Soil organic matter, alkaline extract, sodium salt

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January-February 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

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

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

Test material information

Constituent 1

Reference substance name:

Soil organic matter, alkaline extract, potassium salt

Cas Number:

1175006-56-0

IUPAC Name:

Soil organic matter, alkaline extract, potassium salt

Test material form:

liquid

Specific details on test material used for the study:

Sample ID: 11120 (sample source 620)
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 in 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):

Daphnia magna

Details on test organisms:

Daphnids were selected as the test organism because they are recommended by U.S. EPA and are commonly used in acute freshwater invertebrate toxicity tests. The Daphnia magna used in this toxicity test were obtained from laboratory cultures maintained at Smithers Viscient. Test organisms were ≤ 24 hours old at the initiation of the test. Daphnids were obtained by removing all immature daphnids from the culture vessel, thus isolating mature gravid daphnids ≤ 24 hours prior to initiating the test. Young produced by these organisms were subsequently pipetted into the test beakers. Daphnids were not used for the test if the cultures contain ephippia, if adult daphnids in the cultures did not produce offspring by day 12, if more than 20% of the culture daphnids died 2 days prior to test initiation, if adult daphnids did not produce an average of at least 3 offspring per female per day during the 7-day period prior to test initiation or if the daphnids were used in any part of a previous test.
The culture water was prepared by fortifying well water based on the formula for hard water (U.S. EPA, 1975) and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants. For the two weeks prior to testing, the prepared culture water had total hardness and total alkalinity ranges as calcium carbonate (CaCO3) of 160 to 180 mg/L and 92 to 98 mg/L, respectively, and a conductivity range of 610 to 790 microsiemens per centimeter (μS/cm). A pH range of 7.8 to 8.3, a temperature range of 19 to 22 ºC, and dissolved oxygen concentration range of 6.1 to 9.9 mg/L were measured in the culture solutions during the two weeks prior to test initiation.
The daphnid culture area received a regulated photoperiod of 16 hours of light and 8 hours of darkness. Light at an intensity of 80 to 99 footcandles (860 to 1100 lux) at the surface of the culture solutions was provided by fluorescent bulbs. Daphnids were fed unicellular green algae, Ankistrodesmus falcatus (4 x 107 cells/mL) in addition to a suspension of YCT (yeast, cereal leaves and flaked fish food). In culture, daphnids were fed various amounts based on their age. Daphnids that were 0 to 6 days old were fed 1.0 mL of algae and 0.5 mL of YCT suspension per vessel per day. Daphnids that were 7 to 10 days old were fed 1.5 mL of algae and 0.5 mL of YCT suspension per vessel per day. Daphnids that were > 10 days old were fed 2.0 mL of algae and 0.5 mL of YCT suspension per vessel per day. 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 food samples analyzed, in agreement with ASTM (2002) standard practice. Based on these analyses, the food sources were considered to be of acceptable quality because all analyte concentrations were below limits of concern.

Study design

Test type:

static

Water media type:

other: well water

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Observed for an additional 48 hours after the 48-hour static acute exposure period.

Test conditions

Hardness:

The dilution water used during the definitive test was from the same source as the water in the daphnid cultures and was characterized as having a total hardness and a total alkalinity as CaCO3 of 160 mg/L and 98 mg/L, respectively.

Test temperature:

The test vessels were impartially placed in a temperature-controlled water bath designed to
maintain exposure solution temperatures at 20 ± 2 °C.

pH:

The dilution water used during the definitive test was from the same source as the water in the
daphnid cultures and was characterized as having a pH of 8.1.

Dissolved oxygen:

The dilution water used during the definitive test was from the same source as the water in the
daphnid cultures and was characterized as having a dissolved oxygen concentration of 12 mg/L.

Conductivity:

The dilution water used during the definitive test was from the same source as the water in the
daphnid cultures and was characterized as having a conductivity of 610 μS/cm.

