n AQUATIC TOXICITY TESTING Understanding and Implementing

Your Testing Requirement ll

RECEIVED SEP 1 8 1995

I FACILITIES ASSESSMENT UNcl

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Prepared by the Aquatic Toxicology Unit/Environmental Sciences Branch Water Quality Section

X.C. Division of Environmental Management

FEBRUARY 1994

This L?fomauon parkt has been prepared 2s a mource for NPDES permit holden q u i r e d to perform effluent toxicity rrsting or are utilizing toxicity testing as pan of a toxicity duction progm. T h e document explains many of be concepts and erminology used by a regulatory agency regarding toxiity testing. By carefully reading this informaljon it is hopd that funher communication with either your own en+anmenral staff or an outside consultant w i l l be made rare productive.

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Toxiciry refers to h: pentid for a substance to produce an adverse or hmL%l effect on a living organism. A toxicult is 2n agent (e.g.. .;.hole effluent discharge) that can pmjuce an adverse e%ct in a biological system, seriously dm2,o inp its Sr:cture or function or causing death. The adverse resporce may be dcfrned in terms of a memremtnt that is ouLCi:~ the "normal" range for healthy organisms, such as ahormal mortality, reproduction or growth.

Toxiciv tcsts rn u x l to determine the level of toxicity, if any, present in aa effluent and the duration of exposur, required for the Jxicity to tZ expressed 2s adverse effects. Test organins are exposed in t a t chambers to variou coxenrations oi "ie effluent. Thc criteria for effecu, such as mohality Ed repmduction, art then evaluated by compmg those orgi-isms which are expoKd LO different dilulions of the efhent with those organisms (conuob) :xposed only c 3 nontoxic dilution water.

Acute :ffezts x e t h e s that occur rapidly as a result of short-term exposure. Exposure is consid& relative to the crgznism's life span. The most commonly measured acute effect in aquaac a-ganisms is death. Cbronic effects Kc:; ~~t:; ~7 effluent 0; z~xicant produces adverse effects as a result of a repeat& or long-term exposurt. Chronic efiects inciude lehal 2nd ?~blethzl responses (such as abnormal growth and/or rcprduction).

Srzdstical mzlyses z.d mathemuicd modehg summarize the data ~olltcted $zing a toxicity test The specific application of h e x rourkx map be quite simple or exaemely complex. The frA analysis (after these staristics nave been periorm:dj ho..;?ver, is easily understcd. All sutistical routines are s ~ ~ i f i c a l l y defined for each procedure. it is no; neces:q to comp1et:ly undersmd all of the analyses perfomed by a laboratory in order to ud:ze &z p:odu::d by r x i c i t y t s i n g . Tnis d m x n e n t includes an overview of l e s e data interpretations.

!n n : s ~ + z s 3 2 2 c z 2 roxiciry of an effluent, the objective is to measure a r q e of effluent concentrations ar . - m e s7c.1:: c c x : m z k 5at prcdcces a readily cbs:nfable and quanrifiablen~~nse. The quzndfiable n s p ~

nest C?i tR c5scr\.ed is mxiaiity, which is then used to calculate an LCso value 0: determine if significant B C U I ~ morulity is occurring. i".? LCsois the concenmtion estimated to cause monali: in 50% of the r e s t population 0)':: f q x c i k ! tirnc peC:d. Application facton n a y k applied to 2 mtzsured LC50[o predict the concenuation of t f 5 c m ;;.:lich m y have :.a adverse impacs over m extended duadon ( i.e., no ckonic u'xiciry).

3a:h:: L;.m .;:in: 2". xu;e test with an applicaion iaclor to evaluate chronic toxicity, it is possible to directly n:'2s~g, cr,:cz: :r,p:u :.;h 2 more sophisU~a:ed test procedure. Thest chronic i ~ . t s ZTC more difficult to perform bu: eiiminxc usc ci 3 ~-::~cial rppljczdon facmr. The chronic test measures bo5 sublerhal and lerhzl effects over a. bnge: 1:s; ;XT?LIOE ard ;r.rs_cures rzrpns:s during a sexidve ~ n o d of h e orgar3m's life cycle. . . *.

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G u i d a n c e Documents:

United Slates Environmenial Protectjon Agency. 1991. Methods for Measuring the Acute Toxiciry of Effluents to Freshwartr and Marine Orgznisms. Founh Editioa. EP.4/600/4-90/02?. 293 pp.

North Carolina PassFail Methodolog for Determining Acute Toxicity in a Single Effluent Concenuaion. Nonh Carolina D e p m e n t of Environment, Heahh, and Naturzl Resources, DEM, Water Q d t y Section. December 1987. Revised July 1992. (This document is auacned 2s Appendix A.)

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Hamilton, M.A., Russo, R.C., and Tnurston, R.V. Trimmed Spearman-Karber Method for Estimating Median Leth.d Concenuations in Toxicity Bioassays. Environmentzl Science & TEhnoIogy, Yo1.11, No. 7, July 1977.

Acute toxicity tests determine nhccher some concmuacion of test material or effluent will produce an adverse effect on a group of test organisms during a short-term exposun under connolled conditions. Experimentally, a 50% lethal response (concenuation at which 50% of the test organisms die) is the most reproducible rne2sure of acute toxicity. When the median lethal concentration (LCso) is calculated, the 95% confidence limits associated with that wiue =e also reported. An acute tcxiciry test require by the Division may have a test dunrion of 24,48 or 96 hours. The test species is usually one of the following: the water fleas Duphniu p u l u or Ceriodu.phnia dubia, the fathe20 minnow, Pimepha1espromck.s. or the mysid shrimp, Mysidqon'J buhiu, representing salt water species. 0:her species may be u tilizd to a a k s s a specific concern. l71cse rests are typically static. meaning the orgvlisms are maintzined in rhe original test solutions for the dunrion of the test.

P:ior to ccllcting an effluent s.nplt and performi?g 2 toxicity res4 sampling glassware 2nd stzinless steel or tenon equipment are washed with so?? and hot water, k n r i n s e d in nimc acid, acetone, and ~isrilledldeionited w2rer . to remove toxicants and contaminant. Plastic conrainen and equipment may be used on a one-time or disposable basis, or dedicarcd to use with a ptzicular effluent. Tnc effluent sample used in the static U~XS is collected below chlorinarion as'a grab or 24 hour composite (depending on permit requirements). The sample mast be collected and srored with an 2mount of ice suffcim to maintzi.1 its temperame between 0" and 4OC until receipt at the laboratory. I he cn!y allowzble exception to this sample shipment plicy is the situation where the umc lapse between:the collection of a grab sample and its use in the laboratory that same day does not exceed four (4) hours. P E M defines "use" of a sample as h e i n d u c t i o n of the test organisms into the test solutions. This exctption is based on trn zpproximare time necessary icr a chilled sample to rezch roam or testing remperanue on its own, after being removed from ice or refigeration. I\-here this exception is used, appropriate chain-of-custody documentation should be submitted with test results showing, ai minimum, collection time md date, collector, mehod of collection, sample temperature on receipt in thc labontory, and the tine and date a t which the toxicity t s t on tlis sample was initiated. Ai Iabrarories certified by the Stare of Nonh C v o h a a perfom,toxicity tes~ng ;re rqquirtd to measure szmpie ~empei~ure on receipt in thtk laboratories. Should this ternpram exced Alowzble sfaduds, the smple

- does not quaEy for the periormancz of valid t e a s q d mch resulrs will be rejtxted for use in ATDES compliance de1trminations. Addidonally, the smpie is not to be frozen under any circumsmcts. Frozen samples.will be rejec:ed for L ' S ~ in hTDES compliuct determinations. It is suggwed that coorainztion of rmpling and sample shipment methods be discassed wiin the lzboratory pedorming the malyses so that these crierfa are met. '

I ne effluent samples are pre?zd for testing by being rhorouginly mixed, allowed to rea-h smdard tes t

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- temperz:rre, a d 3 e ~ t e d if dissolved oxygen (DO) is'keiow 4 mg/l. Total residual chlorine ij measured. The effluent is then ciiured with control ~21e7, t>@dly IO f ive con~nuzuons (with the appropriate num'kr of replic2tcs) €tom 0 to 100% efff uent. The tes: vessels z e then f d l d wih the appiopriate volume of test solution. Test organisms are ben tnnsferred to test chmbers in 2 random manner. Initial DO 2nd pH ue measured in sqarate vessels of diluuon and ei9uent solutions. Tie 1 s t is inrubired at 25°C wid3 a 16:8 hour lighcdark cycle. ~MomLiry of the test orgmism is re:c:ded zfier the aefixd test period along with finai pH. dissolved oxygen. and rernpenturc. This test dam is :a be :nt::cj on SEte i o n .\T-l (Appendix B) for submittai to ihe Division.

