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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
effects on growth of green algae
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Justification for type of information:
An acute toxicity test with the green alga Raphidocelis subcapitata was conducted in freshly prepared T/Dp medium, using Fe3P as test substance. For various reasons it is impossible to mimic the speciation profile of dissolved Fe3P, (using soluble P- and Fe- salts as test substances); therefore the test was conducted in freshly generated 7-day Transformation/Dissolution protocol test medium.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Principles of method if other than guideline:
OECD (Organisation of Economic Co-operation and Development) Method 29, “Transformation/Dissolution of Metals and Metal Compounds in Aqueous Media” (OECD 2001) was employed during the period 20 to 27 October 2020 to generate extract aqueous solutions from a sample of Fe3P. Aqueous solutions were generated from loadings of 10 and 100 mg/L Fe3P (and control; 0 mg/L Fe3P) and these were used, in turn, to conduct of a chronic algae toxicity test.
GLP compliance:
not specified
Remarks:
Testing and documentation for the study were carried out in the spirit of U.S. EPA and OECD Good Laboratory Practice (GLP) standards.

Test material

Constituent 1
Chemical structure
Reference substance name:
Triiron phosphide
EC Number:
234-682-8
EC Name:
Triiron phosphide
Cas Number:
12023-53-9
Molecular formula:
Fe3P
IUPAC Name:
Iron (3+); phosphorous (3-)
Test material form:
solid: particulate/powder
Details on test material:
Source: Höganâs
Reported chemical properties as 15.5% as P and 83.0% as Fe
10% below 3.18 µm
50% below 9.89 µm
90% below 17.66 µm
Specific details on test material used for the study:
The test substance, triiron phosphorus (Lot #3105326), was received from Höganäs Sweden AB (Höganäs, Sweden) on 17 August 2020. Fifty gram of test substance was received in a (double) plastic bag under secure, chain-of-custody. The test substance was a solid with a physical appearance best described as a dark grey fine-grained powder. The certificate of analysis was accompanying the test substance, and was provided by Höganäs Sweden AB (reported chemical properties as 15.5% as P and 83.0% as Fe). A summary of XRD analysis of comparable lots of the substance (but not specific lot number used in the study) was also supplied by Höganäs Sweden AB. Following receipt at OSU AquaTox, the test substance was stored at room temperature in the dark in its original packaging until use.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Total Iron: Analytical samples from each treatment were collected for total Fe analysis from waters after the 7-day T/Dp mixing/filtration (prior to the addition of algae nutrients and buffer) and after the addition of algae nutrients/pH buffer at test initiation. Samples taken at these time points are termed “new”. A composite sample of replicates was taken at test termination and is termed “old”. For sampling, approximately 5 mL of sample was drawn into the syringe to rinse the inside of the syringe and then discarded. Next, 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 (BDH Aristar® Plus; VWR Analytical, Mississauga, ON, Canada) to pH < 2 and refrigerated (1 - 4 °C) prior to analysis.

Dissolved Iron: Analytical samples from each treatment were collected for dissolved (filtered through a 0.45 µm PES filter [VWR; Radnor, PA, USA]) Fe analysis at the same time points as the total iron samples (also termed “new” and “old”). Approximately 20 mL was drawn 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. A new filter was used for each sample. Samples were then preserved with trace metal grade nitric acid (BDH Aristar® Plus; VWR Analytical, Mississauga, ON, Canada) to pH < 2 and refrigerated (1 - 4 °C) prior to analysis.

Phosphorus: Analytical samples from each treatment were collected for phosphorus analysis at the same time points as the total and dissolved Fe samples. Samples were not filtered prior to sampling (i.e., not additionally filtered after the T/Dp 0.2 µm filtration). Approximately 80 mL of sample was poured into a 125 mL HDPE bottle and then refrigerated (1 - 4 °C) prior to analysis.

