EPA ORD: Literature Survey of Chitosan & Synthetic Cationic Polymer Toxicity 

November 15, 2011 -- Page 1 Some of the studies and manufacturer data reporting aquatic toxicity for cationic polymers generally and chitosan in particChitosan 

Bullock et al (2000) Toxicity of acidified chitosan for cultured rainbow trout 

 12/15 fish died within 24 h exposed to 0.75 ppm; 6/15 died within 24 h, exposed to 0.075 ppm 

 Mortality at 6 days with exposure to 0.038 ppm: Lowest observed effect – 38 ppb

 No mortality at 14 days with exposure to 0.019 ppm: No observed effect – 19 ppb May be toxicant alters glycoprotein; affecting negative charge on gills; favoring adhesion or ionic imbalance interfering with oxygen uptake 

 Authors suggest further research necessary to determine exact toxic mechanism of solubilized chitosan 

Romoren et al (2002) Immersion delivery of plasmid DNA...rainbow trout

 Mortality depended on chitosan concentration, molecular weight and deacetylation  All fish in one study died when exposed to 15 μg/ml (ppm) of chitosan.  At 10 μg/ml (10 ppm), fish exposed to higher molecular weight chitosan died; at 5 μg/ml (ppm) no lethality observed 

National Site Solutions, LLC, chitosan manufacturer (2004) – Powerpoint presentation 

 Acute toxicity: 1.7 mg/l (ppm) at 96 hours for rainbow trout (chitosan acetate)  Acute toxicity: 6.4 mg/l (ppm) at 96 hours for rainbow trout (chitosan lactate) 

Cationic Polyelectrolyte (including polyacrylamide)

Beisinger & Stokes (1986) – Effect of synthetic polymers...

 10 anionic (one was toxic to daphnids only) & 15 cationic polymers  4 species: daphnids, fathead minnows, midges and gammarids 

 Daphnids (LC50 at 48-hr): 0.09 mg/L (ppm) to >100 mg/l (ppm); (8 cationic polymers were ≤ 1.2 mg/L (ppm)) 

 Fathead minnows (LC50 at 96-hr): 0.88 mg/L (ppm) to >100 mg/L (ppm); (3 cationic polymers were ≤ 1.05 mg/L (ppm)) 

 Many of the cationic polyelectrolytes are very toxic with large differences in toxicity of various formulations; addition of anionic prior to discharge m

Muir et al (1997) Localization, depuration, bioaccumulation and impairment of ion regulation...rainbow trout

 Sublethal doses: 3 cationic polymers (EDP 7200 μg/l (ppb), CYT 307 μg/l (ppb), STK 342 μg/l (ppb)) After 3-hour polymer exposure, saw decreased swimming and mucous-like substance stream from gills  Blood pH decreased from 7.1 to 6.6; sodium and chloride concentrations decreased  Total ammonia and potassium increased  Gills appear to be gas exchange and ion regulation site, suggests toxicity when ion regulation is impaired by the binding of cationic polymers 

Weston et al (2009) Toxicity of Anionic Polyacrylamide Formulations when used for Erosion Control

 Oil-based PAM toxic to 4 of 5 species tested at less than 10 mg/L (ppm) (normal erosion control concentration)  Other PAMs showed minimal toxicity even at 10x normal concentration ; may be due to oil or other ingredients in the formulation
EPA ORD: Literature Survey of Chitosan & Synthetic Cationic Polymer Toxicity 

November 15, 2011 -- Page 2 Author(s) Title Results Chitosan Graham Bullock, Vicki Blazer, Scott Tsukuda Steve Summerfelt 

Toxicity of acidified chitosan for cultured rainbow trout _Oncorhynchus mykiss

 Chitosan dissolved in acetic acid 

 added to a culture system at 1.0 ppm to remove organic solids,  acute toxicity to rainbow trout _Oncorhynchus mykiss controlled experiments to determine toxicity  trout average weight of 120.5 g for all tests  7-day exposure: chitosan acutely toxic at 0.75 and 0.075 ppm 

 12 of 15 died within 24 h at 0.75 ppm; six died within 24 h at 0.075 ppm; one on day 7 at 0.038 ppnone died at 0.019 ppm 

 14 day exposure: 

 At 0.038 ppm trout began dying on day 6 in one tank and on day 8 in two other tanks  No trout died in three tanks exposed at 0.019 ppm 

 Histological examination of gills, skin, muscle, and internal organs indicated significant and consistent 

pathological changes only in gills. Lesions similar to mercury exposure. 