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AII LCJo or concenration of effluent lethal to 50% of the test organisms over the tcst period is calculated from L-:? momlity bra using one of the several methods, preferably b e pmbit or Spearman-lhber analyses, as descrikd i:. -,ie EPA acute lut ing protocols (EPA/600/4-90/027). An insueam waste concentradon (Twc) for the effluent in i.5 receiving sveam is calculated (in percent) using the wastewater mtment system p e r m i d flow and receiving ~r:m 7410 flow. The LC50 and nVC are then used to predict ins- toxicity.

In inslanw where localized effects at the point of discharge art of conkrn, alttrnare protection SUalcgies ma: be rqired. In thest instances it is imponant that no shon term acute effects occur. To a d d r e s s this issue. the Lvision of Environmental Management will often require the uie of an acute test methodology in which acute rr::nality in a specific effluent concenuation, usually 90%, may be statistically dettrmined. The acute W f a i l F.-:cedure is a natic non-renewal toxicity examination generally using the Fathead Minnow (~impha~esprornclat~ fs freshwater or the Mysid Shrimp (MpidopJis bahia ) for salnvalcr dischargers. Two concenmtions art urilizd in L-e procedure with a control population s p e c i f i e d as rreatment one and an effluent m t m e n t Specified Bs treatment I-; 3. The acrurl effluent concentration at which the test is to be performed will be specified in the A'PDES pennibor b: .4dminismtive letter. Each rreaunent is tested using four identical test vessels each containing ten test cgiisrns. AI t es t termination, organisms are identified as alive or dead. Analysis of the data from the acute ~-s/fail piOCCdUe is performed using a Student's t us1 to deurmine if mortality in the effluent treatment (Umtmtnt r A D j is significantly different than the control population. All statistical analyses are performed using arc sine s q w x*:[ uansformtd dam (see referenced EPA document) and rested for signif"cance at a 99% confidence level. Test r t x h a ~ e recorded as "Pass" or "Fail" and are to be reported on Stalc form AT-2 (Appendix C). AU supporring i-ixnacion requured on the AT-2 form must be provided in order for the report to be considered a complete . s,:.-nittal to the Division of Environmental Managepent

khronic Toxiciiv T u

G y i d a n c e Documents:

L'IEPA. Shon-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Myin: 2nd Esmz+e Crganisms. EP.V600/3-87/028. May 1988.

3 ::3 Carolin? Phase I1 Chronic Whole Effluent Toxicity Test P r d u r e . Nonh Carolina Depanment of EnvinnncnL Health, andh'anual Resources, DEM, Water Quality Section. July 1991. F i s dmmen: is anzched ES Appendix D. )

5139 Cvolina Division of Environmental Management. 1985. Revistd Sept 1989. N o d Cvolina CerioduphrA Ctuosic Effluent Bioassay Procdure (Cerioduphniu Mini-Chmnic PassFA Toxicity Test). (This document is 2ri:ned ij Appendix r).

Chronic mic i ty tests allow evaluation of adverse effects of an effluent under conditions of long-term expors. it:sthenin,o ik t s t duration to include one or more complete life cycles or performing the t e ~ t during a sensitivt L:: srrge emp.kizes more subde adverse effects, such as reducaon in growth and reproduction. .Evaluatio.n of t h e s t ::xs from long-ten exposure to the effluent can pnvide a dimt estimate of the effects threshold of the toxicant 2 r i n S iifc cy:!: tests with several ,rpecjes of fish and invenebrates, cenain developmental stages have consistenlly i k n shwn 10 be more sensitive than 0th~~. Use of shoner USIS wilh the early developmental slages can also r-dkt chronic toxicity. Thtst methods have been developed to probide quicker and l e s s costly ways to meaSUlt :.Tonic toxicj? :o aquaric organisms.

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The Division of Environmental Mmzgcment often requires a rhrtc brood Static renewal: test using the cladoce.~, Cir iodgphio ixbio, as the test organism. A stadc renewal test is one in which the test solutions are renewed ;e*>ikally by umsfernng the test orgmisms to chambers wilh freshly prepared solutions. The test is initialed with c..,~qL'ljms "" u.nich zre less b a n 24 h o w old and born within 8 hours of each other.

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The Cerioduphniu chronic toxicicy test m a w s both survival and rtproduction during the test period. The original ntonate (newly born Ccriodaphnio) inuodnced into each e s t container at the beginning of the Wt is monitored for survival as well as for the number of offspring it produces. Exposure of the organisms to differing concentradons of effluent can determine the conmuation of effluent expected to cause signif~cant monaiiry or suppression of reproduction, as compared to conml populations. The endpoints of these multiple concentration tesLs can often be described by the highest concennation which caw no obstrved effect or the NOEC (NO Obsernd Effect Concentration) and by the lowest concentration which causes an observed effect or the LOEC (Lowest Observed Effect Concentration). The geomemc mean of these cor.cenuaaons, termed h e chronic value or ChV. rcpnsenls the effluent concenvation at which obsuved effects begin to appkar. An important effluent concentration to compare to the ChV is the insueam waste concentration or nVC. This represents the percentage of the receiving s w a n comprised by the effluent during periods of low stream flox and maximum permitted effluent flow per the equation:

Maximum Permitted Discharrc Volume x lOQ Maximum Pemiaed Discharge Volume + 7410

The 7QlO is the lowest average 7 day flow in he receiving s m which has a probability of recurrence every ten years. Comparison of the facility W C to the ChV of the ttss can predict whether an impact will occur on sensitive or,oanisms in the receiving sueam. "he multiple concenmtion analysis is typical of procedures such as the EPA described chronic toxicity test and the North Carolina Phase II chronic toxicity analysis referenced previously.

m e sutisticzl comparisons for evaluating the &gnificance of chronic analysis test results are generally . performed 3 outlined in the EPA guidulce documents previously referenced (EPA/603/4-89/001, EPA/600/4-87/018) or by the spcific NC DEM modified method. Srarisucal signifimce may be evaluated in part by calculation of Dunnett's t value. The use of this test is discussed in the EPX d r x h e n t as well as on the back of the State AT-3 form (Apvndix E), the form required for submission of multiple concenuation chronic test dam Significant difference; in monality rates arc determined by use of the Fisher's Exact Test as discussed in the cited EPA documenr

x.. Chronic toxiciry analysis qudity conuol paramtters for conad organisms include avenge total repduction which must equal or exceed 15 offspring per surviving female. A h , mortality grater than 20% in the con001 population w i l l b t considered abnormd, invalidahg the test resde. Other quality conrrol components of the test include intubatin_e the test chambtrs for temperature control. maintaining a photoperiod of 16 hours of light and 8 hours of h k n e s s , use of samples within 72 hours of collecaon urd maintenance of samples between 040C during shipping md storage. This packet includes a checklist of quality control parameters to assist facility personnel in evaluating rest acceptability (Appendix F).

