical to achieve a degree of equilibrationwithin the sediment. Addition of organisms before this time could potentially lead to significant variations in thebioavailability of the test substance, resulting in the generation of different LC50 and body burden data. Pyrenehas a reasonably high log Kow (4.88), and previous investigations (e.g. Gunnarsson et al. 1996, Landrum etal., 1992, Kukkonen and Landrum 1998 and Loonen et al., 1997), have also used chemicals with high log

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Kow/Koc (e.g. PAHs or dioxins). Whether more water soluble substances show a similar behaviour, is notknown.

As the contact time increased, partitioning of a substance, in this case pyrene, into the sediment continuedand a larger fraction became bound in the more inaccessible sites within the sediment particle. Here it wasnon-labile, and partitioning back into the pore water was a kinetically controlled process which would occurover an extended period. Reduction in the pore water concentration leads to a lower rate of uptake throughdirect contact, which could lead to an increase in the significance of uptake via ingestion. Figure 10 illustratesthat the contribution from the dietary uptake route was most marked when sediment has been aged for 1-2months, after which the difference became insignificant. It was most likely that in sediment aged for 1-2months there was a reduction in the pore water concentration of pyrene but the animal was still able to digestthe non-labile fraction and receive a dietary contribution of pyrene. As the ageing process proceeded and thepyrene became bound within even more inaccessible sites the effectiveness of digestive removal declined.

Figure 10 The effect of sediment ageing on the accumulation of pyrene by feeding and non-feedingLumbriculus. Data are averages taken from the accumulation experiments at days 5,6 and 7.Error bars are 95% confidence intervals.

Figure 11 The effect of sediment ageing on the chemical extractability of pyrene from the sediment.Error bars are 95% confidence intervals.

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R2 = 0.91

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Predicting uptake routes of chemicals

Quantitative structure-activity relationships (QSARs) have been shown to provide reliable estimates for someparameters for use in the hazard assessment of organic chemicals. Toxicology-based QSAR methodology isfounded on the premise that the toxicity of a given group of chemicals can be correlated to different moleculardescriptors. Typical molecular descriptors are quantitative parameters for either molecular structure (e.g.molecular weight, molecular volume, surface area, etc.), physicochemical properties (e.g. hydrophobicity) orelectronic properties (e.g. HOMO, LUMO). They may be derived experimentally or estimated using softwarebased mathematical models such as molecular orbital quantum chemical software.

The toxicity exibited by a chemical is a combination of its ability to penetrate into the biophase, transport withinthe organism and the interaction of the chemical toxicant with the site of action (McFarland 1970). Penetrationof a chemical can be modelled by many parameters, the most commonly used is the octanol water partitioncoefficient (Bearden and Schultz, 1997). The interaction term has been quantified by a variety of differentstereo-electronic parameters, such as the dissociation constant (pKa) and the energy of the lowest unoccupiedmolecular orbital (LUMO).

Bioaccumulation of a chemical is primarily dependant on the penetration term rather than the chemicals abilityto interact with a certain receptor. For a chemical to enter the organism it must diffuse through biologicalmembranes. This indicates that biota lipid to water or sediment partitioning is the dominant factor in thephysical process resulting in the uptake and accumulation of lipophilic compounds from these phases (Figure12).

Figure 12 Schematic representation of the accumulation of organic contaminants in a sedimentdwelling organism.

Materials and Methods

Bioaccumulation and route of uptake data was generated for 23 chemicals of which 18 were pesticides.Percent uptake through the dietary route and BSAFs were generated experimentally with the wormLumbriculus variegatus (section 4).

Molecular descriptors were generated using the Oxford Molecular Ltd. Tools for Structure ActivityRelationships (TSAR 3.1) software. Briefly, a three dimensional structure was generated using molecular

Sediment Solids Interstitial water

Desorption/adsorption (Kp )

Aqueous phaseuptake

Aquatic Organism

Gastro-intestinal

Tract

MetabolismStorage inbody lipids

Circulatoryfluid

KowFeeding

Solid phaseelimination Excretion

Uptakesurfaces

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modelling software (Pimms) and a near minimum energy conformation of each chemical was determined usingthe optimisation procedure in TSAR version 3.1.

Structure property calculations (moments of inertia, molar refractivity, log Kow, Verloop parameters, dipolemoment, charge-2), topological, connectivity and shape indices were calculated for all optimised 3-Dstructures. A complete list of the molecular descriptors is given in Appendix B. Regression analysis was usedto search for relationships between the experimental parameters and calculated molecular descriptors.Regression analysis produced an equation describing the relationship between a single independant yvariable, and several explanatory x variables. This equation can then be used to predict values of theindependant variable. The molecular descriptors acted as the dependant variables.

Results and discussion

The experimental variables were not strongly correlated with any of the molecular descriptors. Dietary uptakewas only high for a few chemicals therefore these overly high values biased the regression (panhandle effect).

For chlorinated phenols, pKa seemed to determine the degree of uptake through the dietary route (section 4).For other compounds taken up through feeding, different mechanisms were acting that were probably betterdescribed by a different molecular descriptor. Insufficient compounds from the same class were assessed todetermine these parameters with a high degree of certainty.

Numerous QSARs estimating bioconcentration based on lipophilicity have been published (Neely et al., 1974,Chiou 1985, Nendza 1991). In general an increase in Bioconcentration Factor (BCF) is associated with anincrease in log Kow. If the BSAF data in this study is converted to BCF data using a chemical’s Kp to predictthe amount of dissolved chemical in the pore water a similar trend was observed (Figure 13). Thebioaccumulation process can then be viewed to a first approximation as a partitioning between pore water andbenthic fauna.

Figure 13 Log BCF vs log Kow for the study compounds. BCF data was generated by calculating thesediment pore water concentration using the experimental Kp values and dividing the biotaconcentration by this number.

log BCF = 0.53 log Kow + 1.6.

y = 0.53x + 1.6

R2 = 0.72

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The above relationship forms the basis of many predictive models for the bioaccumulation of substances inbiota. It must be noted however, that the use of a log:log plot of the variables serves to mask potentiallysignificant deviations from the regression line.

Conclusions

A new method for assessing the route of uptake of chemicals by the oligochaete Lumbriculus variegatus wasdeveloped to screen for compounds taken up via ingestion.

