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Ecotoxicological information

Toxicity to aquatic plants other than algae

Administrative data

toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Comparative responses of freshwater organisms to exposures of a commercial naphthenic acid
Ciera M. Kinley, Andrew D. McQueen, John H. Rodgers Jr.
Bibliographic source:
Chemosphere 153 (2016) 170-178

Materials and methods

Test guideline
no guideline followed
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Naphthenic acids
EC Number:
EC Name:
Naphthenic acids
Cas Number:
Molecular formula:
For the acidic (naphthenic) fraction: CnH2n+zO2, where n = carbon number and z = homologous group series number: z = 0 when no ring structures are present, z = -2 when 1 ring is present, z = -4 when 2 rings are present etc. For the non-acidic fraction: not applicable
Naphthenic acids
Test material form:
Details on test material:
Physical state: Viscous liquid
Appearance: Pale yellow to dark amber
Specific details on test material used for the study:
Parameter General characteristic
Identification 1338-24-5 (CAS No)
Color Pale yellow
Physical state Viscous liquid
Molecular weight 210-250 amu (average molecular weight for refined naphthenic acids)
Water solubility 88.1 mg/L at pH 7.5 (alkylated cyclopentane carboxylic acids - mixture)
Vapor pressure 1.1 x 10-07 - 7.1 x 10-06 mm Hg at 25 °C
Partition coefficient octanol/water +/- 4 at pH 1
(Log Kow) +/- 2.4 at pH 7 (<0.1 at pH 10)
Density 0.92 g/mL at 20 °C
Flash point 101 °C
Initial boiling point 106.4 - 333.6 °C
Viscosity 22 mm2/s
pKa 5 to 6

Test solutions

Details on test solutions:
Stock solutions were prepared by mixing 100 mg of Fluka commercial NAs (obtained from Sigma-Aldrich) in 1 L (100 mg/L) of reconstituted formulated moderately hard water (pH 7.7 ± 0.5 SU, alkalinity 65 ±8mg/L as CaCO3, hardness 88 ± 10 mg/L as CaCO3, conductivity 350 ± 20 ms/cm) prepared using reverse osmosis filtered water and reagent grade chemicals based on recommended culture methods (USEPA, 2002). The formulated water contained 5 mg/L CaCO3, 102 mg/L NaHCO3, 48 mg/L MgSO4e7H2O, 33 mg/L CaSO4e2H2O, 65 mg/L CaCl2e2H2O, 2mg/L KCl, 0.8 mg/L KNO3, 0.02 mg/L K2PO4, and 0.002 mg/L of each Cu, Se, and Zn (from aqueous standards). All reagents were obtained from Fisher Scientific (Pittsburgh, PA). A modified water accommodated fraction (WAF) method was used to prepare NA stock solutions, where solutions were mixed with magnetic stir bars for 12-h at a speed sufficient to create a vortex which extended 30-50% of the solution depth (OECD, 2000). Stock solutions were adjusted to pH 10 ± 0.1 S.U. prior to mixing to ensure solubility of NAs. Below pH ~7, NAs would be expected to significantly precipitate out of solution, even after mixing (Headley et al., 2002). After stirring, undissolved fractions were decanted and the remaining dissolved fraction in solution was used for testing.
Experimental concentrations were prepared by serial dilution of the stock solution with moderately hard water. pH of exposures was adjusted to 8.3 ± 0.1 with 0.1 M HCl (Fisher Scientific, Pittsburgh, PA), in all toxicity tests in order to ensure homogenous exposures while remaining within environmental tolerances of organisms. Initial NA concentrations were measured in all treatments using high performance liquid chromatography (HPLC; Dionex, UltiMate-3000; Sunnyvale, CA) according to a derivatization method described in Yen et al. (2004). The HPLC analytical column was an Agilent LiChrospher 100 RP-18 (5 mm particle size, 125 mm x 4 mm) with a guard column packed with 2 mm RP-18 solid phase material. Column temperature was maintained at 40 °C with a sample injection volume of 60 mL mobilized with HPLC grade methanol (Fisher Scientific) at a flow rate of 1.5 mL min 1. Calibration standards were prepared with Fluka NAs usingWAFs as described for stock solutions. The detection limit for this method is approximately 5 mg/L. To measure nominal exposure concentrations below the MDL, stock solutions were prepared at concentrations 10x (or 100x) greater than the targeted (nominal) concentration, NAs were measured from those solutions, and then solutions were diluted 10x (or 100x) with moderately hard water for exposing to organisms.

Test organisms

Test organisms (species):
other: Typha latifolia
Details on test organisms:
common name: common cattail
inflorescences were collected from a ruralwetland site at Clemson University, Clemson, SC (344007.1200N, 8250053.9800W).
Inflorescences were stored in plastic bags and incubated at 20 ± 1 °C until testing.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
7 d

Test conditions

start of the test: 102-114 mg/L CaCO3
Test temperature:
not specified
start of the test: 8.30-8.38
Dissolved oxygen:
start of the test: 7.88-8.13
not relevant
start of the test: 359.8-388.0 µS/cm
Nominal and measured concentrations:
nominal concentrations (mg/L): 0, 20, 40, 60, 80, 100
measured concentrations (mg/L):2.6, 25.2, 49.8, 61.6, 67.1, 100.2
Details on test conditions:
Seeds were separated from bristle hairs by placing in a blender filled with NANOpure® water and blending for 30-s. Seeds that sank to the bottom after blending were considered viable and used for testing (Muller et al., 2001; Moore et al., 1999). Viable seeds were then added to a small volume (about 1 mL) of moderately hard water and incubated for 2-d to induce germination. Toxicity experiments for T. latifolia were initiated by adding 10 germinated T. latifolia seedlings (2-d old) to each replicate 50 mL beaker (three replicates/concentration) under fluorescent lighting
(1500-3000 Lux) with a 16-h light/8-h dark photoperiod at 24 ± 1 °C. Exposure concentrations were pipetted into treatment chambers and volumes were maintained as necessary. Control (untreated) exposures were moderately hard water. After 7-d, seedlings were removed from exposures and preserved in 70% ethanol until analysis. Root and shoot lengths (mm) of seedlings were measured using a Leica® M80 Stereoscope and software (Leica Microsystems®).
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
Key result
7 d
Dose descriptor:
Effect conc.:
25.2 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
7 d
Dose descriptor:
Effect conc.:
49.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
7 d
Dose descriptor:
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI (52.4-60.1)
Results with reference substance (positive control):
7 d NOEC: 0.012 mg/mL
7 dLOEC: 0.067 mg/mL
7 d LC50: 0.503 mg/L (95% CI: 0.139-0.1039)
Reported statistics and error estimates:
No observable effect concentrations (NOECs) and lowest observable effect concentrations (LOECs) of commercial NAs and copper as copper sulfate for T. latifolia root and shoot growth and mortality in animals were determined by statistically significantdifferences relative to untreated controls using one way analysis of variance (ANOVA) and Dunnett's multiple range test (alpha =0.05; JMPPro V.11). Median lethal effect concentrations (LC50s) were estimated using the Probit model (Bliss, 1935). The median effect concentration (EC50) for T. latifolia was estimated using non-linear regression, with a sigmoid logistic fit function. Inflection points calculated are synonymous with EC50 values.

Applicant's summary and conclusion

Executive summary:

Under the presented testing conditions the 7 d NOEC and LC50 of the Fluka commercial NAs were reported to be 25.2 mg/L and 56.2 mg/L, respecyively, for T. latifolia.