For the Nonh Carolina test procedire, effiuent samples art collected twice below chlorination as 24 hour composites, unless othenvise spetifjed by the pemir ?he samplts must be collected and stored with an amount of ice sufficim to maintain a sample's temperature tmwecn 0" and 4'C until receipt at thclaboratoxy. The only zllowable Exception to this sample shipment policy is the situation where the time lapse between the collection of a grab S ~ ~ F I C and its use in the laboratory does not exceed four (4) hours. DEEM defines "use" of samples as the introduction of the test organisms into the t a t solutions. ?his exception is based on an approximate Lime ntCessaxy for a chillxi sample to reach room or tesring tempature on i s om, after being removed from ice or refrigeration. . In such inances appropriate chain-of-custody d m e n t a d o n should be submitted with test results showing, at minimum, collection time and date, colltctor, met;?od of collection. sample temperature on receipt in the laboratoiy, and h e line and date at which the toxicity !est on his sample WS initiated. All laboratories certified by the Stale of Xorh Carolina to perform toxicity taring art rqui+d to measure sample temperawe on receipt in their labomones. Shouid his remperarure exceed allowable smdar& the sample dots not qualify for the performance of valid tests and such resuls will be rejected for use in NPDES compliance determinations. Additionally, the sample is not to be frozen unc:r any circumstances. Frozen samples uill be rejected ior use in NPDES compliance determinanons. Ir is suzgested kat coordinadon of sampling and sample shipment meihods be discussed with the laboratory performing the zndyss so that these criteria are met.

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Tht c c i l ~ ~ i o n of rhese samples should accomnodate the schxiule outlined in the protocol being used. The effiuent =?pies m prepzed for testing by being umoughly mixed, adjusted to smndard test ternperatllre of 25OC. and semi i i dissolved oxygen is.below 5 mg/l. Tne effluent samples are also analyzed for total residual chlorine.

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The co1l:crion of quality toxicity testing data requires good laboratory practices. The EPA guidance downtnts previously cizd discuss quality assurance (QA) methods. QA practices for effluent toxicity tests include dl aSptcf5 of the test thx affect the accuracy and precision of the data such as: effluent sampling and handling; the source and condition of tire test organisms; condition of equipment: test conditions; instrument calibration; use of reference toxicants; record keeping, and data e\aluafon.

Per Nor;? Carolina Adminisuacive Code Title 15,2H Section .110O,'any commercial, indusuial, or publjc taboratory pxfoxming biological toxicity testing as required by an NPDES permit, must be certified by h e Division of Environmcnral blanagement. lfiese Rules, effective October 1.1993, provide that laboratories performing these tcsu may bc :tnified and decenified by the Slate of Konh Carolina. NPDES permittees required to perfom whole effluent toxicity tesring are responsible for the submittal of quality lest data and ensuring that their performing laboratories LZ cemfed to conduct specific tcsts.

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To aid ysu in Iccaung aquatic toxicological resting rervjces, Appendix G provides a list of biological laboralones CTat have been cenifkd by the Division of Environmental Management In accordance with the Regul?uons. k s e libratones have betn evaluated as having the capability t~ adequately perform ctnain aquatic toxicity tesL: for ciims in North Carolina. The list of certified biological laboratories will be kept on file and updzted by C..t Division's Environmental Sciences Branch, and will be available upon request by mail or fax (see DEbl conu:s iistcd at the end of this document).

' Under rast circumsrances, toxkity testing resuh from samples taken prior to the permitted discharge point arc no1 rtqubed ZI be xponed. Such samples include prc-chlorination samples, treatment process samples, and industrial process sam;!~. However, any result from a test pen'ormed on a sample taken from rhe permitted discharge point must be repzxd. Tnis requirement applies even to hose faciliues which do not have mxicity monitoring in their h'PDES p e r i t .

The toxisitp lesl,ing requirement language in NFDES permits states that in addition to including test results on the ficility ;r.anthly monitoring report form "1). toxicity test results must be submitted to Lhe State on the appropriate E M AT f o n within thirty days afur the end of the reporting period for which the repon is made. A copy of the AT-1, AT-2, and AT-3 forms an= attached (Appendix B,C, and E). It is essential that all the information bc provided 5 reqctsted and applicable to the type of test results king submitted. The AT form submittal to the S w e is to ir,:!ude L+ signatures of the facility opemor in responsible Chsge and the labratory supervisor, as provided for cn the form. Please note that exclusion of any of h e necessary information wiIl constitute an incomplete FLbmissjon of toxicity test data to the Division. Dual reportins requirements exist for perminces required to :?nduci toxicity testing by ATDES pennit or adminiscative 1eIfer. Discharge Monitoring Report forms should be r i k d 10 the Division's Central Files while the Aquatic Toxicity Test forms should be sent LO the address localed belo:. A g i n , spccial note should be made that the AT forms and the standard &fR-1 reponing forms are' . sen1 to diffe::;.rt adeesses. This is necessary due to b e exVd degree Of quality B S S U ~ ~ ~ C ~ review given to Lhe aquauc toxicity test 2~ scjrnit1ed to the Environmental Scitnces Branch. You should consider submitting your toxicity self-monitof-qg r e p = via cerufied mail t o ensure h t your repom art received timely by Lhe Environmental Sciences B;.,-.=h. The AT forms shall be sent to:

ATIEhTION: ENVIRONMEA'TAL SCIEKCES BRAXCH NORTH CAROLINA DIVISION OF ENVLRONMEATAL MANAGEMENT . 4 0 1 REEDY CREEK ROAD RALEIGH, NORTH CAROLINA 27607

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Test results may be rejected due to inappropriate sampling. inadequate nnuol organism survival. or in the c a t o i chronic tests. inadequate conuol organism nproduction. Division staff L"I such an analysis a "bad test" (bt). Under lnese circumsunces a follow-up test must be initiated within 30 days of h e initial monitoring event.

A t times the laboratory may be aware of QA problems during or imme2iatcly following a Est thaf will prevent ~ h t d m from being accepted. Additionally, a test may be s c h d u l c d which =?not be completed due to sample collcuon or shipment problems. In such cases the analysis should be reschzjuled within 30 days of the initial monitoring attempr If the analysis cannot be rescheduled durin,o the permit defined monitoring month. a letter should k drafted to thc Environmtnrzl Sciences Branch at the above addrtss which explains why the analysis could not bc completed during the appropriate monitoring monrh and specifjes tht xcheduled date of the analysis. While this letcr does not relieve the facility from compledng the monitoring, it ~ i l help to prevent Notices of Violation for failure to perform the initizl monitoring.

hlost new permi5 issued wirh a quarterly monitoring requirement ~ p e c L ~ that any failurt tD mett a ptrmit h i t incrczccs the monitoring frequency to monthly until a test result is generate2 which meeu the permit limit. Good lin:s of communicauon with the tonoacting laboratory are essenuzl to ensm h a t appropriate follow-up testing is scheoui:d regardless o i the circumsrancts requiring such 1esring.

In Lie event that no discharge of flow occurs from a facility during a mcnth that toxicity testing is required, you should comp1e:e the information block located at h e top of the AT form iniicating the facility name, pennit number, pipe numbcr. county, and the month/year of the subject repon Ycu should write "NO FLOW" on the AT f o n t i d submit IO ~ $ 2 Environmtntzl Sciences Branch following normal plxcedurcs. .

GuidEnce Documents:

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Unittd S ~ t t s Envuonmentzi Protxuon Agency. 1991. Meho-3 for Mcsxing the Acute Toxicity of Effluents to FrEsiiwate; and Maine Orgmisrns. Founh Edition. E?M600/4-90/02'. 293 pp.

. ".- c 2 r ? X . Sho::-'i:n ?!ethods fer Estimating the Chronic Toxicity of Efflut:?s md Receiving W2:ers to Frtshwattr

0:gaisms. 2nd cdirion. E?A/600/4-S9/001 March 1989.

l23.2.. Shon-Tzm ?ieth&s for Estimating the Chronic Toxicity of Efflums 2nd Receiving Waters to biuine urd Esiu&ne Orgmijms. EPPJ6@0/4S7/02S. May 1988.

Hamilion, M.A., Ruxso. R.C., and llurston. R.V. Trimmtd Sp-r;nan-Wcr Method for Estimating Median kLkl Conctntmions in Toxicity Bioassays. Environmend Science i Technolom, Vol 11, No. 7, July 197f.

SE;? 233 form Ai-!(3/87) rev. 9/39 (anzched 2s Appendix B). $:.E 3E3.1 iom AT-2(10/90) (arnthed as Appendix C) S n ~ t 331 form Ai-:(7/91) (artached 25 Appendix E) . - . ..