A total of 27 compounds (mostly pesticides) were tested for porewater-sediment partitioning characteristics,and for 23 of these compounds bioaccumulation data was generated. For many of the pesticides there werelittle or no previous bioaccumulation data.

Sediment partitioning studies with the study compounds showed that the experimental log Koc values were ingood agreement to those published in the literature, and on the whole compared well with predicted valuesgenerated by computer modelling software.

The dietary route of uptake was only significant for 7 of the 23 compounds tested, namely quinclorac, lindane,DODMAC, pentaclorophenol, permethrin, cypermethrin and benzo(a)pyrene. No single molecular descriptorcould be found to link these compounds. This suggested that digestive uptake is a complex process withdifferent mechanisms operating.

For compounds that dissociate (quinclorac and PCP), the pKa of the compound can be used to predict thecontribution from ingestion.

Overall sediment-biota accumulation factors taken at the end of the experiment showed that the phenols(DCP, TCP and PCP) were accumulated to a much higher extent than the other compounds tested (BSAF =206, 396 & 734 respectively). BSAF values for the other compounds ranged between 0.006 and 33.

A sediment ageing study illustrated that bioavailability of pyrene declined by 40-50% within the first 24 hours ofspiking. After seven months of ageing of the sediment, the bioavailability had decreased by 70%, comparedwith only 20% for the chemical extractability in methanol. This illustrated the importance of obtaining a ‘stable’system before introducing test organisms and commencing uptake studies. Premature addition of the testspecies is likely to lead to overestimates of bioavailability and body burden.

Recommendations

It would be prudent to test more compounds from those classes that have been screened during the currentstudy which showed ingestion to be a significant uptake route, namely pesticides that dissociate,organochlorines and pesticides with high Kows.

A variety of classes of pesticides were not tested in this study (e.g. acid herbicides, persistent phenyl ureasetc), it would be beneficial to screen one or two representative compounds from each of these classes toinvestigate whether dietary uptake is of importance.

It is recommended that sediment ageing studies with other persistent compounds of differing physico-chemicalproperties is performed, to investigate the relationship between bioavailability and contaminant residence timein the sediment.

In this study only one freshwater sediment with a fairly average organic matter content was used in order tonormalise the results. It is recommended to take a compound shown in this study to be accumulated viaingestion, and test it in sediments with different particle size distribution, and different amounts of organiccarbon, as ingestion rate and gut residence time may be controlled by these parameters.

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References

ASTM (1995) Standard test methods for measuring toxicity of sediment-associated contaminats withfreshwater invertebrates. E1706-95a. In Annual book of ASTM standards. Vol. 11.05, Philadelphia, PA, pp.1204-1285.

Batterman AR, Cook PM, Lodge KB, Lothenbach DB and Butterworth BC (1989). Methodology used for alaboratory determination of relative contributions of water, sediment and food chain routes of uptake for2,3,7,8-TCDD bioaccumulation in Lake Trout in Lake Ontario. Chemosphere, 19, (1-6), 451-458.

Bearden AP and Schlutz TW (1997). Structure-activity relationships for Pimephales and Tetrahymena: amechanism of action approach. Environmental Toxicology and Chemistry 16(6), 1311-1317.

Belfroid A, Sikkenk M, Seinen W, Vangestel K and Hermens J (1994). The Toxicokinetic Behaviour ofChlorobenzenes in Earthworm (Eisenia-Andrei) Experiments in Soil. Environmental Toxicology & Chemistry13(1), 93-99.

Boxall A, Conrad A and Watts C. (1997) Predicting exposure of benthic infauna to chemicals, particularlypesticides, bound to sediments. WRc interim report to MAFF (10348-0), December 1997.

Boxall A, Conrad A and Watts C. (1998a) Predicting exposure of benthic infauna to chemicals, particularlypesticides, bound to sediments. WRc interim report to MAFF (10348-0), January 1998.

Boxall A, Conrad A and Watts C. (1998b) Predicting exposure of benthic infauna to chemicals, particularlypesticides, bound to sediments. WRc interim report to MAFF (10348-0), December 1998.

Chiou CT (1985). Partition coefficients of organic compounds in lipid-water systems and correlations with fishbioconcentration factors. Environmental Science and Technology 19, 57-62.

Conrad AU, Comber SD and Simkiss K (2000) New method for the assessment of contaminant uptake routesin the oligochaete Lumbriculus variegatus. Accepted by Bulletin of Environmental Contamination & Toxicology.

Davies NA, Edwards PA, Lawrence MA, Taylor MG and Simkiss K (1999) The influence of sediment particlesurfaces on the bioavailability to different species of 2,4-diclorophenol and pentachlorophenol. Environ. Sci.technol. 33, 2465-2468.

DiToro DM, Zarba CS, Hansen DJ, Berry WJ, Swartz RC, Cowan CE, Pavlou SP, Allen HE, Thomas NA andPaquin PR (1991) Technical basis for establishing sediment quality criteria for non-ionic organic chemicalsusing equilibrium partitioning. Environmental Toxicology and Chemistry, 10, 1541-1583.

Driscoll SK and McElroy AE (1996). Bioaccumulation and Metabolism of Benzo(a)pyrene in three species ofpolycheate worms. Environmental Toxicology & Chemistry 15(8), 1401-1410.

Ferraro SP, Lee H, Ozreteich RJ and Specht DT 1990) Predicting bioaccumulation potential: A test of afugacity-based model. Archives of Environmental Contamination and Toxicology, 19, 386-394.

Ferguson PL and Chandler GT (1998) A laboratory and field comparison of sediment polycyclic aromatichydrocarbon bioaccumulation by the cosmopolitan estuarine polycaete Streblospio. Spp. MarineEnvironmental Research, 45, 4/5, 387-401.

Forbes Tl, Forbes VE, Giessing A, Hansen R and Kure LK, (1998). Relative role of pore water versus ingestedsediment in bioavailability of organic contaminants in marine sediments. Environmental Toxicology andChemistry, 17(12), 2453-2462.

Gerstl, Z. (1990) Estimation of organic chemical sorption by soils. Journal of Contaminant Hydrology, 6, 357-375.

