Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
yes
Remarks:
Two minor deviations that did not have any negative impacts on the results were described in the full study report and given in more detail below.
GLP compliance:
yes
Remarks:
GLP compliance statement is included in attached full study report

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Sample ID: 11120
Expiration Date: September 1, 2012
0.9% complex polymeric polyhydroxy acids (based on total organic carbon content of 0.48%)
610 mg/kg Fe
Density: 1.01 g/mL at 25 degrees C
Sample is stable at 60 degrees C for >12 months

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
63, 130, 250, 500, and 1000 mg CP Concentrate per Liter, Stored at room temperature in original container in dark ventilated cabinet.

Test solutions

Vehicle:
no
Details on test solutions:
Test concentrations were selected based on the results of preliminary testing conducted at Smithers Viscient. Based on these results (see Section 3.1) and consultation with the Study Sponsor, nominal test concentrations chosen for the definitive study were 63, 130, 250, 500 and 1000 mg CP Concentrate/L (0.038, 0.079, 0.15, 0.31 and 0.61 mg Fe/L).

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Rainbow trout (Oncorhynchus mykiss) was selected as the test organism because it is recommended by the U.S. EPA and commonly used in freshwater acute toxicity tests. The rainbow trout used during this study (SMV Lot No. 12A01) were obtained from TroutLodge, Inc., a commercial supplier located in Sumner, Washington. Prior to testing, these fish were held in a 500-L fiberglass tank under a photoperiod of 16 hours light and 8 hours darkness. The well water which flowed into this holding tank was characterized as having a total hardness range and a total alkalinity as calcium carbonate (CaCO3) of 68 to 70 mg/L and 28 mg/L, respectively, and a conductivity range of 310 to 350 microsiemens per centimeter (μS/cm). Other parameters monitored in the holding tank were pH with a range of 7.2 to 7.5 and a dissolved oxygen concentration range of 98 to 100% of saturation. The fish used during the definitive exposure were maintained under these conditions for at least 14 days prior to testing. The temperature in the holding tank ranged from 12 to 14 ºC during this 14-day period. The temperature in the holding tank ranged from 12 to 13 ºC during the seven days prior to testing. The fish were fed a dry commercial fish food (Trout Chow), ad libitum, daily. Fish were not fed during the 48-hour period prior to test initiation or during the exposure period. Representative samples of the food source were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds were detected at concentrations that are considered toxic in any of the samples analyzed. Based on these analyses, food sources were considered to be of acceptable quality because all analyte concentrations were below levels of concern. No mortality was observed among the test fish population during the 48-hour period prior to testing. A representative sample (N = 30) of the fish from the test population had a mean wet weight of 0.84 g (range 0.46 to 1.4 g) and a mean total length of 44 mm (range 37 to 50 mm).

Study design

Test type:
static
Water media type:
other: well water
Limit test:
no
Total exposure duration:
96 h

