Naphthenic acids

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to fish, other

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

no guideline followed

Principles of method if other than guideline:

P.promelas organisms (< 24h) were exposed during 7 days under static, non-renewal exposure.

GLP compliance:

no

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

Analytical monitoring:

yes

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 ±8 mg/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 (species):

Pimephales promelas

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

start of the test: 99-128 mg/L CaCO3
end of the test: 94-136 mg/L CaCO3

Test temperature:

not specified

pH:

start of the test: 8.30-8.39
end of the test: 8.03-8.31

Dissolved oxygen:

start of the test: 7.55-8.26
end of the test: 6.87-8.70

Salinity:

not relevant

Conductivity:

start of the test: 321.5-379.4 µS/cm
end of the test: 437.8-551.0 µS/cm

Nominal and measured concentrations:

nominal concentrations (mg/L): 0.1, 0.5, 1, 2, 3, 4
measured concentrations (mg/L): -, 0.4, 1.2, 2.3, 2.8, 3.8

Details on test conditions:

P. promelas were conducted by exposing 30 organisms (<24-h old) per concentration (10 organisms per replicate for 3 replicates) in 250 mL borosilicate beakers.
During exposures, fish were fed once daily with Artemia sp.

Reference substance (positive control):

yes

Remarks:

CuSO4

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CI (0.8-3.2)

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Results with reference substance (positive control):

7 d NOEC: 0.05 mg/mL
7 d LOEC: 0.095 mg/mL
7 d LC50: 0.169 mg/L (95% CI: 0.089-0.284)

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.

Validity criteria fulfilled:

no

Conclusions:

Under the presented testing conditions the 7 d NOEC and LC50 of the Fluka commercial NAs were reported to be 0.4 mg/L and 1.9 mg/L, respecyively, for P. promelas. Only mortality during 7 days were followed.

Reference 2

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

GLP compliance:

no

Specific details on test material used for the study:

Commercial Na-NA was supplied as a 50% (w/v) aqueous solution by Pfaltz–Bauer Inc. (Waterbury, CT; N00910). Embryo rearing medium (ERM, Kirchen and West, 1976) was used to dilute the Na-NA standard solution, and as the control. Water from Gregoire Lake (south of Fort McMurray, AB; 56°27'N, 111°120'E), served as the reference water. Gregoire Lake water had an average conductivity of 106 µS/cm and pH of 7.1, and contained a low measurable background level of NAs. The nominal concentrations of the Na-NA solutions were higher than the actual concentrations measured in the treatments. This reflects the 50% solution of the Na-NA mix, the Na content (approx. 10%), and the impurities within the commercial grade of the chemical.

Analytical monitoring:

not specified

Test organisms (species):

Oryzias latipes

Details on test organisms:

For the experiments using medaka, eggs were obtained from a breeding stock maintained at the Department of Biology, University of Waterloo. Egg clusters were collected from the females and placed in ERM. Eggs were teased apart using forceps, and all fertilized eggs were pooled into one petri dish with fresh ERM and left until development had reached the blastodisc stage. Upon inspection, dead or non-developing eggs were removed.

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Remarks on exposure duration:

exposure duration was until larval hatching

Hardness:

not specified

Test temperature:

25 +/- 1 °C

pH:

Gregoire Lake (reference) 7.06
20 mg/L Na-NA 7.18
10 mg/L Na-NA 7.16
5 mg/L Na-NA 7.1
2.5 mg/L Na-NA 7.17
1.25 mg/L Na-NA 7.19

Dissolved oxygen:

beakers were individually aerated

Salinity:

not relevant

Conductivity:

Gregoire Lake (reference) 107 µS/cm
20 mg/L Na-NA 112 µS/cm
10 mg/L Na-NA 109 µS/cm
5 mg/L Na-NA 110 µS/cm
2.5 mg/L Na-NA 107 µS/cm
1.25 mg/L Na-NA 111 µS/cm

Nominal and measured concentrations:

nominal concentrations (mg/L Na-NA): 1.25, 2.5, 5, 10, 20
measured concentrations (mg/L NA): 0.27, 0.48, 1.1, 2.08, 4.49

Details on test conditions:

Individual embryos were then placed in 2 ml vials, each containing 1 ml of test solution. The serial dilutions of Na-NA solutions were made with ERM. For each of the exposures there were four replicates per treatment, with five vials in each. The vials were capped and placed in a dry incubator set at 25 ± 1 °C, with a 16:8 h light:dark photoperiod cycle. Using a dissecting
microscope, normal development was monitored daily as outlined by Kirchen and West (1976), and embryos were screened for abnormalities, as described by Shi and Faustman (1989). The frequency of deformities was calculated as a percentage of the number of embryos per treatment (n = 20). The lengths of live, non-deformed larvae were measured at hatch. NAs threshold effect concentrations were calculated for each endpoint for the Na-NA standard exposures.

