1171

Environmental Toxicology and Chemistry, Vol. 25, No. 4, pp. 1171–1177, 2006q 2006 SETAC

Printed in the USA0730-7268/06 $12.00 1 .00

A PERSPECTIVE ON THE ENVIRONMENTAL RISK OF HALOGENATEDBY-PRODUCTS FROM USES OF HYPOCHLORITE USING A WHOLE EFFLUENT

TOXICITY BASED APPROACH

IAN JOHNSON,*† JOHN A. PICKUP,‡ and DOLF VAN WIJK§†WRc plc, Frankland Road, Blagrove, Swindon, Wiltshire SN5 8YF, United Kingdom

‡John Pickup Associates, Fairy Cottage, Neenton, BridgeNorth, Shropshire WV16 6RL, United Kingdom§Euro Chlor, Avenue E. Van Nieuwenhuyse 4, Box 2, B-1160 Brussels, Belgium

(Received 21 March 2005; Accepted 15 September 2005)

Abstract—An assessment has been made of the potential for toxic effects to aquatic life that may arise from halogenated organicby-products formed by reaction of hypochlorite with organic matter in various use scenarios as a contribution to the risk assessmentof sodium hypochlorite under the European Union Existing Chemicals legislation. In the study, samples that would represent worst-case models for effluents containing halogenated by-products from a range of uses of hypochlorite were prepared by chlorinatingand then dechlorinating (removing residual chlorine) raw settled sewage (C/D-RSS). This sample was then compared to raw settledsewage that had not been treated (RSS) to assess whether halogenated by-products formed in the chlorination process were toxicor bioaccumulable and persistent. The relative toxicity of the samples was assessed using a series of tests with representatives ofdifferent taxonomic groups (bacteria, algae, and invertebrates). The persistence and potential for bioaccumulation of chlorinatedby-products was assessed by exposing solid-phase microextraction fibers to samples of RSS and C/D-RSS before and after degradationin a Zahn–Wellens test. For all the taxa tested in the study, the mixture of by-products formed in the C/D-RSS sample did notincrease toxicity relative to that measured in the RSS sample. Chlorination of the raw settled sewage did produce additionalpotentially bioaccumulable halogenated substances compared to the raw settled sewage. However, after degradation, the amount ofpotentially bioaccumulable halogenated substances in the RSS and C/D-RSS samples was comparable, indicating that these substanceswere degradable. The results are discussed in the context of the overall risk assessment for sodium hypochlorite.

Keywords—Raw settled sewage Chlorination Whole effluent toxicity Biodegradation By-products

INTRODUCTION

A risk assessment of sodium hypochlorite has been con-ducted under the European Union Existing Chemicals legislationthat has involved considering the potential environmental im-pacts that may arise from halogenated organic by-productsformed by reaction of hypochlorite with organic matter in var-ious use scenarios. The risks of the major identified speciesformed, such as haloacetic acids and trihalomethanes, were as-sessed directly in conventional predicted environmental con-centration/predicted no-effect concentration terms in the riskassessment. However, it was also considered important to assesspotential effects of unidentified halogenated species, and it wasdecided to address this using a whole-effluent testing approach.This would also provide an additional perspective on the iden-tified components. A tiered approach was planned for the testingprogram; a worst-case scenario was to be investigated first withothers being assessed in a sequential manner on the basis of theevaluation of the accumulated data should the worst-case notshow clear results. Chlorination of raw sewage was chosen asa worst case that would cover several other use scenarios wherethe substrates (natural organic matter including proteins, car-bohydrates, and fats) and reaction conditions (pH . 6 withexcess available chlorine) are similar but where the by-productmixtures discharged to the environment are expected to have alower impact, for example, wastes from household bleach usedischarged to a sewage treatment works (STW), wastes fromindustrial and institutional cleaning discharged to an STW, water

* To whom correspondence may be addressed(johnsonpI@wrcplc.co.uk).

from swimming pools discharged to an STW, drinking waterdischarged to an STW, treated cooling waters discharged directlyto a receiving water, and sewage disinfected before dischargeto a receiving water.

The by-product mixture formed by chlorinating raw sewagerepresents a worst case for the previously mentioned scenariosin two senses. First, the concentration of by-products in thetest effluent is higher, often by orders of magnitude, than thosethat are discharged to the environment from these other sce-narios. Second, the wide range of available substrates in rawsewage would mean that the range of by-products potentiallyformed would be likely to be wider than in the other scenarios.In the first four of the previously mentioned scenarios, effluenttoxicity would also be reduced by biodegradation during sew-age treatment.