Prtstnung md jnttrpreung Ecute 2nd chronic toxicity test r s u l r s requi -s the use of srztistical analysis. S c p p ~ i r , ~ SL21js:iCS z e used to evelu?re the level ofconfidtnce !32t mey k sscciated with he test results. The tec; Ssiisics muSI 'w reponed on h e Srate AT-I; .41-2, znd AT-: forms, k t : rtquired forms for submitting toxicity L ~ S I : w i t s . 0Lht:wiS. the submission of test datz will be considzed incorr.plete.

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in 21 ~ t u t e toxicity test. the primay purpose of the test is gentrally an :$timation of the concentration of the 1es1 nzrt;ial or pe:c:nrage of effluent that is lethal to 50% of the 1 s t orgmisrns within a specific !engh of h e . This nezsxe is cal1:d m LCJo. 7-m LCso is chosen in most xute toxicity Ltirs because an esrimate of the median ro1:mnc: (50% kill) for a fixed m p l e site is most reproducible in this rang:. The LC50 is statistically anmated t i z z c s : ii is u n i i k l y that one of h e concenuations selected in th5 txperimea will kill exacuy 50% of the exposed test g u k r i c n . .A :onfidznce i n tend ior t$e uue LCso is compu::d along -4th his point estimate and ZSSCN w i t h

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a pre-specified level of confidence (usually 95%) bat this interval contains b e me LC50 n e test LC50 and

protocols (EPAlboG/1-90x)27) describe several methods for estimating h e LClo and confidence intervals. Alrhough any of the referenced methods are acceplable. the recommended methods are the probit and Speannan-bbw methods beczuse their LCso estimates rely on the data in the more stable, cenual: portion of the tolerance distribution.

* supponing test data are to k reponed on State form AT-l(auached as Appendix B). EPA acute toxicity usting

The Division may require the acute toxicity Pasflail test in instances where additional: protection of a water body r n w be provided to reduce h e likelihood of localired effects due to incomplee mixing. The analysis employs a Student's t test to determine if monality in a single effluent meanent is significantly different than the convol populauon. AU statistical analyses an performed using arc sine square rmt uansfonnd data (see referenced EPA document 60G/4-90/027) and evaluated for significance at a 99% confidence level. Should monality in the effluent ue2unent exceed that of the convol population and the absolute value of the calculaed t value exceed the absolute value of tht tabular t value, then the effluent ueatment is considered as having signiBcant acutt effccu on the test organisms. This would be considered a "Fail." If all vessels within each traunent have the same tnonality but at different levels between uezaents. then a t statistic is not calculable. In this case. if the monaIily is identical b c r w w trea~ment~ th tn the test is considered a "Pass." If the response in the effluent m m e n t is greater thanthe conuol mztment then the test is considtred a "Fail." State form AT-2, the form required to xbmit rtsulu of the zcute P & d Lest procedure. is provided as Appendix C.

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T!x chronic toxicity ttsu which m used and required by the Division deurmin: the effects of whole emuenu on f

the monality and reproduction of a species (Ccrioduphnin) for an exknded period of h e . Mean reproduction and a

pexen: mordity results for the effluent concentration are compared to hose for tht control by performing statistical tess of significsnce. The EPA chronic toxicity tesring protocol (EPA/600/4-89/001) describes mean reproduction as h e summation of L O B I number of young produced per female Ccriodophnio until time of death or end of experiment divided by tht initid numbtr of females exposed.

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t Uistance Available

Guidance Documents:

USEPX. )Je!hods for XCuz!i: Toxicity Idenufication Evaluations: Phast I Toxicity Charactexi~adon Procedures. EPMCW-31iGOj. Februq 1991. Envkonmenrzl Research Laboratoq, Duluth, 3linn.

USE?.% ?hue 11-Toxicity Idmifietion Procedures. EPA 600/R-92/080. September 1993, Environmental Research LZSC;ZiOr)., Duluth, Minn.

USEPX. ?hue 111-Toxicity Confirmarion Procedu~s. EPA 600/R-92/0S1. Scptem'kr 1993, Environmental R e r a c h Lrborztory. Dululh, biinn.

USE?:.. Tosicip R d u c u o n E~zlu2do;l Protocol For hlunicipzl Wutewatrr Treznent Plznts. EPMjKI/2-88/062. Apii 1589, EfA Risk Reoucdcn Engineering Laboratory, Cincinnati. Ohio.

USE?A. Generzlite-d h f e 5 n i d o ~ for Conducting Industrial Toxicity Reduction Evaluations (TFSs). EPA 600@ SS/G70. 1SS9, EPA \V2ter Engineering Resedch Lsbratory. Cincinnati, Ohio. . . . . .

USETA. Technic21 Suppon Document For Water Quality-bastd Toxics Conuol. EP;VS05/2-90-001. 1991. EPA Offict of Yi'aer, \'dashington. D.C.

3

?

AppenCir, H provides 2 listing of contxt persons in each Of LhC Depanment of Environment, Heallh, and Natural Resouxes' Iiegiond Offices. You SI= :ncou:agd IO contact the Regiod Water Qulity Supervisor in your area R t g i o x l oEre for zssisrvlcc in undtrsunding and inplemenring your toxiciii test rrquirement.

S

EhT7RONMENTAL SCIENCES BRANCH NOXTH CAROLINA DIVISION OF EhTlRONMENTAL hcMNAGEME?IT 4401 REEDY CREEK ROAD R a I G H , NORTH CAROLFA 27607

Assistance is also available for inri:.cuies and local governments 10 help identify and apply ways 1O reduce, recycle, and minimize wastes before they become toxic pollutants. The Pollution Prevention Program is a non- regulator\. progam within the D e p m t n t of Environment. Health, and N a n d ResourcCS which provides technical assistance, resevch and education. and zatching grants for such toxiciry reduction efforts.

The Pollurion Prevention Program is an information clearinghouse with access to over 1600 references, cast stuaiei, ma conBcrs on waste reduction options. If addicional infomation is needed, a customized computer literature w r c h can be conducted. Ba& on the production process, an Lidustry-specific q o r t providing economic and technical evduations of available w s i e nduction techniques can be developed. More specific alternatives can be identified rhrough an on-site visit 7"It ?ollution Prevention Program offers matching funds (S5,OOO of a S10,OOO project) to businesses and communitiez krough the Challenge Grants for Waste Reduction and Minimization Projects. Inquiries should be directed 12 rfre Pollution Prevention Program at (919) 571-4100.

9

V

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EPA/600/2-88/062 April 1989

Toxicity Reduction Evaluation " Protocol -

for Municipal Wastewater Treatment Plants

1

John A. Botts Jonathan W. Braswell

Jaya Zyman Engineering-Science, Inc.

Fairfax, Virginia 22030

-?

T

William L. Goodfellow EA Engineering, Science and Technology

Sparks, Maryland 21 152

Samuel B. Moore '

Burlington Research, Inc. Burlington, North Carolina 2721 5

Contract No. 68-03-3431

Project Officer

Dolloff F. Bishop Treatment Assessment Branch

Risk Reduction Engineering Laboratory Cincinnati, Ohio 45268

RISK REDUCTION ENGINEERING LABORATORY '

OFFICE OF RESEARCH AND DEVELOPMENT U.S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268

Introduction A toxicity source evaluation is conducted to locate the sources of influent toxicity or toxics that are contributing to the P O W effluent toxicity. This svaluation is performed in two tiers. Tier I, which is discussed in this section, involves sampling wastewater of indirect dischargers or sewer lines and analyzing the wastewaters for toxics andior toxicity. Tier II, which is described in Section 6, is performed to confirm the suspected sources of toxicity identified in Tier 1 testing.

The flow diagram for the Tier I source evaluation is presented in Figure 5-1. Selection of sampling locations is based on evidence that a source contributes toxicants causing POTW effluent toxicity or diecharges substantial levels of potentially toxic 301' :s. Sampling of wastewaters from indirect disLdrgers is recommended where existing pretreatment program data or TIE results are adequate to indicate that the dischargers may contribute toxic pollutants that cause P O W effluent toxicity., Sewer line sampling can be used to locate toxic sources by process of elimination, if pretreatment program information and TIE data are lasking, or sampling of indirect dischargers is not feasible (e.g., large number of Us).