 Lifting of lamellar epithelial cells occurred in trout exposed to 0.019 and 0.038 ppm.  In trout exposed to 0.75 or 0.075 ppm chitosan, large areas of lamellar fusion and severe gill lesions we

observed. 

 These results show that soluble acidified chitosan is highly toxic to rainbow trout even at low concentra

Kristine Romøren, Beate J. Thu, Øystein Evensen 

I mmersion delivery of plasmid DNA II. A study of the potentials of a chitosan based delivery system in rainbow trout (Oncorhynchus mykiss) fry 

 Toxicity depended mainly on chitosan concentration, but also molecular weight and degree of deacetyla

 Toxicity reduced due to charge reduction when chitosan complexed with DNA 

 In vitro experiments support in vivo observations  Chitosan (1 mg/ml) dissolved in 25 mM Na-acetate buffer  Rainbow trout fry had average weight of about 1 g.  In freshwater tanks, acclimated for 1 week 
EPA ORD: Literature Survey of Chitosan & Synthetic Cationic Polymer Toxicity 

November 15, 2011 -- Page 3 Author(s) Title Results 

 Comparing chitosan with similar molecular weight but different acetylation, highly deacetylated appear

more toxic than less deacetylatd types. Most toxic had high deacetylation and high molecular weight. Adied at chitosan concentration of 15 μg/ml 

 Second set of experiments using polyplexes of chitosan and chitosan with DNA that had reduced charge 

fixed chitosan concentrations at 15 μg/ml 

 Mortality depended on molecular weight of chitosan with shorter survival for higher molecular weight  Mortality likely caused by positively charged chitosan and negatively charged groups on gills 

Rizzo, L., Di Dennaro, A., Gallo, M. and V. Belgiorno 

Coagulation/chlorination of surface water: A comparison between chitosan and metal salts 

 Coagulation and subsequent chlorination are typical for treating surface water to produce drinking wate Toxicity tests compared chitosan and two metal salts, aluminum sulfate and ferric chloride 

 Chitosan: 1.0 mg/L and 0.5 mg/L  Aluminum sulfate and ferric chloride: 40 mg/L and 60 mg/L  Toxicity tests performed before and after chlorination of raw and coagulate/settled water samples 

 Organisms: 24-h newborn Daphnia magna (ISO 66431 method)  Chlorine effect stopped by adding sodium thiosulphate in chlorinated samples 

 Five daphnids in each test beaker with 50 mL effective volume.  Samples were adjusted to pH 6 to make performance of chitosan for comparing chitosan with metal salt Water samples were spiked with humic acid to evaluate effect on acute toxicity in finished water  Experiment conducted in quadruplicate; samples were diluted when immobilization was 100%  All coagulants reduced water sample toxicity from 100% to 0% immobilization of daphnids Spiking coagulated samples with 10 mg humic acids/L increased toxicity to 100%; humic acids may im

metal uptake causing toxicity in daphnids

 Coagulated samples that were chlorinated increased toxicity for all unspiked coagulated samples except 

one with 60 mg/L of alum. Chitosan was the most toxic, followed by ferric chloride and aluminum sulp

 Coagulated/chlorinated samples that were spiked with humic acids showed lower toxic effect  Toxic effects may depend on several factors: type of dissolved metals, initial humic acid concentration, 

coagulant type and dose, residual humic acid content post-coagulation, formation of humate-metal 

complexes, chlorine dose and formation of chlorination by-products 

 Toxicity of chitosan in drinking water treatment may increase, depending on surface water characteristic

Macpherson, J. Natural Site Solutions 2004 

StormKlearTM (Chitosan) Toxicity and Applications Presentation: October 21, 2004 Sacramento, CA 

 Powerful cationic biopolymer 

 Removed in the treatment process  Residual chitosan detected to 100 μl in treated water using field test kit  Liqui-Floc (chitosan acetate) Aquatic Toxicity Results  Toxicity to fish EC50 or IC50 - Oncorhynchus mykiss (rainbow trout) – 1.7 mg/l - 96 h  Acute oral, LD50 (mice) >16g/kg  Gel-Floc (chitosan lactate) Aquatic Toxicity Results 

 Acute oral, LD50 (mice) >10g/kg  Toxicity to fish EC50 or IC50 - Oncorhynchus mykiss (rainbow trout) – 6.4 mg/l - 96 h 

Santa Cruz Biotechnology, Inc. 