Since the purpose of the study was to assess the risks ofby-products and not the effects of residual chlorine that weredealt with separately in the risk assessment, the test procedureincluded a dechlorination step to exclude any toxic effects fromresidual chlorine. In the study, the acute and chronic effectsof the by-products on algae and invertebrates were measured,which, based on toxicity data for individual substances, wereexpected to be the most sensitive taxa. Testing with fish wasnot included in this initial study for ethical and practical rea-sons, but this did not preclude subsequent testing should theresults have shown this to be necessary.

MATERIALS AND METHODS

Sample preparation

On February 16, 2004, a 40-L sample of raw settled sewagefrom Henley STW (Buckinghamshire, UK) was collected in

1172 Environ. Toxicol. Chem. 25, 2006 I. Johnson et al.

two 25-L plastic containers that had been rinsed with the sam-ple before filling. The sample was returned to the WRc lab-oratory, and two 20-L aliquots were dispensed into large glasscontainers. One of the aliquots served as an untreated control(raw settled sewage [RSS]), while the other aliquot was chlo-rinated (with 50 mg chlorine/L for 1 h) and then residualchlorine removed (referred hereafter as dechlorinated) by theaddition of an excess concentration (40 mg/L) of sulfite (cod-ing C/D-RSS).

A 1-L volume of a 1,000 mg/L sodium hypochlorite so-lution was prepared from a stock 10 to 12% Analar sodiumhypochlorite solution by dilution of 10 ml of stock withgroundwater. This 1-L solution was then added to one of the20-L aliquots of raw settled sewage (C/D-RSS). A 1-L sampleof groundwater was added to the other 20-L raw settled sewagealiquot that was acting as the unmodified control (RSS). Thesamples were then stirred for a period of 1 h.

Measurements of free residual chlorine in the samples weremade in the raw settled sewage aliquots before and after theaddition of either groundwater or sodium hypochlorite usingthe diethyl-p-phenylenediamine (DPD) method [1]. Residualchlorine concentrations (mg/L) were determined by adding0.12 g of DPD to 8 ml of each sample. A Merck SQ 118spectrophotometer (Merck, Poole, Dorset, UK) was calibratedat a wavelength of 550 nm and zeroed using a groundwaterblank (2 ml of groundwater in a 1-cm cuvette). Two-milliliteraliquots of the samples were then transferred to a 1-cm cuvette,and the resulting concentration was determined.

The chlorinated C/D-RSS sample was then ‘dechlorinated’by the addition of a 1,000-ml volume of an 800-mg sulfite/Lsolution (1,260 mg sodium sulfite/L) to achieve a final nominalsulfite concentration of 40 mg sulfite/L. A 1,000-ml volumeof groundwater containing 35 mg/L of calcium sulfate wasadded to the unmodified RSS aliquot to balance the increasein sulfate in the C/D-RSS sample resulting from oxidation ofsulfite to sulfate. The samples were again stirred for a periodof 1 h after addition of the sulfite solution. At the end of thisperiod, the residual chlorine level in each test vessel was mea-sured.

The 22-L samples of RSS and C/D-RSS were then eachsubdivided into two aliquots. Seventeen-liter aliquots werecentrifuged at 3,500 rpm for 15 min in a Sigma 6K10 cooledcentrifuge (Sigma Laborzentrifugen, Harz, Germany) to pro-vide material for the bacterial (Vibrio fischeri) biolumines-cence, algal (Pseudokirchneriella subcapitata) growth inhi-bition, and Daphnia magna immobilization/reproduction tox-icity tests. The purpose of centrifuging the samples was toreduce suspended solids concentrations to ,20 mg/L (a levelthat would not affect the conduct of the tests). Five-liter ali-quots were not centrifuged and were used for the biodegra-dation and bioaccumulation studies.

The samples were stored at 28C until the results of an anal-ysis of halogenated organic by-products (as adsorbable organichalide [AOX]) had confirmed that a severalfold increase hadoccurred in the level of these by-products following chlori-nation/dechlorination. A 48-h D. magna immobilization testwas conducted on the freshly prepared samples so that anychanges in toxicity that occurred during storage could be quan-tified.

Chemical analysis

Two 500-ml aliquots of the untreated RSS and chlorinated/dechlorinated C/D-RSS were taken for analysis for biochem-

ical oxygen demand (BOD), chemical oxygen demand (COD),ammonia (NH3), dissolved organic carbon (DOC), total or-ganic carbon (TOC), suspended solids, chloride, and sulfate.Replicate samples (250 ml) of the unmodified and centrifugedRSS and C/D-RSS samples were taken for analysis of orga-nohalogens using the AOX method. Chemical analyses werecarried out according to standardized Standard Committee ofAnalysts (SCA; London, UK) methods.