Test conditions

Hardness:
The dilution water (well water) used during this study was from the same source as the water used to culture rainbow trout and was characterized as having a total hardness and total alkalinity (as CaCO3) of 68 mg/L and 28 mg/L, respectively.
Test temperature:
The test aquaria were impartially placed in a temperature-controlled water bath designed to maintain exposure solution temperatures at 12 ± 1 °C.
pH:
The dilution water (well water) used during this study was from the same source as the water used to culture rainbow trout and was characterized as having a pH of 7.5.
Dissolved oxygen:
Another parameter monitored in the holding tank was dissolved oxygen concentration range of 98-100% saturation.
Conductivity:
The dilution water (well water) used during this study was from the same source as the water used to culture rainbow trout and was characterized as having a conductivity of 310 μS/cm.
Nominal and measured concentrations:
Results for the analysis of iron in the exposure solutions throughout the exposure period are presented in Table 2. Analysis for the concentration of iron at test initiation resulted in recoveries of 0.035, 0.092, 0.17, 0.30 and 0.62 mg Fe/L (ranging from 92 to 120% of nominal concentrations), respectively, for the 0.038, 0.079, 0.15, 0.31 and 0.61 mg Fe/L treatment levels. These results verify that the test solutions were properly prepared. At test termination, analysis resulted in recoveries of 0.036, 0.087, 0.17, 0.29 and 0.58 mg Fe/L (ranging from 94 to 110% of nominal concentrations), respectively, for the 0.038, 0.079, 0.15, 0.31 and 0.61 mg Fe/L treatment levels.
Analysis of the quality control samples resulted in measured concentrations which were consistent with the predetermined recovery range (80 to 120%) and ranged from 91.1 to 113% (N = 6) of the nominal fortified concentrations (0.038, 0.153 and 0.610 mg Fe/L). Based on these results, it was established that the appropriate precision and quality control was maintained during the analyses of the exposure solutions.
Details on test conditions:
Each aquarium was constructed of glass and silicone adhesive and measured 39 x 20 x 25 centimeters (L x W x H). The test was illuminated to a light intensity of 64 to 85 footcandles (690 to 910 lux) using fluorescent bulbs. Light intensity was measured using a VWR Traceable light meter. A 16-hour light, 8-hour dark photoperiod was maintained with an automatic timer.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Because no concentration tested resulted in ≥ 50% mortality, the LC50 value for CP Concentrate and Oncorhynchus mykiss and was empirically estimated.
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Because no concentration tested resulted in ≥ 50% mortality, the LC50 value for CP Concentrate and Oncorhynchus mykiss and was empirically estimated.
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Because no concentration tested resulted in ≥ 50% mortality, the LC50 value for CP Concentrate and Oncorhynchus mykiss and was empirically estimated.
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Because no concentration tested resulted in ≥ 50% mortality, the LC50 value for CP Concentrate and Oncorhynchus mykiss and was empirically estimated.
Details on results:
The nominal concentrations tested, the corresponding percent mortality, and observations recorded during the 96-hour definitive test are presented in Table 3. No mortality or adverse effects were observed in any of the treatment levels tested (63, 130, 250, 500 and 1000 mg CP Concentrate/L; 0.038, 0.079, 0.15, 0.31 and 0.61 mg Fe/L) or the control.
Table 4 presents the 24-, 48-, 72- and 96-hour LC50 values and corresponding 95% confidence intervals. Because no concentration tested resulted in ≥ 50% mortality, the 96-hour LC50 value for CP Concentrate and Oncorhynchus mykiss was empirically estimated to be > 1000 mg CP Concentrate/L (> 0.61 mg Fe/L), the highest nominal concentration tested. The No-Observed-Effect Concentration (NOEC) was determined to be the 1000 mg CP Concentrate/L (0.61 mg Fe/L).
Additional testing to further define the LC50 value was not performed because the highest nominal concentration tested, 1000 mg/L (0.61 mg Fe/L), is the limit dose for this study and satisfies the guideline requirement for testing.
Reported statistics and error estimates:
LC50 values were empirically estimated; therefore, corresponding 95% confidence intervals could not be calculated.

Any other information on results incl. tables

 


PROTOCOL DEVIATIONS


 


1. The protocol states that the temperature of the solutions will be maintained at 12 ± 1 ºC. The temperature of the water bath used in this exposure was inadvertently set at 14 ºC. During this exposure, temperature ranged from 13 to 15 ºC. Because the control performance met the requirements for an acceptable exposure and because the temperature range during the study was within ranges observed in culture, this deviation did not have a negative impact on the results or interpretation of the study.


 


2. The protocol states that QC samples will be prepared at each sampling interval and stored and analyzed with the set of study samples. Because iron is stable in aqueous solutions, the QC samples were prepared at test termination in order to assess the effects of shipping on stability. The test solution analyses and the QC sample results were within acceptable recovery range (80 to 120% of nominal), therefore this deviation did not have a negative impact on the results or interpretation of the study.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The measured concentrations closely approximate the desired nominal concentrations, indicating that the test solutions were prepared correctly. Because no concentration tested resulted in ≥ 50% mortality, the 96-hour LC50 value for CP Concentrate and Oncorhynchus mykiss was empirically estimated to be > 1000 mg CP Concentrate/L (> 0.61 mg Fe/L), the highest nominal concentration tested. The No-Observed-Effect Concentration (NOEC) was determined to be the 1000 mg CP Concentrate/L (0.61 mg Fe/L). Additional testing to further define the LC50 value was not performed because the highest nominal concentration tested, 1000 mg/L (0.61 mg Fe/L), is the limit dose for this study and satisfies the guideline requirement for testing.