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CSA 3907

27

Gobas FA, Clarke KE, Shiu WY and MacKay D (1989) Bioconcentration of polychlorinated benzenes andbiphenyls and related super-hydrophobic chemicals in fish : role of bioavailability and elimination into thefaeces. Environmental Toxicology and Chemistry, 8, 231-245.

Gunnarsson JS, Schaanning MT, Hylland K, Skold M, Eriksen DO, Berge JA and Skei J (1996). InteractionsBetween Eutrophication and Contaminants .3. Mobilization and Bioaccumulation of Benzo(a)Pyrene FromMarine Sediments. Marine Pollution Bulletin, 33(1-6), 80-89.

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Jiminez BD, Cirmo CP and McCarthy JF (1987) Effects of feeding and temperature on uptake, elimination andmetabolism of benzo(a)pyrene in the bluegill sunfish (Lepomis macrochirus). Aquatic Toxicology 10, 41-57.

Kaag NH, Foekema EM, Scholten MC and Vanstraalen NM (1997). Comparison of Contaminant Accumulationin Three Species of Marine Invertebrates With Different Feeding Habits. Environmental Toxicology &Chemistry, 16(5), 837-842.

Kishino T and Kobayashi K (1980) Studies on the metabolism of chlorophenols in fish-XIV. A study on theadsorption mechanism of pentachlorophenol in goldfish relating to its distribution between solvents and water.Nippon Suisan Gakkaishi, 46, 1165-1168.

Kishino T and Kobayashi K (1995) Relation between toxicity and accumulation of chlorophenols at various pH,and their absorption mechanism in fish. Water Research, 29(2), 431-442.

Kolok AS, Huckins JN, Petty JD and Oris JT (1996). The role of water ventilation and sediment ingestion in theuptake of benzo(a)pyrene in gizzard shad (Dorosoma cepedianum). Environmental Toxicology and Chemistry,15(10), 1752-1759.

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CSA 3907

28

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Projecttitle


MAFFproject code

CSA 3907

29

Please press enter

APPENDIX A. Summary Datasheets For Test Compounds

Herbicide quinclorac

CAS number: 084087-01-4

Molecular formula: C10H5Cl2NO2

Chemical family: quinoline

Mode of action: Weak auxin activity. No influence on Hill reaction.

Uses: Control of grass weeds, especially Echinoclea, pre- and post-emergence in transplanted and seeded rice.

Physico-chemical parameters

Specific activity (MBq/mg): 1.5

Supplier: BSAF

Molecular weight: 242

Solubility (mg/l): 76

Henry’s law constant (atm*m3*mole-1): 8.6*E-12

Log Kow: 3.0

Log Koc: 2.6

pKa 4.34

Experimental data

% recovery from sediment: 93±6

Solvent for sediment extraction: 1 M HCl in MeOH

HPLC column: ODS2

HPLC conditions: 70%ACN:30%DIW

Log Koc: 2.2

BSAFtotal: 1.8 ± 0.3

% uptake through feeding: 71

HPLC trace of quinclorac extracted from the sediment

N

CO OH

Cl

Cl X

Partitioning of compound between whole sediment,pore water and overlying water

Uptake of compound by L.variegatus

A) Ratio of body burdens in feeding (F) worms B) Accumulation factors for sediment and their non-feeding (NF) counterparts feeding and non-feeding worms

0

1

2

3

4

5

6

0 5 10 15

Time (days)

Ra

tio

(F

/NF

)

Standard

Day 1

Day 13

0 1 2 3 4 5 6 7 8 9 10

CPM

Time (m inutes)

0

0

0

500

1000

1500

2000

2500

0 2 4 6 8 10 12 14

Time (days)

Wa

ter

(Bq

/ml)

0

500

1000

1500

2000

2500

Se

dim

en

t (B

q/g

DW

)

Overlying water

Porewater

Sediment

0

1

1

2

2

3

0 5 10 15Time (days)

BA

F

feeding

non-feeding

Insecticide cypermethrin

CAS number: 052315-07-8

Molecular formula: C22H19Cl2NO3

Chemical family: pyrethroid

Mode of action: Non-systemic insecticide with contact and stomach action.Also exhibits anti-feeding action.

Uses: Control of a wide range of insects, especially Lepidoptera infruit and vegetable crops. Also used as an animalectoparasiticide.



Supplier: Astra-zeneca


Solubility (mg/l): 0.009


Log Kow: 6.4

Log Koc: 5.0

Experimental data


Solvent for sediment extraction: MeOH/DCM

HPLC column: ODS1


Log Koc: 4.5



HPLC trace of cypermethrin extracted from the sediment

O

O

CH3H3C

CHCCl

Cl

CN

O

X




0

0.5

1

1.5

2

0 3 6 9 12Time (days)

Ra

tio

(F

/NF

)

Day 4

Standard

Day 1

Day 8

Day 11

0 1 2 3 4 5 6 7 8 9 10 11

0

CPM

Time (m inutes)

0

4

8

12

16

0 4 8 12 16

Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

5000

6000

Se

dim

en

t (B

q/g

)

Overlying water

Porewater

Sediment

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 2 4 6 8 10

Time (days)

BA

F

feeding

non-feeding

Insecticide/Fungicide/Herbicide 2, 4-diclorophenol

CAS number: 000120-83-2

Molecular formula: C6H4Cl2O

Chemical family: phenol

Mode of action: fungicide

Uses: Control of termites, as wood perservative to protect againstfungal rots and woodboring insects.



Supplier: Sigma




Log Kow: 2.8

Log Koc: 2.9

pKa 7.85

Experimental data


Solvent for sediment extraction: MeOH

HPLC column: ODS1


Log Koc: 2.9

BSAFtotal: 52 ± 10

% uptake through feeding: -3

HPLC trace of DCP extracted from the sediment

OH

Cl

Cl

X




Standard

Day 1

Day 11

0 1 2 3 4 5 6 7 8 9 10

0

0.5

1

1.5

2


Ra

tio

(F

/NF

)

0

100

200

300

400

500

600

0 2 4 6 8 10

Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

Se

dim

en

t (B

q/g

DW

)

Overlying water

Porewater

Sediment

0

10

20

30

40

50

60

70

0 5 10 15

Time (days)

BA

F

feeding

non-feeding

Non-pesticide DODMAC

CAS number: 000107-64-2

Molecular formula: C38H80ClN

Chemical family: cationic surfactant

Mode of action:

Uses: Main ingredient in conditioner and other washing products



Supplier: Unilever


Solubility (mg/l): 4.3E-9

Henry’s law constant (atm*m3*mole-1): 4.9E-9

Log Kow: 12.5

Log Koc: 10.4

Experimental data



HPLC column: Partisil PAC

HPLC conditions: 90%ACN:10%MeOH+ CTAB

Log Koc: 5.2



HPLC trace of DODMAC extracted from the sediment

Cl-

H3C [CH2]17N+ [CH2]17

CH3

CH3

CH3

+




0

0

2

4

6

8

10

12

0 5 10 15

Time (days)

Ra

tio

(F

/NF

)

Standard

Day 1

Day 13

0 1 2 3 4 5 6 7 8 9 10Time (minutes)

0

0

2

4

6

8

10

0 2 4 6 8 10 12 14

Time (days)

Wa

ter

(Bq

/ml)

0

600

1200

1800

2400

3000

3600

Se

dim

en

t (B

q/g

DW

)

Overlying water Porewater Sediment

0.00

0.02

0.04

0.06

0.08

0.10


BA

F

feeding

non-feeding

Fungicide fenpropidin

CAS number: 067306-00-7

Molecular formula: C19H31N

Chemical family: piperidine

Mode of action: Systemic fungicide with protective and curative action,absorbed mainly by the roots, with translocation acropetally inthe xylem. Inhibits ergosterol biosynthesis.

Uses: Control of powdery mildew, rusts and Rhynchosporium inbarley and wheat.



Supplier: Novartis




Log Kow: 6.4

Log Koc: 5.3

pKa 10.1

Experimental data



HPLC column: ODS2

HPLC conditions: 40%MeOH+10mMol CTAB:60%DIW

Log Koc: 3.8



C

CH3

H3C

CH3

CH2 CH CH2

CH3

N

HPLC trace of fenpropidin extracted from the sediment




0

0.5

1

1.5

2


Ra

tio

(F

/NF

)

Standard

Day 1

Day 11

0 1 2 3 4 5 6 7 8 9 10

CPM

Time (minutes)

0

0

0

20

40

60

80

100

120

140

0 2 4 6 8 10

Time (days)

Wa

ter

(Bq

/ml)

0

2000

4000

6000

Se

dim

en

t (B

q/g

DW

)Overlying waterPorewaterSediment

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0 2 4 6 8 10Time (days)

BA

F

feeding

non-feeding

Insecticide MGK264

CAS number:

Molecular formula: C17H25NO2

Chemical family: carboximide

Mode of action:

Uses: Insecticide synergist for pyrethrins, allethrins, pyrethroids androtenone. Stabilizes and prolongs life of pyrethrins, allethrinsand pyrethroids.



Supplier: MGK




Log Kow: 3.8

Log Koc: 4.1

Experimental data



HPLC column: Partisil PAC

HPLC conditions: 10%ACN:90%MeOH+10mMol

CTAB

Log Koc: 3.9



N CH2 CH2 CH2 CH2CH2 CH2 CH2 CH3

O

O

CH3

HPLC trace of MGK264 extracted from the sediment




0

0.5

1

1.5

2

2.5

0 5 10Time (days)

Ra

tio

(F

/NF

)

Standard

Day 13

0 1 2 3 4 5 6 7 8 9 10 11 12

CPM

Time (minutes)

0

0

20

40

60

80

100

0 2 4 6 8 10 12 14Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

5000

6000

7000

Se

dim

en

t (B

q/g

DW

)


0

1

2

3

4


BA

F

feeding

non-feeding

Herbicide paraquat

CAS number: 001910-42-5

Molecular formula: C12H14N2

Chemical family: bipyridyl

Mode of action: Non-selective contact herbicide, absorbed through foliage withsome translocation through xylem. During photosynthesis,superoxide is generated, which damages cell membranes andcytoplasm.

Uses: Control of annual broadleaved weeds in fruit orchards. Alsoused for general weed control on non-crop land.



Supplier: Amersham


Solubility (mg/l): 10E5


Log Kow: -2.7

Log Koc: 3.2

Experimental data

% recovery from sediment: could not be extracted

Solvent for sediment extraction: n/a

HPLC column: ODS2

HPLC conditions: 80%buffer:20%DIW

Log Koc: 5.5

BSAFtotal: 0.012 ± 0.003


N+ N+ CH3H3Cx

HPLC trace of paraquat standard




0

0.5

1

1.5

2

2.5

3

-1 4 9 14Time (days)

Ra

tio

(F

/NF

)

Standard

0 1 2 3 4 5 6 7 8 9 10 11 12

CPM

Time (minutes)

0

1

2

3

4

5

0 2 4 6 8 10 12 14

Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

5000

6000

Se

dim

en

t (B

q/g

DW

)Overlying water Porewater Sediment

0.00

0.01

0.02

0.03

0 5 10 15

Time (days)

BA

F

feeding

non-feeding

Insecticide/Fungicide/Herbicide pentachlorophenol

CAS number: 000087-86-5

Molecular formula: C6HOCl5

Chemical family: organochlorine, phenol

Mode of action: Insecticide, fungicide, and non-selective contact herbicide.

Uses: Control of termites, as a wood perservative to protect againstfungal rots and woodboring insects, as a pre-harvest defoliantin cotton, and as a general pre-emergence herbicide.



Supplier: Sigma




Log Kow: 4.7

Log Koc: 3.5

pKa 4.75

Experimental data



HPLC column: ODS1


Log Koc: 3.1

BSAFtotal: 184 ± 47


HPLC trace of PCP extracted from the sediment

OH

Cl

Cl

Cl

Cl Cl




Non-pesticide pyrene

0

0.5

1

1.5

2

2.5


Ra

tio

(F

/NF

)

Standard

Day 1

Day 13

0 1 2 3 4 5 6 7 8 9 10 11 12

CPM

Time (minutes)

0

0

0

50

100

150

200

0 3 6 9 12

Time (days)

Wa

ter

(Bq

/ml)

0

500

1000

1500

2000

Se

dim

en

t (B

q/g

DW

)


0

50

100

150

200

250

0 2 4 6 8 10Time (days)

BA

F

feeding

non-feeding

CAS number: 000129-00-0

Molecular formula: C16H10

Chemical family: PAH

Mode of action:

Uses: By-product of combustion of fossil fuels.