Test framework and procedures

Bacterial (V. fischeri) bioluminescence test. The Microtoxt(Strategic Diagnostics Incorporated Europe, Hook, Hampshire,UK) test system utilizes the bioluminescent marine bacteriumV. fischeri and measures the change in bioluminescence onexposure to the sample [2]. The toxicity of the RSS and C/D-RSS samples to the Microtox test system after a 30-min ex-posure period was determined on two occasions (March 9 and10, 2004) according to the manufacturer’s instructions (SDIEurope). Conductivity, temperature, and pH measurementswere taken on the sample submitted for testing to establishthe appropriate degree of salinity and pH adjustment necessaryin order to comply with the Microtox assay protocol. To ensurethat the response of the Microtox reagent was consistent withsimilar studies undertaken previously at WRc, it was also test-ed with the reference substances phenol and zinc. This wasachieved by comparing the results of the reference toxicanttest with the in-house Shewart control charts. All median effectconcentrations (EC50 values) were calculated using the ap-propriate statistical method menu of computer software sup-plied by Microbics Corporation (ver 7.8; US Microbics, Carls-bad, CA, USA).

Algal (P. subcapitata) growth inhibition test. A culture ofP. subcapitata was obtained from the Institute of FreshwaterEcology, Culture and Collection of Algae and Protozoa Centre(Ambleside, Cumbria, UK) on March 5 and was used within6 d of receipt. The algal test was initiated on March 9, 2004,according to the Organization for Economic Cooperation andDevelopment (OECD) test guideline method 201 [3] in a tem-perature-controlled orbital incubator. For each test concentra-tion of the RSS and C/D-RSS samples, four flasks were pre-pared. Three replicate flasks were inoculated with algae, whilealgae were omitted from the fourth, which served as a blankto monitor any background changes in nonalgal particles. Toavoid potential issues of nutrient deficiency, the same volumesof nutrients were added to all test solutions. Algae were addedimmediately after preparing the test solutions. The algal celldensity of the starter culture was determined using an elec-tronic particle counter, and the three replicate algae-containingflasks were inoculated to achieve an initial cell density ofapproximately 10,000 cells/ml. The background particle con-centration and pH of each stock solution contained in the vol-umetric flasks were measured using an electronic particle coun-ter and a pH meter, respectively. All flasks (inoculated anduninoculated) were then plugged with sterile foam bungs andplaced under constant illumination (6,000–10,000 lux) androtation (100–130 rpm) at 23 6 28C. After 24, 48, and 72 hthe particle density of each flask was measured. These mea-surements were taken by removing 0.5 ml of solution fromeach flask using a sterile pipette and adding this to 50 ml ofIsoton before determining the particle density using an elec-tronic particle counter. At the end of the test, the pH of eachsample concentration was measured using the solution from areplicate flask.

Perspective on risks of halogenated by-products Environ. Toxicol. Chem. 25, 2006 1173

Table 1. Analytical chemistry data for a range of determinants in the raw settled sewage (RSS) and chlorinated/dechlorinated raw settled sewage(C/D-RSS) samples

Sample

Biochemicaloxygendemand(mg/L)

Chemicaloxygendemand(mg/L)

Ammonia(mg/L)

Dissolvedorganiccarbon(mg/L)

Totalorganiccarbon(mg/L)

Suspendedsolids(mg/L)

Chloride(mg/L)

Sulfate(mg/L)

RSS 170 349 32.8 43.2 43.2 118 117 50.0C/D-RSS 194 388 23.6 59.4 59.4 158 328 85.7

To ensure that the response of the algae was consistent withsimilar studies undertaken previously at WRc, a test with thereference substance zinc was also conducted at nominal con-centrations of 100, 320, and 1,000 mg Zn/L. This was achievedby comparing the results of the reference toxicant test withthe in-house Shewart control chart. The actual exposure con-centrations were confirmed by chemical analysis. The con-centrations corresponding to the no-observed-effect concen-tration (NOEC), lowest-observed-effect concentration(LOEC), and 50% inhibition of growth (EC50) in the effluentand reference tests was then determined using a statisticalsoftware package ToxCalc5t (Tidepool Scientific, Mc-Kinneyville, CA, USA).