T le choice of chemical-specific anatyses or toxicity tests for source tracking will depend on the quality of tke TIE data on the POTW effluent. Chemical- ssecific investigation is recommended in cases where tke effluent toxicants have been identified and presumably can be traced to the responsible sewer. dischargers. In situations where TIE data indicate that ccecific industrial chemicals such as copper or phenol =re tbe effluent toxicants, the TRE should proceed to the evaluation of local pretreatment limits a s aescribed in Section 8.

Toxicrty tracking is required in situations where TIE 2ata on specific toxicants are not definitive. Prior to tcxicity analysis, sewer samples are afforded the sane level of biological treatment as provided by the POT' '7r its influent wastewaters. If toxicity tracking is SL ,sful in locating sources that are contributing toxicity, the TRE proceeds to the Tier I1 source

evaluation to confirm the toxic indirect dischargers. At this point, the POTW may require the IUS to conduct a TRE to reduce IU wastewater toxicity. It may also be possible in a few instances to correlate the pretreatment program data gathered at the beginning of the TRE (Section 2) with the effluent toxicity results to identify the influent sources of toxicity.

Sampling Location Sampling locations for Ter I testing are established by reviewing the pretreatment program data Fable 2-2) and the TIE results. and selecting the sources that contribute relatively high loadings of potentially toxic pollutants or toxicants identified in TIE tests. Where possible, the selection of sampling locations should be based on TIE results, because the toxicants causing the effluent toxicity are the pollutants that must be controlled. Sampling-may be conducted at the point where the IU discharges its wastewater to the sewer or in the sewer lines of the sewer collection system, if no IU appears to be the source of the toxicants. A plan for sewer line sampling can be devised to track toxic sources through a process of elimination of segments of the coilection system (USEPA, 1983a).

The choice of IU discharge sampling or sewer line sampling will depend on the number of IUS contributing to the POTW, the quality of the pretreatment program data, and the TIE results.. IU discharge sampling is recommended where the:

Number of IUS is small enough so that sufficient . '

test data can be collected with the allocated resources:

Pretreatment program data are sufficient to characterize each of the IUS; and

TIE data indicate .POTW effluent toxicants that can be attributed to selected IUS.

In the Patapsco I R E indirect dischargers were selected for sampling based on evidence that the IUS contribured substantial loadings of toxicity (as

5- 1

, . ,_ . .. -

-: ’ .. . .

Results of

Evaluation (Figure 4-1)

1 1 Toxicity 1;nttficatIon

r I 1 Sample Collectton 1 f i Sewers/Polnt Discharges

”””””

v

I

! 7

j I Chernrcal-Speclfic ! Refractory Toxlcltv lnvestlgatron of I Assessment of Selected ’

i i Selected Porn1 Discharges or 1 ! Pocnt Discharges or I Collection System Co1Iec:lon System

! I

! I

1

i : 1: -5

Figure 5-1. Tier 1: toxicity source evaluation.

measured by MicrotoxTM) and toxicants (non-polar organic materia!) identified by TIE tests of P O W effluent (Botts et.al.. 1987). A description of the Patapsco study is given in Appendix A

Sewer line sampling is recommended where:

e

e

e

The

A sewer line sampling plan can be developed that will allow a more efficient method of toxicity tracking than sampling IU discharges;

Pretreatment program data are limited or unavailable; and

Sources of toxicants ideniified ‘duiing the TIE are not obvious.

Town of Billerica. Massachusetts, utilized a sewer line sampling approach to identify areas within the sewer collecr:on system that contrlbute toxicity to the Town’s wzsiewater treatment plant (Durkin et.al.,

1987). P. description of the Billerica study i s provided in Appendix A.

Whether sampling of IU discharges or sewer lines is conducted, 24-hour flow proportional samples are recommended to characterize daily variations in toxics or toxicity while accounting for variations in flow. Flow data must be gathered in order’ to determine the relative contributions of toxicants or toxicity from the IUS. Other considerations for Tier I sampling are des.cribed in Section 13. .QA/OC sampling requirements are discussed in Section io..

Chemical-Specific Investigation A chemical-specific approach can be used to trace the influent sources of toxics. if definitive TIE data on the specific toxicants causing P O W effluent toxicity are, available. This approach is not recommended in czses where the TIE data only. indicate a broad Class of compound3 (e.g.. polar organic compounds). because the toxicants may be contributed by a large

number and variety of sources which will be dititcult to pinpoint by chemlcal tracking.

The chemical-specific approach involves testing iU discharges or sewer line samples for specific tov:-ants using chemical analysis techniques. In some

aoequate to determine the IUS that are contributing the toxicants. It is likely, however, that further sampling and analysis wiil be necessary, because many toxicants other than those typtcally monitored he., priority pollutants) are present in IU discharges. Existing pretreatment program data can be used to reduce the amount of sampling and analysis by indicating which sources contribute toxtcs that are similar to the effiuent tcxicants.

C existing pretreatment program data may be

Chemical analysis methods for prrority polluiants are described in several E?A documents (USEPA 1979a. 1979b, 1S80, 19SSb) and S:andard Methods for the Exammaiton of VJzier and Wasiewater (APHA, 1985). knaly!ical techntques for non-priority pollutan!s can be found in Amerrcm Soclety for Testtng and fdaterials (ASTM) mawals m a an2lyiicsl cnemlstry journals sucn as the halvtical Chenlstry Journal. I he selected znaly;lcal mernod shouio be vertfied in the laboratory prlc: to sanpllnS and anelysis. Prior to znaiysis, a literaiure search should be made to determine if the iOxlCant could be a biodegradation ?roduct resulting from POTW treatment. Where clear ?Jidence is aailtble to show that the toxicant is a :r?etment by-produci, the sewer sample should be a ~ = ' , ~ ~ e d fo: the precursor fcrm(s) oi rhe toxicant 2s h the toxcant.

In cases where Tier I chsmica! trzcking is successful :n locating the IUS ;hzI ere responsible f3r the P O W e%uer,t toxicats, tf;e T3E p:ocess ctl: move to the seiection and develognent of local pretreatment :e;ulattons (Section 6). If the responsible IUS can not 'Je iocated, the TIE results should be reviewed to cc.?iirm previous ccnciusions. The chemical analysis " ,t:JliS -. should also be carefuliy reviewed to determine :i errors or wastewater matrix effects may have "I, <\.sed ineccura!e !esA:s. In cases where the ~:r~emical-spcclflC E ; ? ~ G E C ~ is ultimately not " " _ d :.*~-ocsful. :tie Tie: I tEsiin2 shcuij be repeared .sing tcxicity ieS:S rn !ieu c i chemicei analysis as x-sc:ic.ed 121er in ;T&:S SEC~IC~:.

-

=retreatment Program Review

only a few IUS wench have rela!lvely non-complex discharges.

The PPR approach was applied a! the Mt. Airey POTW in North Cerolina (Diehl and Moore, 1987) which receives industrial wasiewater from only 2 few sources, predominerely textile industries. In the Mt. Airey TRE, detailed tnformatlon On the manufacturing processes and westewater discharges of the industries was gathered (see Table 2-2). including data on the toxicity and biodegradability of raw and manufac:ured cherntcals as provlded in material safety -data shee!s Iib'iSDS). This information was used 'to identify indusrrtel chemic,?ls with relatively high potenttal toxict!;, that may be present in the POTW effluent. Subsequent chemical analysis of the POTW effluent v ~ c s performed using methods specifically destgneo to measure for the Suspected industrial toxics. The chemical analysis results were compared with water quality criteria and toxicity values for individual compounds. Using this approach, alkyl phenol ethoxylare surfactants, phthalate esters and chlorinated solvents, largely ttirituted to textile industries. were idmi f led as the prlmary toxics caustng :he FOTW EYluent toxicrty.