Nominal and measured concentrations:

Test concentrations were selected based on the results of preliminary testing conducted at Smithers Viscient. Based on these results (see Section 3.1) and in 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).
Results for the analysis of iron in the exposure solutions throughout the exposure period is presented in Table 2. Analysis for the concentration of iron at test initiation resulted in recoveries of 0.037, 0.088, 0.17, 0.28 and 0.58 mg Fe/L (ranging from 93 to 110% 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 0.035, 0.085, 0.17, 0.27 and 0.60 mg Fe/L (ranging from 88 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 five of the six quality control samples resulted in measured concentrations which were consistent with the predetermined recovery range (80 to 120%) and ranged from 88.5 to 114% of the nominal fortified concentrations (0.0380, 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. One of the six QC samples was measured at 78.2% recovery. QC samples can be out of the acceptable range due to a number of factors, some of which are spiking, handling or instrument errors.

Details on test conditions:

The test vessels were impartially placed in a temperature-controlled water bath designed to maintain exposure solution temperatures at 20 ± 2 °C. Test vessels were 600-mL glass beakers, each containing 500 mL of test solution. The test area was illuminated with fluorescent bulbs at an intensity range of 80 to 83 footcandles (860 to 890 lux) at the solutions' surface. The photoperiod during the test was the same as in the culture area. Sudden transitions from light to dark and vice versa were avoided. Light intensity was measured with a VWR Traceable light meter.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Because no concentration tested resulted in ≥ 50% immobilization, the EC50 value for Daphnia magna and CP Concentrate was empirically estimated.

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Because no concentration tested resulted in ≥ 50% immobilization, the EC50 value for Daphnia magna and CP Concentrate was empirically estimated.

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Because no concentration tested resulted in ≥ 50% immobilization, the EC50 value for Daphnia magna and CP Concentrate was empirically estimated.

Effect concentrations 4

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Because no concentration tested resulted in ≥ 50% immobilization, the EC50 value for Daphnia magna and CP Concentrate was empirically estimated.

Details on results:

The nominal concentrations tested, the corresponding cumulative number and percent immobilization, and the observations made during the definitive exposure are presented in Table 3. Following 48 hours of exposure, 5% immobilization was observed among daphnids exposed to the 1000 mg CP Concentrate/L (0.61 mg Fe/L) treatment level. ASTM (2002) recognizes the limitations of acute toxicity testing, i.e., response less than or equal to 10% is allowable in a control population and is considered within the expected range of naturally occurring variability. Therefore, the immobilization (5%) observed in the 1000 mg CP Concentrate/L (0.61 mg Fe/L) treatment level is not considered an adverse response from exposure to the test substance. No immobilization or adverse effects were observed among daphnids exposed to the remaining treatment levels tested or the control.
Table 4 presents the 24 and 48 hour EC50 values and corresponding 95% confidence intervals. Because no concentration tested resulted in ≥ 50% immobilization, the 48-hour EC50 value for Daphnia magna and CP Concentrate 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 Concentrate/L (0.61 mg Fe/L).
Additional testing to further define the EC50 value was not performed because the highest nominal concentration tested, 1000 mg/L (0.61 mg Fe/L), is the limit dose for these studies and satisfies the guideline requirement for testing.

Reported statistics and error estimates:

EC50 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 total dissolved oxygen will not be allowed to drop below 60% or exceed 105% of saturation for the duration of the test. At the 0-hour interval, the total dissolved oxygen was approximately 110% of saturation in all treatment levels tested. Because control survival exceeded acceptable criteria and no adverse effects were observed in the controls, this deviation did not have a negative effect on the results or the interpretation of this 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% immobilization, the 48-hour EC50 value for Daphnia magna and CP Concentrate 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 EC50 value was not performed because the highest nominal concentration tested, 1000 mg/L (0.61 mg Fe/L), is the limit dose for these studies and satisfies the guideline requirement for testing.