Daphnia magna immobilization and reproduction test

The 14-d D. magna immobilization and reproduction testwas initiated on March 10, 2004, according to the OECD testguideline method 211 [4]. The juvenile D. magna used in thetest were obtained from in-house cultures. The concentrationseries used for the RSS and C/D-RSS samples were 0 (control),1.0, 3.2, 10, 32, and 100% sample. Fifty-milliliter aliquots ofeach concentration were added to 100-ml glass beakers, andone juvenile (,24 h old) daphnid was added to each test vessel.Twenty control vessels of groundwater were also used in thetest. Observations of the number of mobile and immobiledaphnids in each vessel were made daily, and the number ofjuveniles in each vessel was noted from day 9 on. Water qualityparameters (pH, temperature, dissolved oxygen, and hardness)were measured at the beginning of the test. If pH and/or dis-solved oxygen levels in the test solutions were outside ac-ceptable test ranges, then modifications were made to ensurethey were within range at the start of the test. The animalswere fed each day with an algal suspension, and the test so-lutions were replaced every Monday, Wednesday, and Fridayprepared from the stored RSS and C/D-RSS samples.

A zinc reference toxicant test accompanied the D. magnaimmobilization and reproduction test to ensure that the sen-sitivity of the test organisms was consistent with previousstudies at WRc using this test organism. This was achievedby comparing the results of the reference toxicant test withthe in-house Shewart control chart. The following nominalconcentrations were used, expressed as zinc: 0.0 (control), 0.2,0.4, 0.8, 1.6, and 3.2 mg/L. The actual exposure concentrationswere confirmed by chemical analysis. The concentrations cor-responding to the NOEC, LOEC, and EC50 in the effluent andreference tests were then determined using the statistical soft-ware package ToxCalc5.

Zahn–Wellens biodegradation test. The Zahn–Wellens testis a static die-away method measuring disappearance of DOC.The method identifies substances that have the potential toundergo ultimate biodegradation in an aerobic environment.The test material is dissolved in a buffered mineral salts me-dium and inoculated with activated sludge not previously ex-

posed to the test material and incubated in darkness at constanttemperature for 28 d. Analyses of DOC are made at intervalsand compared against the concentration present at the start ofthe test. Biodegradation is indicated by DOC removal. Theprocedure and the activity of the inoculum are checked usinga functional control substance whose pattern of biodegradationis well established. The Zahn–Wellens biodegradation test wascarried out according to OECD test guideline 302 B [5]. Du-plicate solutions containing the test substance (RSS and C/D-RSS) mineral nutrients and activated sludge in aqueous me-dium were agitated and aerated at 20 to 258C in the dark for28 d. A blank control (containing activated sludge and mineralnutrients but no test substance) and a reference sample (con-taining activated sludge, mineral nutrients, and diethylene gly-col) were tested in parallel. Mineral medium was preparedaccording to test guideline OECD 302B the day before thetests were initiated and allowed to stand overnight at the testtemperature. For the blank control, 5 ml of the concentratedmineral medium were added to a volumetric flask and madeup to 2,000 ml with distilled water before being transferredto the test vessel. For the reference test, 2.22 g of diethyleneglycol were weighed into a glass weighing dish and made upto 1,000 ml with distilled water (nominal concentration 1,000mg organic carbon per liter). A 200-ml aliquot of the diethyl-ene glycol stock solution and 5 ml of the concentrated mineralmedium were added to a 2,000-ml volumetric flask and madeup to a final volume of 2,000 ml with distilled water beforebeing transferred to the test vessel. Duplicate test vessels wereprepared for each of the test substances (RSS and C/D-RSS),where 5 ml of the concentrated mineral medium were addedto make up to a total volume of 2,000 ml. Each of the testvessels was inoculated with 5 ml/L microorganisms of a mixedpopulation obtained from the activated sludge of Henley Sew-age Treatment Works (Buckinghamshire, UK) on the day oftest initiation. Each of the test vessels contained a large poly-tetrafluoroethylene-coated magnetic follower and was con-nected to an airline that provided an even distribution of finebubbles from the aeration tubes. After recording the pH (andadjusting, if necessary, to within a pH range of 6.5–8.0) foreach of the treatments (day 0), the remaining test solutionswere kept in the dark at a constant temperature (20–258C).

In the test, degradation was followed by analysis of DOC[6] over a 28-d period. Samples were taken for the determi-nation of DOC at test intervals of 0 (after 0 and 3 h), 1, 2, 7,9, and 28 d for the control, reference substance (diethyleneglycol), and each of the test substances (RSS and C/D-RSS).The ratio of eliminated DOC corrected for the blank, after eachtime interval, to the initial DOC value was expressed as thepercentage biodegradation at the sampling time. The pH of thesolutions in each of the test vessels was monitored every work-ing day and adjusted with acid (0.01 M HCl) or alkali (0.01M NaOH) to be within the pH range 6.5 to 8.0.