Material Safety Data Sheet Chitosan 2009 

Refer to data for ingredients, which follows: CHITOSAN: ■ Toxic to aquatic organisms. ■ Do NOT allow product to come in contact with surface waters or to intertidal areas below the mean high water 
EPA ORD: Literature Survey of Chitosan & Synthetic Cationic Polymer Toxicity 

November 15, 2011 -- Page 4 Author(s) Title Results 

Do not contaminate water when cleaning equipment or disposing of equipment wash-waters. Wastes resulting from use of the product must be disposed of on site or at approved waste sites. ■ Cationic substances, and their polymers and those polymers that are reasonably anticipated to become cationic inatural aquatic environment (pH range 4-9) may be environmental hazards. Exempt from this concern are those polymers to be used only in solid phase, such as ion-exchange resins, and whethe FGEW (Functional Group Equivalent Weight) of cationic groups is not 5000 and above. Cationic groups such as alkylsulfoniums, alkylphosphoniums and quaternary ammonium polymers are highly toxfish and other aquatic organisms. Similarly potentially cationic groups such as amines and isocyanates are of conc

Some cationics, however, may fall into the category of PLCs (polymers of low concern) provided they possess low charge density, and/or are not water-soluble or are not self-dispersing polycarboxylates or poly- (aromatic or aliph

sulfonate polymers. Ecotoxicity Ingredient Persistence: LOW Water/Soil Persistence: LOW Air Bioaccumulation Mobility HIGH 

SIGMAALDRICH Material Safety Data Sheet 

Chitosan Version 3.0 Revision Date 04/10/2008 Print Date 06/26/2008 Sigma-Aldrich Catalog: Version 5.3 Revision Date 06/03/2011 Print Date 10/17/2011 

12. ECOLOGICAL INFORMATION 

Toxicity 

 Toxicity to fish LC50 - Oncorhynchus mykiss (rainbow trout) - 0.038 mg/l - 48 h 

Persistence and degradability 

 no data available 

Bioaccumulative potential 

 no data available 

Mobility in soil 

 no data available 

PBT and vPvB assessment 

 no data available 

Other adverse effects 

 no data available 

C. Dautremepuits, S. Paris-Palacios, S. Betoule and G. Vernet 

Modulation in hepatic and head kidney parameters of carp (Cyprinus carpio L.) induced by copper and chitosan 

 Copper used as fungicide in Champagne region in France 

 Chitosan has antifungal and heavy metal ion chelating properties 

 Considered chitosan as biopesticide alternative to copper in vineyards 

 Investigated potential toxicity of chitosan on aquatic animal health and compared its toxicity to copper 

 Water soluble chitosan (Chitogel, Aber Tech, France) added to spring water  Seven treatments: 37.5, 75, 150, 225, 300 and 375 mg/l and a control with no chitosan  Mortality recorded every 12 h for 96 h 

 Median lethal concentration at 96 h using StatPhar 2.4 software  LC50 determined as 30018 mg/l 

 Sublethal effects observed when antioxidant enzymes were measured in the liver and head kidney after 

days exposure 

 Defense against oxidative stress seemed to be dose-dependent at concentrations below the LC50 at 96 ho

of 300 mg/l 
EPA ORD: Literature Survey of Chitosan & Synthetic Cationic Polymer Toxicity 

November 15, 2011 -- Page 5 Author(s) Title Results Y.Y. Mosleh, S. Paris-Palacios, M. T. Ahmed, F.M. Mahmoud, M.A. Osman, and S. BiagiantiRisbourg 

Effects of chitosan on oxidative stress and metallothioneins in aquatic worm Tubifex tubifex (Oligochaeta, Tubificidae) 

 T. tubifex worms collected from sediment in vineyard retention basin; held 6 months before experiment 

 Worms were about 1.5 g 

 Species is very resistant to pollution, usually last macroinvertebrate to disappear from a contaminated si Twenty worms were placed in crystallizing dishes with spring water. Chitosan (chitogel 2.5% San-Quen

Fallavier, Fr) was added for final concentrations of 25, 62 and 125 mg/l and incubated in growth chamb

2, 4 and 4 days. Three dishes for each concentration and for each duration. 