Supplier: Sigma




Log Kow: 4.9

Log Koc: 4.8

Experimental data



HPLC column: Partisil PAC (PAH)


Log Koc: 4.7



HPLC trace of pyrene extracted from the sediment

CPM




Standard

Day 1

Day 11

0 1 2 3 4 5 6 7 8 9 10 11 12

0

0.5

1

1.5

2

0 5 10Time (days)

Ra

tio

(F

/NF

)

0

5

10

15

20

0 2 4 6 8 10 12 14 16Time (days)

Wa

ter

(ng

/ml)

0

1

2

3

4

5

6

Se

dim

en

t (m

g/k

g D

W)

Porewater Overlying water Sediment

0.0

0.5

1.0

1.5

2.0


BA

F

feeding non-feeding

Non-pesticide 2,4,5-trichlorophenol

CAS number: 000088-06-2

Molecular formula: C6H3OCl3


Mode of action:

Uses: Phenolic compounds have a distinct odour and are used indisinfectants, deodorisers, paints, and as anaesthetic for skin.



Supplier: Sigma




Log Kow: 3.5

Log Koc: 3.1

pKa 6.2

Experimental data



HPLC column: ODS1


Log Koc: 3.3

BSAFtotal: 99 ± 3


HPLC trace of TCP extracted from the sediment

OH

Cl

Cl

Cl




0

0.5

1

1.5

2

2.5

0 5 10 15

Time (days)

Ra

tio

(F

/NF

)

Standard

Day 14

0 1 2 3 4 5 6 7 8 9 10 11 12

CPM

Time (minutes)

0

0

50

100

150

200

0 2 4 6 8 10 12 14

Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

Se

dim

en

t (B

q/g

DW

)

Overlying water

Porewater

Sediment

0

20

40

60

80

100

120

0 5 10 15

Time (days)

BA

F

feeding

non-feeding

Herbicide atrazine

CAS number: 001912-24-9

Molecular formula: C8H14ClN5

Chemical family: triazine

Mode of action: Selective systemic herbicide, absorbed principally throughroots. Inhibits photosynthesis and interferes with otherenzymic processes.

Uses: pre- and post- emergence control of annual grass and broad-leaved weeds in a variety of crops.



Supplier: Ciba




Log Kow: 2.8

Log Koc: 2.4

pKa 1.7

Experimental data



HPLC column: ODS1


Log Koc: 2.6


% uptake through feeding: 14%

N N

NNH

Cl

NHCH2H3C CHCH3

CH3

X

HPLC trace of atrazine extracted from the sediment




0

0.5

1

1.5

2

2.5

0 5 10 15

Time (days)

Ra

tio

(F

/NF

)

Standard

Day 13

0 1 2 3 4 5 6 7 8 9 10 11 12

0

CPM

Time (m inutes)

0

250

500

750

1000

0 2 4 6 8 10 12 14

Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

Se

dim

en

t (B

q/g

DW

)

Overly ing water

Porewater

Sediment

0

1

2

3

4

5

6

0 5 10 15

Time (days)

BA

F

feeding

non-feeding

Non-pesticide benzo(a)pyrene

CAS number: 000050-32-8

Molecular formula: C20H12

Chemical family: PAH

Mode of action:

Uses: By-product of combustion of fossil fuels.



Supplier: Sigma




Log Kow: 6.1

Log Koc: 5.9

Experimental data



HPLC column: Partisil PAC (PAH)


Log Koc: 4.9



HPLC trace of benzo(a)pyrene extracted from the sediment




0

0.5

1

1.5

2

2.5

-1 4 9 14Time (days)

Ra

tio

(F

/NF

)

Standard

Day 1

Day 11

0 1 2 3 4 5 6 7 8 9 10 11 12

0

CPM

Time (m inutes)

0

0

2

4

6

8

10

0 2 4 6 8 10 12 14

Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

5000

Se

dim

en

t (B

q/g

DW

)

Overlying water Pore water Sediment

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0 5 10 15

Time (days)

BA

F

feeding

non-feeding

Insecticide cyfluthrin

CAS number: 068359-37-5

Molecular formula: C22H18Cl2FNO3


Mode of action: Non-systemic insecticide with contact and stomach action.Acts on the nervous system, with rapid knockdown and longresidual activity.

Uses: Control of chewing and sucking insects on oilseed rape,ornamentals and vegetables.







Log Kow: 5.7

Log Koc: 5.3

Experimental data



HPLC column: ODS1


Log Koc: 4.7



O

O

CH3H3C

CHCCl

Cl

CN

O

X

F

HPLC trace of cyfluthrin extracted from the sediment




0

1

2

3

0 5 10 15

Time (days)

Ra

tio

(F

/NF

)

Day 4

Standard

Day 1

Day 8

Day 11

0 1 2 3 4 5 6 7 8 9 10 11

0

CPM

Time (m inutes)

0

1

2

3

4

5

6

0 2 4 6 8 10 12 14 16

Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

5000

Se

dim

en

t (B

q/g

DW

)

Overly ing water

Porewater

Sediment

0.0

0.5

1.0

1.5

2.0

2.5

0 5 10 15

Time (days)

BA

F

feeding

non-feeding

Arcaricide fenazaquin

CAS number: 120928-09-8

Molecular formula: C20H22N2O

Chemical family: quinazoline

Mode of action: Selective acaricide with contact and stomach action.

Uses: Control of various pests and mites of vegetable and fruitcrops.



Supplier: DOW




Log Kow: 5.8

Log Koc: 4.9

Experimental data



HPLC column: C-8


Log Koc: 4.6



N

N

O CH2 CH2 C CH3

CH3

CH3

HPLC trace of fenazaquin extracted from the sediment




0

0.5

1

1.5

2

2.5

0 2 4 6 8 10 12

Time (days)

Ra

tio

(F

/NF

)

Standard

Day 13

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

CPM

Time (minutes)

0

0

3

6

9

0 2 4 6 8 10 12

Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

Se

dim

en

t (B

q/g

DW

)

Overlying water

Porewater

Sediment

0

1

2

3

4

5

6

0 5 10 15

Time (days)

BA

F

feeding

non-feeding

Insecticide fenthion

CAS number: 000055-38-9

Molecular formula: C10H15O3PS2

Chemical family: organophosphorus

Mode of action: Insecticide with contact, stomach and respiratory action.Cholinesterase inhibitor.