Assessment of bioaccumulation potential using solid-phase

1174 Environ. Toxicol. Chem. 25, 2006 I. Johnson et al.

Table 2. Summary of the adsorbable organic halide (AOX) values on freshly prepared samples

Sample Code

Uncentrifuged samples

Mean AOX (mg/L)Range of AOX values

(mg/L)

Supernatants

Mean AOX (mg/L)Range of AOX values

(mg/L)

Raw settled sewage A 210 200–220 180 170–190B 174 148–200 153 151–155

Chlorinated/dechlorinated raw set-tled sewage A 1,355 1,300–1,410 1,025 950–1,100

B 1,223 1,149–1,296 1,100 1,075–1,125

Table 3. Bacterial (Vibrio fischeri) bioluminescence test data

Test number Substance

Mean effective concentration (EC50) value (95% confidence intervals)(%v/v sample)

5 min 15 min 30 min

1 Raw settled sewage (RSS) 7.1 (6.5–7.9) 4.2 (3.6–4.9) 5.9 (5.2–6.6)(March 9, 2004) Chlorinated/dechlorinated

raw settled sewage(C/D-RSS)

37.3 (34.3–40.6) 33.0 (30.9–35.2) 33.1 (31.3–34.9)

Zinc — — 0.45 (0.42–0.47)Phenol — 16.1 (15.5–16.7) —

2 RSS 10.1 (9.5–10.7) 7.4 (6.5–8.3) 8.5 (7.7–9.4)(March 10, 2004) C/D-RSS 35.3 (32.6–38.1) 27.2 (26.1–28.4) 27.9 (26.6–29.2)

Zinc — — 0.42 (0.40–0.44)Phenol — 18.0 (16.9–19.1) —

microextraction fibers. At the start (day 0) and end (day 28)of the biodegradation tests on the RSS and C/D-RSS samples,the presence of halogenated by-products with the potential tobioaccumulate was investigated using solid-phase microex-traction (SPME) fibers. This involved using polyacrylate fibers(Supelco, Bellafonte, CA, USA) having a length of 1 cm andan internal diameter of 55 mm. The fiber had a coating of 100mm polydimethylsiloxane. The volume of the polydimethyl-siloxane phase was 0.621 ml. The fibers were conditioned be-fore use according to the manufacturer’s instructions [7]. Onday 0, replicates of the RSS and C/D-RSS samples were dis-pensed into 25-ml glass bottles. One fiber was placed in themiddle of each sample bottle. No headspace existed in thebottle, and the fibers were brought into the solution througha seal in the cap of the bottle. The extraction period lasted for24 h, and the solutions were continually stirred during thisperiod. After the exposure period, the fiber was removed fromthe bottle, dried gently with a tissue, and stored at 48C in asealed container until analysis. The same procedure was re-peated on RSS and C/D-RSS samples after 28 d of degradation.One of the replicate fibers from both the RSS and the C/D-RSS exposure on days 0 and 28 was analyzed for AOX, whilethe other fibers were analyzed for total halogenated by-prod-ucts by direct injection into a gas chromatograph–mass spec-trometer in electron capture mode. The concentrations of totalhalogenated by-products on the SPME fibers were quantifiedby comparison with areas under the curve for SPME fibersexposed to known concentrations of chlorinated paraffins.

RESULTS

Sample preparation phase

Chlorine (free and residual) measurements. Analysis of thefree chlorine concentration in the sodium hypochlorite solutionused to chlorinate the RSS resulted in a value of 980 mg/Lsuch that the initial free chlorine concentration in the test ves-

sels was estimated to be 46.7 mg/L. Following reaction of thesodium hypochlorite solution with RSS for an hour, the re-sulting level of residual chlorine was 8.2 (8.0–8.4) mg/L. Theaddition of 40 mg/L sulfite concentration resulted in a reducedresidual chlorine concentration in the C/D-RSS sample (0.56mg/L) that was similar to that in the RSS (0.58 mg/L).

Chemical analysis. Table 1 summarizes the analytical chem-istry data for a range of determinands, BOD, COD, NH3, TOC,DOC, suspended solids, AOX, chloride, and sulfate, in theRSS and C/D-RSS samples. The C/D-RSS sample had a lowerammonia concentration than the RSS. Higher concentrationsof BOD, COD, DOC, TOC, and suspended solids were mea-sured in the C/D-RSS compared to the RSS. Because of thechlorination, larger molecules and polymers (e.g., proteins)will have become degraded to smaller molecules that may havebeen measured as BOD and COD.