Reference substance (positive control):

no

Dose descriptor:

NOEC

Effect conc.:

1.51 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: NA concentration

Basis for effect:

other: embryo deformity

Details on results:

Japanese medaka embryos in all treatments appeared to develop normally past the gastrulation stage. When the embryos reached the stage of early heart formation, developmental problems became apparent in the 20 mg/L commercial Na-NA solution. As development progressed, embryos in other treatments began to show signs of circulatory distress. Problems observed included sluggish circulation, blood pooling at the tail and blood islands over the yolk. The more severe circulatory abnormalities were associated with the occurrence of pericardial edema and tube-heart. Irregularities in the head region included decreased optic cup pigmentation, anisophthalmia and microphthalmia, and mandible malformations. Decreased yolk utilization was observed, suggesting possible metabolic complications. Due to their large yolk sacs, several embryos could not move to properly distribute hatching enzymes, or rupture chorion vestiges, resulting in incomplete hatching (Depeche and Billard, 1994; Vines et al., 2000). All medaka embryos in the 20 mg/L Na-NA standard solution died before hatching. Many of the deformities observed in the medaka were associated
with the circulatory system and osmoregulation. It appears that the medaka may have experienced arrested development during cardiogenesis. The decrease in available energy to osmoregulate may have been aggravated by the surfactant properties of NAs acting on the egg chorion.

Validity criteria fulfilled:

yes

Conclusions:

Signifcant effects of NA on early lifes stages were observed. The NOEC was 1.51 mg/L for embryo deformity.

Reference 3

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Eggs were dry fertilesed with the pooled milt and tehn treatment soluion was added. 24h later sub-samples of 100-200 eggs were taken to determine fertilization. after 6d and 9 d, 50-100 eggs were monitored for development.

GLP compliance:

no

Specific details on test material used for the study:

Commercial Na-NA was supplied as a 50% (w/v) aqueous solution by Pfaltz–Bauer Inc. (Waterbury, CT; N00910). Treatment dilutions of the Na-NA standard for the perch exposures were prepared with water from Gregoire Lake (south of Fort McMurray, AB; 56°27'N, 111°120'E), that also served as the reference water. Gregoire Lake water had an average conductivity of 106 µS/cm and pH of 7.1, and contained a low measurable background level of NAs.

Analytical monitoring:

not specified

Test organisms (species):

Perca flavescens

Details on test organisms:

For the perch exposures, adult yellow perch were captured from Syncrude’s Mildred Lake using trap and fyke nets. Mildred Lake (ML) is a 200 ha reservoir on the Syncrude
site that was created from an existing lake to supply water to the extraction plant. The lake’s water source is theAthabasca River and contains no process-affected waters. Milt was collected and pooled from over 15 male perch and stored at 4 °C until needed. Eggs were stripped from six ripe females, and the egg mass from each was evenly divided among treatments. One yellow perch egg mass contains 2000–90000 eggs that can easily be divided into sections using scissors. The eggs themselves have transparent chorions that allow embryonic development to be monitored (Mansueti, 1964).

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

24 h

Remarks on exposure duration:

exposure duration was until larval hatching

Hardness:

not specified

Test temperature:

10 +/- 1 °C

pH:

Gregoire Lake (reference) 7.06
20 mg/L Na-NA 7.18
10 mg/L Na-NA 7.16
5 mg/L Na-NA 7.1
2.5 mg/L Na-NA 7.17
1.25 mg/L Na-NA 7.19

Dissolved oxygen:

beakers were individually aerated

Salinity:

not relevant

Conductivity:

Gregoire Lake (reference) 107 µS/cm
20 mg/L Na-NA 112 µS/cm
10 mg/L Na-NA 109 µS/cm
5 mg/L Na-NA 110 µS/cm
2.5 mg/L Na-NA 107 µS/cm
1.25 mg/L Na-NA 111 µS/cm