Uses: Control of fruit flies, leafhoppers, leaf miners, leaf-eatinglarvae and other insect pests in fruit, vegetables and othercrops. Control of insect pests in public health situations.



Supplier: Bayer




Log Kow: 4.1

Log Koc: 3.4

Experimental data



HPLC column: ODS1


Log Koc: 3.5



OPO

S

OH3C

H3C

CH3

S CH3

HPLC trace of fenthion extracted from the sediment




0

0.5

1

1.5

2

0 5 10Time (days)

Ra

tio

(F

/NF

)

Day 3

Standard

Day 1

Day 14

0 1 2 3 4 5 6 7 8 9 10

CPM

Time (minutes)

0

0

0

0

100

200

300

400

500

0 2 4 6 8 10 12 14 16

Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

5000

Se

dim

en

t (B

q/g

DW

)

Overlying waterPorewaterSediment

0

1

2

3

0 2 4 6 8 10

Time (days)

BA

F

feeding

non-feeding

Insecticide permethrin

CAS number: 052645-53-1

Molecular formula: C21H20Cl2O3


Mode of action: Non-systemic insecticide with contact and stomach action,having a slight repellent effect.

Uses: Control of larvae of chewing lepidopterous and coleopterousinsect pests in a variety of crops.







Log Kow: 7.4

Log Koc: 5.3

Experimental data



HPLC column: ODS1


Log Koc: 4.6



O

O

CH3H3C

CHCCl

ClX

O

HPLC trace of permethrin extracted from the sediment




0

0.5

1

1.5

2

2.5

3

3.5


Ra

tio

(F

/NF

)

Day 4

Standard

Day 1

Day 8

Day 11

0 1 2 3 4 5 6 7 8 9 10 11

CPM

Time (minutes)

0

0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14

Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

5000

6000

Se

dim

en

t (B

q/g

DW

)

Overlying waterPorewaterSediment

0

1

2

3

4

5

6

0 2 4 6 8 10

Time (days)

BA

F

feeding

nonfeeding

Non-pesticide phenol

CAS number: 000108-95-2

Molecular formula: C6H6O


Mode of action:

Uses: This compound is used as an intermediate, generaldisinfectant, in the manufacture of colourless or light-colouredartificial resins, in many medical and industrial organiccompounds and in dyes. It is also used topically as ananaesthetic in pruritic skin conditions and internally andexternally as an antiseptic.



Supplier: Sigma




Log Kow: 1.5

Log Koc: 2.4

pKa 9.99

Experimental data



HPLC column: ODS1


Log Koc: 2.3

BSAFtotal: nd

% uptake through feeding: nd

OH


0

60

120

180

240

300

0 2 4 6 8 10 12 14

Times (days)

Wa

ter

(Bq

/ml)

0

200

400

600

800

Se

dim

en

t (B

q/g

DW

)Overlying water

Porewater

Sediment

Herbicide simazine

CAS number: 000122-34-9

Molecular formula: C7H12ClN5

Chemical family: triazine

Mode of action: Selective systemic herbicide, absorbed principally throughroots. Photosynthetic electron inhibitor.

Uses: Control of most germinating annaul grasses and broad-leavedweeds in fruit crops and as an aquatic herbicide and algicidefor control of algae and submerged weeds in ponds



Supplier: Ciba




Log Kow: 2.4

Log Koc: 2.2

pKa 1.62

Experimental data



HPLC column: ODS1


Log Koc: 2.6



N N

NNH

Cl

NHCH2H3CCH2

CH3

X

HPLC trace of simazine extracted from the sediment




0

0.5

1

1.5

2


Ra

tio

(F

/NF

)

Standard

Day 13

0 1 2 3 4 5 6 7 8 9 10 11 12

CPM

Time (minutes)

0

0

600

1200

1800

0 2 4 6 8 10 12 14

Time (days)

Wa

ter

(Bq

/ml)

0

500

1000

1500

2000

2500

Se

dim

en

t (B

q/g

DW

)

Overlying water

Porewater

Sediment

0

2

4

6

8

10

12

0 5 10 15

Time (days)

BA

F

feeding

non-feeding

CDEA

CAS number: 002315-36-8

Molecular formula: C6H12ClNO

Chemical family: acetamide

Mode of action:

Uses:



Supplier: Amersham




Log Kow: 1.1

Log Koc: 1.8

Experimental data


Solvent for sediment extraction: 1 M NaOH in DIW

HPLC column: ODS2


Log Koc: 2.7

BSAFtotal: ND

% uptake through feeding: ND

Cl CH2XC

O

NCH2

CH2

CH3

CH3

HPLC trace of CDEA extracted from the sediment


Day 8

Standard

Day 1

Day 13

0 1 2 3 4 5 6 7 8 9 10

0

CPM

Time (minutes)

0

0

0

250

500

750

1000

1250

1500

0 2 4 6 8 10 12 14

Time (days)

Wa

ter

(Bq

/ml)

0

2000

4000

6000

8000

Se

dim

en

t (B

q/g

DW

)


Fungicide cyproconazole

CAS number: 113096-99-4

Molecular formula: C15H18ClN3O

Chemical family: triazole, conazole

Mode of action: Systemic fungicide with protective, curative, and eradicantaction. Ergosterol biosynthesis inhibitor.

Uses: Control of powdery mildew, rusts, Rhynchosporium, and leafspot disease on cereals.



Supplier: Novartis




Log Kow: 3.3

Log Koc: 4.2

pKa 10.3

Experimental data



HPLC column: ODS1

HPLC conditions: 10%ACN:90%MeOH+10mMol

CTAB

Log Koc: 3.0



Cl C

OH

HC

CH2

CH3

N

N

NX

HPLC trace of cyproconazole extracted from the sediment




0

0.5

1

1.5

2

0 5 10 15

Time (days)

Ra

tio

(F

/NF

)

Standard

Day 1

Day 11

0 1 2 3 4 5 6 7 8 9 10

0

CPM

Time (minutes)

0

0

100

200

300

400

500

0 5 10 15

Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

5000

6000

Se

dim

en

t (B

q/g

DW

)

Overlying water

PorewaterSediment

0.0

0.2

0.4

0.6

0.8

0 2 4 6 8 10 12 14

Time (days)

BA

F

feeding

non-feeding

Herbicide diquat

CAS number: 000085-00-7

Molecular formula: C12H12N2

Chemical family: bipyridyl

Mode of action: Non-selective contact herbicide, absorbed through foliage withsome translocation through xylem. During photosynthesis,superoxide is generated, which damages cell membranes andcytoplasm.