Orthophosphate: Analytical samples from each treatment were collected for orthophosphate analysis at the same time points as the phosphorus samples. Approximately 80 mL of sample was collected in a 125 mL HDPE sample bottle and immediately frozen at (-20 °C) prior to analysis. Samples were not additionally filtered prior to sampling after the T/Dp 0.2 µm filtration or from the “new” waters in the toxicity test but were filtered through a 0.7 µm GF/F filter (Whatman; Maidstone, UK) in the “old” waters during the toxicity test water-renewal and test termination. These filtrations occurred immediately after defrosting and before analysis.

Test solutions

Details on test solutions:
Toxicity test media was prepared in accordance with the Guidance Document on Transformation/Dissolution of Metals and Metal Compounds in Aqueous Media (Guidance 29; OECD 2001). Sections 2.4.1 to 2.4.8 provide the details of the protocol. Figure 2-1 shows the setup during the T/Dp period and Section 3 (Table 3-1) includes water quality monitoring during the period.
2.4.1 T/Dp - MEDIA
The composition of the T/Dp media was a 10x dilute OECD laboratory water (Table 2-2). The reconstituted water was prepared by adding reagent grade salts to laboratory deionized water (17.6-18.0 Ω), followed by mixing, and aerating (bubbled with air). Following this period, the water was filtered through a 0.2 µm filter (Pall, Supor 47 mm PES Lot#42357613). After filtration, the water was conditioned by bubbling a specialty-produced gas mixture consisting of 0.5% CO2 : 95% compressed air (Airgas, Portland, OR; Lot #16-401918638-1; Appendix E) through polyethylene tubing and an air stone. Conditioning of the reconstituted water with the specialty gas occurred for 25 hours. The media was then used for the loadings of the Fe3P.