A description of PPR methods is provided in Appendix A. The meihods described involve a direct comparison of IU chemical data to P O W effluent toxrcity. ti is imponat to emphasize that drawing preiiminsry conclusicns based on PFR resultscan be ntsleadinc, because IU monitoring informaticn 'could be incomplete. chemical analysis techniques may not be sensitive to low levels of effluent toxics, and the estimated toxicity of individual compounds may not reflect the whole efiluent toxicity. Due to these factors, comparisons of suspected toxicants to effluent toxicity nay y ie ld fa lse correlat ions. -

Whenever possible, results of TIE testing should be used in lieu of PPS results. because tne TIE test directly measures the relaftve toxrciry ai the effluent constituents.

Refractory Toxicity Assessment Influent toxicity trackrng is cecess2ry when TIE testing is cn!y ab!e tc rden:i!y a bread class of ._ toxicants. rather than specific compcunds, that are causing the POTW e?dent toxicty. Toxicity. packing - . c a y also be reculres tn s w a r m s 'where there are -a ..

!age numoer of effluent toxicants and !he occurrence cf :hese toxicants ir: the P O W effluent is highly vzrtable. In inis case X?xlc!ty testlns may be more . c:s:-etiec::ve !>an ch;.~~cal analysis.

: .,

-r 1 "1 .* I? 'i

. *

of toxicity in a sewer discharge which could potentially pass through the P O W must be estimated by treating sewer samples in a simulation of the P O W process prior to toxicity analysis. Based on the experience to date, a simulation of activated sludge treatment has been developed for predicting the potential for a sewer discharge to Contribute to the P O W effluent toxicity. This treatment step accounts for the toxicity removal provided by the P O W . A toxicity tracking approach can be applied to IU discharges and sewer line wastewaters to locate the sources contributing either acute or chronic toxicity that is refractory to P O W treatment.

The. refractory toxicity assessment (RTA) approach involves treatmg sewer -samples in ijerobic batch bioreactors and testing the resulting effluents . for toxicity. Batch bloreactors have been used by several researchers to screen wastewaters fcr activated sludge inhibition (Grady, 1985, Adams e! al.. 1981, Philbrook and Grady, 1987, and Kang et al.. 1983) and non-biodegradable aquatic toxicity loague and Hagelstein, 1984, Lankford et ai., 1987, and Sullivan et at.. ,987). Oague and Hzgelstein (1 584) and Lankford et ai. (1987) have found ihat toxicity measurements coupled with bioreactor tests can be a pragmatic way to evaluate refractory wastewater toxicity.

The RTA protocol was develcped in the Patapsco TRE (Botts et ai.. 1987) to evaluate the potential for indirect dischargers to contribute toxicity that was refractory to treatment provided by the P O W . This protocol involves treating sewer samples in a bench- scale batch reactor that is designed to simulate, as close as possible, the operating characteristics of the POTW's activated sludge process (e.g., MLSS concentration, DO level and F/M). Acute andlor chronic toxicity measurements of the batch effluent indicate the amount of refractory toxicity in the sewer sample. In the protocol, coarse filtratiorl of the decant from the batch reactor is used to produce the batch effluent. Decant-fiitration more closely simulates sedimentation in lhe full scale plant-because batch settling alone is not as efficient as the P O W settling process.

A general description of the RTA procedure is presented as follows. A step by step protocol for applying the RTA test to sewer wariewaters is provided in Table 5-1. It is imponant to note that the RTA procedure was developed based upon the experience to date and additional research is in procress to further refine the protocol. ?ublic works managers should recognize that variations of the RTA protocol can be used io address site-specific circuns:ances. 9esi professional judccsnt will be important in aGclylng the procedurgs and in in;arpretmg the results.

Biomass Tcxicity Measurement During the Patapsco TRE, filtrate from coarse filtration of the P O W return activated sludge (RAS) was found to be acutely toxic to Ceriodaphnia (Eons et al., 1987). The high level Of toxicity from residual biomass in the filtrate masked the measurement of batch effluent toxicity. and thereby reduced the effectiveness of the RTA tesi for monitoring tu refractory toxicity. The existing data on the toxicity of sewage sludges is not sufficient to determine how widespread is the occurrence of biomass filtrate toxicity. The following discussion provides information on how to proceed. if the P o r n biomass filtrate toxicity presents an interierence in the RTA test.

Additional testing was conducted during the Patapsco TRE to determine if the biomass toxicity interference could be removed. TIE results fGund that the effluent toxicity was caused mainly by non-polar organic compounds which preferentially adsorb onto sewage solids. Thus, tes:; were performed to determine if solids removal would reduce rhe Patapsco RAS toxicity. These tests demonstrated that toxicity in the RAS coarse filtrate could be removed by filtering the coarse filtrate through a 0.2 pm pore size 'filter to remove colloidal material from the liquid (Botts et.al., 1987). Alternatively, centrifugation of the coarse filtrate at 15,000 xg for 10 minutes also substantially reduced the biomass toxicity. Although biomass toxicity can be removed by applying these treatment steps to RTA test effluents. the resulting effluent toxicity will only indicate the soluble refractory toxicity of the IU wastewater, instead of the total refractory toxicity (i.e., soluble and particulate).

Prior to conducting the RTA, aquatic toxicity tests of the POTW activated sludge should be performed to determine if the sludge is toxic. This testing involves filtering the activated sludge through a coarse glass fiber filter, which is the same type of filter used for SS analysis, and testing the filtrate toxicity (Mount and Anderson-Carnahan, 1988a). If :he biomass coarse filtrate is obseryed to have toxicity that is equal to or less than that of the POTW effluent, the P O W biomass can be used in RTA testing. Small partide filtration or centrifugation of the resulting RTA batch effluents will not be required:

I f the biomass coarse filtrate is observed to be more toxic than the POFN effluent. the authors suggest the use of a non-toxic biomass such as ano:her P O W biomass or a commercially available freeze-dried preparation. The non-toxic biomass wil l not be acclimated to the influent wastewaters of !he POTW. but its use may allow an esimafe of the non- biodegradaole toxicity of the sewer discharge. The authors also recommend that ihe POTW actlvated sludge be used in a parallel series of RTA tests to determine :he amount o i soluble refractory :oxicity in the s w e r wastewater. The use of toxic POTW biomass is suggesied because It !s acc1ima:ed io the

. :

"_ "_ . ..

Table 5-1. Tier I - Retranory Toxtctry Assessment

B ~ o m s s Toucny Measurement 0 Coflect 5 llters of fresh return acwated sludge (RAS) md aerate vrgorously for 15 mtnules. 0 Prepare glass f ibe r filter [ s a m e type used for SS anatysts (APHA, 1985)] by rlnstng (M, 50 mi volumes of hlgh purtty water through

0 Filter RAS to yeld 200 ml of fitate.' 0 Test RAS filtrate lor aMe toxlclty using me prccedure described by Mount and Anderson-Camahan (1988aI.Or f o r chrontr t o x c ~ t y

0 Repeat above steps on several RAS samples. 0 ll R4S filtrate o m e toxtc Iran the PCTW effluen: obtarn non-toxic blomass (e.g,. another POTW biomass or a freeze-tire9

me fdter.

uyng the methods p m e d by Horntng and W e e r (19eS). . . . - . . preparatton)

Sample Cdkcuon (wlumes based on sngle sewer sample):

0 Dbtarn 24-hour composlte samaes of sewer dlscharge (1.e.. IU effluent or sewer llne was;ewater) and Pow primary effluenl Lag cdrecton of prmary eflluent,sampte by the estrmated travel trme of sewer wmewater 10 P O W . ' I ...

0 Refrigerate 6 bters of tewer sample at 4% until use. Determine the maximum holding tIme by measurtng JMlW t D x 0 t y over bma t uslng methods described by Mount and Anderson-Carman (1988a). -:'$

0 Reirrge:ate 5 llters of prlmary effluent sample at 4.C untd use. Determlne the maxtmum holdlng tlme as descflbed above. .-4

- .

( 4 - 4

0 H31d 3 llters of tap waler for 2 days to dlstoate chlonne. 0 Collect 10 hers oi RAS (and non-mxtc bomass) on oay of test and aerate vigorously lor 15 mnules before use.