 Minimum of 3 independent experiments in triplicate. Data are presented as the mean ± standard deviatio



M. Sekine, A. Takeshita, N. Oda, M. Ukita, T. Imai and T. Higuchi 

On-site treatment of turbid river water using chitosan, a natural organic polymer coagulant 

 Field study in Imou River, Yamaguchi Prefecture, Japan at river construction site  FLONAC #250 (1:1 mix of chitosan flake and acetic acid) 

 Jar test: optimum concentration – 1.5 mg/L; continuously injected for two hours  “Strange” fish surfacing behavior observed in high turbidity area of stream about 50 m downstream of 

station where injected. Fish didn’t take breath at surface but stayed calm and quiet below surface with hslightly up. 

 No dead fish, no quickly moving fish observed 

 No other behavior determined strange at other stream sections 

 Toxicity test conducted based on observation 

 Acute toxicity test on larva of ten Oryzias latipes(Japanese rice fish) in petri dish with 25 mL test water

cultivated 48 h at 25oC 

 Dead and abnormal larvae counted at 1, 2, 3, 6, 12, 24 and 48 hours 

 Control test conducted with water treated with activated carbon  Chitosan shows low but constant abnormal behavior 

 Fins of larva seem to cling to body 

 Toxicity test inconclusive for chitosan toxicity 

Cationic & Anionic Polymers Beisinger, K.E. and G. N. Stokes 

Effects of synthetic polyelectrolytes on selected aquatic organisms 

 Acute Bioassay: 

 fathead minnows and daphnids  polyelectrolyte concentration of 100 mg/L (10 anionic and 15 cationic) 

 if toxic, then used less sensitive gammarids and midges 

 used 5-8 concentrations of polyelectrolytes, along with controls 
EPA ORD: Literature Survey of Chitosan & Synthetic Cationic Polymer Toxicity 

November 15, 2011 -- Page 6 

 Tests for gill damage in fish (30-day-old) in 1 L of water  Water and polyelectrolyte solutions renewed daily  LC50

 Daphnids (48-hr): 0.09 mg/L to >100 mg/l; 8 polymers were ≤ 1.2 mg/L 

 Fathead minnows (96-hr): 0.88 mg/L to >100 mg/L; (3 polymers were ≤ 1.05 mg/L) 

 Hypothesize suffocation of fish  Different sensitivies between species suggest different modes of action; may also be caused by oligome

impurities 

 Anionic polyelectrolytes could be added before discharging to a wastewater to detoxify cationic 

polyelectrolytes 

Muir, M.M., Kosteretz, K.G., and J.J. Lech 

Localization, depuration, bioaccumulation and impairment of ion regulation associated with cationic polymer exposure in rainbow trout (Oncorhynchus mykiss) 

Experiment 

 Static exposure to three commercial 14C-labelled cationic polmers:  EDP, epichlorhydrin-deimethylamine; CYT, polyacrylamide ester; and STK, polyacrylamide amide  14C concentrated in gill tissue  Trout hatched and then raised in-house, in flowing, dechlorinated water  20 liter glass aquaria  N=9 fish and tissue samples pooled in groups of three fish  Concentrations high enough to cause morbidity but not mortality;  Blood pH, and blood ions (Na+, total NH3 and Cl-) were measured 

Results 

 Sequential exposure did not result in any cumulative levels of 14C concentrated in gill tissue  At end of each 3 hour polymer exposure, fish exhibited decreased swimming and mucous-like subs

streaming from gill area 

 During first CYT exposure one fish died.  Four fish died after the first SYT exposure  Blood pH decreased from 7.1 to 6.6  Total ammonia increased two exposures, but a 2-fold increase was only statistically significant in one 

exposures 

 Sodium and chloride concentrations decreased in both exposures but was significant in only one o

exposures 

 Potassium concentrations increased  Data from tissue distribution studies showed three cationic polymers are limited to the gills  In addition to gas exchange gills appear to be ion regulation site  Freshwater fish such as rainbow trout are hypertonic to water and get sodium and chloride from water  Decrease in blood sodium and chloride as well as increased total ammonia are consistent with interrupte

regulation 

 Studies indicate humic acid additions to water reduce cationic polymer toxicity  14C concentrations were significantly reduced in gill tissue by proponed exposure to polymer-free wate

polymer-free water with HA. 