Table 2 2 Reconstituted Water recipe for transformation/dissolution media
Chemical Media Concentrations 1
NaHCO3 6.5 mg/L
KCl 0.58 mg/L
CaCl2 x 2H2O 29.4 mg/L
MgSO4 x 7H2O 12.3 mg/L
CO2 concentration (balance is air) in test 0.50% (Section 2.4.4)
Calculated pH 6.09
1 Recipe represents a 10x dilution of full-strength ISO media for pH 6 (OECD 2001).
2.4.2 T/Dp - VESSEL
The T/Dp vessels consisted of 2-Liter glass media bottles (VWR Part #10754-822; 136 mm bottom diameter, 260 mm height), pre-cleaned (hydrochloric acid soaked, deionized water rinsed, oven dried) with a loosely placed holed-silicone stopper. The hole in the stopper allowed the CO2 gas/air mixture to be injected into the water (section 2.4.4).
2.4.3 T/Dp - LOADINGS
Two nominal loading rates of the Fe3P were used: 10 mg/L and 100 mg/L. The test substance was weighed into a plastic weigh boat on a calibrated analytical balance with a readability of 0.1 mg and a repeatability ±0.01 mg. In order to obtain a sufficient quantity of water for both the ecotoxicity test and the associated analytical chemistry, a total of 1.9 L of water was prepared (range from 1.900 to 1.923 L to achieve exact loading targets of the test substance). Prior to adding the Fe3P to the test vessel, a measured volume of pre-conditioned media was added to the vessel. Following this, the weighed Fe3P was added to the vessel, followed by the addition of the final volume of water to rinse the Fe3P from the weigh boat and to also achieve the exact target loading rates (10 mg/L and 100 mg/L based upon the actual weight of the substance). In addition to the 10 mg/L and 100 mg/L Fe3P loadings, a 0 mg/L (no Fe3P; control) was prepared. A total of three batches of T/Dp loadings were prepared: Batch #1 (Fish acute toxicity testing; reported under separate cover; OSU 2021), Batch #2 (Algae toxicity testing; reported here), and Batch #3 (Fish acute toxicity testing solution renewal; reported under separate cover; OSU 2021).
2.4.4 T/Dp - pH CONTROL
To control pH at the target T/D pH of 6.09 (± 0.2 SU) during the course of the 7-day T/D mixing period, a specialty-produced gas mixture consisting of 0.5% CO2 - balance air (Airgas, Portland, OR; Lot #16-401918638-1; Appendix E) was bubbled into the aqueous media through polyethylene tubing and pre-cleaned glass pipettes. Each vessel “batch” had a separate line for the specialty produced gas mixture. A rotameter was used to monitor gas flow rate, in addition to a digital flow-meter (7000 flowmeter; Ellutia, Charleston, SC, USA).
2.4.5 T/Dp - AGITATION
T/Dp vessels were agitated on an orbital shaker table (VWR DS2-500E-2) with a 2” (5.1 cm) orbit set at 100 rpm. The agitation was optimized to maintain the flow of media over the test substance while maintaining the integrity of the surface of the test substance. Agitation continued for the 7-day dissolution period.
2.4.6 T/Dp – TEMPERATURE CONTROL
Temperature was controlled by conducting the 7-day mixing period in a temperature-controlled environmental chamber at 23 ± 2°C. A surrogate temperature vessel (maintained on the same test system) was used to measure temperature daily.
2.4.7 T/Dp – DAILY MONITORING
During the course of the T/D mixing period, temperature was measured using a standard laboratory thermometer. Dissolved oxygen was measured using a HACH HQ40d meter with a calibrated LDO probe. pH was measured using a HACH HQ40d meter with a calibrated electrode. Probes were pre-cleaned prior to and between submersions into each vessel for measurements. Agitation was briefly stopped during the measurements.
2.4.8 T/Dp - FILTRATION
Following the 7-day mixing period, the contents of each vessel were filtered through 0.2 μm hydrophilic polyethersulphone membrane filters (PALL Supor, 47 mm, Lot #42357613), that had been pre-rinsed with deionized water and air dried, to obtain a solution essentially free of solids. Due to the large quantity of solution needing to be filtered for performance of the toxicity tests, T/Dp media was filtered using an oil-free vacuum pump and multiple filters were used.
2.5 TOXICITY TEST SOLUTION
To perform the algae toxicity test in T/Dp media, nutrient stocks were added to each treatment. Nutrient stock solutions were prepared as per OECD methodology (Table 2-3). Each nutrient stock solution was sterilized by filtration using a vacuum filtration apparatus with a 0.20 µm filter (nylon membrane, Whatman, Maidstone, England). The OECD Na2EDTA x 2(H2O) stock was replaced by a stock prepared with dissolved organic carbon (DOC; added as Aldrich Humic Acid). This stock has been previously used for algae toxicity testing with copper (De Schamphelaere et al. 2003), zinc (Heijerick et al. 2002), and cobalt (OSU 2015). The omission of EDTA from the media removes the potential for complexation of metal. Minimal background DOC from Stock #2 (AHA-DOC) was 0.032 mg/L. To perform the algae toxicity test in T/Dp media at the target pH of 6, pH control was achieved through the use of a buffer (Section 2.7). Water quality parameters were measured and are reported in Tables 3-2 and 3-3. The hardness and alkalinity were measured

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
SOURCE:
R. subcapitata was cultured at OSU AquaTox using standard OECD algal medium with EDTA (OECD 2011). The cultures were originally obtained from Aquatic Biosystems (Fort Collins, CO, USA) and have been cultured at OSU AquaTox. At test initiation, the culture was 5 days old and in log-phase growth.

ORGANISM HEALTH:
In order to assess organism health prior to the conduct of each test, monthly reference toxicant tests were performed using cadmium chloride as the toxicant. Test acceptability criteria, appropriate concentration-response relationships, and test sensitivity were achieved in the reference toxicant tests verifying that organism health was optimal prior to the conduct of the test. Prior to test initiation, algal cell health was confirmed by microscopic assessment of algae cell shape and to confirm an axenic culture.