Nominal and measured concentrations:

nominal concentrations (mg/L Na-NA): 1.25, 2.5, 5, 10, 20
measured concentrations (mg/L NA): 0.41, 0.69, 1.12, 2.5, 4.89

Details on test conditions:

For each treatment, eggs were dry fertilized (Hurley, 1972) with the pooled milt, and then treatment solution was added. Each sample was incubated in a 1 mm mesh bottom sample cup suspended in a 250 ml beaker containing 200 ml of treatment water. Beakers were arranged in a water bath at 10 ± 1 °C and individually aerated. Twentyfour
hours later sub-samples of 100–200 eggs were taken and preserved in 10% neutral buffered formalin (BDH Inc., Mississauga, ON) to determine percent fertilization. Further sampling of 50–100 eggs was conducted on days 6 and 9 to monitor development as described by Mansueti (1964). The frequency of deformed embryos was calculated as a percentage of the number of embryos in the subsample. The lengths of live, non-deformed larvae were measured at hatch and NA threshold effect concentrations
were calculated.

Reference substance (positive control):

no

Dose descriptor:

LOEC

Effect conc.:

4.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

larval development

Remarks:

deformity

Dose descriptor:

NOEC

Effect conc.:

1.67 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

larval development

Details on results:

Although eggs in 20 mg/L Na-NA standard were successfully fertilized, they all died before gastrulation was complete. Many embryos sampled on day 6 were deformed. The predominant types of lesions observed in the perch embryos included a variety of optic-cephalic abnormalitiesand spinal deformities. The optic irregularities ranged from a decrease in the number of eye pigmentation granules to synophthalmia and full cyclopia. Lateral and dorsal curvatures were common spinal deformities. Many of the severely deformed embryos had little or no tail. The calculated deformity threshold effect concentration for NA in the Na-NA standard was 1.67 mg/L. Above this threshold, there were positive relationships between the frequency of deformed perch embryos and the concentration of NA in the Na-NA standard treatments. In test exposures where fertilization occurred, the 10 mg/L Na-NA standard (NAs = 4.9 mg/L) was the only treatments that had significantly more deformities than the control ( p < 0.001). The conductivity and the ionic content of the water used in these experiments suggest that the fertilization. and early mortality in the yellow perch were not directly related to high sulphate salinity. The conductivity of the 20 mg/L Na-NA treatment was similar to the control, yet all embryos died. It is possible that there are salinity or conductivity-NA interactions that might result in higher mortality, particularly during water hardening. All perch larvae without obvious deformities were measured within 24 h of hatching and showed an inverse relationship between NA exposure concentration and larval length. For the Na-NA solutions, concentrations of NAs of 1.12 mg/L or greater produced smaller larvae at hatch relative to controls (p < 0.001).

Validity criteria fulfilled:

not applicable

Conclusions:

Signifcant effects of NA on early lifes stages were observed. The NOEC and LOEC were 1.67 mg/L and 4.9 mg/L for embryo deformity.

Reference 4

Endpoint:

long-term toxicity to fish, other

Remarks:

two types of tests are decribed to assess sub-lethal effects: short term reproduction assay and short-term toxiicty on embryo and larval stages

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 229 (Fish Short Term Reproduction Assay)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)

GLP compliance:

no

Specific details on test material used for the study:

The method described by Frank et al. (2006) was used to extract NAs (and other organic acids) from OSPW. Briefly, OSPW was collected from Syncrude Canada Ltd.’s (Syncrude) West In-pit settling basin. This was acidified to pH 2, the aqueous layer was decanted, and the precipitate (slurry) was centrifuged. The pellet was then dissolved in a 0.1 M NaOH solution before being passed through a bed of the weak anion exchanger diethylaminoethyl-cellulose to remove humic-like substances. The filtered NAE was washed with dichloromethane to remove neutral organics such as PAHs. The extract was again acidified and the precipitated acids were collected on a 0.2 µm Teflon filter. The acid precipitate was then redissolved in 0.05 M NaOH. The concentration of the resulting NAE stock solution (2408 mg/l of NAs in a 0.05 M NaOH) used in the toxicity test.