 Presence of HA in polymer-free depuration water didn’t markedly increase rate of 14C disappearance 

gills after exposure to two polymer but did decrease for one polymer (EDP). 

Conclusions 

 Gills appear to be the only cationic polymer binding site in fish  Binding of polymers appears to be reversible in polymer-free water with or without negatively-ch

organic matter 

 Toxic effects data suggest that the gills are an ion regulation site and that gill epithelial cellular mem

mechanisms responsible for ion regulation are impaired by binding cationic polymers 
EPA ORD: Literature Survey of Chitosan & Synthetic Cationic Polymer Toxicity 

November 15, 2011 -- Page 7 Rowland, C.D., Burton, G.A., and S.M. Morrison. 

Implication of Polymer Toxicity in a Municipal Wastewater Effluent 

Montgomery, OH Wastewater Treatment facility 

 Final effluent 24-h composited samples collected in borosilicate glass jars; just prior to entering stream 

 Acute (near-field) and chronic (far-field) mixing zones and upstream receiving water grab samples colle

in plastic and stored on ice 

 Upstream samples used for dilution water and field reference control 

 Laboratory culture water control test accompanied WET tests 

 Ceriodaphnia dubia toxicity tests with toxicant elimination steps included extensive effluent characteriz

and manipulation 

 C-380 dry cationic polyacrylamide polymer  Hyperfloc, a water-in-oil emulsion cationic acrylamide copolymer 

 Both products were proprietary and no toxicity data was available 

Toxicity Test Results 

 C18 extraction column effluent samples from plastic containers exhibited 100% C. dubia survival after 

hours of exposure 

 Same effluent from glass jars had a markedly reduced survival rate of 44% (±0.53) within the first 24 ho

and none survived after 48 hours 

 Effluent manipulation ruled out surfactants as a toxicant 

 In all effluent samples collected, reduced toxicity was observed for plastic containers.  Appears to be potential for sorption of cationic polymers to plastic containers, reducing toxicity 

 Glass containers are suggested for storage of effluent samples where polymer toxicity is suspected 

Weston, D.P., Lentz, R.D., Cahn, M.D., Ogle, R.S., Rothert, A.K., and M.J. Lydy. 

Toxicity of Anionic Polyacrylamide Formulations when Used for Erosion Control in Agriculture 

 Toxicity Test Results 

 Freshwater amphipod, midge, daphnia, fathead minnow, freshwater algae 

 Results indicate toxicity depends on product formulation  From standpoint of aquatic toxicity it appears that solid and water based products are preferable to oil-b

forms 

 Granular form: four of five species no toxicity; daphnia reproductive IC50 5mg/L and LC50 of 29 mg/L  Tablet form: no toxicity in one species tested: LC50 > 100 mg/L 

 Water –based form: no toxicity in most species. At 100 mg/L toxic to algae. 

 Oil-based form: toxic to amphipod, midge and daphnia; fathead minnow had statistically significant 

mortality at 1.5 mg/L and LC50 of 16.6 mg/L; algae seemed unaffected 

Sojka, R.E., Bjorneberg, D.L, Entry, J.A., Lentz, R.D., and W.J. Orts 

Polyacrylamide in Agriculture and Environmental Land Management 

 PAM refers to a broad class of compounds sold since 1995 to reduce irrigation-induced erosion and enh

infiltration with increasing non-agricultural applications 

 Nonionic and cationic PAMs risky to aquatic organisms at low concentrations; anionic are not 

 PAM toxicity lower in natural waters due to humic substances and other dissolved organic compounds 

 Increased germination when PAM mixed with grass seed  PAM increases infiltration at optimal application rate; applying too much can restrict infiltration 

 Careful control of PAM concentration is important  Results from field and lab tests showed Chitosan not as effective as PAM for erosion control 

Albassam,M., Moore, J., and A. Sharma 

Ultrastructural and Clinicopathological Studies on the Toxicity of Cationic Acrylamide-based Flocculant to Rainbow Trout 