ACCLIMATION
R. subcapitata are cultured at OSU AquaTox in standard OECD (2011) algae media with EDTA. The culture media under ambient conditions has a hardness of 32 mg/L as CaCO3, an approximate pH of 7.5, and a DOC of < 0.5 mg/L. Algae cultures were not specifically acclimated to the water quality parameters of the T/D media. The algae were cultured at 25 ± 2 ºC under continuous fluorescent lighting (at 400 ± 60 ft. candles) and were hand-shaken twice daily. Axenic cultures were maintained and used in the toxicity testing.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg/L as CaCO3
Test temperature:
23-24°C
pH:
6.03-6.19
Nominal and measured concentrations:
nominal: 1, 10, 100 mg Fe3P/L
measured: see any other information on results
Details on test conditions:
The test included two Fe3P loadings (10 mg/L and 100 mg/L), a T/Dp control media (no Fe3P), and a concurrent OECD algae media control with no buffer. Each test treatment was prepared as a batch and then equally distributed to the test chambers, maintaining homogenization as replicate test chambers were filled and analytical samples taken. Three replicate chambers were prepared for each treatment and concurrent algae media control. Six replicate chambers were prepared for the control (0 mg/L Fe3P) treatment. An additional replicate was also added without algae for background blank absorbance measurements through the test.
Test treatments were prepared by filling a graduated cylinder to 80% capacity with T/Dp media (either 0 mg/L (Control), 10 mg/L Fe3P, or 100 mg/L Fe3P), adding the appropriate volume of algal nutrients, adding the appropriate volume of buffer, and then completing the volume with the respective T/Dp media. The pH of each test solution was then individually adjusted using small aliquots of dilute NaOH to the target pH of 6.

pH CONTROL
In order to maintain the target pH of 6.0 in the toxicity test, pH control was obtained through the use of a non-metal-complexing buffer.
The synthetic buffer, MES (2-(n-morpholino) ethanesulfonic acid, monohydrate) was used to control pH. The buffer (CAS# 145224-94-8, Lot #0000149136) was manufactured by J.T. Baker (Phillipsburg, NJ, USA). A stock solution of 200 mM MES was prepared by addition of the buffer to deionized water (18Ω). The stock was then stored in a plastic container in the dark at 1 - 6°C. An aliquot of the buffer was then warmed to test temperature and pH adjusted to 6.00 (utilizing dilute NaOH) to the target pH immediately prior to use.
Aliquots of buffer were added to achieve a final concentration of 5 mM MES. After buffer addition, the pH of each test solution was then re-checked and adjusted using dilute NaOH to the target pH of 6.

TEST CHAMBERS
Test chambers were 250 mL glass Erlenmeyer flasks filled with 100 mL test solution. All chambers were loosely capped with polypropylene soufflé cups to prevent contamination and allow CO2 exchange.

TEST CONDITIONS
The test chambers were housed in a temperature-controlled environmental chamber designed to maintain the test temperature at 25 ± 2 °C. Test chambers were assigned a location according to a computer-generated randomization sheet. Test chambers were re-randomized daily to account for any variability in environmental conditions within the chamber. Lighting was controlled to provide continuous illumination of 400 ± 60 foot-candles (ft-c) using cool-white fluorescent lights (Philips Silhouette F24T5/841/HO). Illumination was measured at test initiation and termination by a light-meter (VWR, Radnor, PA, USA). To keep algae in suspension and for CO2 gas exchange, the test chambers were hand shaken three times daily (a.m., during biological measurements, and p.m.).

TEST INITIATION
Algal inoculum was harvested from a culture that was in log-phase growth (5 days old). Approximately 50 mL of the stock culture was centrifuged for approximately 30 minutes. The resulting clear supernatant was decanted and the algae were resuspended using OECD algae media. This centrifugation, decantation, resuspension process was repeated two additional times. The cell density of the resulting algae suspension was determined using a hemacytometer. The algae suspension was then diluted with the test dilution water to achieve a target inoculum density of approximately 1.0 x 10^6 cells/mL (actual: 0.884 x 10^6 cells/mL). The test was initiated by delivering the appropriate volume of algal inoculum (1130 µL) to each test chamber containing 100 mL of test solution to achieve a nominal cell density of 10,000 cells/mL per chamber.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 14.8 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (total fraction)
Remarks:
total Fe
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 7.7 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Remarks:
dissolved Fe
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 426 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (total fraction)
Remarks:
phosphorus
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 285 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other:
Remarks:
dissolved orthophosphate
Basis for effect:
growth rate
Details on results:
For the testing, the control (0 mg/L Fe3P control) (without EDTA, with pH control) was compared to a concurrent control media (standard OECD media, without EDTA and no pH control) using an Equal variance t-test. Although the concurrent control had one replicate identified as an outlier, there was no difference in outcomes (no difference between concurrent algae media and 0 mg/L Fe3P exposure) whether the outlier was included or removed. On average, the 10 mg/L Fe3P test solution and the 100 mg/L Fe3P test solution performed better (more substantial growth) than the concurrent control media.

