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Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
according to guideline
other: United States Environmental Protection Agency (USEPA). 2002. Short-term methods for estimating the acute toxicity of effluents and receiving waters to freshwater organisms. Fourth edition. Office of Water, US-EPA, Washington, DC 20460. EPA-821-R-02-012.
according to guideline
other: ASTM. 2007. Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians. Method E729 - 96. West Conshohocken, PA, USA.
GLP compliance:
not specified
Testing and documentation for the study were carried out in the spirit of U.S. EPA Good Laboratory Practice (GLP) standards.

Test material

Constituent 1
Reference substance name:
Reaction mass of diiron carbide, diiron phosphide and triiron phosphide
EC Number:
Cas Number:
not applicable
Molecular formula:
not applicable (multi constituent substance)
Reaction mass of diiron carbide, diiron phosphide and triiron phosphide
Test material form:
solid: particulate/powder
Details on test material:
The test substance, triiron phosphorus (Lot #1580165), was provided by Höganäs Sweden AB (Höganäs, Sweden). One plastic jar with 1 kg of test substance was received. The certificate of analysis reported the chemical properties as 15.8% as P and 0.82% as C and Fe as base.
Specific details on test material used for the study:
The test substance, triiron phosphorus (Lot #1580165), was provided by Höganäs Sweden AB (Höganäs, Sweden). The certificate of analysis reported the chemical properties as 15.8% as P and 0.82% as C and Fe as base.

Sampling and analysis

Analytical monitoring:
Details on sampling:
Total Iron
Analytical samples from each treatment were collected for total recoverable iron analysis from newly prepared waters at test initiation and from a composite of old waters from the test chambers at test termination. Approximately 5 mL was drawn into the syringe to rinse the inside of the syringe and then expunged to waste. Then 15 mL of sample was drawn into the syringe and injected into a 15 mL polypropylene conical tube. Samples were preserved with trace metal grade nitric acid (AR-ACS grade, Mallinckrodt Chemical, Hazelwood, MO, USA) to pH < 2 and refrigerated (0 - 4 °C) prior to analysis.

Dissolved Iron
Analytical samples from each treatment were collected for dissolved (filtered through a 0.20 μm Acrodisc Supor PES filter, [Pall Life Sciences; Ann Arbor, MI, USA]) iron analysis from newly prepared solutions at test initiation and Day 2 and from old waters (a composite sample of each replicate within a treatment) on Day 2 and at test termination. Sampling occurred by drawing approximately 20 mL into the syringe of which 5 mL was pushed through the filter to waste and the remaining 15 mL was collected into a 15-mL polypropylene conical test tube. Samples were then preserved with trace metal grade nitric acid (AR-ACS grade, Mallinckrodt Chemical, Hazelwood, MO, USA) to pH < 2 and refrigerated (0 - 4 °C) prior to analysis.

Orthophosphate (Phosphorus, Reactive)
Analytical samples for the analysis of orthophosphate were sampled at the same sampling schedule as that of the dissolved metals.

Test solutions

Details on test solutions:
Due to the insoluble nature of each of the test substances, water-soluble fractions (WSFs) were prepared. A stock solutions was prepared by combining the test substance (at a concentration of 100 mg/L) with 4.5 Liters of moderately- hard reconstituted water (adjusted with dilute HCl to a pH of 6) in a glass aspirator bottle and mixed on a stir plate (set at approximately 200 revolutions per minute [rpm]) at 20°C for 7- days. The aspirator bottle was covered with multiple layers of parafilm to achieve little to no headspace in the bottle. The pH of the mixture was checked daily and if needed, re-adjusted to a pH of 6 with dilute HCl/NaOH. Minimal pH drift was observed over the 7-days resulting in only small additions of dilute HCl/NaOH. Following the 7-day mixing period, the mixture was filtered through a 0.45 μm filter to remove the undissolved fraction of the substances.
All concentrations within this report are expressed as nominal percent WSF (%).

Test organisms

Test organisms (species):
Pimephales promelas
Details on test organisms:
The test was initiated with 4-day old larval fish. The fish were hatched in-house using moderately-hard reconstituted water as the culture medium (nominal hardness, alkalinity, and pH of approximately 100 mg/L as CaCO3, 70 mg/L as CaCO3, and 8.0, respectively (USEPA 2002). In order to acclimate the fish to the lower pH 6 test conditions prior to test initiation, 80% water renewals occurred with a pH 7.5 water occurred on the first day, renewal with pH 7 water on the second day, and renewal with pH 6.5 water on the third day. Based upon visual observation, the organisms appeared healthy at test initiation.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
96 h