Abstract. Rainbow trout (Sa/rno gairdnerz) of two age groups were exposed to a cationic acrylamide-based 

ftocculant at various concentrations in static bioassay chambers. At lethal concentrations the flocculant produced severe gill alterations in all fish. The principal alterations were necrosis and separation of the respiratory epithelia

cells covering secondary lamellae. Many necrotic chloride cells were also seen, their apical plasma membrane was destroyed, and mitochondria were swollen with separated cristae. An influx of a large amount of fluid into 

the interstitial spaces caused partial or complete separation of subepithelial spaces from the covering epithelial cells and basement membranes of underlying blood vessels. Clinicopathological alterations included marked 
EPA ORD: Literature Survey of Chitosan & Synthetic Cationic Polymer Toxicity 

November 15, 2011 -- Page 8 

decreases in blood pH, partial pressure of oxygen, bicarbonate and plasma sodium, and chloride concentrations. Hematocrit, total protein, and blood glucose were increased. Fish exposed to sublethal concentrations had gill 

alterations characterized by hypercellularity and thickening of the secondary lamellae. These were due to undifferentiated cell proliferation and macrophage and lymphocyte infiltration between the covering epithelial cells and the underlying blood vessels. Macrophages and undifferentiated cells had large phagolysosomes contain

cytoplasmic organelles, an indication of cell injury and increased turnover. 

Bolto, B., and J. Gregory 

Review Organic polyelectrolytes in water treatment 

Abstract. The use of polymers in the production of drinking water is reviewed, with emphasis on the nature of the 

impurities to be removed, the mechanisms of coagulation and flocculation, and the types of polymers commonly available. There is a focus on polymers for primary coagulation, their use as coagulant aids, in the recycling of filt

backwash waters, and in sludge thickening. Practicalities of polymer use are discussed, with particular attention to 

polymer toxicity, and the presence of residual polymer in the final drinking water. The questions of polymer degradation and the formation of disinfection by-products are also addressed. 

Liber, K., Weber, L.,and C. Le ́vesque 

Sublethal toxicity of two wastewater treatment polymers to lake trout fry (Salvelinus namaycush) 

Abstract. Lake trout fry (Salvelinus namaycush) were exposed in laboratory experiments to two wastewater treatm

polymers, one anionic (MagnaFloc 156) and one cationic (MagnaFloc 368; Ciba Speciality Chemical), to determi

these chemicals which are used and discharged by mining operations in Canada’s North pose a significant hazard 

juvenile fishes. The cationic polymer was substantially more toxic to lake trout fry than the anionic polymer, with 

96-h LC50 estimates of 2.08 and >600 mg/l, respectively. Separate 30-d exposure experiments yielded no observe

and lowest observed effect concentrations, respectively, of 0.25 and 0.5 mg/l for MagnaFloc 368, and 75 and 150 mg/l for MagnaFloc 156. In both cases, behavioural responses, especially startle response, were the most sens

test endpoints. Histopathological assessment revealed that gill pathology appeared within a few days of exposure 

both polymers, apparently as a result of localized hypoxia. Acute (4 d) effects included cloudy swelling of epithelcells, increased gill vascularization, and thickening and shortening of the gill lamella. Chronic (30 d) polymer expproduced only slightly greater pathological effects than acute exposure, with comparable responses observed only >1.0 mg/l MagnaFloc 368 and 150 mg/l MagnaFloc 156, suggesting that the fish displayed some level of both 

behavioural and physiological adaptation to the respiratory stress imposed by the two polymers. 
EPA ORD: Literature Survey of Chitosan & Synthetic Cationic Polymer Toxicity 

November 15, 2011 -- Page 9 Performance Comparisons of Chitosan to PAM in Agriculture and Construction SOURCE: Sojka, R.E., Bjorneberg, D.L., Entry, J.A., Lentz, R.D. and W.J.Orts. (2007). Polyacrylamide in Agriculture and Environmental LanAgronomy (2007). Vol 92: 75-162 Note: PAM used (18% anionic, 12-15 Mg/mol); 10 ppm 

SOURCE: Tobiason, S., Jenkins, D., Molash, E., and Rush, S. (2001). Polymer use and testing for erosion and sediment control on constructio

<Accessed 25 Oct, 2011: www.erosioncontrol.com/forms/print-19178.aspx> 

Note: PAM used was Chemco 9836A granular ionic polyacrylamide