Uses: Control of annual broadleaved weeds. Control of emergentand submerged aquatic weeds.



Supplier: Amersham


Solubility (mg/l): 1.3*E5


Log Kow: -2.8

Log Koc: 3.3

Experimental data

% recovery from sediment: could not be extracted

Solvent for sediment extraction: n/a

HPLC column: ODS2

HPLC conditions: 70%buffer:30%DIW

Log Koc: 5.7

BSAFtotal: 0.008 ± 0.002


N+ N+

X

HPLC trace of diquat standard




0

0.5

1

1.5

2

2.5


Ra

tio

(F

/NF

)

Standard

0 1 2 3 4 5 6 7 8 9 10 11 12

CPM

Time (m inutes)

0

0.5

1

1.5

0 2 4 6 8 10 12 14

Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

5000

Se

dim

en

t (B

q/g

DW

)

Overlying water

Pore water

Sediment

-0.01

0.00

0.01

0.02

0.03

0 5 10 15

Time (days)

BA

F

feeding

non-feeding

Herbicide ethofumesate

CAS number: 026225-79-6

Molecular formula: C13H18O5S

Chemical family: benzofuran

Mode of action: Selective systemic herbicide, absorbed by the emergingshoots (grasses) and roots (broad-leaved plants), withtranslocation foliage. Inhibits the growth of meristems, retardscellular division and limits formation of waxy cuticle.

Uses: Pre- and post-emergence control of annual grasses andbroad-leaved weeds in a variety of crops.



Supplier: Amersham




Log Kow: 2.9

Log Koc: 2.5

Experimental data



HPLC column: ODS2


Log Koc: 2.6



OSH3C

O

O

CH3

CH3

OO CH2 CH3

HPLC trace of DODMAC extracted from the sediment




0

0.5

1

1.5

2

2.5


Ra

tio

(F

/NF

)

Standard

Day 11

0 1 2 3 4 5 6 7 8 9 10 11 12

CPM

Time (minutes)

0

0

200

400

600

800

0 2 4 6 8 10 12

Time (days)

Wa

ter

(Bq

/ml)

0

500

1000

1500

2000

2500

3000

Se

dim

en

t (B

q/g

DW

)

Overlying water

Porewater

Sediment

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 5 10 15

Time (days)

BA

F

feeding

non-feeding

Insecticide lindane

CAS number: 000058-89-9

Molecular formula: C6H6Cl6

Chemical family: organochlorine

Mode of action: Insecticide with contact, stomach and respiratory action.

Uses: Control of a broad spectrum of phytophagous and soil-inhabiting insects



Supplier: Cyanamid




Log Kow: 4.3

Log Koc: 3.5

Experimental data



HPLC column: ODS2


Log Koc: 3.6



Cl

ClCl

Cl

Cl Cl

X

HPLC trace of lindane extracted from the sediment




0

0.5

1

1.5

2

2.5

0 5 10

Time (days)

Ra

tio

(F

/NF

)

Standard

Day 1

Day 11

1 2 3 4 5 6 7 8 9 10 11 12

CPM

Time (minutes)

0

0

0

50

100

150

200

0 2 4 6 8 10 12

Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

Se

dim

en

t (B

q/g

DW

)

Overlying water

Porewater

Sediment

0

1

2

3

4

0 2 4 6 8 10

Time (days)

BA

F

feeding

non-feeding

Herbicide linuron

CAS number: 000330-55-2

Molecular formula: C9H10Cl2N2O2

Chemical family: urea

Mode of action: Selective systemic herbicide, absorbed principally by the rootsbut also by the foliage, with translocation primarily acropetallyin the xylem. Inhibits photosynthesis.

Uses: Pre- and post-emergence control of annual grass and broad-leaved weeds in a variety of crops.



Supplier: AgrEvo




Log Kow: 2.9

Log Koc: 2.5

Experimental data



HPLC column: ODS1


Log Koc: 3.4

BSAFtotal: nd


Cl

Cl

NH C N

O

CH3

CH3O

X

HPLC trace of linuron extracted from the sediment


Standard

Day 4

Day 13

0 1 2 3 4 5 6 7 8 9 10 11 12

CPM

Time (minutes)

0

0

0

5

10

15

20

0 2 4 6 8 10 12

Time (days)

Wa

ter

(Bq

/ml)

0

50

100

150

200

250

Se

dim

en

t (B

q/g

DW

)

Overlying water

Porewater

Sediment

Insecticide/Arcaricide pirmiphos-methyl

CAS number: 029232-93-7

Molecular formula: C11H20N3O3PS

Chemical family: organophosphorus

Mode of action: Broad-spectrum insecticide and acaricide with contact andrespiratory action. Penetrates the leaf tissue and exhibitstranslaminar action.

Uses: Control of a wide range of insects and mites in warehouses,stored grain, animal houses and industrial premises. Controlof sucking, chewing insects and mites on a variety of crops.







Log Kow: 3.5

Log Koc: 2.1

pKa 3.71

Experimental data



HPLC column: ODS1


Log Koc: 2.4

BSAFtotal: nd


N

N

CH3

N

O

CH2

CH2

PO

O

S

H3C

CH3

H3C

H3CX

HPLC trace of pirmiphos-methyl extracted from the sediment


0 1 2 3 4 5 6 7 8 9 10 11

Day 4

Standard

Day 1

Day 8

Day 11

CPM

Time (minutes)

0

0

400

800

1200

0 4 8 12Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

5000

Se

dim

en

t (B

q/g

DW

)


Fungicide difenconazole

CAS number: 119446-68-3

Molecular formula: C19H17Cl2N3O3

Chemical family: triazole, conazole

Mode of action: Systemic fungicide with preventive and curative action. Actsby inhibition of demethylation.

Uses: Control of a wide range of fungal diseases includingAscomycetes and several seed-borne pathogens of wheat andvegetables.