Analytical monitoring:

not specified

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name:
fathead minnow
- Source:
purchased from Aquatic Research Organisms (Hampton, NH, USA)
- Age at study initiation: 5 days old

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food during acclimation:
- Feeding frequency during acclimation:
- Health during acclimation (any mortality observed):

FEEDING DURING TEST
- Food type:
- Amount:
- Frequency:

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

25 ± 2 °C

Nominal and measured concentrations:

nominal NA concentrations (mg/L): 10, 14.7, 21.5, 31.6, 46.4, 68.1, 100

Details on test conditions:

TEST SYSTEM
- Test vessel:
500 ml glass pyrex dishes containing 250 ml of test solutions
- Renewal rate of test solution (frequency/flow rate):
daily renewal
- No. of organisms per vessel:
10
- No. of vessels per concentration (replicates):
3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:

OTHER TEST CONDITIONS
- Adjustment of pH:
pH was adjusted with 18 M H2SO4 to a pH of 8.6 ± 0.2
- Photoperiod:
16 h light:8 h dark photoperiod

Key result

Duration:

9 d

Dose descriptor:

LC50

Remarks:

embryo exposure

Effect conc.:

32.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CI (28.4-36.9)

Table 1. Assay 1: Effect of NAE exposure on reproduction after 21 days.

Note: Values with an asterisk (*) are signifcantly different (p < 0.05) when compared to controls

Treatment Average fecundity  (eggs/female/day +/- SE) mean number of eggs spawned per female (+/- SE)  male testosterone concentration (ng/mL)  male 11-ketotestosterone concetration (ng/mL)  female testosterone concentration (ng/mL)  female 17 beta-estradiol concentration (ng/mL) Control  12.2 +/-1.2 8.8 +/-0.8  11.6  42.6  5.33  4.75 NaOH control  10.6 +/- 1.0 7.0 +/-0.8  17.3  40  4.66  4.17  5 mg/L NAE 6.9 +/- 1.7 * 5.5 +/-0.9 *  10.1  12.9 *  6.5  5.2 10 mg/L NAE  3.9 +/- 0.7 *  2.8 +/- 0.5 *  7.84 *  17.2 *  4.29  5.42

Table 2. Assay 2: Effect of NAE + NaHCO3 exposure on reproduction after 21 days.

Note:Values with an asterisk (*) are signifcantly different (p < 0.05) when compared to controls.

Treatment  Average fecundity (eggs/female/day +/- SE)  mean number of eggs spawned per female(+/- SE)  male testosterone concentration (ng/mL)   male 11 -ketotestosterone concentration (ng/mL)  female testosterone concentration (ng/mL)  female 17 beta-estradiol concentration (ng/mL)  Control 8.9 +/- 0.6   3.8 +/- 0.5  10.2  62.3  4.65  4.82  NaOH control  9.5 +/- 1.6  5.3 +/- 0.6  12.8  53.8  5.06  5.55  NaHCO3 control  9.6 +/- 1.2  4.0 +/- 0.4  10.2  55.5  5.55  3.92  10 mg/L NAE  0.0 +/- 0.0  0.0 +/- 0.0 3.4 *   22.2 *  6.12  2.94  10 mg/L NAE + NaHCO3  1.39 +/-0.4  1.3 +/- 0.4  9.4  21.3 *  4.49  3.43

Table 3. Condition factor (CF), gonadosomatic indices (GSI), liver somatic indices (LSI), and spleen-somatic indices (SSI) for fathead minnows used in Assays 1 and 2.

Note: Values are means ± SE. Values with an asterisk (*) are significantly different (p < 0.05) when compared with control and “–” indicates that the parameter was not measured.