Table 2 summarizes the AOX measurements in RSS andC/D-RSS. Separate analysis of replicate samples confirmedthat a 6.5- to 7.0-fold increase in AOX had occurred in theC/D-RSS sample relative to the RSS sample. The AOX levelsin the centrifuged RSS and C/D-RSS samples were measuredat the start of the toxicity tests and were found to be compatiblewith those in the freshly prepared samples. The AOX concen-tration in the centrifuged RSS was 110 mg/L and 900 mg/L inthe C/D-RSS.

Initial D. magna immobilization test. The initial D. magnaimmobilization test on the freshly prepared samples showedthat the toxicity of the RSS sample (48-h EC50 value 5 17.9%)was slightly greater than that of the C/D-RSS sample (48-hEC50 value 5 47.6%). The test can be considered valid sincethe results of the accompanying zinc reference toxicant testwere consistent with the mean EC50 values from the in-houseShewart control chart for this test.

Test proceduresBacterial (V. fischeri) bioluminescence test. Table 3 sum-

marizes the results of the bacterial bioluminescence tests car-

Perspective on risks of halogenated by-products Environ. Toxicol. Chem. 25, 2006 1175

Table 4. Algal (Pseudokichneriella subcapitata) growth inhibition test data for test started onMarch 9, 2004

Substance ConcentrationMean growth rate

(per day)

% Inhibition(relative to

control)

Raw settled sewage Control1.0%3.2%

10%32%

1.131.100.980.760.31

—2.7

13.333.372.6

Chlorinated/dechlorinatedraw settled sewage

Control1.0%3.2%

10%32%

1.131.121.020.800.47

—0.99.7

29.258.4

Zinc reference 100 mg/L320 mg/L

1,000 mg/L

1.100.860.24

0.723.978.8

Table 5. Daphnia magna immobilization and reproduction test for test started on March 10, 2004

Testconcentration(%)

Immobilization (%) after 48 h

RSSa C/D-RSSa

Reproduction data

Time of first brood (d)

RSS C/D-RSS

Mean no. of juvenilesper surviving adult

RSS C/D-RSS

Control1.03.2

1032

100

00000

80

00000

100

98888

—

98888

—

36.4115.1127.2146.6

86.6—

36.4116.7132.4141.6117.2

—

a RSS 5 raw settled sewage; C/D-RSS 5 chlorinated/dechlorinated raw settled sewage.

ried out on the RSS and C/D-RSS samples on March 9 and10, 2004. Good agreement was observed between the resultingEC50 values from the two tests. On both occasions, the RSSsample showed slightly greater toxicity (lower EC50 values)than the C/D-RSS sample. The results of both tests can beconsidered valid since the results of the zinc and phenol ref-erence toxicant tests were consistent with the mean EC50 val-ues from the in-house Shewart control chart for this test.

Algal (P. subcapitata) growth inhibition test. Table 4 sum-marizes the results of the algal growth inhibition tests carriedout on the RSS and C/D-RSS samples between March 9 and12, 2004. From the data, it is evident that the responses forboth samples were similar with a reduction in algal growthbeing evident at concentrations exceeding 3.2% volume pervolume (v/v). A greater than 50% reduction in algal growthwas evident in both samples at concentrations of 32% v/v. Nogrowth occurred in the 100% v/v concentrations of either theRSS or the C/D-RSS samples, indicating that toxicants wereprobably not responsible for the observed effects. Instead, theabsence of growth was probably due to reduced light attenu-ation in the test vessels due to the presence of fine particulates.The resulting 72-h NOEC and 72-h LOEC for the RSS andC/D-RSS samples were the same, being 1.0% and 3.2% v/v,respectively. The 72-h EC50 values for the RSS and C/D-RSSsamples were similar, being 19.5% (95% confidence intervals5 15.9–24.0%) and 25.7% (95% confidence intervals 5 12.5–44.8%), respectively. The results of the test can be consideredvalid since the result of the zinc reference toxicant test (EC505 642 mg/L with 95% confidence intervals of 570–742 mg/

L) was consistent with the mean EC50 values from the in-house Shewart control chart for this test.

Daphnia magna immobilization and reproduction test

Table 5 summarizes the results for the D. magna immo-bilization and reproduction test on the RSS and C/D-RSS sam-ples that was started on March 10, 2004. The extent of theimmobilization of daphnids was similar for both samples withno effects being observed at 32% v/v and high levels of im-mobilization at 100% v/v. The results for the C/D-RSS sampleare similar to those from the initial test on the freshly preparedsample. The RSS sample showed a slightly lower toxicity afterstorage. For both samples, production of juveniles was evidentafter 8 to 9 d. The numbers of juveniles per surviving adultshowed a similar pattern for both samples, with increasingnumbers from 1.0 to 10% v/v sample followed by a reductionat 32% v/v back to the numbers found at 1.0% v/v sample.The test can be considered valid since the results of the ac-companying zinc reference toxicant test were consistent withthe mean EC50 values from the in-house Shewart control chartfor this test.