No significant effect on algae growth (based upon the two response variables of cell yield and average specific growth rate) was observed after exposure to solutions of Fe3P at loading rates of 10 mg/L and 100 mg/L Fe3P generated from the transformation/dissolution protocol.


Reported statistics and error estimates:
Statistical analysis was performed using the measured loading rate concentrations. Differences in algae growth (average specific growth rate and cell yield) were evaluated using a statistical computer package (Comprehensive Environmental Toxicity Information System [CETIS], version 1.8.7.4, Tidepool Scientific Software, McKinleyville, CA, USA) following the USEPA statistical decision tree (USEPA 2002). As the data met the assumptions of normality and homogeneity, the NOEC and 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 (0 mg/L loading). The CETIS package was also used for the determination of effect concentrations to reduce growth by 10% and 50% relative to control performance (EC10/EC50).
For the test, the control water (0 mg/L Fe3P T/Dp, with OECD algal nutrients without EDTA and buffer for pH control) was compared to a concurrent control media (OECD media without EDTA and no pH control) using an Equal variance t-test.

Any other information on results incl. tables

Total and Dissolved Iron

In the T/Dp control (0 mg/L Fe3P loading), measured concentrations for both total and dissolved iron were below the detection limit (< 0.9 µg Fe/L). Concentrations were minimally higher in the 10 mg/L Fe3P loading batch (1.9 µg total Fe/L, 1.0 µg dissolved Fe/L) and 13.8 µg total Fe/L and 11.7 µg dissolved Fe/L in the 100 mg/L Fe3P loading batch. In the T/Dp solutions, when compared to their total Fe counterparts, dissolved Fe concentrations were 53% of total Fe for the 10 mg/L Fe3P loading, and 85% of total Fe for the 100 mg/L Fe3P loading. In the toxicity test, values are reported under two categories: “new” and “old.” “New” waters were sampled directly from the freshly prepared batch of solutions after the addition of algae nutrients and pH controlling buffer. “Old” waters consisted of a composite of replicate solutions at test termination. When compared to their total Fe counterparts, dissolved Fe concentrations ranged from 99 % of total Fe for the 10 mg/L Fe3P loading in the “new” waters, and 75% of total Fe for the 100 mg/L Fe3P loading in the “new” waters. Higher concentrations of total and dissolved Fe were measured in the new waters, while reduced concentrations were measured in the old waters. No visible flocculent or precipitate was observed in the toxicity test.

Method blanks were run with each analysis and consisted of deionized water treated identically as the samples through the entire process including acidification. Blank measurements were all below the detection limit(<0.9 µg/L Fe). Quality control samples were analyzed with duplicate samples, standard concentrations, and an over-spike of a known addition of Fe and analyzed to calculate % recovery for the samples. Quality control samples of 50 and 500 µg Fe/L resulted in recoveries of 97.3 to 99.3%, duplicate samples resulted in 98.7 – 109.5% recovery, and standard additions of 1000 µg Fe/L resulted in 97.3 – 100.4% recoveries, all within the acceptability criteria of 85-115% recovery (USEPA 1994).

Phosphorus

In the T/Dp solution control (0 mg/L Fe3P loading), background concentrations of phosphorus and orthophosphate were 20µg/L and 14µg/L, respectively. For the T/Dp solutions, phosphorus concentrations were 56µg/L in 10 mg/L Fe3P loading and 62µg/L in 100 mg/L Fe3P loading. Inthe toxicity test, concentrations of phosphorus primarily were due to the added essential nutrients for algae toxicity testing (364 µg P/L, added as KH2PO4), with measured values in the control media, ranging from 390 to 392 µg/L. Phosphorus concentrations ranged from 427 to 432 µg/L in the 10 mg/L Fe3P loading and 421 to 431 µg in the 100 mg/L Fe3P loading. Concentrations remained similar between the new test water and the old test waters. Quality control samples remained within+/- 10% of their theoretical values.