*t

Samole Characienzetlon (pedwmeo on oay of sample CO(ktr0nJ: .?4

B 8 3

Analyze sewer w2snwater f o r TKN. TP, TDS, COD. SCOD, pH.

0 Use historical ratio of CODBODS of sewer wastewatsr. If available. to esllmate BODS. . e . Prepare glass fiber filter as sated above. Filter RAS 10 ylelE 200 mi of filtrate.' Test filtrzte for acute loxrcrty (MoJnI anb A n j e r m - g

.!

I

CamaMn. 10882) or chrcnlc : D X J C I I ~ (Homlng and VJew:. lW5). 0 Determine percent volume of sewer wastewater tn POTW influent based on f!ow data

L

. . . -

S s m k Preparatton: 3 Atd numents to sewer szmp:e io adjust BODc.TKNRTP ralro to 1OO:S:l. Adjust pH of sewer SaTOIe 13 average pH value of P O W influent . .

0 Ter! umple loxlcny (Mount and Anderson-Carnahan, 1988a) after nutrient addttlon and pH adJUSlment to Oetermlne i f these Steps &a the sample toxlary. f

0 Warm gll refrlgerated smp:es to r w m temperature uslng 30.C waler bath. Do not overwarm. 0 Selen volume of RAS (V,) 13 yleld a MLSS concentranon in 1.5 hters of balch mtxlure that is equal to the averzae Pofw MLSS.

11 RAS IS IOXIC [,.e.. more tcxc than POTW eiiluent). also select appropriate volume of nontoxlc blomass (VNs).

0 Add RAS volume (V,) to three 2-liter beakers. a6d difiused alr (use air stone). and Qently aerate. If AAS is toxlc (i.e., more Ioxic ;. tnan POTW efiluent), add non!oxc biomass (ktNB) to three addltlonal beakers and aerate. :

Prepare 2 llters of synthetc wastewater soiutlon csmg StOcK synthellc wastewzter (Table 5-2) and the tap water. Add volume Ol . ~: m k that wdl yleld a solution COD esual to the prlrnary effluent COD. Measure acute loxlclty of the synthetcc solutlon using the f prOteoure oescrlbed Qy Mount and Anderson-Carnshan (1S98a). Chrontc tOxlCity c a n be measured using the methods descnbed by Hornlng and Weber (1985). ,' 3

0 Mezsure sewe: szmple volume (VW) that will yleld I percent vclume in 1.5 liters equal to 10 times the nomnal percent volume Of sewer wastewaer n the P O W mfluent.

t

'$

,i: 7

. .:

(cont~nuedl

5-5

Table 5-1. Conttnued

Periormance ol Batch Tests (loa batch volume equals 1.5 Irlers): ~ d d Vw and prlmary effluent to one beaker mtaintng Vg. If RAS is touc (!.e., more toxic chan P O W eMuent). ~ISO add vw i prlmaty effluent lo one beaker contalntnD VNB.

Add VW and synthet~c waslewaler 10 one bedter fMtatning Vg. I f RAS is toxic (1.e.. more toxic man pow effluent). abo eod 1.

and synthebc wastewater to one beaker conmntng VNB.

Add Prlmary effluent 10 one beaker COnOtning Vg. If RAS is toxrc (i.e., mote toxic than P O W effluent), also red pnrnary effiuez. me remalnlng beaker contavung VNB.

0 Perlcdlcally check the batch reactor pH. Adjust pH io 6-9 range, i f necessary. Note: b a m tests should be performed at room temperature.

Einuent Toxlclry Analysts:

0 St00 aerabon after the requlred reacrion penod and allow me Vg (and V N ~ ) to settle for 15 mtnutes.

0 Decant 200 ml of clarified batch supernatant f r o m each beaker. Rinse glass fiber biters as stated above. Filter each ba suoernatant using sewra!e fitters.' Wash fil:er apparatus between each Sample liltratton usmg 10% HNO3, 8cetone and hlQh pu: -? water. Eatcb filtrates that were treated with toxlc biomass (Vg) must be elther filtered Ihrougn prewashed 0.2 pm ~ l a u Pwrs centrifuged at 10.000 xg for 10 IO 15 m n to remove collotdal stze pamcles. VISCOUS mtnures may requtre h e r or cenrrdugatlon (ASM, 1981).

Prepare filter blanks for each filter type using dilutton water from toxlctty test procedure. < .

0 Analyze n e batch frttrates. centrates. and finer blanks tor zcuie toxicity usng tne plocwure descnbcd by Mount and Amem .<

Camanan (1988a). Chrontc toxicity ctn also be measured (Homing and Weber. 1985) .. . t'

Hornmg and Weoer (19SS).

POTW influent wastewaters and will therefore provide a level of batch' treatment that is more similar to the treatment efficiency of the POTW than that of the unacclimated alternate biomass. In this case, small particle filtration or centrifugation is required to remove t h e interfering biomass particles. By pen'orming RTA tests with P O W biomass in parallel with RTA tests with alternate biomass, both the soluble and total refractory toxicity of the IU wastewater may be estimated.

Sample Collection Wastewater and activated sludge samples should be collected according to the procedures described in Section 13. R e t u r n activated sludge (RAS) is recommended for use in batch testing, because it is in a concentrated form that can be easily diluted to the correct MLSS concentration. Mixed liquor from the P O W ' S aeration basins can be used in lieu of RAS: however, the activated sludge will need to be thtckened to the same SS concentration as that of the RAS before use.

Sample Characterization and Preparation The sewer sample should be analyzed for total and soluble COD. total kjeldahl nitrogen (TKN). total

5-6

phosphorus (TP). total dissolved solids (TDS) and on the day of sample collection. An estimate of sample BOD5 can be calculated using historical c on the COD/BODs ratio of the wastewater. Based these results, the BODSRKNITP concentration r; of the sewer sample should be adjusted, if necess; to 100:5:1 which is the typical ratio for rnunic: sewage. This BODs/TKNTTP ratlo will ensure t; sufficient nutrients are av~ilable for consistent ba- treatment of the IU wastewaters. Phosphorus sho be added in the form of three parts KH2PO4 to f c parts K2HP04. Nitrogen should be added a s ur nitrogen.

Tollowing nutrient addition, the .pH of - the seh :i sample should be adjusted to the average pH value A the POTW influent. Sulfuric acid. and sodic 9. 'i

hydroxide can be used for pH adjustment. . .. 3

'?

.;

Following nutrient addition and pH adjustment, tr Sewer sample toxicity should be measured determine if the nutrients or pH adjustment cause change in sample toxicity. Substantial differencr between the initial toxicity and the adjusted samF toxicity may indicate the presence of specific types toxicants. A discussion of the use of pH adjustme

for toxicity cbracterization is given by Mount and Anderson-Carnahan (1 988a).

p-oparation of Batch Test Mixtures

,,,s volume of RAS biomass (VB), to be used in batch testing, should yield a batch MLSS concentration that is equal to the average MLSS concentration in the P O W aeration basins. The amount of RAS to be added to the total batch volume of 1.5 liters is calculated as follows:

P O W MLSS RAS SS x 1.5 liters.

The same 'equation is used to determine the alternate (non-toxic) biomass volume (VNB).

A total of three batch influent solutions are prepared for each sewer sample: sewer sample spiked into synthetic sewage, sewer sample spiked into POTW influent (primary effluent) and primary effluent alone. The synthetic wastewater provides a standard non- toxic substrate that will allow consistent batch treatment of the IU wastewaters and will permit a determination of the refractory toxicity of the IU wastewaters. The composition of this synthetic wastewater reflects the soluble COD (SCOD) and nutrient content of typical domestic sewage (Table 5-2). Similar synthetic preparations have been used

supplements in biodegradation studies (Kirsh et.& L. A ) in lieu of domestic sewage.

Table 5-2. Synthetic Wastewater Composition.