In the D. magna reproduction/immobilization test, the bell-shaped response for juvenile production was probably the re-sult of the competition between two factors: the presence ofincreasing levels of nutrients and/or food items with increasingsample concentration and the presence of increasing levels ofcontaminants with increasing sample concentration.

At the exposure concentrations of 3.2 and 10%, the en-hanced effects of the nutrients and/or food items on juvenile

1176 Environ. Toxicol. Chem. 25, 2006 I. Johnson et al.

Table 6. Mean measured dissolved organic carbon in the biodegradation test over the 28-dexposure period

Treatment

Dissolved organic carbon concentration (mg carbon/L) over the 28-dexposure period

Day 0(0 h)

Day 0(3 h) Day 1 Day 2 Day 7 Day 9 Day 28

Control blank 5.0 7.0 2.8 1.4 2.9 2.0 2.5Raw settled sewage 21.7 18.7 12.6 16.1 15.5 17.3 11.1Chlorinated/dechlorinated

raw settled sewage 45.6 38.9 16.7 16.0 15.4 14.9 12.0Diethylene glycol

reference test 94.6 96.1 67.6 86.0 76.0 78.4 18.7

Table 7. Summary of the data from the analysis of solid-phase microextraction (SPME) fibers

TreatmentDay of

biodegradation test

Lipophilic organichalide from SPME

fiber (mg/fiber)

Total chlorinatedorganics from SPME

fibers (mg/L)

Raw settled sewage 028

160100

0.190.11

Chlorinated/dechlorinatedraw settled sewage

028

510100

1.030.06

production appear to outweigh the effects of the contaminantspresent. However, in contrast, at 32% v/v, the effects of thecontaminants appear to outweigh the effects of the nutrientsand/or food items. The levels of juvenile production observedin all the controls were markedly lower than in RSS andC/D-RSS treatments, presumably reflecting the greater levelsof nutrients and/or food items present in the treatments.

Zahn–Wellens biodegradation test. After sample collectionand preparation, the RSS and C/D-RSS samples were storedat 48C for 20 d while confirmatory AOX analyses were per-formed. During this period, a loss of DOC occurred becauseof degradation. In the RSS sample, DOC decreased from 43.2to 21.7 mg/L and in the C/D-RSS sample DOC decreased from59.4 to 45.6 mg/L. Table 6 shows the DOC (mg/L) values fromthe test vessels, the blank control, and the reference test after0, 1, 2, 7, 9, and 28 d, which were determined by ultraviolet–persulfate oxidation/nondispersive infrared. While the initialDOC analysis results for the RSS and C/D-RSS treatmentswere different (21.7 and 45.6 mg/L, respectively), the samplestaken on day 28 show that degradation has occurred and thatthe remaining DOC concentration was similar in both treat-ments (11.1 mg/L for RSS and 12.0 mg/L for C/D-RSS). Inthe diethylene glycol reference test, the DOC levels decreasedfrom an initial level of 94.6 mg/L to 18.7 mg/L after 28 d,indicating that .80% degradation of the test material had oc-curred during the test. No marked changes in DOC were ev-ident in the control blank throughout the exposure period.

Assessment of bioaccumulation potential using solid-phasemicroextraction fibers. Table 7 summarizes the results of theanalysis of the SPME fibers that had been exposed to RSS andC/D-RSS solutions before (day 0) and after 28 d degradation.The analysis of lipophilic organic halide (TOX) on the SPMEfibers showed that the TOX concentration on the fiber exposedto the C/D-RSS sample on day 0 (510 mg) was higher comparedto the concentration on the fiber exposed to the RSS (160 mg).After the RSS and C/D-RSS samples had been degraded for28 d in the Zahn–Wellens test, the TOX concentrations on thefibers exposed to them were the same (100 mg), indicating that

the additional potentially bioaccumulable halogenated by-products that were present in the C/D-RSS sample at day 0were biodegradable. In the analysis of the TOX on the SPMEfibers, the variability associated with the measurements of theRSS day 0, RSS day 28, and C/D-RSS day 28 samples washigher than for the C/D-RSS day 0 sample (Table 7). However,this level of variability does not preclude drawing conclusionsas to the changes in TOX between exposure regimes. Theanalysis of the total extractable halogenated organics on theSPME fibers by their direct injection into a gas chromatographin electron capture mode provided data that were consistentwith that for TOX. This showed that though the fiber exposedto the undegraded C/D-RSS sample contained more haloge-nated organics than the fiber exposed to the unchlorinated RSSsample, levels in the fibers exposed to the two samples afterbiodegradation were similar.