Assay	Treatment	Length (cm)	Mass (g)	CF	GSI (%)	LSI (%)	SSI (%)
Males
1	Control	6.90 +/- 0.08	4.33 +/- 0.08	1.29 +/- 0.02	1.19 +/- 0.12	1.63 +/- 0.13	-
1	NaOH control	7.03 +/- 0.25	4.46 +/- 0.38	1.28 +/- 0.06	1.19 +/- 0.16	2.30 +/- 0.15	-
1	5 mg/L NAE	7.06 +/- 0.12	4.77 +/- 0.24	1.33 +/- 0.03	1.09 +/- 0.16	2.50 +/- 0.31 *	-
1	10 mg/L NAE	7.09 +/- 0.37	4.54 +/- 0.59	1.26 +/- 0.09	1.20 +/- 0.07	2.37 +/- 0.13 *	-
2	Control	5.41 +/- 0.07	2.22 +/- 0.10	1.40 +/- 0.04	1.30 +/- 0.09	2.21 +/- 0.24	0.38 +/ 0.18
2	NaOH	6.10 +/- 0.30	3.12 +/- 0.52	1.34 +/- 0.09	1.62 +/- 0.19	2.59 +/- 0.13	0.48 +/- 0.07
2	HCO3^-	5.76 +/- 0.11	2.72 +/- 0.15	1.40 +/- 0.12	1.10 +/- 0.08	2.22 +/- 0.07	0.33 +/- 0.07
2	10 mg/L NAE	5.90 +/- 0.11	2.58 +/- 0.31	1.24 +/- 0.09	1.25 +/- 0.14	2.80 +/- 0.26	0.25 +/- 0.07
2	10 mg/L NAE + HCO3^-	5.63 +/- 0.11	2.25 +/- 0.15	1.30 +/- 0.12	1.32 +/- 0.08	2.25 +/- 0.07	0.27 +/- 0.07
Females
1	Control	5.23 +/- 0.08	1.60 +/- 0.07	1.10 +/- 0.02	12.1 +/- 1.5	2.33 +/- 0.11	-
1	NaOH control	5.43 +/- 0.05	1.79 +/- 0.09	1.11 +/- 0.03	12.5 +/- 1.4	2.61 +/- 0.26	-
1	5 mg/L NAE	5.44 +/- 0.05	1.79 +/- 0.08	1.11 +/- 0.05	11.4 +/- 2.2	2.14 +/- 0.23	-
1	10 mg/L NAE	5.25 +/- 0.11	1.73 +/- 0.05	1.17 +/- 0.04	14.6 +/- 0.99	2.62 +/- 0.17	-
2	Control	4.36 +/- 0.13	1.14 +/- 0.08	1.36 +/- 0.04	10.6 +/- 1.4	2.57 +/- 0.20	0.41 +/- 0.13
2	NaOH control	4.27 +/- 0.02	1.09 +/- 0.04	1.37 +/- 0.03	11.4 +/- 0.81	2.44 +/- 0.07	0.38 +/- 0.10
2	HCO3^-	4.29 +/- 0.13	1.05 +/- 0.10	1.30 +/- 0.02	9.30 +/- 0.69	2.19 +/- 0.10	0.51 +/- 0.12
2	10 mg/L NAE	4.38 +/- 0.11	1.13 +/- 0.10	1.32 +/- 0.03	8.65 +/- 1.69	2.81 +/- 0.16	0.29 +/- 0.04
2	10 mg/L NAE + HCO3^-	4.35 +/- 0.13	1.12 +/- 0.09	1.33 +/- 0.07	8.75 +/- 1.35	2.38 +/- 0.19	0.29 +/- 0.05

Validity criteria fulfilled:

yes

Conclusions:

The 96-hour LC50 of NAE was reported to be 51.8 mg/L for larval P. promelas under the presented testing conditions. Fathead minnows exposed to a 10 mg/L naphthenic acid extract (NAE) for 21 days spawned signifcantly fewer eggs and males had reduced expression of secondary sexual characteristics. Male fathead minnows exposed to a 5 mg/l NAE had lower plasma levels of 11-ketotestosterone whereas those exposed to a 10 mg/l NAE had lower concentrations of both testosterone and 11-ketotestosterone. Since OSPW also contains high concentrations of salts, this study also investigated whether they modify the toxicity of NAEs. Spawning was significantly reduced in fathead minnows exposed to a 10 mg/l NAE alone and in combination with NaHCO3 (700 mg/l), typical of concentrations in OSPW. Interestingly, the addition of NaHCO3 reduced the inhibitory effects of the NAE on the numbers of reproductive tubercles and plasma testosterone levels. Further studies showed that NaHCO3 acted by reducing the uptake of the NAE to the fish. NaHCO3 but not NaCl or Na2SO4 reduced the acute toxic effects of the NAE on fathead minnow embryo and larvae mortality. Collectively, these studies show that the NAs in OSPW have the potential to negatively affect reproduction in fathead minnows and that HCO3- reduces the acute and chronic toxicity of NAs.

Reference 5

Endpoint:

fish early-life stage toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Description of key information

There are three literature studies available that determine the long-term toxicity to fish and are used as supporting studies because standard guideline studies were not followed. Based on this information, a long-term toxicity to fish study should not be performed.

A conservative approach was therefore applied and it was decided to derive the aquatic PNEC using the acute key data on the most sensitive species only (i.e. fish).