potassium ferrite

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

430-010-4

EC Name:

-

Cas Number:

12160-44-0

Molecular formula:

K2Fe22O34

IUPAC Name:

hydrate iron potassium hydride

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report):Potassium ferrite
- Physical state: Reddish brown powder
- Expiration date of the lot/batch: 6 August 1997
- Storage condition of test material: Room temperature, dark, desiccated

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples of media for chemical analysis were taken via the tap of the preparation vessel at the start of the test period, followed by addition of stock culture, and where appropriate sulphuric acid (0.1 M) for those samples requiring pH adjustment (i.e. 100, 220, 460 and 1000 mg/L loading rates). At the end of the 72 h test period, one of the replicate test flasks from each of the nine treatments (used for algal cell counting) and a control were used for analysis. There was constant agitation of the test media during the lest and therefore little likelihood of any coating of Potassium Ferrite on the botiom of the lest vessels. Media were sampled into conical flasks, which were then stoppered and stored prior to analysis.

Test solutions

Vehicle:

no

Details on test solutions:

As Potassium Ferrite was expected to be sparingly soluble in water, the test media were produced using WAF methodology. A preliminary study was carried out specifically to determine the most suitable mixing regime for the preparation of the test media using this approach. Based on the findings of a preliminary study, WAFs were prepared by adding measured amounts of test substance to measured volumes of test medium and using a fast stir rate (2-3 cm vortex depth) for approximately 24 hours to ensure even dispersion through the test media. After stirring the contents of the vessels were left to stand for approximately 24 hours to allow the majority of the undissolved material to separate out. The aqueous phases - the WAFs - were then drawn off for use in the tests. Control media were subject to the same regime but did not contain Potassium Ferrite. The test media (loading rate >100 mg/L) were alkaline due to the presence of KOH and/or K2C03.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Procedure for the culture of Raphidocelis subcapitata (Nygaard):
Axenic stock cultures of R. subcapitata (formerly Selenastrum capricomutum) are maintained on agar plates. These are used to inoculate liquid cultures which, while in exponential phase growth, are in turn used to inoculate test solutions.

- Source:
The axenic culture of R. subcapitata (strain number CCAP 278/4) was obtained from the Institute of Freshwater Ecology, Windermere.
- Maintenance of cultures:
Agar plates are prepared by adding 1.5% mass/volume agar to liquid medium prior to autoclaving. On cooling the agarified medium is poured into 9 cm diameter sterile plastic petri dishes and allowed to set. The algal cultures are streaked onto these plates and maintained under continuous illumination at nominally 21-25°C in a Gallenkamp vertical incubator. The cultures are renewed approximately every fortnight. Cultures in liquid medium are initiated with cells transferred on a sterile loop from an agar plate. The cultures are grown as 100 mL batch cultures in 250 mL Erlenmeyer flasks for up to 4 days. The cultures are maintained at nominally 21-25°C in a Gallenkamp orbital incubator under constant illumination.
- lnoculation of test cultures:
The culture inoculum volume introduced at the start of the test is sufficient to give an initial concentration of 5000 cells/mL in the vessels. The cell concentration of an exponentially growing starter culture is measured using a Coulter Counter or Coulter Multisizer and the required inoculum volume calculated. All flasks used for a particular test are inoculated from the same starter culture.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Not reported

Test temperature:

23.2-23.7°C

pH:

6.7-11.3 (maximum change recorded in any treatment group: 2.8)

Dissolved oxygen:

Not reported

Nominal and measured concentrations:

- Nominal: 0, 46, 100, 220, 460 and 1000 mg/L.
- Measured: No dissolved iron was detected in test media at the start or end of the study at or above the Iimit of detection of 0.1 mg/L except for one value (0.1 mg/L) for a freshly prepared medium from the highest loading (1000 mg/L) which involved pH adjustment. The concentrations of iron in suspension were essentially dose-related ranging from 0.2 mg/L (loading rate of 46 mg/L) to approximately 1 mg/L (loading rate of 1000 mg/L). Little decrease in suspended material, probably as a result of the constant agitation, was evident over the 72 h test period.