DISCUSSION

In the study, chlorination of the raw settled sewage didproduce additional potentially bioaccumulable halogenatedsubstances compared to the raw settled sewage control, asevidenced by the elevated levels of halogenated organics mea-sured on SPME fibers. It is likely that these substances prob-ably included some of the major typically identified by-prod-ucts, that is, haloacetic acids (such as chloroacetic acid, di-chloroacetic acid, and trichloroacetic acid) and trihalometh-anes. However, the by-products formed during chlorinationapparently did not bioconcentrate in the test species used inthe toxicity tests (bacteria, algae, or invertebrates) to levelsthat were sufficient to result in enhanced toxic effects sincesimilar responses were observed in the toxicity tests of theRSS and C/D-RSS samples.

The absence of enhanced toxicity in the whole-effluent testson the chlorinated/dechlorinated RSS also needs to be consid-ered in the light of predicted toxic effects resulting from thepresence of halogenated by-products, such as trihalomethanesand haloacetic acids. Previous studies [8] have shown that inan operating sewage disinfection plant, with chlorine residuals

Perspective on risks of halogenated by-products Environ. Toxicol. Chem. 25, 2006 1177

maintained around 55 to 58 mg/L, average chloroform levelsrose from 4 mg/L in the unchlorinated effluent to 71 mg/Lfollowing chlorination, an increase of 67 mg/L (equivalent to60 mg/L AOX). Other THM levels rose from 0.8 to 3.3 mg/L,an increase of 2.5 mg/L (equivalent to ;2.4 mg/L AOX). Thetotal AOX levels rose from an average of 91 mg/L in unchlo-rinated effluent to 801 mg/L following chlorination, an increaseof 710 mg/L. In laboratory experiments using 40 mg/L chlorinefor 1 h, carried out during the same series of studies, estimatesof the formation of dichloroacetic acid and trichloracetic acid(detected by gas chromatography–mass spectrometry as meth-yl ester) were 19 and 17 mg/L (equivalent in each case to 10mg/L AOX), while the average AOX level rose from 188 to625 mg/L, an increase of 437 mg/L. On the basis of ratios seenin other scenarios, concentrations of haloacetic acids other thanTCA and DCA are likely to be around 10% of the combinedDCA and TCA concentration, that is, another 2 mg/L AOX[9]. The increases in AOX levels in these studies are consistentwith those measured in the present study. The concentrationsof haloacetics produced in chlorinated effluents are also evi-dently dependent on the chlorine dose used. In another studyusing a dose of 10 mg/L but with a contact time of 24 h, 7mg/L DCA and 2 mg/L TCA were produced in the chlorinatedeffluent [9].

Although this study showed that the chlorination/dechlo-rination of raw settled sewage resulted in increased levels ofhalogenated by-products, it also showed that the substancesformed by chlorination of the raw settled sewage were de-gradable. As a result, the amount of potentially bioaccumulablesubstances in the RSS and C/D-RSS samples after degradationwas comparable.

The data obtained in the current study were also consistentwith whole-effluent toxicity data for the freshwater inverte-brate Ceriodaphnia dubia and the fathead minnow (Pime-phales promelas) from an assessment of the use of hypochlo-rite for household laundry bleaching [10]. Chronic bioassaysconducted with the treated bleached laundry wash water re-sulted in no toxicity to test organisms that could be attributedto laundry AOX, even at concentrations 40 times higher thanthe predicted environmental AOX concentration. The studyalso explored whether the by-product mixtures generated dur-ing laundry bleaching might contain significant quantities ofpotentially bioaccumulative species after sewage treatment.Based on a draft U.S. Environmental Protection Agency meth-od [11], samples were fractioned by high-performance liquidchromatography and subsequently analyzed by capillary gas

chromatography with full-scan electron impact ionization massspectrometry at a detection limit of 10 ng/L. This provided nopositive or tentative identifications of chlorinated substancesin the potentially bioaccumulative fraction. Similarly, no peakscorresponding to any of the compounds of highest concernwere observed as listed by the U.S. Environmental ProtectionAgency. Comparison with unbleached laundry effluents in-dicated that the range of other potentially bioconcentratablecompounds present was similar regardless of the use of bleach.It was concluded that use of hypochlorite in laundering isunlikely to generate persistent bioconcentratable compounds.

Acknowledgement—The authors acknowledge the support of A. Ber-ends, H. Berger, and P. Lemaire in the conduct of this study.

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