Test conditions

92 mg/L as CaCO3
Test temperature:
20°C +/- 2°C
During preparation of WSF: 5.92-6.21
During the test: 6.0 - 6.3
Dissolved oxygen:
> 60% of saturation
new medium: 8.0-8.9 mg/L
old medium: 6.6-7.8 mg/L
Total residual chlorine: < 0.50 mg/L
new medium: 372-400 µS/cm
old medium: 371-399 µS/cm
Nominal and measured concentrations:
Nominal: 100% WSF + dilutions of this WSF-stock
Details on test conditions:
Organisms were tested in 4 ounce glass jars containing approximately 4 ounces of test solution. Each test chamber was capped with a Teflon lined lid with little to no headspace within the chamber.
The test chambers were housed in a temperature-controlled environmental chamber
The test was conducted under a 16:8 hour light:dark cycle using cool-white fluorescent lights at ~100 foot candles. The test chambers were randomized according to a computer generated randomization sheet.

At test initiation, ten larval fish were randomly placed into each test chamber/replicate. Each concentration had four replicates for a total of 40 fish per concentration. Each test chamber was randomly assigned to a location within a temperature controlled environmental chamber. After 48-hours, each test chamber was opened and fish were fed 0.20 mL per chamber of a concentrated suspension of brine shrimp (Artemia salina) nauplii (Brine Shrimp Direct, Ogden, UT, USA). Following four hours of feeding, a water renewal was performed on each chamber by siphoning out approximately 80% of test solution and replacing it with freshly prepared waters.

Results and discussion

Effect concentrations
Key result
96 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: WSF of 100 mg Fe3P/L was tested
Details on results:
Control: 97.5% survival
100% WSF: 84.4% survivial

A 97.5% control survival in the Fe3P toxicity test, the control acceptability criterion was achieved (criterion ≥ 90% survival in control). A small but statistically significant difference in survival was observed between the control and the 100% WSF exposure, Median lethal concentrations (LC50s) is >100% WSF.
Reported statistics and error estimates:
Statistical analysis was performed using nominal percent WSF. Differences in survival were evaluated using a statistical computer package (Comprehensive Environmental Toxicity Information System [CETIS], Tidepool Scientific Software, McKinleyville, CA, USA). If the data met the assumptions of normality and homogeneity, the no observable effect concentration (NOEC) and lowest observable effect concentration (LOEC) were estimated using an analysis of variance to compare (p = 0.05) organism performance in the experimental treatments with that observed in the control. Estimation of a median-lethal concentration (LC50) (if relevant) is estimated using Trimmed Spearman-Karber.

Any other information on results incl. tables

Sublethal observations / clinical signs:

100% Fe3P WSF:

new: 3893.5 µg Fe/L (dissolved)

old: 1279.0 µg Fe/L (dissolved)

0.33 mg/L reactive phosphorus

Applicant's summary and conclusion

Validity criteria fulfilled:
The Transformation dissolution test on Fe3P showed low dissolution levels. However, the lack of speciation of the P form could not exclude the potential availability of the extreme ecotoxic phosphine form. Acute standard fish tests according to OECD were therefore conducted on 7 d transformation dissolution waters in order to conclude on this outstanding critical question. The 7d dissolution waters used for the toxicity test yielded much higher Fe and P concentrations than the more standardized TDp results given the more stringent conditions. The obtained WSF solutions can therefore be considered as conservative.
The acute fish experiment on Fe3P proved that even at high loadings (100 mg/l) only very mild effects were noted for Fe3P, the latter presumably more corresponding with physical effects at the fish gill due to very high Fe concentrations.
In line with the Transformation dissolution results the Fish validation studies therefore conclude that Fe3P would not require acute environmental classification due to the absence of a toxic P species.
Executive summary:

The reported study describes the acute toxicity of water-soluble fraction (WSFs) of triiron phosphorus (Fe3P), tested at a pH of 6, on the fathead minnow, Pimephales promelas. Due to the anticipated low solubility of Fe3P, a WSF was prepared separately using a loading rate of 100 mg compound per liter of moderately hard reconstituted laboratory water (at a pH of 6.0). The solution was mixed with little to no headspace for 7 days (while maintaining pH 6) followed by filtration through a 0.45 μm filter. P. promelas were exposed to a series of WSF concentrations for 96 hours, starting with 4-day old larval fish. The study was conducted according to the standard Organisation of Economic Co-operation and Development (OECD, 1992), ASTM (2007), and United States Environmental Protection Agency (USEPA, 2002) methodology on acute toxicity testing with fish. Survival was the endpoint analyzed. During the course of the test, samples were taken for analysis of total and dissolved iron and orthophosphorus. Analytical measurements (as measured total Fe, dissolved Fe [0.2 um filtered] and orthophosphate), resulted in values up to 4976 μg/L total Fe, 4721 μg/L dissolved Fe, and 0.5 mg/L PO43 for the 100% Fe3P WSF. Survival was calculated based upon % nominal WSF. The LC50 was >100% Fe3P WSF.