Details on test conditions:

- Equilibration studies:
Prior to the toxicity tests an equilibration study was performed to determine the time needed to equilibrate a 1000 mg/L sample of Potassium Ferrite and water under conditions similar to those used to prepare the WAFs for use in toxicity tests. Five glass flasks were each filled with approx. 2.4 litres of reconstituted fresh water. Potassium Ferrite was added to four of the replicates at a nominal loading rate of 1000 mg/L. The fifth vessel was used as a control. Stirring of one vessel containing Potassium Ferrite was stopped at nominal periods of 24, 48, 72 and 96 hours. The contents of each vessel were then left to settle for approximately 24 hours before drawing off a sample of the aqueous phase (WAF) for chemical analysis. The control vessel was sampled repeatedly along with the treated vessels. Only two glass aspirators were filled with 22 litres of mains borehole water. Potassium Ferrite was added to one vessel at a nominal loading rate of 1000 mg/L. The remaining vessel was used as a control. The vessel containing Potassium Ferrite was continuously stirred for 96 hours and 100 mL samples of the aqueous phase (WAF) were Iaken at approximately 24, 48, 72 and 96 hours. These samples were allowed to settle for 24 hours, prior to taking the required volume for chemical analysis. The control vessel was sampled for chemical analysis at the same time and using the same method as the treated vessels. Following the results of the equilibration study, a nominal stirring period of 20-28 h with a settling period of 20-28 h was adopted for the production of WAFs in the subsequent definitive toxicity studies.

- Definitive toxicity test:
A growth inhibition test was carried out in 287 mL full volume Erlenmeyer flasks. Four flasks containing sodium bicarbonate (2 mL) and test medium (98 mL) were prepared for each WAF of Potassium Ferrite. At four of the loading rates the test medium was significantly more alkaline than the control medium. The test was therefore conducted with and without pH adjustment. For the set requiring pH adjustment, sulphuric acid (0.1 M) was used to adjust a portion of these WAFs to within 0.3 (pH value) of the control medium, except for the lowest loading rate which was within range. Both sets of WAFs (adjusted and non-adjusted) were tested in this study. Seven flasks containing algal growth medium and sodium bicarbonate solution were also prepared to serve as controls. Three out of each set of four flasks containing test substance and six of the control flasks were inoculated with sufficient R. subcapitata to give an initial concentration of 5000 cells/mL. The remaining ten flasks were not inoculated but used to determine background particle counts in the absence of R. subcapitata. The flasks were sealed with glass stoppers and placed in a cooled orbital incubator (100 cycles/min) under constant illumination (4380 Iux). A nutrient medium is prepared by dissolving Analargrade salts in reverse osmosis filtered water. Nutrient concentrations are according to standard except that boric acid is present at 105 µg/L (in stead of 184 µg/L), and the final concentration of sodium bicarbonate is 50 mg/L (instead of 15 mg/L). The medium (excluding sodium bicarbonate) is autoclaved at 1.0 kg/cm2 for 15 min and allowed to cool. When used in toxicity tests, the sodium bicarbonate solution is added directly to each control and test flask. CeII counts were made, using a Coulter Multisizer, on samples Iaken from each flask at the start of the test and then at approximately 24 h intervals.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: the exposure Ievels in the tests have been expressed in terms of loading rates i.e. the mass of test substance per unit volume of test medium (mg/L)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: the exposure Ievels in the tests have been expressed in terms of loading rates i.e. the mass of test substance per unit volume of test medium (mg/L)

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: the exposure Ievels in the tests have been expressed in terms of loading rates i.e. the mass of test substance per unit volume of test medium (mg/L)

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: the exposure Ievels in the tests have been expressed in terms of loading rates i.e. the mass of test substance per unit volume of test medium (mg/L)

Details on results:

The ranges within which the estimated 72-h EbL50 and ErL50 values lie for both the pH adjusted and non-adjusted tests are 220-460 and 46-100 mg/L for EbL50 and 460-1000 and 220-460 mg/L for ErL50. In the non-adjusted test, the statistically derived 72-h NOEL for growth rate was 46 mg/L , for biomass it was less than the lowest loading rate of 46 mg/L reflecting the high precision of the test as the percentage reduction in biomass was only 8% at the lowest loading rate. In the pH adjusted test the statistically derived 72 h NOELs for growth rate and biomass were both 100 mg/L.

Reported statistics and error estimates:

Williams' test was used to compare differences in areas under the growth curves and average specific growth rates between control and treated cultures. The highest concentration at which there was no significant difference (p ≤ 5%) between treated and control cultures was identified as the NOEL.