Orthophosphate (Soluble Reactive Phosphorus)

In the T/Dp solution, the control (0 mg/L Fe3P loading) had a measured orthophosphate concentration of 14 µg P/L (shown as soluble reactive phosphorus in µg/L). For the T/Dp solutions, orthophosphate concentrations were 24µg/L in 10 mg/L Fe3P loading and 11µg/L in 100 mg/L Fe3P loading. In the toxicity test concentrations of orthophosphate (soluble reactive phosphorus) were also due to the added essential nutrients for algae toxicity testing ranging from 390 µg/L, 402, and 390 µg/L in the control, 10 mg/L Fe3P, and 100 mg/L Fe3P test solutions, respectively.Concentrations remained similar between the new test water and the old test waters. Concentrations in the “old” test waters were approximately half of their “new” water counterparts, lowering to187 µg/L, 199, and 179 µg/L in the control, 10 mg/L Fe3P, and 100 mg/L Fe3P “old” test solutions, respectively

Quality control samples remained within+/- 10% of their theoretical values.

Table: Summary of Measured Concentrations from T/Dp

Analyte

Measured Concentration *

0 mg/L (Control)

10 mg/L Fe3P Loading

100 mg/L Fe3P Loading

Batch #2 (Algae testing)

Batch #2 (Algae testing)

Batch #2 (Algae testing)

Total Iron (µg/L)

BMDL

< 0.9

1.9

13.8

Dissolved Iron (µg/L)

BMDL

< 0.9

1.0

11.7

Phosphorus

(µg/L)

20

56

62

Orthophosphate (SRP)

(µg/L)

14

24

11

BMDL = Below method detection limit

* T/Dp Batch #1 and #3 were used in fish toxicity testing and are reported separately (OSU 2021).

 


 

Table: Summary of Measured Iron Concentrations from toxicity test

Loading

Total Iron (µg/L)

Dissolved Iron (µg/L)

0 hrs

72 hrs

Average

Std Dev.

0 hrs

72 hrs

Average

Std Dev.

Concurrent algae media

3.0

1.1

2.1

1.3

3.0

BMDL

< 0.9

1.7

1.8

0 mg/L Control

3.5

BMDL

< 0.9

2.0

2.2

3.0

BMDL

< 0.9

1.7

1.8

10 mg/L Fe3P loading

6.9

1.6

4.3

3.7

6.8

BMDL

< 0.9

3.6

4.5

100 mg/L Fe3P loading

20.0

9.5

14.8

7.4

15.0

BMDL

< 0.9

7.7

10.3

BMDL = Below method detection limit. Sample assigned a value of half the detection limit (0.45 µg/L) to determine average.

 

 

Table: Summary of Measured Phosphorus and Orthophosphate Concentrations from toxicity test

Loading

Phosphorus (µg/L)

Orthophosphate (µg/L)

0 hrs new

72 hrs old

Average

Std Dev.

0 hrs new

72 hrs old

Average

Std Dev.

Concurrent algae media

Not measured

Not measured

0 mg/L Control

392

390

391

1

390

187

289

144

10 mg/L Fe3P loading

427

432

430

4

402

199

301

144

100 mg/L Fe3P loading

421

431

426

7

390

179

285

149

 


Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Samples (for total Fe, dissolved Fe [0.45 um filtered], phosphorus, and orthophosphate [soluble reactive phosphorus]) resulted in measured concentrations of < 0.9 µg/L total Fe, < 0.9 µg/L dissolved Fe, 20 µg/L P, and 14 µg/L orthophosphate (as soluble reactive phosphorus) in the Fe3P control T/Dp, measured concentrations of 1.9 µg/L total Fe, 1.0 µg/L dissolved Fe, 56 µg/L P, and 24 µg/L orthophosphate in the 10 mg/L Fe3P T/Dp , and measured concentrations of 13.8 µg/L total Fe, 11.7 µg/L dissolved Fe, 62 µg/L P, and 11 µg/L P as orthophosphate in the 100 mg/L Fe3P T/Dp solution. For toxicity testing with algae, nutrients were added to each loading of the T/Dp solutions per OECD methodology. This nutrient addition included approximately 3.5 µg/L total Fe and 372 µg/L P as essential nutrients. From the nutrient addition, measured concentrations of phosphorus and orthophosphate at test initiation in the control media and 10 mg/L and 100 mg/L Fe3P loadings ranged from 392 to 427 µg/L P and 390 to 402 µg/L orthophosphate (as soluble reactive phosphorus). Therefore the majority of the phosphorus (and orthophosphate) were a result of the essential OECD nutrients added to the T/Dp solutions for algae growth in the toxicity test.
The study reported here demonstrates that chronic exposure of the green algae, R. subcapitata, to Fe3P at loading rates of 10 and 100 mg/L in transformation/dissolution protocol media resulted in no observable toxicity.
Executive summary:

The study reported herein describes the chronic toxicity of triiron phosphide (Fe3P), tested at a pH of 6, on the freshwater green algae, Raphidocelis subcapitata. In order to prepare solutions for testing, the standard Organisation of Economic Co-operation and Development Guideline 29 Transformation/Dissolution protocol (T/Dp; OECD 2001) was employed, using loading rates of 10 mg/L and 100 mg/L Fe3P, and a control. The T/Dp was performed in 10x dilute media, at a pH of 6.09, with an agitation period of 7 days. Both the T/Dp and the toxicity test were performed at a pH of approximately 6 to ensure maximum solubility and bioavailability of the Fe3P. In order to perform the toxicity test in T/Dp media following the 7 day T/Dp procedure, essential algae nutrients were added to each exposure concentration. In addition, a non-metal complexing buffer was added to the solutions to control pH during the test. R. subcapitata were exposed to the Fe3P T/Dp solutions (0, 10, and 100 mg/L Fe3P loadings) for 72 hours under static test conditions.The study was conducted according to the standard OECD Guidance 201 (OECD 2011) on chronic toxicity testing with algae. Average specific growth rate and cell yield were the evaluated endpoints. Following the 7-day T/Dp duration and during the course of the toxicity test, samples were taken for analysis of total and dissolved iron (Fe), phosphorus (P) and orthophosphate. Analytical samples directly from the T/Dp solutions (as measured total Fe, dissolved Fe [0.45 um filtered], phosphorus, and orthophosphate [soluble reactive phosphorus]) resulted in measured concentrations of < 0.9 µg/L total Fe, < 0.9 µg/L dissolved Fe, 20 µg/L P, and 14 µg/L (orthophosphate; as soluble reactive phosphorus) in the Fe3P control T/Dp, measured concentrations of 1.9 µg/L total Fe, 1.0 µg/L dissolved Fe, 56 µg/L P, and 24 µg/L orthophosphate in the 10 mg/L Fe3P T/Dp , and measured concentrations in the 100 mg/L Fe3P T/Dp solutions of 13.8 µg/L total Fe, 11.7 µg/L dissolved Fe, 62 µg/L P, and 11 µg/L P as orthophosphate. For toxicity testing with algae, nutrients were added to each loading of the T/Dp solutions per OECD methodology. This nutrient addition included approximately 3.5 µg/L total Fe and 372 µg/L P as essential nutrients. From the nutrient addition, measured concentrations of phosphorus and orthophosphate in the control media (and 10 mg/L and 100 mg/L Fe3P) ranged from 392 to 427 µg/L P and 390 to 402 µg/L orthophosphate (as soluble reactive phosphorus). Therefore the majority of the phosphorus (and orthophosphate) were a result of the essential OECD nutrients added to the T/Dp solutions for algae growth in the toxicity test. No effect on algae growth was observed in any Fe3P T/Dp solutions when compared to the control, resulting in a no observable effect concentrations (NOEC) at 10 mg/L Fe3P and 100 mg/L Fe3P loadings and an EC10 and EC50 of >100 mg/L Fe3P loading.