Supplier: Novartis




Log Kow: 5.2

Log Koc: 4.4

Experimental data



HPLC column: ODS1


Log Koc: 3.9



Cl O

Cl

O

O

H2C Me

N

N N

HPLC trace of difenconazole extracted from the sediment




0

0.4

0.8

1.2

1.6

2


Ra

tio

(F

/NF

)

Standard

Day 14

0 1 2 3 4 5 6 7 8 9 10 11 12

CPM

Time (minutes)

0

0

40

80

120

160

200

0 2 4 6 8 10 12 14

Time (days)

Wa

ter

(Bq

/ml)

0

2000

4000

6000

8000

10000

Se

dim

en

t (B

q/g

DW

)


0.0

0.2

0.4

0.6

0.8

0 5 10 15

Time (days)

BA

F

feeding

non-feeding

Herbicide

trifluralin

CAS number: 001582-09-8

Molecular formula: C13H16F3N3O4

Chemical family: dinitroaniline

Mode of action: Selective soil herbicide, which acts by entering the seedling inthe hypocotyl region, and disrupting cell division. Also inhibitsroot development.

Uses: Pre-emergence control of many annual grasses and broad-leaved weeds in vegetables.



Supplier: DOW




Log Kow: 5.3

Log Koc: 4.0

Experimental data



HPLC column: ODS1


Log Koc: 3.7



C

N

N

N

CH2

CH2 CH2

CH2

F

F

F

OO

OO

CH3

CH3

X

HPLC trace of trifluralin extracted from the sediment




0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 5 10Time (days)

Ra

tio

(F

/NF

)

Day 4

Standard

Day 1

Day 11

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

CPM

Time (m inutes)

0

0

0

0

10

20

30

40

0 2 4 6 8 10 12Time (days)

Wa

ter

(Bq

/ml)

0

1000

2000

3000

4000

Se

dim

en

t (B

q/G

DW

)

Pore water

Overlying water

Sediment

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 5 10

Time (days)

BA

F

feeding

non-feeding

APPENDIX B. Table of physico-chemical properties of test compounds

Table 1 Test Compound Physico-chemical Properties

Compounds Molecularformula

CAS # Supplier Molecularweight

WaterSolubility@ 25oC

(calc.) mg.l-1*

HLC(calc.)

atm.m3.mole-1*

Log Koc(pred)*

LogKow

(calc.)

Dipolemoment*

*

PesticidesAtrazine C8H14ClN5 001912-24-9 Ciba 216 214 4.5 x 10-9 2.4 2.8 3.6Cyfluthrin C22H18Cl2FNO3 068359-37-5 Astra-zeneca 434 0.008 9.2 x 10-7 5.3 5.7 5.0Cypermethrin C22H19Cl2NO3 052315-07-8 Astra-zeneca 416 0.009 7.9x 10-7 5.0 6.4 2.6Cyproconazole C15H18ClN3O 113096-99-4 Novartis 292 148 1.7 x 10-10 4.2 3.3 3.5Difenconazole C19H17Cl2N3O3 119446-68-3 Novartis 406 1 1.7 x 10-11 4.4 5.2 3.6Diquat C12H12N2 000085-00-7 Amersham 184 552 1.4 x 10-7 2.9 2.4 2.6Ethofumesate C13H18O5S 026225-76-6 Amersham 286 73 1.2 x 10-9 2.5 2.9 2.3Fenazaquin C20H22N2O 120928-09-8 DOW 306 0.22 4.3 x 10-8 4.9 5.8 2.5Fenpropidin C19H31N 067306-00-7 Novartis 273 0.6 1.2 x 10-5 5.3 6.4 1.0Fenthion C10H15O3PS2 000055-38-9 Bayer 278 5 1.4 x 10-6 3.4 4.1 5.1Lindane C6H6Cl6 000058-89-9 Cyanamid 291 4 1.3 x 10-4 3.5 4.3 2.2Linuron C9H10Cl2N2O2 000330-55-2 AgrEvo 249 44 1.2x 10-8 2.5 2.9 5.0MGK 264 C17H25NO2 MGK 275 9 2.9 x 10-7 4.1 3.8 2.2Paraquat C12H14N2 001910-42-5 Amersham 257 1000000 3.2 x 10-13 3.2 2.7 1.1PCP C6HOCl5 000087-86-5 Sigma 266 3.1 2.1 x 10-7 3.5 4.7 0.9Permethrin C21H20Cl2O3 052645-53-1 Astra-zeneca 391 0.001 2.9 x 10-7 5.3 7.4 2.3Pirmiphos-methyl C11H20N3O3PS 029232-93-7 Astra-zeneca 305 3 2.5 x 10-6 2.1 3.4 6.0Quinclorac C10H5Cl2NO2 084087-01-4 BSAF 242 76 8.6 x 10-12 2.6 3.0 6.6Simazine C7H12ClN5 000122-34-9 Ciba 202 590 3.4 x 10-9 2.2 2.4 3.5Trifluralin C13H18F3N3O4 001582-09-8 DOW 335 0.2 2.1 x 10-4 4.0 5.3 1.1Non-pesticidesCDEA C6H12ClNO 002315-36-8 Amersham 147 10000 3.4 x 10-8 1.8 1.1 3.7Benzo(a)pyrene C20H12 000050-32-8 Amersham 252 0.01 5.1 x 10-7 5.9 6.1 0.01Pyrene C16H10 000129-00-0 Sigma 202 0.02 5.6 x 10-6 4.8 4.9 0.01Phenol C6H6O 000108-95-2 Sigma 94 2616 6.1 x 10-7 2.4 1.5 1.3DCP (2,4) C6H4Cl2O 000120-83-2 Sigma 163 614 3.9 x 10-7 2.9 2.8 2.0TCP (2,4,5) C6H3OCl3 000088-06-2 Sigma 197 121 3.2 x 10-7 3.1 3.5 1.0DODMAC C38H80ClN 000107-64-2 Unilever 586 4.3 x 10-9 4.9 x 10-9 12.5 10.4 23.6* predicted using the SRC software, ** calculated using the TSAR programme

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Final Project Report - Science Searchsciencesearch.defra.gov.uk/Document.aspx?Document=OC9607_172_… · Final Project Report ... compound of interest, ... were decapitated, and duplicate