Cons'hent Concentratton (911)

Bacto Peptone 32.0

Beef Emact 22.0

Urea 6.0 N&I 1.4

CaC12.2 Hz0 0.8

MgSO4.7 Hz0 0.4

KHzPOd 3.5 U$IPOd 4.5

'SCOD of me SlOCk solution IS 64,000 rnw

Prior to use in RTA testing, the synthetic wastewater, which hzs an SCOD of 64,000 mgn, is diluted .in dechlormated tap water. The synthetic Hiastewater snould be diluted to an SCOO concentration that is equal to the average SCOD concentration of the POnill primary effluent. The required stock synthetic wastewater volume can be calculated as follows:

Stock Synthetic Wastewater Volume = Primary EffZuent SCOD 64,000 mgllSCOD

x 1.5 liters

The amount of sewer sample to be used in batch testing should reflect the percent volume of sewer wastewater in the P O W influent. In some cases, the toxicity in sewer wastewater from small contributors may not be readily observed when the wastewater is mixed by percent volume with synthetic wastewater or P O W influent. Thus, the authors recommend using a volume of sewer wastewater (Vw) equal to ten times the percent volume of sewer wastewater typically found in the POTW influent. For example. if the sewer wastewater flow is 0.5 mgd and the P O W influent flow is 50 mgd, Vw would equal 10% of the batch influent solution. In cases where the sewer wastewater flow is L 10% of the P O W influent flow, the sewer sample would comprise 100% of the batch influent.

Performance of Batch Tests In the batch tests, the samplekynthetic solution is used to measure the refractory toxicity of the sewer sample. The sample/primary eff bent solution provides an indication of the interactive effects (e.g., additive or antagonistic) that can occur when the sewer wastewater- and P O W influent *are combined. The third batch solution, primary effluent, serves as a control for the sample'primary effluent test by providing a measure of refractory toxicity in the primary effluent.

The batch influent solutions are mixed with RAS (Ve) to yield a total batch volume of 1.5 ,liters and diffused air is applied to the mixture. The diffused aeration must be performed in appropriate laboratory fume hoods to prevent exposure of laboratory staff to any toxic vapors stripped from the wastewater samples (Section 1 l).The aeration rate is adjusted to ensure complete mixing in the batch reactor and to maintam a DO concentration above 2 mgA.

The organic loading to .the batch reactors can vary substantlally depending on the type of .sewer wastewater being tested. To allow comparable treatment of the various sewer wastewaters, the food-to-microorganism ratio of the ba.tch reactor (FiMe) can be standardized by varying the time of aeration. F/MB should be made equal to the FiM (based on SCOD) of the P O W bioreactors. The required batch test period can be calcu1at.ed as follows:

Batch Influent SCOD CmgiO

MLVSS (mglD x FiM, Test Period (days) =

. .

5-7

A typical test period for a sewer sample with COD < 1000 will be appropriately 2 to 4 hours.

Toxic* Measurement

Either acute or chronic refractory toxicity can be measured in RTA testing. Procedures for acute

. . toxicity measurement should follow the methods described by Mount and Anderson-Carnahan (1 988a). In order to obtain comparable acute toxicity results, RTA !esting should utilize the same species that was used for TIE tests. Other acute toxicity analyses such as bacterial bioluminescence tests (e.g., MicrotoxTM) can be used in conjunction with

information. Chronic toxicity testing should utilize the same species that was used for TIE testing. Chronic toxicity test methods are described by Horning and Weber (1 985).

The batch test mixtures are prepared for toxicity analysis by allowing the mixed liquors to settle, decanting the clarified supernatant, and filtering the supernstznt .through a coarse glass fiber filter. The coarse filtration step is used to more closely simulate the P O W clarification process because the batch senling alone is not as efficient as the P O W settling process. If toxic biomass is used in the RTA tests, further particulate removal is required to measure the soluble refrac?ory toxicity in the sewer wastewater. In this case, the coarse filtrate c t n be filtered through a 0.2 pm pore size glass filter to remove colloidal size particles from the wastewater. Membrane filters such as cellulose nitrate filters are not recommended because some soluble organic .constituents may adsorb onto the filter. Prior to sample filtration, all filters should be washed and filter blanks should be prepared using the steps described in Section 10. Alternatively, the coarse filtrate can be centrifuged at 10.000 xg for 10 to 15 min to separate colloidal material from the wastewater (ASM, 1981).

Data Evaluation

Results of RTA testing are used to locate the sources that are contributing refractory toxicity to the P O W . A discussion of the evaluation of RTA results is provided as follows.

Results of RTA Tests if P O W Biomass is NOn- Toxic - In cases where the POTW biomass filtrate is determined to have toxicity that is equal to or less than that of the P O W effluent, RTA tesis will utilize P O W biomass and wastewater samples. Results for each IU sample analysls wlil consist of data on three batch :ests: one test of sample!synthetic sewage solution. one test of sample!primary effluent solution, and one !est of primary effluent. The batch test of the

I the preferred test species to provide additional

I

' * + 1 _.

sewer sample'synthetic wastewater solution reveals the amount of refractory toxicity in t h e sewer wastewater excluding the effects of other influent wastewaters. Perhaps the most important batch tesx is the anaiysis of the sewer samplelprimary effluent solution, because test data will indicate the toxicity that would realistically occur upon mixture of the sewer wastewater with P O W influent. Results of this combined wastewater test are compared to results of the primary effluent batch test to determine if combining the wastewaters decreases the refractory toxicity (i.e.. antagonistic effect) or increases the refractory toxicity (additive effect) of the primary effluent.

If the effluent toxicity of the sewer sampleiprimary effluent test is greater than the effluent toxicity of the primary effluent test, the sewer wastewater source can be presumed to be a contributor of refractory toxicity. A list of toxic sewer sources should be '

prepafed for further evaluation. In sltuations where sewer line tracking is being conducted, this list can be compared to a sewer collection system map to identify possible toxic IU dischargers on the sewer lines.

Results of RTA Tests if P O W Biomass is Toxic - In situations where the P O W biomass filtrate is found to De more ioxic :!?an the PSlW eifluent. RTA. tests will utilize .alternate (nontoxic) biomass and wastewater in addition to tests with POT& biomass and wastewater. The data on each IU sample analysis will consist of results of three batch tests using alternate biomass (i.e., one test cf sample/synthetic sewage, one test of samplelprimary effluent, and one test of primary effluent); and results of three batch tests using P O W (toxic) biomass (i.e., using same wastewaters as above). The results of tests that use alternate biomass may provide an estimate of non- biodegradable toxicity of the sewer wastewater. The disadvantage of these tests is that the biomass is not acclimated to the POTW influent wastewaters, therefore the level of toxicity reduction'may not reflect the treatment efficiency of batch tests using the POTW acclimated biomass. Nonetheless, the non- toxic biomass tests can give an indication of the relative level of refractory toxicity being contributed by the sewer waste&ater sources.

Batch tests using toxic POTW biomass better reflect the treatment efficiency of the activated sludge process: however, manipulation of the batch effluent (i.e., centrifugation or small panicle filtering) removes panicles that -normally, are present in the POTW effluent. Batch effluent treatment is necessary to remove ' the interfering toxic biomass, but this treatment causes artificial changes in the batch effluent toxicity. The advantage of toxic biomass tests is that the soluble refractory toxicity of source

~ ~ ~~

w2stev;ate:s can be determned. The nontoxic bromass tes!s Canns: prcvlof 25 good an estimate of soluble toxrclty, beczuse !nls biomass i s not z"l Lb,~mzie5 tc tne P3rVd rnfiue,x wastewaters.

F,TA ip-c:-c which utrllze POTW toxic biomass and ~11e:r:t;s ~ : s m a s s can provide Information on both the soiGSle and total refractory toxiclty of t h e IU wa_ctev;ater. Fur ther studies are in progress to i q r a v e ;be u!ihty Of the RTA tesi for toxicity source evaluation. These s tud les V;ill f o c u s on tne

. d e v e k p e n : of procedures tc a x o u n : fo: t h e toxlclty in;e*erences cause3 by toxl: t c ! ~ ~ , a ~ e S sludpe.

RTC, Conclusions :' :+E GTA tes:ln2 IS successful in locatrng the C Z C : C ~ S tt re ! rz : :q :OX\C i i ' j , i u r t h e r teSi\nl; I C